PT89683B

Anthracycline immunoconjugates having a novel linker and methods for their production.

Abstract

The present invention relates to novel immunoconjugates, methods for their production, pharmaceutical compositions and method for delivering cytotoxic anthracyclines to a selected population of cells desired to be eliminated. More particularly, the invention relates to immunoconjugates comprised of an antibody reactive with a selected cell population, having a number of anthracycline molecules linked to its structure. Each anthracycline molecule, having a keto group at the C-13 position, is conjugated to the antibody via a linker arm and is bound to that linker arm via an acid-sensitive acylhydrazone bond at the 13-keto position of the anthracycline. The immunoconjugates and methods of the invention are useful in antibody-mediated drug delivery systems for the preferential killing of a selected cell population in the treatment of diseases such as cancers and other tumors, non-cytocidal viral or other pathogenic infections, and autoimmune disorders.

PT89683B, drawing sheet 1 — Sheet 1 of 26

Term

Term ended

Expired 10 February 2004, 22.6 years ago.

10 claims: 4 independent, 6 dependent

1
REIVINDICAÇÕES 1. - Processo para a preparação de um imunoconjugado comportando cerca de 4 a 10 moléculas de antraciclina ligadas a um anticorpo reactivo com uma população celular escolhida que deve ser morta, caracterizado pelo facto de se ligar um grupo ceto no átomo de carbono em posição 13 e de ligar ao anticorpo através de um ligando unido por covalência, através de uma ligação de acil-hidrazona na posição 13-ceto, à antraciclina.
2
- Processo de acordo com a reivindicação 1, caracterizado pelo facto de o ligando comportar adicionalmente uma ligação dis84 sulfureto ou tio-éter.
3
- Processo para a preparação de un imunoconjugado que comporta pelo menos uma molécula de antraciclina com um grupo ceto no átomo de carbono em posição 13 ligado por meio de um ligando a um anticorpo reactivo com uma população celular escolhida que deve ser morta, caracterizado pelo facto de se unir o ligando ã antraciclina por covalência através de uma ligação de acil-hidrazona na posição 13-ceto da antraciclina e de se incluir, adicionalmente, uma ligação dissulfureto ou tio-éter.
4
- Processo de acordo com a reivindicação 1, caracterizado pelo facto de se escolher a molécula de antraciclina no grupo constituído por adriamicina, daunomicina, detorubicina, carminomicina, idarubicina, epirubicina, esorubicina, 4'-THP-adria micina, AD-32 e 3 ' -desamino-3 1 - (3-ciano-'4-morfolinil) -doxorubicina.
5
- Processo de acordo com uma qualquer das reivindicações 1 ou 3, caracterizado pelo facto de a antraciclina ser adriamicina ou daunomicina.
6
- Processo de acordo com uma qualquer das reivindicações 1 ou 3, caracterizado pelo facto de o anticorpo ser reac tivo para células de tumor. • · »
7
- Processo de acordo com a reivindicação 6, caracterizado pelo facto de o anticorpo ser reactivo com um antigénio associado com carcinomas, melanomas, linfomas ou sarcomas do tecido ósseo ou dos tecidos moles.
8
- Processo de acordo com uma qualquer das reivindicações 1 ou 3, caracterizado pelo facto de o anticorpo ser reactivo com o antigénio CD37 que se encontra em linfomas de células B.
9
- Processo de acordo com a reivindicação 1, caracterizado pelo facto de o anticorpo ser um anticorpo monoclonal
10
- Processo de acordo com uma das reivindicações 1 ou 3, caracterizado pelo facto de o anticorpo monoclonal ser 5E9, 3A1, L6, G28.1 ou G28.5 e como antraciclina a adriamicina,

Independent claims10

479 paragraphs in 21 sections, as filed

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel anthracycline immunoconjugates and methods for their preparation. More particularly the present invention relates to immunoconjugates consisting of an antibody reactive with a cell population selected to be deleted, said antibody having several anthracycline cytotoxic molecules covalently linked to its structure. Each anthracycline molecule is conjugated to the antibody via a linker (linker), and anthracycline is attached to that linker via an acid-sensitive acylhydrazone linkage at the 13-keto position of anthracycline. A preferred aspect of the present invention relates to an adriamycin immunoconjugate wherein adriamycin is coupled to the coupling element via an acylhydrazone bond at the 13-keto position. 0 element of

The coupling additionally contains a disulfide or thioether bridge as part of the coupling of the antibody to the immunoconjugate. On the other hand, according to the present invention, novel anthracycline ac1-hydrazone derivatives are synthesized and used for the preparation of the immunoconjugates of the present invention.

The acid sensitive acylhydrazone binding of the immunoconjugates of the present invention allows the release of anthracycline from the immunoconjugate into the external or internal acidic environment of the target cell. Accordingly, the immunoconjugates and methods of the present invention are useful in systems for delivering antibody-mediated drugs for preferential annihilation of a selected cell population in the treatment of diseases such as cancers or other tumors, non-cytocidal viral infections or other conditions. pathogenic infections and in cases of autoimmune diseases.

BACKGROUND OF THE INVENTION

Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Several studies have shown that anthracyclines can eventually annihilate cells in a variety of ways including: 1) intercalating drug molecules into a cell's DNA thereby inhibiting DNA-dependent nucleic acid synthesis; 2) drug production of free radicals which then react with cell macromolecules to damage cells or 3) interactions of drug molecules with cell membrane [see, for example, C. Peterson et al., Transport and Storage of Anthracyclines in Experimental Systems and Human Leukemia, in Anthracycline Antibiotics in Cancer Therapy, FM Muggia et al. (Ed.s), p. 132 (Martinus Nijhoff Publishers 1982); see also, NR Bachur, Free Radical Damage, id. at pp. Due to their cytotoxic potential anthracyclines have been used to treat various types of cancer such as leukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma and sarcomas [see, for example, PH Wiernik, Current Status Of Adriamycin And Daunomycin In Cancer Treatment, Anthracyclines: Current

Status And New Developments, ST Crooke et al., Ed. 273-94 (Academic Press 1980)]. Commonly used anthracyclines include adriamycin and daunomycin.

Although these compounds may eventually be useful in the treatment of neoplasms and other disease states in which a targeted cell population is to be eliminated, their therapeutic efficacy is often limited by the dose-dependent toxicity which is a factor associated with their administration. For example, in the treatment of tumors, myelosuppression and cardiotoxicity are typical adverse side effects [see ST Crooke, Goals For Anthracycline Analog Development At Bristol Laboratories, Anthracyclines: Current Status And New Developments, supra, p. 11]. As a result they have been

Attempts have been made in the treatment of tumors to improve the therapeutic effects of these compounds by binding anthracyclines to antibodies directed against tumor associated antigens. By this process the drug may be delivered to or directed to the tumor site and its toxic side effects on normal cells throughout the body may be diminished. Immunoconjugates consisting of adriamycin (ADM) or daunomycin (DAU) anthracyclines coupled with polyclonal or monoclonal antibodies directed against tumor associated antigens are known in the art [see, for example, J. Gallego et al., Preparation Of Four Daunomycin-Monoclonal Antibody 791T / 36 Conjugates With Anti-Tumor Activity, Int. J. Cancer, 33, p. 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, pp. 9-14. 5-27 (1982)].

The most frequently used approaches for coupling an anthracycline to an antibody have used a binding to the amino sugar radical of anthracyclines. For example, the amino sugar radical is oxidized by treatment with sodium periodate and is directly coupled to the lysine residues in the antibody by forming a Schiff base [see, for example, E. Hurwitz et al., The Covalent Binding Of Daunomycin And Adriamycin To Antibodies, With Retention Of Both Drug And Antibody Activities, Cancer Res. 1182-86 (1975)]. Alternatively, anthracyclines have been linked to

antibodies through carbohydride-mediated joints of the amino-sugar radical of anthracyclines to the carboxyl groups of the antibody [see, for example, E. Hurwitz et al., supra]. In addition, anthracyclines also

<td>have been linked</td><td>to</td><td>antibodies by</td><td colspan="2">radical interconnection</td>

<td>amino sugar</td><td colspan="2">drug and groups</td><td>amino</td><td>of antibody with</td>

<td>glutaraldehyde [</td><td>to see,</td><td>for example,</td><td>M.</td><td>Belles-Isles and</td>

<td>contributors,</td><td>In</td><td>Vitro Activity</td><td>Of</td><td>Daunomycin-Anti-</td>

-AlphaFetoprotein Conjugate On Mouse Hepatoma Cells, Br. 841-42 (1980)]. However, studies with immunoconjugates whose amino sugar radical of the anthracycline molecule was modified by antibody binding indicate a loss of cytotoxic activity of the conjugated drug [see, for example, R. Arnon et al., Supra, at pp. 7-8], On the other hand, studies of anthracycline analogs indicate that modifications of anthracyclines in their amino sugar radicals result in a decrease in the cytotoxic activity of the analogous drug compared to the parent drug [see, for example, K. Yamamoto et al., Antitumor Activity Of Some Derivatives Of Daunomycin At The Amino And Methyl Ketone Functions, J. Med. Chem. 872-75 (1972)].

Other immunoconjugates have also been prepared wherein the anthracycline known by the name daunomycin was bound directly to an antibody at the 14-position of the drug carbon atom (C-14). However, the selective cytotoxic activity of these immunoconjugates on tumor cells was not easily reproducible and only revealed

<td colspan="2">consistent</td><td>for</td><td>concentration of</td><td> 20</td><td>pg / ml</td><td>[see J.</td>

<td>Gallego e</td><td colspan="2">contributors,</td><td>above].</td><td></td><td></td><td></td>

<td></td><td> 0</td><td>request of</td><td>japanese patent</td><td>noQ</td><td> 274658</td><td>reveals and</td>

<td>describe</td><td>The</td><td>conjugation</td><td colspan="2">of an anthracycline</td><td>to one</td><td>antibody</td>

through an acylhydrazone union at the 13-keto position. This conjugation was performed using methods involving antibody transformation and subsequent reaction of that derivative with an anthracycline. These methods are not advisable since transformation of the antibody involves undesirable non-specific reactions and very low anthracycline: antibody ratios are obtained.

According to the first method the antibody was treated with a carbohydride in the presence of hydrazine to provide a derivative of the hydrazide group antibody which was then reacted with anthracycline such that such anthracycline was directly bound to the structure of the hydrazine. antibody. However, the resulting immunoconjugates are prone to aggregation of antibody molecules. On the other hand, since this method requires the presence of carboxylic acid groups in the antibody molecule whose number is limited, these immunoconjugates exhibit weak anthracycline: antibody ratios (approximately 1.1-1.3).

The second method involves reacting the antibody with succinic anhydride to provide an amino acid derivative of the antibody. This derivative is then reacted with hydrazine to provide a hydrazine antibody derivative which is then reacted with an anthracycline, the daunomycin compound. This second approach has the disadvantage that such reaction of the antibody derivative with hydrazine is of a non-specific type, leading to the production of a mixture of different antibody derivatives in addition to the desired hydrazid derivative. Thus, as indicated in patent specification no.Q 274658, the molar ratio between anthracycline and antibody is quite low (approximately 1, see Japanese patent application, page 264, column 1).

Finally, there are other anthracycline hydrazones disclosed and described by GL Tong et al., J. Med. Chem. 732-37 (1978); T. Smith et al., J. Med. Chem. 280-83 (1978); and RTC Brownlee et al., J. Chem. Soc., Pp. 659-61 (1986). See also U.S. Patent Application No. 4112217 which discloses and describes daunomycin and adriamycin bishydrazones.

According to other studies, anthracyclines have been linked to high molecular weight carriers such as dextran or polyglutamic acid for the purpose of enhancing cytotoxic activity and reducing drug toxicity [see, for example, R. Arnon et al., Supra, p. 5 and E. Hurwitz et al., Soluble Macromolecules As Carriers For Daunorubicin, J. Appl. Biochem., 2, pp. 25-35 (1980)]. These carrier-linked anthracyclines have also been covalently

linked to antibodies directed against tumor associated antigens to form immunoconjugates that target the cytotoxic drug specifically to tumor cells. For example, adriamycin has been linked to one of these anti-tumor antibodies via a carboxymethyl dextran bridge of a hydrazide wherein the adriamycin molecule has been linked to a carboxymethyl dextran group hydrazine derivative on the side chain of the carbonyl group in C-L3 of the adriamycin tetracycline ring to form a hydrazone. 0 The antibody was then ligated to the glutaraldehyde dextranhydrazide derivative to provide an adriamycin-dextran-antibody conjugate [see, R. Arnon et al., Monoclonal Antibodies As Carriers For

Immunotargeting of Drugs, in Monoclonal Antibodies For Cancer Detection And Therapy, RW Baldwin et al., Ed. 365-83 (1985) and E. Hurwitz et al., A Conjugate Of Adriamycin And Monoclonal Antibodies To Thy-1 Antigen Inhibits Human Neuroblastoma Cells In Vitro, Ann. NY Acad. I know., 417, pp. 125-36 (1983)].

However, the use of carriers has some disadvantages. For example, immunoconjugates containing carrier vehicles are quite large in size and are rapidly removed by the reticuloendothelial system in vivo [see, for example, RO Dillman et al., Preclinical Trials With Combinations And Conjugates Of T101 Monoclonal Antibody And Doxorubicin, Cancer Res. , 46, pp. 4886-91 (1986)]. This rapid removal of immunoconjugates that

Containing a carrier may possibly not be advantageous for therapeutic purposes since the conjugated drug may eventually never reach its intended site of action, that is, the selected group of cells to be annihilated. On the other hand, the presence of a high molecular weight vehicle can negatively affect the stability of the immunoconjugate and has been shown to reduce antibody binding activity to the conjugate [see, for example, MJ Embleton et al., Antibody Targeting Of Anti-Cancer Agents, in Monoclonal Antibodies For Cancer

Detection And Therapy, RW Baldwin et al., Ed. 323-24 (1985)]. Moreover, in tumor cell studies, there was no evidence that monoconjugates containing high molecular weight vehicles are capable of localizing to tumor cells in vivo. Compare with CHJ Ford and colleagues, Localization And Toxicity Study Of A Vindesine-Anti-CEA Conjugate In Patients With Advanced Cancer, Br. J. Cancer, 47, 35-42 (1983). 0 which demonstrates the localization of drug-antibody conjugates conjugated directly to tumor cells in vivo.

Thus conjugation of anthracyclines with antibodies was disclosed and described using specific linkages and vehicles. As evidenced above the use of these immunoconjugates imposes distinct disadvantages that depend on the specific binding or vehicle used.

SUMMARY OF THE INVENTION

Accordingly the present invention provides

a novel chemical variant for the binding of various cytotoxic anthracycline molecules via a coupling to an antibody directed against a targeted cell population that is the target to be annihilated. In accordance with the present invention each anthracycline molecule is linked to an antibody via a coupling element and anthracycline is attached to that coupling via an acylhydrazone bond at the 13-keto position of anthracycline to provide the novel ones. immunoconjugates of the present invention. For example, a preferred embodiment of the present invention involves the synthesis of a novel adriamycin hydrazone derivative (ADM-HZN) which was then condensed with a thiolated antibody, resulting in the coupling of anthracycline to the antibody via a coupling element. The acylhydrazone bond formed at position C-13 of adriamycin (ADM) serves as a site for coupling the ADM to the coupling element. In addition, there is a disulfide bridge contained in the coupling element that constitutes the coupling site to the antibody. In another preferred embodiment, the ADM-HZN derivative was reduced to generate a sulfhydryl group and the resulting new hydrazone derivative was condensed with a maleimide-transformed antibody. This procedure resulted in the formation of a coupling element having an acylhydrazone bond which forms the coupling site of that coupling member to the ADM position C-13 and which has a thioether bond contained in the coupling member forming part of that coupling.

binding element to the antibody. As is apparent from these variants the present invention provides novel acylhydrazone derivatives from anthracyclines useful for the preparation of the immunoconjugates of the present invention.

The immunoconjugates of the present invention have anthracycline: antibody molar ratios of approximately 4 to 10 and both retain the antibody and cytotoxic activity of the drug which allows to annihilate the selected target cells. The acid-sensitive hydrazone bond which exists at the anthracycline coupling site to the immunoconjugate coupling element and additionally the disulfide or thioether bridges contained in that coupling according to preferred embodiments of the present invention are ideally suited for drug release It is active under reducing and acidic conditions such as those typically found within a cell, for example in lysosomal vesicles.

The immunoconjugates of the present invention may be used in pharmaceutical compositions such as those incorporating a pharmaceutically effective amount of at least one immunoconjugate of the present invention and a pharmaceutically acceptable carrier. The present invention also relates to methods for the selective delivery of cytotoxic drugs to a selected target cell population to be eliminated and further relates to methods for treating a mammal by a pharmaceutically acceptable process using an amount

Pharmaceutically effective L of a composition of the present invention.

Advantageously, the immunoconjugates, pharmaceutical compositions and methods now described and disclosed provide a useful approach for targeting cytotoxic anthracycline drugs to a selected population in order to achieve preferential annihilation of such target cells in the treatment of disease. such as cancers and other tumors, non-cytocidal viral infections or other pathogenic infections and even in the case of autoimmune diseases.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a schematic form of the synthesis of the novel ADM-HZN derivative used for the preparation of the immunoconjugates of the present invention.

Figure 2 is a schematic form of the immunoconjugate synthesis of one embodiment of the present invention in which a monoclonal antibody (MAB) (or ACM) was first thiolated using alternatively SPDP (N-succinimidyl-3- (2- pyridyl dithio) or 2-IT (2-iminothiolane) and then thiolated antibody was reacted with ADM-HZN to provide an immunoconjugate of the present invention, with a 13-keto hydrazone bond of the ADM compound and a disulfide bond contained in the coupling member.

allows

Figure 3 is a scatterplot comparing a number of reactive thiol groups

substituted on the monoclonal antibody (SH / MAB ratio) with the final ADM / MAB molar ratio achieved in the immunoconjugates produced by the condensation of monoclonal antibodies 5E9 and 3A1, thiolated with the SPDP compound, with the ADM-HZN derivative.

Figure 4 is a scatterplot comparing the SH / MAB ratio to the final ADM / MAB molar ratio achieved in immunoconjugates produced by reacting antibodies 5E9 and 3A1 with compound 2-IT with derivative ADM-HZN .

Figure 5 is a scatterplot showing the relationship between the ADM / MAB molar ratio and protein yield of the immunoconjugates of the present invention prepared using alternatively SPDP compound-thiolated antibodies or compound-thiolated antibodies

2-IT.

Figure 6 is a scatter plot comparing the ADM / MAB molar ratio versus protein yield obtained in immunoconjugate preparations using both IgGi isotype (e.g. 5E9 and 3A1) and IgG2 isotype (e.g. , L6). These antibodies were thiolated with the SPDP compound.

Figure 7 is also a scatterplot comparing the ADM / MAB molar ratio versus protein yield of immunoconjugates having IgG? Or IgG2 isotype antibodies (as in Figure 6) except that these antibodies were thiolated with? compound 2-IT.

X

Figure 8 is a graph of the binding curves of the two immunoconjugates of the present invention compared to the binding curves of their unconjugated monoclonal antibodies.

Figure 9 is a high pressure liquid chromatography (HPLC) chromatogram demonstrating the stability of an immunoconjugate of the present invention at pH values between 4 and 7. This chromatogram demonstrates the acid sensitivity of the acylhydrazone bond of present invention as indicated by the increased release of the free ADM compound from the immunoconjugate as the pH becomes more acidic.

Figure 10 is a CLEP chromatogram demonstrating the release of the ADM radical from an immunoconjugate of the present invention following treatment with the DTT compound.

Figure 11 is a graph of the selective cytotoxicity of the immunoconjugates of the present invention relative to the Daudi cell line using a soft agar colony formation assay. These immunoconjugates were prepared using thiolated antibodies with compound 2-IT.

Figure 12 is a graph of the selective cytotoxicity of the immunoconjugates of the present invention relative to Namalwa cells and the increased potency of the immunoconjugates compared to the free ADM compound using a limiting dilution assay.

Figure 13 is a graph of the selective cytotoxicity of the immunoconjugates of the present invention relative to Daudi cells using the soft agar colony formation assay. In the present case immunoconjugates were prepared using thiolated antibodies with the SPDP compound.

Figure 14 is a graph of the selective cytotoxicity of another immunoconjugate of the present invention using the thiolating agent SPDP compound. This immunoconjugate was cytotoxic to antigen-positive Namalwa and Daudi cells but was not cytotoxic to antigen-negative HSB-2 cells using a soft agar colony assay.

Figure 15 is the selective cytotoxicity diagram of the 5E9 and 3A1 immunoconjugates of the present invention relative to a human colon carcinoma cell line (5E9 +, 3A1-) using a colony formation assay.

Figure 16 is a graph of the lack of Daudi cell toxicity of immunoconjugates prepared by coupling ADM to monoclonal antibodies via a leu-ala dipeptide linker.

Figure 17 schematically depicts the synthesis of an immunoconjugate of the present invention wherein the novel ADM-HZN derivative of the present invention has been reduced and then

reacted with an SMPB (succinimidyl-4- (p-maleimidophenyl) butyrate) treated antibody to provide an immunoconjugate having a thioether bridge contained in its structure.

Figure 18 is a graph of the cytotoxicity of an immunoconjugate of the present invention having, in addition to the 13-keto acyl hydrazone bond, a thioether bridge contained in its coupling member.

This immunoconjugate demonstrated greater potency relative to the free ADM compound on the cells of

Namalwa using a 3-thymidine incorporation assay.

Figure 19 is a graph of the cytotoxicity to the immunoconjugate HSB-2 cells of Figure 18 using the same H-thymidine incorporation assay.

Figure 20 is a graph of the selective cytotoxicity of an immunoconjugate of the present invention to antigen-positive cells compared to antigen-negative cells using the H-thymidine incorporation assay, which has the immunoconjugate in addition. of the acylhydrazone bond at the 13-keto position, a thioether bridge contained in its coupling member.

Figure 21 is a graph of the in vivo immunoconjugate anti-tumor activity of the present invention on Daudi tumor cell xenografts of origin.

ί ί

human in mice. Such an immunoconjugate demonstrated greater anti-tumor activity than is observed when using an equivalent dose of free ADM compound.

Figure 22 is a graph of the in vivo anti-tumor activity of ADM compound on human Daudi tumor cell xenografts in mice over time and at varying doses of this ADM compound using a treatment-type scheme. Q7Dx3 and intravenous administration (iv).

Figure 23 is a graph of the in vivo anti-tumor activity of an immunoconjugate of the present invention on human Daudi tumor cell xenografts in mice compared to the anti-tumor activity of an optimized dose of free ADM compound (administered by intravenously via a Q7Dx3 type treatment schedule and at a dose of mg / kg / inj.). This immunoconjugate demonstrated anti-tumor activity.

Figure 24 is a table showing the in vivo anti-tumor activity of ADM compound on human Ramos tumor cell xenografts in mice using intravenous administration but varying the schedule and doses of treatment.

Figure 25 is a graph of the in vivo anti-tumor activity of ADM compound on Ramos human tumor cell xenografts in mice over time and at varying doses of ADM compound, from 10-11 to greater.

using the single injection treatment schedule and intravenous administration.

Figure 26A is a graph of the in vivo anti-tumor activity of an immunocoupled of the present invention on human Ramos tumor cell xenografts in mice compared with the anti-tumor activity of an optimized dose of free ADM compound (administered via intravenously following a QlDxl treatment regimen, the dose being 16-18 mg / kg / inj). Such immunoconjugate demonstrated greater anti-tumor activity than free ADM compound.

Figure 26B depicts the in vivo anti-tumor activity of the immunoconjugate over time at different conjugate doses, demonstrating that the anti-tumor effect of the conjugate is dose dependent in nature. Dosages of immunoconjugates tested in Figures 26A and B are referenced in terms of anthracycline present in the conjugate, with corresponding antibody values in parentheses.

DETAILED DESCRIPTION OF THE INVENTION

In order that the present invention now described and disclosed may be more fully understood, the following detailed description is given below.

The present invention relates to anthracycline immunoconjugates, novel anthracycline-acylhydrazone, methods for their preparation, pharmaceutical compositions and methods for providing novel cytotoxic anthracycline derivatives to a selected cell population to be deleted for treating diseases such as cancers and other tumors, non-cytocidal viral infections and other pathogenic infections, and autoimmune diseases. More particularly, the present invention relates to immunoconjugates consisting of an antibody directed against a selected cell population, said antibody having a number of anthracycline molecules attached to its structure. Anthracycline molecules are covalently linked to the antibody such that a splicing element is formed between each drug molecule and the antibody, the splicing moiety being coupled to the anthracycline by an acylhydrazone bond at the 13-keto position of the antibody. anthracycline. This process can be carried out progressively by the initial formation of a new anthracycline-hydrazone derivative which is then reacted with an antibody of appropriate specificity [see, for example, RR Hardy, Purification And Coupling Of Fluorescent Proteins For Use In Flow Cytometry ”in Handbook Of Experimental Immunology, Volume 1: Immunochemistry, DM Weir et al., Ed. 31.4-31.12 (4th Ed. 1986) for a discussion of conventional antibody coupling techniques]. The length of the coupling element that binds the anthracycline and antibody components of the immunoconjugate may vary as long as the coupling point of that anthracycline coupling element is in the form of an acylhydrazone bond at the position.

it may contain another bond such as a disulfide, thioether, amide, carbamate, ether or ester type bond, along its length and situated between the coupling points from the drug to the antibody.

The anthracyclines constituting the immunoconjugates of the present invention may optionally be any type of anthracyclines which contain a certain group at the 13-position of the carbon atom (C-13). Such anthracyclines include, but are not limited to, adriamycin, daunomycin, detorubicin, carminomycin, idarubicin, epirubicin, esorubicin, 4'-THP-adriamycin, AD-32 and 3'-deaminine -3 '- (3-cyano-4morpholinyl) -doxorubicin (see AM Casazza, Experimental Studies On New Anthracyclines in Adriamycin: Its Expanding Role In Cancer Treatment, M. Ogawa and colleagues (eds.), Pp. 439-52 (Medical Excerpt 1984)].

The antibodies constituting the immunoconjugates of the present invention may optionally be any antibodies reactive with a specific cell population to be eliminated or annihilated. Examples of such antibodies include, but are not limited to, antibodies that bind to tumor associated antigens such as antigens on carcinomas, melanomas, lymphomas, bone or soft tissue sarcomas, and other tumors. , antibodies that bind to virus associated antigens or other pathogens

and antibodies that bind to abnormal cell surface antigens. These antibodies may optionally be polyclonal or preferably monoclonal and may be made using techniques well known in the art [see, for example, RA DeWeger et al., Eradication Of Murine Lymphoma And Melanoma Cells By Chlorambucii-Antibody. Complexes, Immunological Rev., 62, pp. 29-45 (1982) (tumor-specific polyclonal antibodies produced and used in conjugates) and M. Yeh et al., Cell Surface Antigens Of Human Melanoma Identified By Monoclonal Antibody, Proc. Natl. Acad. I know., 76, pp. 2927-31 (1979) and JP Brown et al., Structural Characterization Of Human Melanoma

Associated Antigen p97 With Monoclonal Antibodies, J. Immunol., 127 (No.2), pp. 539-46 (1981) (tumor specific monoclonal antibodies produced)]. For example, the human lung carcinoma cell-specific monoclonal antibody L6 or the osteogenic sarcoma cell-specific monoclonal antibody 791T / 36 may be used. On the other hand, non-internalizing antibodies or preferably internalizing antibodies may be used. 0 The term antibody used herein includes intact antibody molecules or fragments containing the active binding region of the antibody molecule, for example, Fab or F (ab ') 2 fragments. antibodies can be without it

It is any limitation, whether human in nature or derived from mice or chimeric antibodies.

Thus, antibodies of the immunoconjugates of the present invention act to provide anthracycline molecules to the particular cell population with which the antibody is reactive. For example, an antibody directed against a tumor cell surface antigen will bind to and supply its anthracyclines to those tumor cells, or an antibody directed against an AIDS-causing human immunodeficiency virus (HIV) protein will provide their cytotoxic anthracyclines to HIV-infected cells. Release of the drug into or within the particular cell population with which this antibody reacts results in the preference of such particular cells. It is therefore apparent that the immunoconjugates of the present invention are useful for treating any diseases in which it is desired to eliminate a specific cell population, said cell population having an antigen on the cell surface which allows binding of the immunoconjugate. Diseases for which the immunoconjugates of the present invention are useful include, but are not limited to, cancers and other tumors, non-cytocidal viral infections or other pathogenic infections such as AIDS, herpes, CMV (cytomegalovirus), EBV Epstein Barr) and PEES (subacute sclerosing panencephalitis) and rheumatoid arthritis.

Without any theoretical foundation, admitting annihilation is

are if

antibody-bound anthracycline molecules, i.e., in the form of the immunoconjugate of the present invention, are provided to target cells to be annihilated by antibody specificity and therefore may eventually enter the cell via the same endocytic pathway. leading to the internalization of antibodies and unconjugated membrane-bound ligands [see, for example, I. Pastan et al., Pathway Of Endocytosis, in Endocytosis, I. Pastan and collaborators (eds.), Pp. 1-44 (Plenum Press 1985)]. Once inside the cell, the endocytic vesicles containing the immunoconjugate fuse with the primary lysosomes to form secondary lysosomes [see, for example, MJ Embleton et al., Supra, p. 334]. Since anthracycline molecules are linked to the immunoconjugate antibody via acid sensitive acy1-hydrazone bonds, exposure of the immunoconjugate to the acidic environment of endocytic vesicles and lysosomes results in the release of anthracycline from the immunoconjugate. On the other hand, the released anthracycline is believed to be a relatively unmodified drug capable of providing total cytotoxic activity. Thus, the acid-sensitive immunoconjugate hydrazone binding is highly advantageous for the release of the cytotoxic drug into the target cells, increasing the immunoconjugate cytotoxicity with respect to such cells. Alternatively, the hydrazone bond may be cleaved under acidic conditions and /

reducing agents in the immediate external or surrounding environment of the target cells, for example at the tumor site, and eventually the released drug may be absorbed by the tumor cells.

The immunoconjugates of the present invention and the methods for their preparation are exemplified by the

<td>aspects</td><td colspan="2">preferences</td><td>according to which</td><td>adriamycin</td><td>gives</td>

<td>class</td><td>of</td><td colspan="2">anthracyclines was conjugated</td><td colspan="2">with several</td>

<td colspan="2">antibodies.</td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>first</td><td>Instead, it was synthesized</td><td>the derivative</td><td>in</td>

adriamycin hydrazone in a two step reaction. Heterobifunctional reagent SPDP (N-succinimidyl-3- (2-pyridyl dithio) propionate) was allowed to react with hydrazine to afford 3- (2-pyridyl dithio) -propionyl hydrazide and then reacted with hydrazine. adriamycin hydrochloride (ADM-HCl) to provide a novel acyl-hydrazone group ADM derivative containing a disulphide radical protected by a pyridyl group. Optionally, an acid catalyst such as trifluoroacetic acid may be used to facilitate the formation of hydrazone. The derivative that is formed is 13- {3- (2-pyridyl-dithio) propionyl} -hydrazone adriamycin hydrochloride (ADM-HZN) (see Figure 1).

The new ADM-hydrazone derivative was then reacted with a radonoclonal antibody that had previously been thiolated with the SPDP compound and then reduced or thiolated with the 2-IT compound (2-iminothiolane) with the monoclonal antibody. coupled to the C-13 position of an acylhydrazone bond, additionally an antibody coupled bridge (see (see Figure 2). 0 immunoconjugated with ADM molecules conjugated by means of a linker to each ADM molecule containing that disulfide linker through which Figure 2).

Another aspect of the present invention involves the synthesis of another novel adriamycin hydrazone derivative wherein the previously described ADM-HZN compound has been further treated with DTT (dithiothreitol) or tributylphosphine reducing agents to provide compound 13 - {3 -

- (mercapto-propionyl)} adriamycin hydrazone (see Figure 17). This derivative was then reacted with a monoclonal antibody to which maleimide groups had been coupled, for example, by reacting the antibody with the compound SMPB (succinimidyl-4- (p-maleimidophenyl) butyrate). As shown in Figure 17 an immunoconjugate was formed which had a coupling element coupled by a hydrazone bond to the C-13 position of each ADM molecule and also having a thioether bridge as part of its coupling to the antibody. Thus it is evident that the drug-antibody-binding moiety may optionally be formed of various constituents and joints insofar as such joints include the acid-sensitive hydrazone bond at the 13-keto position of anthracycline.

It is also evident that the present invention

provides novel 13α-hydrazone binding derivatives of the 13-keto anthracyclines having the general formulas I, II or III:

on what:

Formula represents a group CH3, CH2OH, CH2OCO (CH2) 3CH3 or

CH2OCOCH (OC2H5)2;

R represents a group of the formula or wherein χ = Η, NO2 or halogen;

R represents an OCH 3 or OH group or the hydrogen atom;

R represents NH2, NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethylamine or 1-cyano-2-group methoxy ethyl amine;

R4 5 represents an OH or O-THP group or a hydrogen atom; R2 represents an OH group or a hydrogen atom provided

5th wherein R is not OH when R is OH or O-THP; en represents an integer from 1 to 10 inclusive;

on what:

R represents a group CH3, CH2OH, CHgOCO (CH2) 3CH3 or

CH2OCOCH (OC2H5)2;

R represents an OCH 3 or OH group or a hydrogen atom;

R represents NH2, NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethylamine or 1-cyano-2-group methoxy-ethyl-amine;

R represents an OH or O-THP group or a hydrogen atom;

represents an OH group or a hydrogen atom, provided that R does not represent the OH group when R represents the OH or O-THP group; en represents an integer from 1 to 10 inclusive; and

on what:

r! represents a CH3 group,

CH2OCOCH (OC2H5)2;

R represents a group of formula ch2oh overall

CH2OCO (CH2) 3CH3 or

4-x or where x = H, N02 or halogen;

R3 represents an OCH3 or OH group or a hydrogen atom;

R and R independently represent a hydrogen atom or an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl 47 or substituted aralkyl group; or R, R and N together form a ring having from 4 to 7 sides, which ring may optionally be optionally substituted;

R2 represents an OH or O-THP group or a hydrogen atom; represents an OH group or a hydrogen atom provided

eX does not represent the OH group when it represents the OH or O-THP group; en represents an integer from 1 to 10 inclusive.

The anthracycline-acylhydrazone class compounds disclosed above represent novel intermediates for the preparation of the Î ± -unconjugates of the present invention and are exemplified by the compounds ADM-HZN and 13- (3- (mercaptopropionyl)} adriamycin, respectively described in the preferred embodiments herein. descriptive.

As can be seen from the foregoing formulas, the acy1-hydrazone group intermediates of the present invention encompass the hydrazones selected from a variety of known anthracyclines such as adriamycin, daunomycin and carminomycin. In addition, these intermediates encompass the acylhydrazones transformed at specific sites of the anthracycline structure (e.g., 4'-THP-adriamycin hydrazone and 3'-deamino-3 '- (3-cyano-4-morphhoinyl) compounds. ) adriamycin hydrazone). These latter intermediates may be synthesized by first transforming anthracycline to provide a desired analog and then using that analog for the preparation of the hydrazone intermediate of the present invention. Known anthracycline analogs include those described in U.S. Pat.çNos. 4,446,429 and 4,330,177, referred to as (3'-deamino-3 '- (4-morpholynyl) - or 3'-deamino-3' - (3-cyano-4-morpholynyl) -anthracycline), in the following

X

U.S. patents5 s

4202967 and

4314054 (3'-deamino-3 '- (1-piperdinyl) - or 3'-deamino-3' - (4-methoxy-1-piperidinyl) anthracillin), U.S.s 4250303 (N-benzyl- or N, N-dibenzyl-anthracycline) in U.S. Patent No. 4,350,303.5 4591637 (N-methoxymethyl or N-cyanomethyl anthracycline) and U.S.Q 4303785 (acetal group anthracycline analogs). These known anthracycline analogs may be subjected to reactions as described above (see Figure 1) to provide novel acylhydrazones which may then be conjugated to an antibody of a desired specificity as described herein.

Alternatively, an unprocessed acylhydrazone intermediate according to the present invention as described herein can be produced first from an unprocessed anthracycline such as adriamycin, daunoraicin or carminomycin and then It is possible to transform this new intermediate to provide a new substituted acyl-hydrazone as desired. For example, compound ADM-HZN may undergo transformation to its amino sugar radical by reducing amination with 2,2'-oxyacetaldehyde according to the procedure described in U.S.Q 4,446,529 to provide the 3'-deamino-3 '- (3-cyano-4-morpholinyl) adriamycin hydrazone compound. Similarly, the ADM-HZN compound may be subjected to a /

transformation into its amino-sugar radical to provide novel acylhydrazone derivatives such as 3'-deamino-3 '- (4-morpholinyl) -ADM-hydrazone compounds (see U.S. Patent No. 4,594,981).2 4301277), 3'-deamino-3 '- (1-piperidinyl) -ADM-hydrazone (see U.S. Patent No. 4,530,177).The 4202967), 3'-deamino-3 '- (4-methoxy-1-piperdinyl) -ADM-hydrazone (see U.S. Pat.s 4,331,454), N-benzyl-ADM-hydrazone and

N, N-dibenzyl-ADM-hydrazone (see U.S. Pat.The 4250303) or N-methoxymethyl-ADM-hydrazone and N-cyanomethyl-ADM-hydrazone (see U.S. Pat.The 4591637). In addition, the ADM-HZN compound can be transformed into position R5 of formulas I-III as described in U.S. Pat.Q4303785 to provide acetal group hydrazone derivatives such as 4'-THP-ADM-hydrazone compound.

It will be appreciated that these novel procedures for the transformation of the acylhydrazone compounds of the present invention may use different anthracycline hydrazones as the starting material such as daunomycin or carminomycin as starting materials to provide novel compounds such as N-benzylhydrazones. daunomycin hydrazone or 3'-deamino-3 '- (4-morpholinyl) carminomycin hydrazone, which are also encompassed within the scope of the present invention.

Evaluation of anthracycline-antibody immunoconjugates prepared in accordance with the present invention demonstrated that such immunoconjugates retained antibody binding activity and exhibited antibody-driven cellular annihilation power for both lymphoma and carcinoma cells under conditions of diversified essay. Thus, cells bearing the antigen to which the conjugate antibody was directed were efficiently annihilated by anthracycline whereas cells lacking the appropriate antigen were not annihilated. Indeed, in several experiments it was found that anthracycline provided by the antibody was more potent than equivalent amounts of unconjugated anthracycline. Differences between the mechanisms of incorporation in the tumor cells and the mechanisms of intracellular transport may possibly be responsible for the power differences observed between the action of free drug and the action of antibody-conjugated drug.

On the other hand, studies using human tumor xenografts in mice have demonstrated the ability of the immunoconjugates of the present invention to inhibit tumor growth in vivo, in some cases providing complete tumor regression. Immunoconjugates have been shown to have greater potency and inhibit tumor growth more intensely than unconjugated anthracycline. In addition, immunoconjugates were tolerated by animals to a much higher degree than free drug, and these immunoconjugates were at least 10 times less toxic than unconjugated anthracycline alone.

The binding and cytotoxicity properties of the immunoconjugates apparently of the present invention are an improvement on literature-referenced immunoconjugates in which anthracyclines were directly bound to the antibody via the anthracycline amino sugar radical. Such amino-sugar-linked immunoconjugates often contained smaller molar ratios of anthracycline and antibody and exhibited reduced cytotoxicity relative to free drug and exhibited reduced binding properties to co-workers, contributors to antibody [see, for example, R. Arnon and Immunological Rev., 62, supra; E. Hurwitz and Cancer Res., 35, supra; and R. Yamamato et al., supra]. On the other hand, stability studies performed on the immunoconjugates of the present invention indicated that anthracycline was released from immunoconjugates under reducing and acidic conditions similar to those in a cellular environment. Thus, retention of the high cytotoxic activity of the drug observed with the immunoconjugates described herein can be explained by the fact that a relatively unmodified drug was provided to the target cells.

In addition, reaction conditions could be optimized to achieve anthracycline: antibody molar ratios of approximately 4 to 10, using various antibodies of different isotypes for this purpose. The amount of protein recovered after condensation with the ADM-HZN derivative dropped dramatically when trying to reach molar ratios greater than 10. The major limitation to obtain immunoconjugates with molar ratios greater than 10 is believed to be due to the reduced solubility of the conjugates in aqueous solution and the physical association of anthracycline with the protein.

Studies now carried out in vivo demonstrating improved anti-tumor activity of the immunoconjugates of the present invention relative to the free drug and also demonstrating their reduced systemic toxicity indicate an increased therapeutic index for such conjugates. Accordingly, the present invention also encompasses pharmaceutical compositions, combinations and methods for treating diseases such as cancers and other tumors, non-cytocidal viral infections or other pathogenic infections and even autoimmune-like diseases. More particularly, the present invention encompasses methods for treating mammalian diseases which consist in administering to the host mammal at least one immunoconjugate containing an anthracycline by a pharmaceutically acceptable method.

Alternatively, variants of the methods of the present invention encompass the simultaneous or sequential administration of several different immunoconjugates, i.e. immunoconjugates carrying different anthracyclines or different antibodies, for use in combined chemotherapeutic methods. For example, according to one of its aspects the present invention may

may involve the use of various anthracycline immunoconjugates varying the specificity of the antibody component of the conjugate, that is, various immunoconjugates are used each having an antibody that specifically binds to an antigen or to different sites or epitopes thereof. antigen present in the target cell population. The anthracycline component of these immunoconjugates may optionally be the same or may vary. For example, this variant may perhaps be especially useful in treating some tumors in which the amounts of the various antigens on the surface of a tumor are unknown or in which the tumor cell population is heterogeneous with respect to antigen expression when desired. Ensure that a sufficient amount of drug is routed to all tumor cells at the tumor site. The use of various conjugates bearing different antigen or epitope specificities for the tumor increases the possibility of obtaining a sufficient amount of drug at the tumor site. In addition, this aspect is important to achieve a high degree of tumor specificity since the likelihood that normal tissue also has all tumor-associated antigens is low [cf., I. Hellstrom et al., Monoclonal Antibodies To Two Determinants Of Melanoma-Antigen p97 Act Synergistically In Complementary Dependent Cytotoxicity, J. Immunol., 127 (No. 1), pp. 157-60 (1981)].

Alternatively, several different immunoconjugates may be used wherein only the anthracycline component of the conjugate varies. For example, a particular antibody may be attached to the adriamycin compound to form an immunoconjugate and such an antibody may be attached to the daunomycin compound to form a second immunoconjugate. Both conjugates may then be administered to a host to be treated, such conjugates being localized due to antibody specificities at the locus of the selected cell population to be deleted. Both drugs will then be released at this location. This aspect may possibly be quite important whenever there is some uncertainty such as drug resistance by a particular cell population, for example a tumor, as this method allows for the release of several different drugs locally or within of the target cells. A further aspect encompasses the conjugation of more than one anthracycline with a particular antibody to provide an immunoconjugate carrying a variety of different anthracycline molecules distributed on its surface - all of which are bound to the antibody via an 13-position acylhydrazone bond. Keto. Administration of the immunoconjugate of this variant results in the release of several different drugs at or within the target cells.

The anthracycline immunoconjugates of the present invention may be administered in the form of compositions /

pharmaceutical agents using conventional methods of administration or including, without limitation, intravenous, intraperitoneal, oral, intralymphatic administration or direct on-site administration of a selected cell population such as, for example, a tumor. Intravenous administration is preferable. In addition, for in vivo treatment, it may optionally be useful to use immunoconjugates incorporating antibody fragments such as Fab or F (ab ') 2 chimeric antibody fragments.

Pharmaceutical compositions of the present invention incorporating anthracycline immunoconjugates may optionally be presented in a wide variety of dosage forms including, but not limited to, solid, semi-solid and liquid dosage forms such as tablets. , polymeric pills, powders, solutions or suspensions, suppositories, microcapsules or microvesicles, liposomes and solutions for injection or infusion administration. The preferred form depends on the mode of administration and therapeutic application.

The pharmaceutical compositions may optionally also incorporate conventional pharmaceutically acceptable carriers known in the art such as whey proteins, for example human serum albumin, buffering substances such as phosphates, water or salts or electrolytes.

The most effective mode of administration and dosage regimen for the immunoconjugate compositions of the present invention depend on the severity and course of the disease, the patient's health and response to treatment, and the judgment of the attending physician. Therefore, the dosages of immunoconjugates and any other concomitantly administered compounds should be titrated according to the individual patient. However, an effective anthracycline immunoconjugate dose according to the present invention may contain the anthracycline component in an amount from 1 to 100 mg / m and the antibody component 2 in an amount from 500 to 5000 mg / m 2.

The following are some examples for the purpose of providing a better understanding of the invention described herein. It will be appreciated that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

EXAMPLE 1 The following example demonstrates the production of a novel anthracycline immunoconjugate according to the present invention wherein the drug is bound directly to a monoclonal antibody via a hydrazone bond at the 13-keto position of the drug.

The particular aspect described in this example involves conjugating the ADM compound with a monoclonal antibody to provide an immunoconjugate having a coupling element with an ac1-hydrazone bond at its coupling point to the immunoconjugate ADM compound molecule, <3 further having this bonding element a disulfide bridge as part of its coupling to the antibody. This variant also provides a novel acylhydrazone derivative and

ADM (ADM-HZN).

SUMMARY OF AN ADRIAMICINE-HYDRAZONE DERIVATIVE

As an initial step for the preparation of the immunoconjugate of the present invention, an ADM-hydrazone derivative was first synthesized as follows: a solution of 0.3 ml of 1 M hydrazine, i.e. NHgNHg in isopropyl alcohol, was prepared and To a cooled solution of SPDP (70 mg, 0.22 mmol) in 3 mL of THF (tetrahydrofuran) was added. After stirring for 20 minutes at 0 ° C, the product was extracted with CH2 Cl2, washed with brine and dried over K2 CO3. The residue obtained after evaporation of the solvents was chromatographed on neutral alumina (5% MeOH, 95% CH 2 Cl 2) to provide 21 mg (41%) 3- (2-pyridyl dithio) propionyl hydrazide (compound 2 in Figure 1).

This hydrazide and adriamycin hydrochloride (obtained from Sanraku Inc., Japan) (48 mg, 0.083 mmol) was dissolved in 5 ml MeOH and then stirred at room temperature and for 6 days. The reaction progress was verified by reverse phase thin layer chromatography (TLC) (MeOH: H2O = 2: 1 containing 3% w / v NH4OAC). After this time the solvent was evaporated and the residue was subjected to column chromatography (H2 O = 3: 2 containing 3% w / v NH 4 OAC). Fractions and

lyophilized and then excess NH 4 OAC was removed under reduced pressure. The residue was taken up in MeOH and precipitated by the addition of acetonitrile to afford 45 mg (72%) of adriamycin-13- (3- (2-pyridylditio) propionyl} hydrazone hydrochloride, hereinafter ADM). -HZN (compound 4 in Figure 1) This ADM-HZN compound exhibits the following characteristics:

<td>mp> 125 °,</td><td>the color</td><td colspan="4">becomes darker and poorly defined;</td><td>NMR</td>

<td>(acetone</td><td>in, -)</td><td>1.25 (s, 3H, J = 6Hz),</td><td> 1,77</td><td>(m, 1H)</td><td>t</td><td> 2,06</td>

<td colspan="2">1016, 988, 698 cm '1</td><td>; EM-BAR (Glycerol)</td><td>m / e 755</td><td>(M + 1)</td><td>t</td><td> 737,</td>

TIOLATION OF MONOCLONAL ANTIBODIES

Before reacting ADM-HZN compound prepared as described above with a monoclonal antibody of interest, that antibody had to be thiolated, that is, reactive sulfhydryl groups had to be introduced into the antibody molecule.

The monoclonal antibodies used were: 1) 5E9, a transferrin receptor reactive IgGi antibody in all dividing human cells and interreactive with various histological cell types.

<td>monoclonal</td><td>5E9</td><td>and T33A1</td>

<td>of Cultures</td><td>Type</td><td>(ATCC).</td>

<td>puri</td><td>The</td><td>leave</td>

<td>mice</td><td>gives</td><td>strain</td>

<td>procedure</td><td>in</td><td>Ç.</td>

<td>Purification</td><td>Of</td><td>Mouse</td>

cancerous; 2) T33A1 (hereinafter referred to as 3A1), an IgG4 antibody reactive with the 40Kd human T-cell antigen and also present in various leukemias of the cells.

T; 3) G28.5, an IgGg antibody reactive with the 50Kd human B cell antigen and also reactive with human B cell lymphomas; 4) G28.1, an IgGi antibody reactive with the 39Kd human B cell antigen and also reactive with B cell lymphomas; and 5) L6, an antibody reactive with a glycolipid antigen in human lung alveolar cell carcinomas.

Hybridomas secreting the antibodies now obtained from the American Collection and the corresponding antibodies were from ascites fluid produced in strain BALB / c according to? C. Bruck and co-workers, One-Step

Monoclonal Mouse Antibodies From Ascitic

Fluid By DEAE-Affigel Blue Chromatography, J. Immun. Methods, 5b, pp. 313-19 (1982). Purified antibodies G28.5, G28.1 and L6 were provided by Drs. J. Ledbetter and I. Hellstrom (Oncogen, Seattle, WA). Hybridomas secreting monoclonal antibodies L6 and G28.5 were deposited with the ATCC institution on December 6, 1984 and 22 May 1986 respectively, corresponding to them at the ATCC institution the accession numbers HB 8677 and HB 9110. Monoclonal antibody G28.1 is one of several antibodies known in the art because it is

reactive with a major epitope of the CD37 antigen and was suitably characterized by AJ Michael (ed.), Leukocyte Typing III, Oxford University Press (UK 1987). There are several such commercially available anti-CD37 antibodies.

Either of these antibodies were thiolated with SPDP as follows: SPDP (Pierce Chemical Co., IL) (50 mM) was dissolved in ethanol and added to a monoclonal antibody solution 5E9 (5-10 mg / ml) in PBS (phosphate buffered saline, pH 7.2) is selected to provide a final concentration of 5 to 10 mM. The reaction mixture was incubated for 30 minutes at 30 ° C. Unreacted SPDP compound was separated from SPDP-transformed antibody by gel filtration chromatography using a PD-10 type column (Pharmacia). The thiopyridyl protecting groups were removed by reduction with excess DTT. Reduced antibodies were passed through a PD-10 type column and antibodies containing free thiol were used for condensation with the ADM-HZN derivative (see Figure 2).

Reactive thiol groups were also introduced into the antibody protein using compound 2-IT: the antibody (5-10 mg / ml in 50 mM triethylamine, 50 mM NaCl, 1 mM EDTA at pH 8.0) was mixed. ) with compound 2-IT (Pierce Chemical Co., IL) to a final concentration of 5-10 mM. The reaction was allowed to proceed for 90 minutes at

at 4 ° C and then thiolated antibodies were separated using a PD-10 column equilibrated with 2M NaCl / PBS.

The number of reactive thiol groups incorporated into the antibodies was determined using compound DTNB (5,5'-dithio-bis (2-nitrobenzoic acid) (E412 = 14150) according to the procedure described by GL Ellman, Arch. Biochem. Biophys., 82, pp. 70-77 (1959).

CONJUGATION OF TIOLATED MONOCLONAL ANTIBODIES

WITH ADM-HZN COMPOUND

Subsequently, several conjugations were performed wherein the thiolated monoclonal antibodies as described above were individually coupled to the ADM-HZN compound (see Figure 2).

Compound ADM-HZN was dissolved in methanol and this solution was added to SPDP-thiolated antibodies in PBS or to 2-IT-thiolated antibodies in 2M NaCl / PBS. In a typical experiment, 10 equivalents of the ADM-HZN compound was added to the monoclonal antibodies containing between 10 and 20 reactive thiol groups. The conjugation reaction was allowed to incubate overnight at 4 ° C. The reaction mixture was centrifuged at 10,000 x g and then the conjugated ADM compound was separated from the unreacted ADM compound by passing through a PD-10 type column. The amount of antibody-bound conjugated anthracycline was determined by absorbance analysis at 495 nm (Ε4θ5 = 8030). The amount was determined

of antibody protein by absorbance at 280 nm (1 mg / ml = = 1.4 OD units). To correct the absorbance overlap of the ADM compound at 280 nm, the following expression was used:

THE280 - (°-72 shah495) Antibody (mg / ml) = ------------------- 1.4

Immunoconjugates were subjected to analysis for the presence of unconjugated ADM compound or ADM compound derivatives by HPLC analysis. HPLC was performed using a 5 micron IB-SIL C18 bead-loaded Phenomenex column. Unconjugated ADM-HCl, ADM-HZN (0.1 μπιοΙθΞ) or immunoconjugates containing 0.5 to 5 μπιοίεε drug equivalent were applied to a column and eluted with methanol and ammonium phosphate. 10 mM at pH 4.5 (70:30) at a rate of 1.5 ml / min. All immunoconjugates produced contained non-significant amounts (<1%) of unconjugated drug as verified by CLEP analysis.

CHARACTERIZATION OF IMMUNOCIATES OF THIS INVENTION

The immunoconjugates thus produced were composed of 13-keto-conjugated ADM compound molecules with a linker that formed a bridge between the drug and its monoclonal antibody. On the other hand, the addition of the free thiol group monoclonal antibody to the ADM-HZN derivative which contained a thiopyridyl protected disulfide bridge led to the

formation of a disulfide bridge in the coupling that binds the ADM compound to the antibodies (see Figure 2). Immunoconjugates produced in this embodiment include, but are not limited to, immunoconjugates 5E9-ADM-7.5, 3A1-ADM-7.0, L6-ADM-9.0 and G28.1-ADM-9.0, where the first part of the designation represents the monoclonal antibody used to form the conjugate, the second part of the designation represents antibody-bound anthracycline and the numeric part of the designation represents the ADM / antibody molar ratio in the particular immunoconjugate.

The ADM / antibody molar ratios achieved in this embodiment depend on the number of thiol groups introduced into the monoclonal antibody and the amount of ADM-HZN derivative introduced into the thiolated antibody. The dispersion diagrams of Figures 3 and 4 show that ADM / antibody molar ratios of 3 to 4 were achieved when condensing the ADM-HZN compound with either 5E9 or 3A1 monoclonal antibodies containing approximately eight thiol groups. Typically, in these reactions, ADM-HZN was added 10 times in molar excess to the protein. ADM / antibody ratios increased to between 8 and 10 when antibodies having between 18 and 25 thiol groups were used. No significant differences in ADM / antibody ratios were observed when using SPDP or 2-IT compounds to thiolate the monoclonal antibody (compare Figures 3 and 4). However, the final protein yields after

Drug-antibody conjugation was apparently somewhat higher with SPDP compound-thiolated antibodies compared to compound 2-IT thiolated antibodies (see Figure 5). Protein yields of between 50 and 80% were often obtained for SPDP-thiolated antibodies, for example with antibodies 5E9 and 3A1, while yields of between 20 and 50% for thiolated antibodies were obtained. with compound 2-IT. In addition, slightly better yields were obtained in the preparation of immunoconjugates using IgG1 isotype monoclonal antibodies such as antibodies 5E9 and 3A1 for conjugations performed with SPDP or 2-IT compounds (see Figures 6 and

The binding activity of the immunoconjugates of the present invention was determined using a competition assay involving the use of labeled antibody.

5th 6 with I. Antigen positive and antigen negative (1 x 10) cell suspensions were prepared in 0.1 ml RPMI 1640 medium containing 2% FBS and mixed with 0.1 ml immunoconjugate solutions or unconjugated monoclonal antibodies sequentially serially diluted at ratio 2 to concentrations starting at 50 μ9 / ιυ1. These cell suspensions were incubated in duplicate under shaking at 4 ° C for 1 hour. The cells were then washed twice and prepared once / twice.

It is suspended in 0.1 ml of medium containing the homologous antibody labeled with

125.

5 pg / ml (specific activity 1-50 x 10 10 cpm / pg antibody protein). The samples were incubated at 4 ° C for 1 hour and then placed into 0.15 ml of a 1: 1 mixture of dibutyl phthalate: dinonyl fatlate cooled to 4 ° C. Samples were centrifuged at 10,000 xg for 1 minute at 4 ° C and pulse counts from the cells (aggregate) were determined using an LKB gamma ray computer.

Retention of immunoconjugate activity of the present invention is shown in Table 1 below.

connection of

Table 1

Estimation of Relative Binding Affinity After ADM-HZN Conjugation

<td>Inhibitor</td><td>Molar Ratio</td><td>Signal</td><td>x10 ~9M</td><td>x10 ~9M</td><td>x107 L / M</td>

SPDP connector

<td></td><td>2-IT connector</td><td></td><td></td><td></td><td></td><td></td>

The[It] = Molar concentration of antibody conjugate providing 50% antibody inhibition B [Tj-] = Molar antibody concentration providing 50% inhibition of locating antibody.

Kconj = Relative affinities (K) were calculated using the formula:

K conj = [Tt] KAb locator.

[it]

KAb θ The Unconjugated MAb (or AcM) equilibrium constant determined by Scatchard analysis.

As shown in the table, 5E9 immunoconjugates prepared using the SPDP compound and having molar ratios of between 3.5 and 8.5 retained more than 80% of their original binding activity compared to unconjugated 5E9 antibody. 5E9 immunoconjugates prepared using compound 2-IT also retained high binding activities. 3A1 immunoconjugates prepared using the SPDP compound demonstrated some loss of antibody binding activity. In general, conjugation of the ADM compound with these and other antibodies resulted in the loss of antibody binding activity to relatively small degrees.

Figure 8 shows the binding curves of two immunoconjugates of the present invention, 5E9-ADM-7.5 and 3A1-ADM-7.0, compared to the binding curves of unconjugated 5E9 and 3A1 monoclonal antibodies. To obtain these curves, immunoconjugates were incubated at 4 ° C in 0.1 ml of complete growth medium containing 1 x 10θ antigen-positive HSB-2 target cells. After 1 hour the cells were washed in said medium and incubated for a further 30 minutes in 0.1 ml of medium containing a 1:40 dilution of conjugated mouse anti-IgG in a FITC labeled goat (Boehringer -Mannheim). The cells were then analyzed on a Coulter Epics V-type fluorescence cell analyzer.

similarly diluted conjugates. As shown in the Figure, the binding activity of each of the immunoconjugates was conserved as demonstrated by the fact that the concentration of immunoconjugate that was required to saturate antigen-positive cells was at most greater than twice the dilution corresponding to the concentration. required for unconjugated antibody. Differences in levels of fluorescence intensity levels between unconjugated and immunoconjugate antibodies were found to be due to the reduced binding of the FITC-labeled goat-conjugated mouse anti-IgG secondary reagent to the immunoconjugate compared to the unconjugated antibody. .

The stability of an immunoconjugate according to this variant - an L6-ADM conjugate - was studied at various pH values ranging from 4.0 to 7.0 by HPLC analysis. L6-ADM-9.0 conjugate was incubated in phosphate buffer solutions at each of the pH values indicated for 24 hours at 37 ° C. Each of these solutions was then applied to a CLEP column and the amount of unconjugated drug determined. As shown in Figure 9, the only product detected after 24 hours of incubation at different pH values exhibited a column retention time similar to that of the standard compound ADM-HCl. The amount of material released from the immunoconjugate after 24 hours increased when the pH value decreased from 7 to 4. The untreated control represents the chromatography of the conjugate stored at -20 ° C in phosphate buffer to pH 7.4. . Thus, the immunoconjugate is believed to have an acid sensitive coupling group which results in the release of the ADM compound from the antibody protein. These results are consistent with the existence of a hydrazone bond that joins the ADM compound to the coupling element as described in Figure 2.

In the embodiment of this example, ADM compound was coupled to the antibody via a linker that also contained a disulfide bridge (see Figure 2). Therefore it should be possible to release the ADM radical by reducing an immunoconjugate of this variant using the DTT compound. Therefore, the L6-ADM conjugate, specifically L6-ADM-9.0, was treated with the 10-fold excess DTT compound, incubated at room temperature for 15 minutes and applied to a CLEP column. Experiments with ADM-HCl and ADM-HZN standard compounds were performed simultaneously and the peaks obtained by chromatography are shown in Figure 10. L6-ADM-9.0 conjugate showed no detectable unconjugated drug peak prior to the addition of DTT. CLEP analysis demonstrated the emergence of a single peak that exhibited a column retention time similar to that of ADM-HCl as opposed to ADM-HZN derivative (see Figure 10). The amount of ADM compound released after DTT treatment was approximately 99% of the initial equivalent amount of antibody-bound ADM. The experimental results shown in Figures 9 and 10 demonstrate that the ADM-like radical is released from the immunoconjugates of the present invention under physiological conditions, that is, acidic and reducing conditions typical of the cellular environment.

CYTOTOXIC ACTIVITY OF IMMUNOCIATES OF

PRESENT INVENTION

The immunoconjugates of the present invention were tested in vitro for cytotoxicity using various assay systems. According to a soft agar colony formation assay Daudi cells (Burkitt's lymphoma) (phenotype: 5E9 +, 3A1-) obtained from the ATCC institution were grown in complete medium [RPMI 1640 medium containing 10% of fetal bovine serum]. 1 χ 10 exposure was5 cells in 1 ml medium for 1.5 hours to sequentially diluted solutions of 5E9-ADM or 3A1-ADM immunoconjugates or unconjugated ADM compound. For each dilution the triplicate determinations were made. Controls consisted of cells treated identically but not exposed to drugs. The cells were then washed and suspended with RPMI 1640 medium containing 15% PBS and 0.3% agar (Marine Colloid). Then 1 ml of the cell suspension (1 x

10 'cells) on a 0.4% agar layer in 6-well microtiter plates (Costar). It was done

The resulting colonies were labeled with 0.5 ml violet p-iodo-nitrotetrazolium at a concentration of 1 mg / ml (Sigma) for 48 hours. Colonies were counted using an Optimax 40-10 image analyzer and inhibition of colony formation was determined by comparing observable values with drug treated or immunoconjugate treated cells to the untreated control.

Figure 11 compares the cytotoxic activity of 5E9-ADM conjugate, specifically 5E9-ADM-7.5 and 3A1-ADM conjugate, specifically 3A1-ADM-7.0, after 1.5 hours exposure on a Daudi cell line Burkitt) positive on 5E9 antigen and negative on 3A1 antigen. These two immunoconjugates were prepared by thiolation with compound 2-IT. Comparison of dose / response curves shows that the 5E9-ADM-7.5 conjugate that retained 93% of the original binding activity relative to antigen-bearing target cells (see Figure 8) was significantly more painful than the control conjugate. non-binder 3A1-ADM-7.0.

A limiting dilution assay, which provides a measure of the annihilated cell logarithm, was used to test the cytotoxic pharmacological activity of the two aforementioned immunoconjugates using a longer exposure format (24 hours). This assay was performed using Namalwa cells (phenotype: 5E9 +, 3A1-) essentially as described by

M. Colombatti et al., Selective Killing Of Target Cells By Antibody-Ricin A Chain Or Antibody-Gelonin Hybrid Molecules: Comparison Of Cytotoxic Potency And Use In Immunoselection Procedures, J. Immunol., 131, pp. 3091-95 (1983). These cells obtained from the ATCC institution were incubated by the immunoconjugates for 22 hours and then washed and then the log number of the annihilated cells was determined. The logarithm of the number of annihilated cells was calculated based on the estimated plaque efficiency values relative to the portion of wells without growth at limiting cell concentrations.

As shown in Figure 12, the 5E9-ADM-7.5 conjugate resulted in a cellular annihilation corresponding to a 1-2-fold higher logarithm at the tested concentrations compared to the non-ligand 3A1-ADM-7.0 conjugate. Cell annihilation was measured to 5-fold logarithm for the highest dose of 5E9-ADM-7.5 conjugate. In addition, although cytotoxic activity was detected for the non-binding 3A1 immunoconjugate, the level of cytotoxicity was lower than for an equivalent amount of unconjugated ADM compound. However, the immunoconjugate activity of 5E9 at various concentrations was greater than that of an equivalent dose of free ADM compound.

As specified above, immunoconjugates 5E9-ADM-7.5 and 3A1-ADM-7.0 were synthesized using thiolation.

evidence from prepared institution ATCC compound 2-IT as thiolating agent. Immunospecific immunoconjugate cytotoxicity was also observed which were prepared using SPDP compound as the agent of Figure 13 shows the selective cytotoxic activity of the 5E9-ADM and 3A1-ADM immunocojugates prepared using the SPDP compound as a thiolating agent in Daudi cells. For this purpose the soft agar colony formation assay described above has been used. Other selective cytotoxicity of immunoconjugates is shown in Figure 14 using the SPDP compound, wherein the G28.1-ADM-9.0 immunoconjugate was tested on two G28.1 antigen positive cell lines, namely

Daudi and Namalwa and on a G28.1 antigen negative human T-cell leukemia cell line, designated HSB-2, using a soft agar colony assay. HSB-2 cells were obtained as shown in Figure, the immunoconjugate was cytotoxic to the two antigen positive cell lines but was not cytotoxic to the antigen negative cell line.

Preferred antigen-positive cell annihilation was observed by immunoconjugate 5E9-ADM-7.5 in a colony-forming assay using the HCT116 attachment-dependent human colon carcinoma cell line obtained from Dr. M Brattain [Bristol-Baylor Labs, Houston, TX].

Carcinoma cell monolayer cultures were removed from the trypsin-EDTA culture flasks (GIBCO) and washed and further passed through a 22 gauge needle to provide a single cell suspension. The conjugates were serially diluted.

5E9-ADM-7.5 or 3A1-ADM-7.0 or the unconjugated ADM compound in 5

0.2 ml medium containing 1 x 10 6 carcinoma cells. Each dilution was performed in triplicate. Controls were established based on untreated or antibody-treated cells. The cells were incubated for 3 hours and washed once with said medium and then placed in ~ 3 12-well microtiter plates (Costar) 1 x 10 5 cells in 1 ml medium. The plates were incubated for 7-10 days at 37 ° C and fixed with absolute methanol for 10 minutes. Colonies were stained with crystal violet and counted using an Optimax 40-10 image analyzer. As shown in Figure 15, greater cytotoxicity was observed when carcinoma cells were exposed to 5E9-ADM-7.5 conjugate compared to values observed when exposed to 3A1-ADM-7.0 conjugate.

Since there are many reports demonstrating that the antibody-bound ADM compound in the amino sugar moiety of the drug yielded immunoconjugates exhibiting significant loss of pharmacological activity, the cytotoxicity of the immunoconjugates prepared by coupling the ADM compound to the amino moiety was tested.

-sugar drug through a leu-ala dipeptide linker using the well-known soft agar colony assay system. As shown in Figure 16, none of the 5E9-ADM-4.0 or 3A1-ADM-3.9 peptide-linked conjugates have been shown to be cytotoxic in Daudi cells. 0 The ADM-leu-ala derivative used for the preparation of the conjugates was less powerful by a value whose logarithm is 2 times lower compared with equivalent amounts of the unconjugated ADM compound.

EXAMPLE 2

This example describes the preparation of an anthracycline immunoconjugate according to the present invention wherein the ADM compound is conjugated to a monoclonal antibody via a coupling element having an acylhydrazone bond at its coupling site to the ADM compound molecule. and which additionally has a thioether bridge as part of its coupling to the antibody. This variant also provides a novel ADM acylhydrazide derivative.

PREPARATION OF IMMUNQUSTERS WHO HAVE A THIOETER BRIDGE IN THE UNION ELEMENT

Monoclonal antibody 5E9 (2.5 mg in 2.5 ml phosphate-buffered saline) was reacted with SMPB (succinimidyl-4- (p-maleimidophenyl) butyrate) (59.5 pg in 100 μΐ tetrahydrofuran). hydrofuran) at 30 ° C for 30 minutes the pH was adjusted to 6.0 using sodium citrate buffer, the mixture was passed through a PD-10 gel filtration column (Pharmacia) to separate from

unreacted materials the antibody containing the maleimide compound. The ADM-HZN derivative (1 mg) prepared as described in Example 1 was then dissolved in 1 ml of a MeOH / H 2 O mixture (9: 1) and ADM-HZN 0.5 μπιοΙθΞ was reacted with 0.5 mmol of tri-n-butyl phosphine in a 4: 1 mixture consisting of acetone: water to provide a novel reduced ADM-HZN compound (see Figure 17). After 10 minutes a 0.1 M solution of sulfur in toluene was added to destroy the remaining phosphine. The reduced ADM-HZN compound was then mixed with a 5E9 antibody containing the maleimide compound. Immunoconjugates thus produced were purified by passage through a PD-10 gel filtration column. In some cases, when the reduction of the toluene solvent was not complete, a layer of organic solvent was separated by dragging some protein from the reaction mixture. A gentle air stream was used to remove the solvent and the denatured protein was removed by centrifuging the mixture for 2 minutes at 16000 x g. The clear supernatant containing the immunoconjugates was then gel filtered and analyzed in PBS medium to pH 7.4. The ADM / antibody molar ratio was determined by spectrophotometry using DO28O values.It's from495 as described in Example 1. A typical reaction provided immunoconjugates at molar ratios of 3 to 4.

CYTOTOXIC ACTIVITY OF IMMUNOCONJUGUSTS WHO HAVE

A THIOETER BRIDGE

Several immunoconjugates prepared according to the variant of this example were subjected to tests for their cytotoxicity against antigen-positive and antigen-negative tumor cell lines for comparative purposes, For this purpose, an H-thymidine incorporation assay was used to measure inhibition of DNA synthesis according to this assay.

100 μΐ of each well dilution in 96-well microtiter plates. Each dilution was performed in triplicate. With the tumor cells a suspension was prepared in said medium and then 100 μΐ containing 1 χ 10 was introduced into each well.5 cells These cells were incubated for 24 hours at room temperature.

37 ° C in a humid atmosphere containing 5% CO2. To each well was added 50 μ 3 containing (6H] -thymidine (1 μθί) (New England Nuclear, 15Ci / mmole) and incubated for 4 hours at 37 ° C. The cells were transferred to microtiter plates (Millipore) and precipitated with cold 25% trichloroacetic acid (ATC) .The precipitates were washed 10 times with cold 5% ATC. The filters were dried and poured and scintillated in Econofluor liquid (New England Nuclear). All ι

-counts were corrected by subtracting the counting units corresponding to ambient noise.

An immunoconjugate in this embodiment, designated 5E9-ADM-3.9, was highly cytotoxic to 5E9 antigen positive Namalwa and HSB-2 cells (see Figure 18). Immunoconjugate was more potent than equivalent concentrations of unconjugated ADM compound.

In another experiment it was found that immunoconjugate 3A1-ADM-6.0 at ADM concentrations below 0.1 μg / ml was cytotoxic to HSB-2 cells positive for 3A1 antigens but not cytotoxic to Namalwa negative cells on 3A1 antigens (see Figure 20). At higher concentrations the immunoconjugate cytotoxicity was about the same for both cell lines.

EXAMPLE 3

IN VIVO ANTI-TUMOR ACTIVITY OF IMMUNOCIATES OF THIS INVENTION

Next, tests were performed on the in vivo anti-tumor activity of the immunoconjugates of the present invention.

More particularly, the ability of immunoconjugates to inhibit the growth of human B lymphoma tumors in mice was tested.

Daudi and Ramos primary solid tumors (Burkitt's lymphoma) were induced in shaved BALB / c mice by subcutaneous (sc) inoculation of lymphoid cells

kept in tissue culture. Ramos' cell line was obtained from the ATCC institution. Daudi and Ramos tumors were then sequentially infused in vivo into 4-6 week-old BALB / c (nu / nu) mouse females with a mass of 20 to 25 grams (Harlan 7

Sprague-Dawley) using 1 x 10 6 tumor cells / 0.1 ml in PBS medium for subcutaneous implantation in the flank of mice. Both tumor lines exhibited a linear growth rate between 200 and 4000 mm. The average time for a tumor to double in volume during the exponential growth period was 6.9 ± 0.8 days for Daudi tumors and 4.4 ± 0.6 days for Ramos tumors. Tumor volumes (V) were calculated as follows:

V = where c = length (mm) and 1 = width (mm).

When tumor volumes reached 400-600 mm, 3 for Daudi tumors, and 250-400 mm for Ramos tumors, mice were randomly assigned to groups of 5-10 animals for treatment with ADM- HCl (i.e. drug free), the ADM immunoconjugates of the present invention, the unconjugated monoclonal antibody or a mixture of the monoclonal antibody and ADM compound. The specificity of cellular annihilation has been demonstrated by comparing the anti-tumor activity obtained with the immunoconjugates tested (i.e., whose antibody component is reactive with the tumor cells that

intended to annihilate) with respect to the activity obtained using non-ligand conjugates (i.e. conjugates that are not reactive with that tumor population). The antibody plus free drug mixture was a control demonstrating the need for covalent coupling of the drug to the antibody.

Results were expressed in terms of tumor growth inhibition (TC) or tumor duplication delay (RDT) which was estimated from the delay in tumor volume doubling time (TDVT) when comparing growth curves of the tumors. cured groups of uninoculated control groups. The RTD value was calculated using the following form:

TC

RTD = ---------- TDVT x 3, 3 where T = time (number of days) for tumors in a treated group to reach 3000 mm, C = time (days) for tumors in a treated group control group reached 3000 mm and TDVT (tumor volume doubling time) = time (days) for tumor volume in control (untreated) mice 3 to increase from 1500 to 3000 mm. Each point represents the average volume of a tumor in the experimental group.

In these studies the anti-tumor activity of ADM immunoconjugates in Daudi or Ramos tumors was compared to: a)

obtained when using the free drug at an equivalent dose, route and timing and b) the activity obtained when using the free drug administered at its optimal dose, route and timing.

In all studies now described, ADM-HCl compound treatment was performed by adding 50-100 X DMSO to the powdered drug, diluting the dissolved drug in PBS to a particular dosage (mg / kg / inj) on day 1. injection and inoculating it into the tumor-bearing mouse either intravenously (iv, tail vein) or intraperitoneally (ip). The immunoconjugates used in these studies were prepared as described in Example 1 and all retained more than 90% of the original antibody binding activity. Specifically, monoclonal antibodies 5E9 and G28.1 were used as antibody components of immunoconjugates in these studies. ADM immunoconjugates were stored at 4 ° C in PBS and used before two weeks after their preparation. All immunoconjugates tested as well as unconjugated antibody controls were administered intraperitoneally (ip).

On the other hand, as used in this specification, the notation corresponding to the Q7Dx3 treatment schedule identifies a treatment schedule in which each mouse in the group of that drug received 3 injections each spaced 7 days, ie one weekly injection. for 3 weeks. Similarly, the notation

Q5Dx2 identifies a treatment schedule in which mice in this group received a total of 2 injections of drug or conjugate with a 5 day interval. The QlDxl notation identifies a single injection. Thus, treatment scheme notations are defined such that the first notation number indicates the time interval (in days) of the injections and the last number represents the total amount of injections of each treatment scheme.

Accordingly, the anti-tumor activity of the ADM immunoconjugates of the present invention was first evaluated compared to the activity of the free drug ADM-HCl at the same or equivalent dose, route of administration and treatment schedule. The anti-tumor activity on Daudi tumors of a 5E9-ADM immunoconjugate specifically designated 5E9-ADM-1.8 (molar ratio = RM = 1.8 ADM / MAB molecules) was compared with the activity of a) unconjugated ADM 'HCl compound at identical drug dose (4.1 mg / kg / inj), b) monoclonal antibody 5E9 at identical antibody dose (630 m9 / kg / inj), c) a 5E9 antibody mixture plus ADM-HCl compound (4.1 mg ADM + 630 mg 5E9) and d) a non-binding immunoconjugate designated L6-ADM-8.6 (4.1 mg / kg / inj ADM) as a control element.

Mice (5 mice / group) were dosed intraperitoneally (ip) at 20 and 25 days after tumor implantation (i.e. according to a type scheme).

Q5Dx2) when the initial tumor dimensions reached 3 values between 800 and 1100 mm. The dose used in this experiment was the maximum tolerated dose (DMT), ip for the free drug, ie the drug dose administered by any given route or according to a given schedule giving a DL] value (lethal dose to 10 % of animals) (see Table 1 below).

As Figure 21 indicates, significant anti-tumor activity was obtained with the 5E9-ADM conjugate. On the other hand, this anti-tumor activity was greater than that observed for an equivalent dose of free drug. As Table 4 below indicates, 3 of the 5 conjugate treated mice exhibited complete tumor regression (cures) which corresponds to an RTD value> 1.5. In contrast, ADM-HCl compound and unconjugated 5E9 antibody and non-ligand conjugate L6-ADM exhibited no anti-tumor activity. Some inhibition of tumor growth was observed using compound ADM-HCi mixed with antibody 5E9, but this effect was transient and represented only a statistically insignificant RTD value = 0.2.

In this experiment the free drug was dosed at 4.1 frogg / kg / inj due to free drug-associated toxicity when administered intraperitoneally (ip) at doses greater than 4-5 mg / kg. For these poor dose values both free ADM compound and mixtures of ADM compound plus monoclonal antibody were inactive. However, ί-Υ.

As Figure 21 shows, even at this low dose, ADM immunoconjugate was still active in inhibiting tumor growth.

Next, the anti-tumor activity of ADM immunoconjugates on Daudi tumors was compared with the anti-tumor activity obtained when using the unconjugated drug administered at its optimal dose, route of administration and schedule. Therefore, the dose, route of administration and administration schedule of the free ADM-HCl compound providing maximum anti-tumor activity on Daudi cells had to be determined. For this optimization study the mice were treated with the ADM-HCl compound using different dosages, schemes and routes of administration. The interval between inoculations depended on the treatment schedule used. Then the RTD values were determined as described below.

The results of this optimization study are summarized in Table 2 below. As shown in Table 2, the intravenously applied (iv) Q7Dx3 scheme provided an optimal anti-tumor response, both in terms of tumor growth retardation and tumor regression rates at doses corresponding to 11 mg / kg / inj, which was also the DMT value for the drug when the Q7Dx3 regimen was administered intravenously (iv).

Table 2

ADM-HCl Compound Anti-Tumor

Daudi Tumor Xenografts

About the

Activity

Dose (mg / kg) - Tumor Inhibition— Toxicity0

<td>Scheme</td><td>mj</td><td>cum</td><td>TC</td><td>RC</td><td>Cures</td><td>RTD</td><td>M / T</td><td> (%)</td>

<td>QlDxl</td><td> 20</td><td> 20</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 7/7</td><td> (100)</td>

Q4Dx3 5 15

Q7Dx3 10 30

6/9 ( 67)

8/8 (100) 6/9 ( 67)

Results of groups of an individual drug.

^ TC: represents the time delay in days for the drug treated group (T) until reaching 3000 mrt? compared to untreated controls (C).

Complete Regression (CR): Temporary reduction of tumor volume below palpable tumor dimensions.

Cures: Complete regression without any evidence of new tumor development.

Q —Μ / T: total number of deaths relative to the total number of animals in a group. Drug-induced deaths were recorded up to 55 days after the last dose of drug.

Free drug anti-tumor activity on Daudi tumor cells is also depicted in Figure 22. Using the Q7Dx3 scheme by intravenous administration (iv) it was possible to significantly inhibit the growth of Daudi tumor xenografts by a dose dependent process respectively. 9, 10 or 11 mg / kg / injection after treatment with ADM-HCl. control mice were not treated. Inhibition of tumor growth (TC) to the DMT value of 11 mg / kg / inj. Occurred at 28 days which corresponds to a

RTD 1.1.

Several schemes were also tested using intraperitoneal (ip) administration. As noted above, the ADM-HCl DMT of the intraperitoneal route (ip) was determined to be between 4 and 5 mg / kg / inj. As can be seen from Table 2B, the free drug is inactive in Daudi cells for their DMT when administered peritoneally (ip). Thus, the optimal anti-tumor activity for the free drug has been determined to be obtained by intravenous (iv) administration wherein the DMT is 11 mg / kg / inj, a dose of drug that inhibits tumor cell development. from Daudi.

From these experiments it was therefore determined that the optimal dose of ADM-HCl compound for anti-tumor activity with respect to Daudi tumors was approximately 11 mg / kg / inj and the optimal schedule was also found to be the type scheme Q7Dx3 and that the route

The optimal administration route is intravenously (iv).

The anti-tumor activity on Daudi tumors of the ADM immunoconjugates of the present invention was then compared with the anti-tumor activity of the free drug ADM-HCl administered under optimal conditions as determined above. A G28.1-ADM immunoconjugate specifically designated G28.1-ADM-7.6 (RM = 7.6 drug / MAB) dosed according to a Q5Dx2 intraperitoneal (ip) administration schedule was compared with compound ADM-HCl dosed at 10, 11 and 12 mg / kg / inj according to a Q7Dx3 intravenous (iv) administration schedule. As shown in Figure 23 and Table 3 below, the free drug was active causing a 28 day delay in tumor development at a dose of 11 mg / kg (its DMT) exhibiting two mice in eight a complete tumor regression. (cures). At the highest dose tested (18.7 mg ADM, Q5Dx2, ip), the immunoconjugate was well tolerated (no deaths or weight loss) and exhibited slightly higher anti-tumor activity than free drug with the finding that three out of eight treated animals had complete tumor regression. Again it was found that there was no anti-tumor activity associated with non-binding L6 immunoconjugate, unconjugated G28.1 antibody or a mixture of unconjugated G28.1 antibody plus ADM-HCl compound. Thus, ADM immunoconjugates were found to inhibit tumor development more intensely than

This could be achieved using the unconjugated drug at its optimal intravenous (iv) or intraperitoneal (ip) dose and schedule.

Table 3

Anti-Tumor Activity of MAB (or AcM) -ADM Conjugate (Q5Dx2; ip) Compared to Compound ADM-HCl under Optimal Conditions (Q7Dx3; iv) Over Daudi Tumor Xenografts

<td rowspan="2">Dose ADM</td><td rowspan="2">(mg / kg) - MAB (or Ac)</td><td colspan="3">Tumor Inhibition -</td><td colspan="2">Q Toxicity</td>

<td>M) TC</td><td>RC</td><td>Cures</td><td>RTD</td><td>M / T (%)</td>

* See the caption of Table 2.

Table 4 summarizes the anti-tumor activity obtained using different immunoconjugate preparations of

5E9 and G28.1 on Daudi tumor xenografts in athymic mice. The highest response rate was consistently obtained at antibody doses of 500 mg / kg or higher. At these dose values anti-tumor activity was obtained using conjugates having molar ratios of 1.8 to 8.6. Apparently the anti-tumor activity is dose dependent on the antibody rather than dose dependent on the conjugate drug, as evidenced by the fact that as the monoclonal antibody dose increased there was a corresponding increase in both the RTD value, i.e. inhibition of tumor development and regression rates. In all experiments no anti-tumor activity was observed with non-ligand L6-ADM conjugates that were tested in parallel with equivalent doses of conjugated drug and antibody (data not shown). In addition, this table illustrates the increased potency of the immunoconjugates of the present invention compared to the free drug which was inactive at equivalent doses of this drug (compare with Table 2 above).

Table 4

Anti-Tumor Activity of ADM Immunoconjugates

Daudi's Tumor Xenografts

<td rowspan="2">Conjugate-RM</td><td>Healing Dose</td><td colspan="2">(mg / kg)</td><td rowspan="2">TCB(Days)</td><td rowspan="2">Cures</td><td rowspan="2">RTD</td>

<td>Ad scheme</td><td>.: Q5Dx2</td><td>Via :</td><td colspan="2">ip RM: ratio</td><td>molar</td><td>in between</td>

drug / MAB (or AcM) molecules BCT: represents the time delay in days for the drug-treated group (T) to 3000 mn? compared to untreated controls (C).

'Cures: Cures / number of animals treated. 5E9-ADM-4. 2 tested in three doses.

G28.1-ADM-7.6 tested at three doses.

'deaths in the control group.

Table 5 below demonstrates the reduced toxicity achieved using the ADM immunoconjugates of the present invention compared to the unconjugated drug. As concluded, immunoconjugates were at least 10 times less toxic than intraperitoneally administered (ip) free ADM compound.

Toxicity of and to MAB Conjugate (or AcM)

Tumors

Painting

Free ADM Compound

-ADM in Shaved Mice

ROM (mg / kg)Ç %

<td>Compound</td><td>N</td><td>Ad scheme</td><td>Via</td><td>inj</td><td>Cumulative</td><td colspan="2">M / T Death</td>

<td>ADM</td><td> 4</td><td>QlDxl</td><td>i. v.</td><td> 18</td><td> 18</td><td> 14/32</td><td> 44</td>

<td>ADM</td><td> 3</td><td>QlDxl</td><td>i. v.</td><td> 16</td><td> 16</td><td> 3/31</td><td> 10</td>

<td>ADM</td><td> 1</td><td>QlDxl</td><td>i. v.</td><td> 14</td><td> 14</td><td> 0/8</td><td> 0</td>

<td>Mice ^ N = Number</td><td colspan="3">bearers of tumors of experiences</td><td>from Daudi</td><td>or of</td><td>Branches</td><td></td>

<td colspan="4">ÇADM = quantity administered,</td><td>free or</td><td colspan="2">in conjunction with</td><td>MAB</td>

(or MCA) ^ D / T = # deaths / total treated

Finally Figure 26A and Table 6 represent the anti-tumor activity of G28.1-ADM conjugates on Ramos tumors in humans. In this case the anti-tumor effect of the immunoconjugates on Ramos tumors was also compared with the effect observed using the free ADM-HCl compound under conditions that provided optimal results which were previously determined and corresponded to a single dose injection of 16-18 mg / kg / inj (see Figures 24 and 25). For the highest immunoconjugate dose that was tested (10.6 mg / kg), the anti-tumor activity of the conjugate was greater than that obtained using the free drug at a dose of 18 mg / kg (25% lethality) in 0.5 RTD and the activity of the ADM * HCl compound at a dose of 16 mg / kg (12% lethality) was higher by 1.0 RTD. Conjugate administered at this dose was well tolerated with no deaths or weight loss in all treated animals. The anti-tumor activity of the G28.1-ADM conjugate was also found to be dose dependent as shown in Figure 26B. Thus, the decrease in conjugate dose resulted in decreases in RTD value and number of complete regressions. L6-ADM (non-binder) conjugate administered at a comparable dose (10.6 mg / kg) was inactive.

Anti-Tumor Activity of MAB (or AcM) -ADM Conjugate (QdDx4;

ip) Compared With ADM * HC1 Compound under Conditions

Optimal (QlDxl; iv) On Ramos Tumor Xenografts

Table 6

<td rowspan="2">Dose ADM</td><td rowspan="2">(mg / kg) - MAB (or AcM</td><td colspan="3">Tumor Inhibition—</td><td rowspan="2">RTD</td><td colspan="2">Toxicity0</td>

<td>) TC</td><td>RC</td><td>Cures</td><td>M / T</td><td> (%)</td>

the dose per injection is the caption of Table 2

Q see caption in Table 2

The foregoing examples demonstrate the preparation of novel anthracycline immunoconjugates in which a drug of a cytotoxic anthracycline was conjugated to an antibody via a novel acid-sensitive acy1-hydrazone bridge. Such immunoconjugates simultaneously retain antibody binding activity (i.e. target cell specificity) and cytotoxic pharmacological activity and allow release of unmodified free drug under acidic and reducing conditions typical of the cell environment of the target cells. The anti-tumor activity of these conjugates was demonstrated both in vitro and in vivo and found to be superior to the activity obtained with unconjugated free anthracycline. In addition, these immunoconjugates were tolerated in vivo in much greater amounts than unconjugated drug. Thus, the immunoconjugates of the present invention exhibit a better therapeutic index (anti-tumor activity relative to toxicity) and are therefore particularly useful for providing cytotoxic drugs to a selected cell population for preferential annihilation of such cells in the treatment of diseases such as cancers and other tumors, non-cytocidal viral infections and other pathogenic infections and even in the case of autoimmune diseases.

While various aspects and variants of the present invention have been described and disclosed, it is evident that this basic description may be altered to provide other variants in which the immunoconjugates and methods of the present invention are used. For this reason it will be appreciated that the scope of the present invention is much better defined by the appended claims than by the specific aspects set forth above by way of example.

Contents21

40 members in 17 offices

This recordMember, by publication datePriority claim7 more offices are in the list below

Priority claims2

Priority claims
Document	Office	Kind	Date
15518188	United States of America	A	1988-02-11
27050988	United States of America	A	1988-11-16

Members40

Family members, oldest first
Document	Office	Kind
DK63989D0	Denmark	D0	1989-02-10
NO890593D0	Norway	D0	1989-02-10
IE890434L	Ireland	L	1989-08-11
DK63989A	Denmark	A	1989-08-12
FI890599A	Finland	A	1989-08-12
FI890599A7	Finland	A7	1989-08-12
NO890593L	Norway	L	1989-08-14
NO950099L	Norway	L	1989-08-14
EP0328147A2	European Patent Office (EPO)	A2	1989-08-16
IL89220D0	Israel	D0	1989-09-10
KR890012658A	Republic of Korea	A	1989-09-18
JPH01246295A	Japan	A	1989-10-02
PT89683A	Portugal	A	1989-10-04
ZA89938B	South Africa	B	1989-11-29
NZ227911A	New Zealand	A	1990-11-27
CA2010164A1	Canada	A1	1991-08-15
EP0328147A3	European Patent Office (EPO)	A3	1991-11-13
US5122368A	United States of America	A	1992-06-16
IL106992D0	Israel	D0	1993-12-28
IL89220A	Israel	A	1994-02-27
PT89683BThis record	Portugal	B	1994-03-31
EP0328147B1	European Patent Office (EPO)	B1	1994-05-11
AT105486T	Austria	T	1994-05-15
ATE105486T1	Austria	T1	1994-05-15
DE68915179D1	Germany	D1	1994-06-16
IL106992A	Israel	A	1994-06-24
MY104944A	Malaysia	A	1994-07-30
ES2053828T3	Spain	T3	1994-08-01
DE68915179T2	Germany	T2	1994-10-06
NO950099D0	Norway	D0	1995-01-10
IE64650B1	Ireland	B1	1995-08-23
NO178229B	Norway	B	1995-11-06
NO178229C	Norway	C	1996-02-14
KR960015398B1	Republic of Korea	B1	1996-11-13
JP2740841B2	Japan	B2	1998-04-15
NO303225B1	Norway	B1	1998-06-15
FI102355B	Finland	B	1998-11-30
FI102355B1	Finland	B1	1998-11-30
CA2010164C	Canada	C	2000-08-29
DK175174B1	Denmark	B1	2004-06-21

2 legal events, as the office reported them to INPADOC

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Legal events, newest first
	Event	Code
2009-02-18	Annulment/lapse due to non-payment of fees, searched and examined patentLapsedMAXIMUM VALIDITY LIMIT REACHEDMM4A	MM4A
1994-03-31	Patent granted, date of grantingGrantedFG3A	FG3A

Anthracycline immunoconjugates having a novel linker and methods for their production.

Abstract

Term

TECHNICAL FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF DRAWINGS

X

DETAILED DESCRIPTION OF THE INVENTION

X

SUMMARY OF AN ADRIAMICINE-HYDRAZONE DERIVATIVE

TIOLATION OF MONOCLONAL ANTIBODIES

CONJUGATION OF TIOLATED MONOCLONAL ANTIBODIES

WITH ADM-HZN COMPOUND

CHARACTERIZATION OF IMMUNOCIATES OF THIS INVENTION

CYTOTOXIC ACTIVITY OF IMMUNOCIATES OF

PRESENT INVENTION

EXAMPLE 2

PREPARATION OF IMMUNQUSTERS WHO HAVE A THIOETER BRIDGE IN THE UNION ELEMENT

CYTOTOXIC ACTIVITY OF IMMUNOCONJUGUSTS WHO HAVE

A THIOETER BRIDGE

EXAMPLE 3

IN VIVO ANTI-TUMOR ACTIVITY OF IMMUNOCIATES OF THIS INVENTION

TC

Priority claims2

Members40

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Titles2

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