RU2471499C2 - New synergetic effects - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a method of treating or modulating malignant cell population growth involving the introduction into an individual suffering a malignant tumour of an effective amount of lenalidomide, at least one corticosteroid, and at least one immunoconjugate, wherein the immunoconjugate contains at least one cell-binding agent which binds CD56, and at least one antimitotic agent wherein the cell-binding agent is an antibody, a one-chain antibody or an antigen-binding antibody fragment.

EFFECT: invention provides a therapeutic synergy or improved therapeutic index in cancer therapy as compared with separately used immunocojugate, chemotherapeutic agent used separately or in a combination with the other chemotherapeutic agent with no immunoconjugate added.

53 cl, 7 ex, 8 dwg, 7 tbl

Description

This application claims priority to Provisional Application US No. 61/061886, filed June 16, 2008, the full disclosure of which is expressly incorporated into this description by reference.

Technical field

The present invention relates to anti-cancer combinations containing their pharmaceutical compositions and their use in the treatment of cancer. In particular, the present invention is based on the discovery that the administration of a combination comprising at least one conjugate of a drug with a cell-binding agent (e.g., immunoconjugate) and one or more chemotherapeutic agents selected from proteasome inhibitors (e.g., bortezomib), immunomodulators / antiangiogenic agents (e.g. thalidomide or lenalidomide) and DNA alkylating agents (e.g. melphalan), with optional further addition of a corticosteroid (e.g. dexamethasone) therapeutic synergy or improves the therapeutic index in treating cancer, as compared with treatment with anti-cancer (s) agent (s). The present invention also provides methods for modulating the growth of selected cell populations, such as cancer cells, by administering a therapeutically effective amount of such a combination.

State of the art

In preclinical studies, the effect of a combination of anti-cancer drugs can be studied in vitro on cell lines or in vivo on various tumor models. Anticancer drugs are usually combined with various mechanisms of inducing cell death, i.e. targeting various targets in the cell. In such experimental systems, it was observed that two anticancer drugs with independent targets (mutually exclusive drugs) act in an additive, synergistic, or antagonistic manner. Chou and Talalay (Adv. Enzyme Regul. 1984, 22: 27-55) developed a mathematical method that accurately describes the experimental data obtained in both qualitative and quantitative form. They have shown that for mutually exclusive drugs, the generalized isobole equation (curve of the same effect) is applicable to any degree of severity of the effect (see Chou and Talalay, p. 52). An isobole or isobologram is a graphical representation of all dose combinations of two drugs that have the same degree of effect, for example, combinations of two cytotoxic drugs will induce the same degree of cell death, for example, 20% or 50% cell death. The equation is valid for any degree of expression, and the graphical representation will have the same form (Chou and Talalay, p. 54, line 1), which is shown in Fig. 11D (Chou and Talalay, p. 5). In isobolograms, a straight line indicates an additive effect, a concave curve (a curve below a straight line) represents synergistic effects, and a convex curve (a curve above a straight line) represents antagonistic effects. These curves also indicate that a combination of two mutually exclusive drugs will exhibit the same effect over the concentration range for additive, synergistic, or antagonistic combinations. Most drug combinations exhibit an additive effect. However, in some cases, the combinations exhibit an effect that is smaller or larger than the additive. Such combinations are called antagonistic or synergistic, respectively. Antagonistic or synergistic effects are not predictable, as are the obtained experimental data. Combinations show therapeutic synergy if they are superior from a therapeutic point of view to one or the other of the components used in the optimal dose. See Corbett et al., Cancer Treatment Reports, 66, 1187 (1982). Tallarida RJ (J Pharmacol Exp Ther. 2001 Sep; 298 (3): 865-72), which also states: “Two drugs that have an obviously similar effect will sometimes have an enhanced or weakened effect when used concomitantly. Quantification is needed to distinguish these cases from a simple additive action. "

The unpredictability of antagonistic or synergistic effects: is well known to the person skilled in the art and has been demonstrated in several other studies, for example Knight et al. See Navy Cancer 2004, 4:83. In this study, the authors measured the activity of gefitinib (also known as the Press) against a wide range of solid tumors, including breast, colon, rectal, esophageal and ovarian cancers, carcinomas with unknown primary tumor location, melanoma of the skin and choroid of the eyeball, non-small cell lung cancer (NSCLC) and sarcoma.

They found that there is heterogeneity in the degree of inhibition of tumor growth (IRO) observed when testing tumors against a single agent, gefitinib. Significant inhibition of tumor growth was observed for 7% (6/86) tumors, but most often a moderate reaction was found, leading to a low degree of RTI. Interestingly, it had both positive and negative effects when used in combination with various cytotoxic drugs. For 59% (45/76) of the studied tumors, the addition of gefitinib appears to enhance the effect of a cytotoxic agent or combination (of which, for 11% (5/45), a> 50% decrease in Index _SUM was observed). For 38% of tumors (29/76), the RTI decreased with the combination of gefitinib + cytotoxic drug in comparison with the use of the cytotoxic drug alone. For the remaining 3% (2/76), no change was observed.

The authors conclude that heftinib in combination with various cytotoxic agents (cisplatin; gemcitabine; oxaliplatin; threosulfan and “threosulfan + gemcitabine”) is a double-edged weapon: its effect on the growth rate may make some tumors more resistant to concomitant cytotoxic chemotherapy, at that time how exposure to cytokine-mediated survival mechanisms (anti-apoptotic) of cells can potentiate the sensitivity of tumors from other individuals to the same drugs. See conclusion on page 7; see also fig. 3. Knight et al., Navy Cancer 2004, 4:83.

Thus, this study proves that two compounds with known suitability for the same purpose, combined for this purpose, do not necessarily achieve this goal.

However, the search for highly effective combinations, i.e. synergistic mixtures of active agents are promising. Intuition is not an effective means, since the number of potential combinations of means is extremely large. For example, there are trillions of possible 5-component combinations of even a relatively small palette of 5,000 potential remedies. The potential of another obvious discovery strategy by deductively inferring potential combinations based on knowledge of the mechanism is also limited, since many biological pathways in living organisms are affected by multiple pathways. These paths are often not known, and even when they are known, the ways in which such paths interact to provide the ultimate biological effect are often not known.

We previously demonstrated a synergistic combination of a maytansinoid immunoconjugate containing a maytansinoid compound bound to a monoclonal antibody with that of a taxane compound, an epothilone compound, a platinum compound, an epipodophyllotoxin compound and camptothecin compound.

The synergistic use of a combination of drugs, even if it has been previously demonstrated, does not eliminate the need for new synergistic combinations, since the synergistic effects are unpredictable and the experimental data obtained are unpredictable. For example, in the treatment of autoimmune deficiency syndrome (AIDS), which includes highly active antiretroviral therapy (NAAOT), it is believed that a cocktail of HIV virus reverse transcriptase (OT) and viral protease (VP) inhibitors exhibits synergistic inhibition of virus replication. Later, synergy was also observed in an intriguing manner within two classes of OT inhibitors - nucleoside OT inhibitors (NRTIs) demonstrated synergy with non-nucleoside OT inhibitors (NNRTIs) in the absence of VP inhibitors. For example, NRTIs AZT (zidovudine) and NNRTIs nevirapine show synergy when administered in combination (Basavapathruni A et al., J. Biol. Chem., Vol. 279, Issue 8, 6221-6224, February 20, 2004). Thus, there is still a need to find drug combinations that exhibit synergism and can be effectively used to treat and prevent disabling diseases such as cancer.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the administration of a combination comprising at least one drug / cell conjugating drug conjugate (eg, immunoconjugate), hereinafter “conjugate”, and at least one chemotherapeutic agent selected from proteasome inhibitors (eg bortezomib), immunomodulators / antiangiogenic agents (e.g. thalidomide or lenalidomide) and DNA alkylating agents (e.g. melphalan), with optional additional corticosteroid (e.g. dex amethasone) exhibits therapeutic synergy or improves the therapeutic index in the treatment of cancer, in comparison with a single immunoconjugate or chemotherapeutic agent used alone or in combination with another chemotherapeutic agent, without adding an immunoconjugate.

In a preferred embodiment, the conjugate and chemotherapeutic agent (s) are (a) administered in combination with a corticosteroid such as dexamethasone. For example, an immunoconjugate, such as the humanized N901-maytansinoid antibody conjugate (huN901-DM1), is administered in combination with a combination of thalidomide / dexamethasone, lenalidomide / dexamethasone or bortezomib / dexamethasone, where such a combination exhibits a therapeutic synergistic or therapeutic index compared with a single immunoconjugate, a chemotherapeutic agent used alone or in combination with another chemotherapeutic agent, without adding an immunoconjugate.

In another embodiment, two or more chemotherapeutic agents are used in combination with an immunoconjugate. For example, bortezomib and lenalidomide are used in combination with a huN901-maytansinoid conjugate, in the presence or absence of a corticosteroid such as dexamethasone, where such a combination exhibits therapeutic synergy or improves the therapeutic index in the treatment of cancer compared to the separately used immunoconjugate chemotherapeutic agent used alone or in combination with another chemotherapeutic agent, without the addition of an immunoconjugate.

As used herein, the term “therapeutic synergy” refers to a combination of a conjugate and one or more chemotherapeutic agents having a more pronounced therapeutic effect than the additive effect of a combination of a conjugate and one or more chemotherapeutic agents.

As another object of the present invention, methods are described for alleviating or treating cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of at least one conjugate (e.g., immunoconjugate) and one or more chemotherapeutic agents (e.g., proteasome inhibitor, immunomodulator / antiangiogenic agent or DNA alkylating agent), with optional further addition of a corticosteroid (e.g., dexamethasone) so that the combination exhibits ter pevticheskuyu synergy or improves the therapeutic index in treating cancer as compared to anti-cancer (s) agent (s) used alone or in combination, without added immunoconjugate.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising an effective amount of a conjugate (e.g., immunoconjugate) and one or more chemotherapeutic agents (e.g., proteasome inhibitor, immunomodulator / antiangiogenic agent or DNA alkylating agent), optionally together with a pharmaceutically acceptable carrier.

The present invention also provides the use of a conjugate (e.g., immunoconjugate) and a chemotherapeutic agent (e.g., proteasome inhibitor, immunomodulator / anti-angiogenic agent or DNA alkylating agent) with the optional addition of a corticosteroid to produce a drug for combination therapy with simultaneous, sequential or separate administration in the treatment of cancer or any disease resulting from abnormal cell proliferation. For example, the conjugate and drug (s) can be administered on the same days or other days using the optimal administration schedule for each agent. For example, in one embodiment, two compounds may be administered at intervals of 10 days, in another embodiment, within an interval of 5 days, and in another embodiment, within 24 hours or even simultaneously. Alternatively, huN901-DM1, chemotherapeutic agent (s), a corticosteroid, or any combination thereof, may be administered on every 2nd day, every other day, on a weekly basis, or at intervals ranging from 0-7 days (e.g., days 0, 1, 2, 3, 4, 5, 6, or 7) or within 0-4 weeks (e.g., weeks 0, 1, 2, 3, or 4, including days that may be added between 1 or more weeks). In some cases, it may be preferable to administer a chemotherapeutic agent immediately after the conjugate. For example, bortezomib is administered on day 0, after which huN901-DM1 is administered on day 3. Periods for drug administration can be determined by a person skilled in the art in accordance with the requirements of the clinical situation.

The present invention also provides methods for modulating the growth of selected cell populations, such as cancer cells, by administering a therapeutically effective amount of at least one conjugate (e.g., immunoconjugate) and one or more chemotherapeutic drugs (e.g., proteasome inhibitor, immunomodulator / antiangiogenic agent or alkylating DNA agent) with the optional addition of a corticosteroid in such a way that the combination exhibits therapeutic synergy or improves therapeutically the index in the treatment of cancer compared with anticancer (s) drug (s), used alone or in combination, without the addition of an immunoconjugate. The conjugate may include cell-binding agents and at least one therapeutic agent that causes the death of selected cell populations.

These and other aspects of the present invention are described in detail in this description.

Brief Description of the Figures

FIG. 1A shows the effect of the combination of “huN901-DM1 plus melphalan” on xpografts of multiple myeloma Molp-8.

FIG. 1B is a table (Table 1) showing data.

FIG. 2A shows the effect of the “huN901-DM1 plus thalidomide" combination on x-ray transplants of multiple Mylp-8 myeloma.

FIG. 2B is a table (Table 2) showing data.

FIG. 3A shows the effect of the combination “huN901-DM1 plus bortezomib" on the xenografts of multiple myeloma OPM2.

FIG. 3B is a table (Table 3) showing data.

FIG. 4A shows the effect of the combination “huN901-DM1 plus bortezomib" (low dose) on large x9 grafts of multiple myeloma H929.

FIG. 4B is a table (Table 4a) showing data.

FIG. 4C shows the effect of the combination “huN901-DM1 plus bortezomib" (high dose) on large x9 grafts of multiple H929 myeloma.

FIG. 4D is a table (Table 4b) showing data.

FIG. 5A shows the effect of the combination “huN901-DM1 plus lenalidomide" on the xenografts of multiple myeloma OPM2.

FIG. 5B is a table (Table 5) showing data.

FIG. Figure 6 shows the dependence of the anti-tumor activity of huN901-M1 with bortezomib on the administration schedule.

FIG. 7A shows the effect of the triple combination “huN901-DM1 plus lenalidomide plus a low dose of dexamethasone” on MOLP-8 multiple myeloma xenografts.

FIG. 7B is a table (Table 7a) showing data.

FIG. 8A shows the results of an immunohistochemical analysis of a marker of apoptosis, caspase-3 in xenografts of multiple myeloma MOLP-8 after treatment with the triple combination of “huN901-DM1 plus lenalidomide plus a low dose of dexamethasone”.

FIG. 8B shows a statistically significant synergistic increase in apoptosis in tumor cells on MOLP-8 multiple myeloma xenografts after treatment with the triple combination of huN901-DM1 with lenalidomide plus a low dose of dexamethasone compared to treatment with any of the therapeutic agents alone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected discovery that administration of at least one conjugate (e.g., immunoconjugate) and at least one chemotherapeutic drug (e.g., proteasome inhibitor, immunomodulator / antiangiogenic agent or DNA alkylating agent), with optional additional corticosteroid (dexamethasone) exhibits therapeutic synergy or improves the therapeutic index in the treatment of cancer compared to a separately used immunoconjugate, chemotherapy vticheskim tool used alone or in combination with another chemotherapeutic agent, without the addition of the immunoconjugate. Suitable conjugates and chemotherapeutic agents are described herein.

Conjugates

The conjugates of the present invention include at least one therapeutic agent that causes the death of selected cell populations associated with a binding agent to the cell.

A therapeutic agent causing the death of selected cell populations is preferably an antimitotic agent. Antimitotic agents that are known in the art cause cell death, inhibiting the polymerization of tubulin and thus the formation of microtubules. Any anti-mitotic agent known in the art can be used in the present invention, including, for example, maytansinoids, vinca alkaloids, dolastatins, auristatins, cryptophycin, tubulizine and / or any other agent that causes cell death, interfering with the polymerization of tubulin. Preferably, the antimitotic agent is maytansinoid.

An agent that binds to cells may be any suitable agent that binds to a cell, usually and preferably with an animal cell (e.g., a human cell). The cell binding agent is preferably a peptide or polypeptide. Suitable cell binding agents include, for example, antibodies (e.g., monoclonal antibodies and fragments thereof), lymphokines, hormones, growth factors, nutrient carrier molecules (e.g., transferrin). Below, therapeutic agents that cause the death of selected populations of cells and agents that bind to cells that may be part of an immunoconjugate are described in more detail.

Maytansinoids

Maytansinoids that can be used in the present invention are well known in the art and can be isolated from natural sources in accordance with known methods or synthetically prepared in accordance with known methods.

Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Examples of suitable maytansinol analogs include those containing a modified aromatic ring and containing modifications at other positions.

Specific examples of suitable maytansinol analogues containing a modified aromatic ring include:

(1) C-19-dechlor (U.S. Patent 4,256,746) (obtained by reconstituting ansamitocin P2 with PAH);

(2) C-20-hydroxy (or C-20-desmethyl) +/- C-19-dechloro (US Patent Nos. 4,316,150 and 4,307,016) (obtained by demethylation using Streptomyces or Actinomyces or dechlorination using PAH); and

(3) C-20-desmethoxy, C-20-acyloxy (-OCOR), +/- dechloro (US Pat. No. 4,294,757) (obtained by acylation of acid chlorides).

Specific examples of suitable maytansinol analogues containing modifications at other positions include:

(1) C-9-SH (US Pat. No. 4,424,219) (obtained by the reaction of maytansinol with H ₂ S or P ₂ S ₅ );

(2) C-14-alkoxymethyl (desmethoxy / CH ₂ OR) (US Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH ₂ OH or CH ₂ OAc) (US Pat. No. 4,450,254) (obtained from Nocardia);

(4) C-15-hydroxy / acyloxy (U.S. Patent 4,364,866) (obtained by the conversion of maytansinol Streptomyces);

(5) C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudifloráa);

(6) C-18-N-desmethyl (U.S. Patent Nos. 4,326,663 and 4,323,248) (obtained by desmethylation of Streptomyces maytansinol); and

(7) 4,5-deoxy (US Pat. No. 4,371,533) (obtained by reduction of maytansinol with titanium trichloride / PAH).

The synthesis of thiol-containing maytansinoids suitable in accordance with the present invention is fully disclosed in US Pat. Nos. 5,208,020, 5,416,064, 6,333,410, 7,276,497 and 7,301,019).

It is expected that all maytansinoids containing a thiol moiety at position C-3, position C-14, position C-15, or position C-20 will be suitable. The C-3 position of maytansinol is preferred, and the C-3 position is particularly preferred. Maytansinoids with an N-methyl-alanine-containing C-3 thiol moiety and maytansinoids with an N-methyl-cysteine-containing C-3 thiol moiety, as well as analogues of each of them, are also preferred.

Specific examples of maytansinoid derivatives with an N-methyl-alanine containing C-3 thiol moiety suitable for the present invention are represented by formulas M1, M2, M3, M6 and M7.

where l is an integer from 1 to 10; and may is a maytansinoid.

where R ₁ and R ₂ represent H, CH ₃ or CH ₂ CH ₃ and may be the same or different;

m is 0, 1, 2 or 3; and

may is a maytansinoid.

where n is an integer from 3 to 8; and

may is a maytansinoid.

where l is 1, 2 or 3;

Y ₀ represents Cl or H; and

X ₃ represents H or CH ₃ .

where R ₁ , R ₂ , R ₃ , R ₄ represent H, CH ₃ or CH ₂ CH ₃ and may be the same or different;

m is 0, 1, 2 or 3; and

may is a maytansinoid.

specific examples of maytansinoid derivatives with N-methyl-cysteine containing a C-3 thiol moiety suitable for the present invention are represented by formulas M4 and M5.

where o is 1, 2 or 3;

p is an integer from 0 to 10; and

may is a maytansinoid.

where o is 1, 2 or 3;

q is an integer from 0 to 10;

Y ₀ represents Cl or H; and

X ₃ represents H or CH ₃ .

Preferred maytansinoids are described in US Pat. Nos. 5,208,020; 5,416,064; 6333410; 6,441,163; 6,716,821; RE 39151 and 7276497.

Vinca alkaloids (e.g., vincristine), dolastatin compounds and cryptophycin compounds are described in detail in WO 01/24763. Auristatins include auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethylauristatin E (MMAE), described in US patent No. 5635483, Int. J. Oncol. 15: 367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No.8, pp. 921-932 (2004); US application No. 11/134826. US Publication Nos. 20060074008, 2006022925. Compounds of tubulizine are described in US Publication No.20050249740. Many of the agents listed under this heading can also be used as chemotherapeutic agents, if they are intended for such use.

Cell Binding Agents

The cell-binding agents used in this invention are proteins (e.g., immunoglobulin and non-immunoglobulin proteins) that specifically bind to target antigens on cancer cells. Such cell-binding agents include the following:

- antibodies, including:

- restored antibodies (US patent No. 5639641);

- humanized or fully human antibodies (humanized or fully human antibodies are selected, but not limited to, from huMy9-6, huB4, huC242, huN901, DS6, CD38, IGF-IR, CNTO 95, B-B4, trastuzumab, bivatuzumab, sibrotuzum pertuzumab and rituximab (see, for example, US patents Nos. 5639641, 5665357 and 7342110; U.S. Temporary Patent Application No. 60/424332, International Patent Application WO 02 / 16,401, Publication of US Patent No. 20060045877, Publication of US Patent No. 20060127407, Publication USA No. 20050118183, Pedersen et al., (1994) J. Mol. Biol. 235, 959-973, Roguska et al., (1994) Proceedings of the National Academy of Sciences, Vol 91, 9 69-973, Colomer et al., Cancer Invest, 19: 49-56 (2001), Heider et al., Eur. J. Cancer, 3 IA: 2385-2391 (1995), Welt et al, J Clin. Oncol 12: 1193-1203 (1994), and Maloney et al., Blood, 90: 2188-2195 (1997).); And

epitope-binding antibody fragments, such as sFv, Fab, Fab ', and F (ab') ₂ (Parham. J Immunol. 131: 2895-2902 (1983); Spring et al, J Immunol. 113: 470-478 (1974); Nisonoff et al, Arch. Biochem. Biophys. 89: 230-244 (I960)).

Additional cell-binding agents include other cell-binding proteins and polypeptides, for example, but not limited to:

- Ankyrin repeating proteins (DARPins; Zahnd et al., J Biol. Chem., 281, 46, 35167-35175, (2006); Binz, HK, Amstutz, P. & Pluckthun, A. (2005) Nature Biotechnology, 23 , 1257-1268) or ankyrin-like repeating proteins or synthetic peptides described, for example, in US Patent Publication No. 20070238667: US Patent No. 7,101,675; WO / 2007/147213; and WO / 2007/062466);

- interferons (e.g., α, β, γ);

- lymphokines, for example, IL-2, IL-3, IL-4, IL-6;

- hormones, such as insulin, TBH (thyrotropin-releasing hormone), MSH (melanocytostimulating hormone), steroid hormones such as androgens and estrogens; and

- growth factors and colony stimulating factors such as EGF, TGF-α, IGF-1, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984)).

If the cell-binding agent is an antibody (e.g., a single-chain antibody, a fragment of an antibody that binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody or a fragment of a monoclonal antibody, a chimeric antibody, a chimeric antibody fragment, a domain antibody, a domain antibody fragment, restored antibody, a restored single chain antibody or a fragment of a restored antibody, a human antibody or a fragment of a human antibody, humanis antibody or a restored antibody, a humanized single chain antibody, or a fragment of a humanized antibody), it binds to an antigen, which is a polypeptide and may be a transmembrane molecule (e.g., a receptor), or a ligand, such as a growth factor. Examples of antigens include molecules such as renin; growth hormone, including human growth hormone and bovine growth hormone; growth hormone release factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; alpha-1-antitrypsin; Insulin A chain; Insulin B chain; proinsulin; folliculotropic hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as vmc factor, factor IX, tissue factor (TF) and von Willebrand factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activator, such as urokinase or human urine or tissue plasminogen activator (t-PA); bombazine; thrombin; hematopoietic growth factor; tumor necrosis factor alpha and beta; enkephalinase; RANTES (regulated by activation, normally expressed and secreted by T cells); human inflammatory macrophage protein (MIP-1 alpha); serum albumin, such as human serum albumin; Muellerian inhibitory substance; Relaxin A chain; Relaxin B chain; prorelaxin; murine gonadotropin-linked peptide; microbial protein, such as beta-lactamase; DNase; IgE; cytotoxic T-lymphocyte bound antigen (CTLA) such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; protein A or D; rheumatoid factors; a neurotrophic factor, such as bone neurotrophic factor (BDNF), neurotrophin -3, -4, -5 or -6 (NT-3, NT4, NT-5, or NT-6), or nerve growth factor, such as NGF- β platelet growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor I and II (IGF-I and IGF-II); des (1-3) -IGF-I (IGF-I of the brain), proteins that bind to insulin-like growth factor, EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptors, EphB receptors folate receptor, FOLR1, mesothelin, crypto, integrin, VEGF, VEGFR, tarusferrin receptor, IOTA1, IOTA2, IOTA3, IOTA4, IOTA5; CD proteins, such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD23, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44 , CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic protein (MBK); interferon, for example alpha, beta and gamma interferon; colony stimulating factors (CSFS), for example M-CSF, GM-CSF and G-CSF; interleukins (IL), for example from IL-1 to IL-10; superoxide dismutase; T cell receptors; membrane surface proteins; decay acceleration factor; viral antigen, for example part of an HIV envelope; carrier proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens, for example, the HER2, HER3 or HER4 receptor; and fragments of any of the above polypeptides, an antibody that mimics adnectins (US application 20070082365), or an antibody that binds to one or more tumor-associated antigens or cell surface receptors disclosed in US Publication No. 20080171040 or: US Publication No.20080305044, incorporated by links in their entirety.

Additionally, GM-CSF, which binds to myeloid cells, can be used as an agent that binds to affected cells in acute myelogenous leukemia. IL-2, which binds to activated T cells, can be used to prevent transplant rejection, to treat and prevent graft versus host disease, and to treat acute T-cell leukemia. MSH, which binds to melanocytes, can be used to treat melanoma. Folic acid can be used to target the folate receptor expressed on ovarian and other tumors. Epidermal growth factor can be used to target various types of squamous cell carcinoma, such as lung cancer and head and neck cancer. Somatostatin can be used to target neuroblastoma and other types of tumors.

To target various types of breast and testicular cancer, estrogen (or estrogen analogues) or androgen (or androgen analogs), respectively, can serve as a cell-binding agent.

Preferred antigens for the antibodies included in the present invention include CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD18, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138 and CD152; members of the ErbB receptor family, such as the EGF receptor, the HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, EpCAM, integrin alpha ₄ / beta ₇ and integrin alpha _v / beta ₃ , including its alpha or beta subunit (e.g., anti-CD11a, anti-CD18 or -CD11b antibodies); growth factors such as VEGF; tissue factor (TF); TGF-β; alpha interferon (alpha IFN); interleukin such as IL-8; IgE; Apo2 blood group antigens, death receptor; flk2 / flt3 receptor; obesity receptor (OB); mpl receptor; CTLA-4; protein C etc. The most preferred targets in accordance with this description are IGF-IR, CanAg, EphA2, MUC1, MUC16, VEGF, TF, CD19, CD20, CD22, CD33, CD37, CD38, CD40, CD44, CD56, CD138, CA6, Her2 / neu , EpCAM, CRIPTO (a protein whose elevated levels are produced in most human breast cancer cells), darpins, integrin alpha _v / beta ₃ , integrin alpha _v / beta ₅ , TGF-β, CD11a, CD18, Apo2 and C242 or an antibody, which binds to one or more concomitant tumor antigens or cell surface receptors disclosed in US Patent No. 20080171040 or US Publication No. 200880305044, which are incorporated by reference ki in their entirety.

Preferred antigens for the antibodies included in the present invention also include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, CD37, CD38, CD46, CD56 and CD138; members of the ErbB receptor family, such as the EGF receptor, the HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Macl, p150.95, VLA-4, ICAM-I, VCAM, EpCAM, integrin alpha ₄ beta ₇ and integrin alpha _v / beta ₃ , including its alpha or beta subunit ( for example, anti-CD11a, anti-C018 or anti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF); TGF-β; alpha interferon (alpha IFN); interleukin such as IL-8; IgE; blood group antigens: Apo2, death receptor; flk2 / flt3 receptor; obesity receptor (OB); mpl receptor; CTLA-4; protein C etc. The most preferred targets in accordance with this description are IGF-IR, CanAg, EphA2, MUC1, MUC16, VEGF, TF, CD19, CD20, CD22, CD33, CD37, CD38, CD40, CD44, CD56, CD138, CA6, Her2 / neu , EpCAM, CRIPTO (a protein whose elevated levels are produced in most human breast cancer cells), integrin alpha _v / beta ₃ , integrin alpha _v / beta ₅ , TGF-β, CD1a, CD18, Apo2 and C242.

Methods for producing monoclonal antibodies allow the production of specific cell-binding agents in the form of monoclonal antibodies. Particularly well-known methods in the art for generating monoclonal antibodies produced by immunizing mice, rats, hamsters, or any other mammal with a target antigen, such as an intact target cell, antigens isolated from a target cell, a whole virus, an attenuated whole virus and viral proteins such as viral envelope proteins. Sensitized human cells can also be used. Another way to create monoclonal antibodies is to use sFv phage libraries (single chain variable region), specifically human sFv (see, for example, Griffiths et al., US Pat. No. 5,885,793; McCafferty et al., WO 92/01047; Liming et al WO 99/06587).

The selection of a suitable cell-binding agent is a matter of choice, which depends on the particular cell population to be targeted, but in general monoclonal antibodies and their epitope-binding fragments are preferred if a suitable antibody is available.

For example, the My9 monoclonal antibody is a mouse IgG2a antibody specific for the CD33 antigen found on acute myeloid leukemia (AML) cells (Roy et al. Blood 77: 2404-2412 (1991)) and can be used to treat AML patients. Also, an anti-B4 monoclonal antibody is murine IgGi that binds to the CD19 antigen on B cells (Nadler et al, J Immunol. 131: 244-250 (1983)) and can be used if the target cells are B- cells or diseased cells that express a given antigen, for example with non-Hodgkin lymphoma or chronic lymphoblastic leukemia. Antibody N901 is a murine monoclonal IgGi antibody that binds to CD56 found on small cell lung carcinoma cells and on cells of other neuroendocrine-derived tumors (Roy et al. J Nat. Cancer Inst. 88: 1136-1145 (1996)); huC242 is an antibody that binds to the CanAg antigen; trastuzumab is an antibody that binds to HER2 / neu; and the anti-EGF receptor antibody binds to the EGF receptor.

Chemotherapeutic agents

Medicines that can be used in the present invention include chemotherapeutic agents. A "chemotherapeutic agent" is a chemical compound suitable for the treatment of cancer. Preferred examples of chemotherapeutic agents are proteasome inhibitors, immunomodulatory agents, antiangiogenic agents, alkylating agents, or combinations thereof.

Proteasome inhibitors

Proteasome inhibitors are drugs that block the action of proteasomes, cell complexes that break down proteins. In one embodiment of the invention, the proteasome inhibitor is selected from the group consisting of: a) natural proteasome inhibitors, including peptide derivatives containing a C-terminal epoxyketone structure, β-lactone derivatives, aclacinomycin A, lactacystin, clastolactacysteine; b) synthetic proteasome inhibitors, including modified peptide aldehydes, such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-lecinal (also referred to as MG132 or zLLL), or derivatives of boronic acid and MG232, N-carbobenzoxy- Leu-Nva-H (also denoted MG115), N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (also denoted LLnL), N-carbobenzoxy-Ile-Glu (OBut) -Ala-Leu-H (also denoted by PS1); c) peptides containing the α, β-epoxyketone structure, vinyl sulfones, such as carbobenzoxy-L-leucinyl-L-leucinyl-L-leucine-vinyl-sulfone or 4-hydroxy-5-iodo-3-nitrophenylacetyl-L-leucinyl- L-leucinyl-L-leucine-vinyl sulfone (NLVS); d) glyoxal or boric acid residues such as pyrazyl-CONH (CHPhe) CONH (CH isobutyl) B (OH) ₂ and dipeptidyl derivatives of boric acid; e) pinacol esters, such as benzyloxycarbonyl ester (Cbz) -Leu-labor-Leu-pinacol. The proteasome inhibitors described in J Clin Pathol 116 (5): 637-646, 2001 or US Application No. 10/522706 (filed July 31, 2003) are also included in the context of the present invention. In a preferred embodiment, the proteasome inhibitor is PS-341 / bortezomib (Velcade ™).

Immunomodulating agents

By “immunomodulatory (s) drug (s)” is meant, for example, agents that act on the immune system directly or indirectly, for example, by stimulating or inhibiting cellular activity; cells of the immune system, such as T cells, B cells, macrophages or antigen presenting cells (APCs), or by acting on components outside the immune system, which in turn stimulates, inhibits or modulates the immune system, such as hormones, agonists or receptor antagonists and neurotransmitters; immunomodulators may be, for example, immunosuppressants or immunostimulants. By "anti-inflammatory drugs" is meant, for example, drugs that treat inflammatory reactions, i.e. tissue reaction to damage, for example, drugs that treat the immune, vascular, or lymphatic systems.

Anti-inflammatory or immunomodulatory drugs or agents suitable for use in this invention include, but are not limited to, interferon derivatives, for example betaseron, β-interferon; prostane derivatives, for example, compounds disclosed in PCT / DE93 / 0013, for example iloprost, cicaprost; glucocorticoids, for example cortisol, prednisone, methylprednisolone, dexamethasone; immunosuppressive agents, for example cyclosporin, FK-506, methoxalen, thalidomide, sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitors, for example, zileuton, MK-886, WY-50295, SC-45662, SC-41661A, BI-L-357; leukotriene antagonists, for example, the compounds disclosed in DE 40091171 German patent application P42 42 390.2; WO 9201675; SC-41930; SC-50605; SC-51146; LY 255283 (D.K. Herron et al., FASEB J. 2: Abstr. 4729, 1988); LY 223982 (D. M. Gapinski et al. J. Med. Chem. 33: 2798-2813, 1990); U-75302 and analogs, for example, described by J. Morris et al., Tetrahedron Lett. 29: 143-146, 1988, C. E. Burgos et al., Tetrahedron Lett. 30: 5081-5084, 1989; B. M. Taylor et al., Prostaglandins 42: 211-224, 1991; compounds disclosed in US patent No. 5019573; ONO-LB-457 and analogues, for example, described by K. Kishikawa et al., Adv. Prostagl. Thombox. Leucotriene Res. 21: 407-410, 1990; M. Konno et al., Adv. Prostagl. Thrombox. Leucotriene Res. 21: 411-414, 1990; WF-11605 and analogues, for example, disclosed in US patent No. 4963583; compounds disclosed in WO 9118601, WO 9118879; WO 9118880, WO 9118883, anti-inflammatory substances, for example, NPC 16570, NPC 17923, described by L. Noronha-Blab. et al, Gastroenterology 102 (Suppl.): 672, 1992; NPC 15669 and analogues described by R. M. Burch et al, Proc. Nat. Acad. Sci. USA 88: 355-359, 1991; S. Pou et al., Biochem. Pharmacol 45: 2123-2127, 1993; peptide derivatives, for example, ACTH and analogues; soluble TNF receptors; TNF antibodies; soluble interleukin receptors, other cytokines, T-cell proteins; antibodies against interleukin receptors, other cytokines and T-cell proteins. Additional examples of immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cyclosporin, and antibiotics (e.g., Macrolide FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin desimesitoris, mucofolizimezit, m brequinar, malononitrile amides (e.g. leflunomide), T-cell receptor modulators, and cytokine receptor modulators. For more information on T-cell receptor modulators and cytokine receptor modulators, see section 3.1. Examples of T cell receptor modulators include, but are not limited to, anti-T cell receptor antibodies (e.g., monoclonal anti-CD4 antibodies, monoclonal anti-CD3 antibodies, monoclonal anti-CD8 antibodies, monoclonal antibodies against CD40 ligand, monoclonal anti-CD2 antibodies ) and CTLA4-immunoglobulin. Examples of cytokine receptor modulators include, but are not limited to, soluble receptors (e.g., TNF-alpha receptor extracellular domain or fragment thereof, IL-6 receptor extracellular domain or fragment thereof), cytokines or fragments thereof (e.g., interleukin (IL) -2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12, IL-15, TNF-alpha, interferon ( IFN) alpha, IFN-beta, IFN-gamma and GM-CSF), antibodies against cytokine receptors (e.g. antibodies against IL-2 receptor, antibodies against IL-4 receptor, antibodies against IL-6 receptor, antibodies against IL receptor -10, antibodies against re IL-12 receptor), anti-cytokine antibodies (e.g., anti-IFN receptor antibodies, anti-TNF-alpha antibodies, anti-IL-1β antibodies, anti-IL-6 antibodies and anti-IL-12 antibodies). The funds are listed in US patent application No. 11/454559 (filed June 16, 2006). Preferred immunomodulatory drugs are those that are effective in the treatment of multiple myeloma, cancer, affecting blood, plasma or bone tissue. In a preferred embodiment, the immunomodulatory agent is selected from thalidomide (Thalomid) and lenalidomide (Revlimid).

Antiangiogenic agents

Antiangiogenic agents include, but are not limited to, receptor tyrosine kinase (RTKi) inhibitors described in more detail in US Patent Application No. 11/612744 (filed December 19, 2006) or 10/443254 (filed May 22, 2003); angiostatic cortisens; MMP inhibitors; integrin inhibitors; PDGF antagonists; antiproliferative agents; HIF-1 inhibitors; fibroblast growth factor inhibitors; epidermal growth factor inhibitors; TIMP inhibitors; insulin-like growth factor inhibitors; TNF inhibitors; antisense oligonucleotides; anti-VEGF antibody, VEGF trap, NSAIDs, steroids, SiRNA and the like, and prodrugs of any of the above agents. Other agents that will be useful in the compositions and methods of the invention include an anti-VEGF antibody (i.e., bevacizumab or ranibizumab); VEGF trap; siRNA molecules, or a mixture thereof, targeting at least 2 tyrosine kinase receptors; glucocorticoids (i.e., dexamethasone, fluoromethalone, medrisone, betamethasone acetonide, triamcinolone, triamcinolone, prednisone, prednisolone, hydrocortisone, rimexolone and their pharmaceutically acceptable salts, predicarbate, deflazacort, halomethasone, thixocortol, prednilidene diacetone diacetone diacetone) , methylprednisolone, meprednisone, mazipredone, isoflupredone, halopredone acetate, galcinonide, formocortoal, flurandrenolide, fluprednisolone, fluprednidine acetate, fluperolone acetate, fluorocortolone, fluocortin butyl, fluocinone n acetonide, flunisolide, flumethasone, fludrocortisone, fluclorinide, enoxolone, difluprednate, diflucortolone, diflorazone diacetate, deoxymethasone, desonide, descinolone, cortazazole, corticosterolonclozonolone clozonoloneclozonolone, cortisone clolzone clonzoloneclozonolone, allopregnan acetonide, alklomethasone, 21-acetoxypregnenolone, thralonide, diphlorazone acetate, desacylcortivazole, RU-26988, budesonide and desacylcortivazole oxetanone); naphthohydroquinone antibiotics (i.e. rifampicin); and NSAIDs (i.e., nepafenac, amfenac). In a preferred embodiment, the antiangiogenic agent is selected from thalidomide (Thalomid) and lenalidomide (Revlimid). Many of the antiangiogenic agents, such as lenalidomide and thalidomide, also act as immunomodulatory agents, i.e. they have a dual mechanism of action.

Alkylating agents

DNA alkylating agents or DNA alkylating agents act by damaging the DNA. DNA damage can be achieved using any of the following mechanisms. In the first mechanism, an alkylating agent attaches alkyl groups to the bases of DNA. This modification leads to DNA fragmentation by repair enzymes in an attempt to replace alkyl bases. The second mechanism by which alkylating agents damage DNA is the formation of cross bridges, bonds between atoms in DNA. During this process, two bases are bonded together by an alkylating agent that contains two DNA binding sites. Crosslinking interferes with DNA separation for synthesis or transcription. The third mechanism of action of alkylating agents causes abnormal pairing of nucleotides, leading to mutations.

There are 6 groups of alkylating agents: mustard nitrogen products; ethylenes; alkyl sulfonates; triazenes; piperazines and nitrourea derivatives. Examples of alkylating agents include, but are not limited to, thiotepa and cyclophosphamide (CYTOXAN ™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocvon, carmustine, meturedof and uredof; ethyleneimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene phosphoramide thiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs, adocelesin, carcelesin, and bicelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8). Preferred alkylating agents are selected from melphalan and cyclophosphamide.

Corticosteroids

Corticosteroids are drugs related to cortisol, a natural hormone produced by the adrenal cortex (the outer layer of the adrenal gland). Corticosteroid drugs include betamethasone (Celestone), budesonide (Entocort EC), cortisone (Cortone), dexamethasone (Decadron), hydrocortisone (Cortef), methylprednisolone (Medrol), prednisolone (Prelone), prednisone (Deltasone) and triamcinolone ) A preferred corticosteroid is dexamethasone (including derivatives such as, but not limited to, dexamethasone sodium phosphate and dexamethasone acetate). Corticosteroids can be administered orally, intravenously or intramuscularly, applied at a specific location on the skin, or administered by direct injection (for example, into inflamed joints). Corticosteroids can be used in combination with other drugs and are prescribed for short-term and long-term use (for example, in the form of pulse therapy, when the drug is administered for a short period of time, repeating the introduction at certain intervals). The recommended dose of corticosteroids can vary from 0.5 to 100 mg / day. For example, dexamethasone can be administered in the range of doses from 0.5 to 100 mg / day, more preferably from 10 to 80 mg / day, even more preferably from 15 to 70 mg / day or, most preferably, from 20 to 60 mg / day for administration on the same day or on other days, for example, on days 9-12 and 17-20 of each 28-day cycle for the first 4 cycles of therapy, and then 40 mg / day orally on days 1-4 every 28 days . The route of administration may be maintained or modified based on clinical and laboratory data. For example, initially a sufficiently high dose can then be gradually reduced to nothing, or vice versa, or at the beginning a higher or lower dose may be prescribed than the recommended one, and the dose may depend on the body weight of the patient-mammal (e.g., human).

Conjugates of drugs can be obtained by biochemical methods. A linker group is used to conjugate a drug or prodrug to an antibody. Suitable linker groups are well known in the art and include disulfide groups, acid-sensitive groups, photosensitive groups, peptidase-sensitive groups, thioether groups and esterase-sensitive groups. Preferred linker groups are disulfide and thioether groups. For example, a conjugate may be constructed using a disulfide exchange reaction between a suitably modified antibody and a drug or prodrug, or by reacting a thiol-containing drug with a modified antibody containing a maleimide group. Alternatively, the drug may contain a maleimide group, and a thiol moiety in the antibody. Methods for the preparation of conjugates are described in this field (see US Pat. a carrier molecule such as serum albumin.

According to the invention, the cell-binding agent is modified by reacting a bifunctional cross-linking reagent with a cell-binding agent, which leads to the covalent attachment of the linker molecule to the cell-binding agent. As used herein, a “bifunctional cross-linking reagent” is any chemical moiety that covalently attaches an agent that binds to cells to a drug, such as the drugs described herein. In a preferred embodiment of the invention, a portion of the linker moiety is provided with a drug. In this regard, the drug includes a linker moiety, which is part of the larger linker molecule used to attach the cell-binding agent to the drug. For example, to form the maytansinoid DM1 or DM4, the side chain of the ester at the C-3 position of the maytansine is modified to contain a free sulfhydryl group (SH) as described in US Pat. Nos. 5,208,020; 6333410; 7276497. This thiolated form of maytansine can react with a modified cell-binding agent to form a conjugate. Thus, the final linker is assembled from two components, one of which is provided with a cross-linking reagent, while the other is provided with a side chain of DM1 or DM4.

Any suitable bifunctional cross-linking reagent can be used in connection with the invention as long as the linker reagent ensures the preservation of therapeutic, for example cytotoxicity, and targeting characteristics of the drug and cell-binding agent, respectively. Preferably, the linker molecule attaches the drug to the cell-binding agent by chemical bonds (as described above) so that the drug and the cell-binding agent chemically bind (e.g., covalently bonded) to each other. Preferably, the linker reagent is a cleavable linker. More preferably, the linker cleaves under mild conditions, i.e. conditions within the cell that are not affected by the activity of the drug. Examples of suitable cleavable linkers include disulfide linkers, acid-sensitive linkers, photosensitive linkers, peptidase-sensitive linkers, thioether linkers and esterase-sensitive linkers. Disulfide-containing linkers are linkers that are cleaved through the exchange of disulfide, which can occur under physiological conditions. Acid sensitive linkers are linkers that break down at acidic pH values. For example, certain intracellular compartments, such as endosomes and lysosomes, are characterized by acidic pH values (pH 4-5), which provide conditions suitable for the cleavage of acid-sensitive linkers. Photosensitive linkers are suitable for use on the body surface and in many body cavities that are accessible for illumination. In addition, infrared light can penetrate tissue. Peptidase-sensitive linkers can be used to cleave certain peptides inside or outside the cells (see, for example, Trouet et al., Proc. Natl. Acad. Sci. USA, 79: 626-629 (1982), and Umemoto et al. Int. J. Cancer 43: 677-684 (1989)).

Preferably, the drug is bound to the cell-binding agent via a disulfide bond or a thioether bond. The linker molecule includes a reactive chemical group that can react with an agent that binds to cells. Preferred reactive chemical groups for reaction with a cell-binding agent are N-succinimidyl ethers and N-sulfosuccinimidyl ethers. Additionally, the linker molecule includes a reactive chemical group, preferably a dithiopyridyl group, which can react with the drug to form a disulfide bond. Particularly preferred linker molecules include, for example, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (see, for example, Carlsson et al., Biochem. J., 173: 723-737 (1978)), H -succinimidyl-4- (2-pyridyldithio) butanoate (SPDB) (see, for example, US Pat. No. 4,563,304), N-succinimidyl-4- (2-pyridyldithio) pentanoate (SPP) (see, for example, registration number CAS 341498 -08-6), and other reactive cross-linking linkers as described in US Pat. No. 6,913,748, incorporated herein by reference in its entirety.

Although cleavable linkers are preferably used in the method of the invention, a non-cleavable linker can also be used to prepare the conjugate described above. A non-cleavable linker is any chemical moiety that is capable of binding a drug, for example maytansinoid, vinca alkaloid, dolastatin, auristatin or cryptophycin, with a stable, covalent bond binding agent to the cells. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide cleavage under conditions in which the drug or cell-binding agent remains active.

Suitable cross-linking reagents that form non-cleavable linkers between the drug and the cell-binding agent are well known in the art. Examples of non-cleavable linkers include linkers containing a fragment of N-succinimidyl ether or N-sulfosuccinimidyl ether for reaction with a cell-binding agent, as well as a fragment based on maleimido or haloacetyl for reaction with a drug. Crosslinking reagents containing a maleinimido-based moiety include N-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidocapro which is a “long chain” analogue of SMCC (LC-SMCC), κ-maleiminimounoundecanoic acid N-succinimide ester (KMUA), γ-maleiminobutyric acid N-succinimide ester (GMBS), ε-maleiciminimidocaproic acid N-hydroxysuccinimide ester, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N- (α-maleinimidoacetoxy) -sucin imide ester (AMAS), succinimidyl-6-β-maleinimidopropionamido) hexanoate (SMPH), N-succinimidyl-4- (β-maleimidophenyl) butyrate (SMPB) and N- (n-maleimidophenyl) isocyanate (PMPI). Crosslinking reagents containing a halogenacetyl moiety include propionate-N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl bromoacetamido) (SBAP).

Other cross-linking reagents in which there is no sulfur atom and which form non-cleavable linkers can also be used in the method according to the invention. Such linkers can be prepared from dicarboxylic acid moieties. Suitable fragments based on dicarboxylic acid include, but are not limited to, α, ω-dicarboxylic acids of the general formula (IX):

,

,

where X represents a linear or branched alkyl, alkenyl or alkynyl group containing 2-20 carbon atoms, Y represents a cycloalkyl or cycloalkenyl group containing 3-10 carbon atoms, Z represents a substituted or unsubstituted aromatic group containing 6-10 atoms carbon, or a substituted or unsubstituted heterocyclic group, where the heteroatom is selected from N, O or S, and where each of 1, m and n is 0 or 1, provided that at the same time not all of 1, m and n are equal 0.

Many of the non-cleavable linkers disclosed herein are described in detail in US Patent Application No. 10/960602. Other linkers that may be used in the present invention include charged linkers or hydrophilic linkers and are described in US patent applications Nos. 12/433604 and 12/433668, respectively.

Alternatively, as disclosed in US Pat. The reaction of such maytansinoids containing an activated linker fragment with a cell-binding agent is another method for producing a cleavable or non-cleavable conjugate of a maytansinoid with a cell-binding agent.

The immunoconjugates and chemotherapeutic agents of the present invention can be administered in vitro, in vivo and / or ex vivo to treat patients and / or modulate the growth of selected cell populations, including, for example, lung, blood, plasma, breast, colon, rectal, prostate cancer , kidney, pancreas, brain, bone, ovary, testis and lymphatic organs; autoimmune diseases such as systemic lupus, rheumatoid arthritis and multiple sclerosis; transplant rejection, for example, renal transplant rejection, liver transplant rejection, pulmonary transplant rejection, heart transplant rejection and bone marrow transplant rejection; graft versus host disease; viral infections such as CMV infection, HIV infection and AIDS; and parasitic infestations, such as hyardiasis, amoebiasis, schistosomiasis, and the like. Preferably, the immunoconjugates and chemotherapeutic agents of the invention are administered in vitro, in vivo and / or ex vivo to treat cancer in a patient and / or modulate the growth of cancer cells, including, for example, cancer of blood, plasma, lung, breast, colonorectal region , prostate, kidney, pancreas, brain, bone, ovary, testis and lymph organs; preferably, the cancer cells are breast cancer cells, prostate cancer cells, ovarian cancer cells, colonorectal cancer cells, multiple myeloma cells, ovarian cancer cells, neuroblastoma cells, neuroendocrine cancer cells, stomach cancer cells, squamous cell cancer cells small cell lung cancer, or ovarian cancer cells, or a combination thereof.

“Modulating the growth of selected populations” of cells includes inhibiting the proliferation of selected populations of multiple myeloma cells (eg, cells: MOLP-8, OPM2 cells, H929 cells, etc.), preventing the formation of more cells; decrease in the degree of increase in cell division rate compared, for example, with cells in the absence of treatment; inducing the death of selected cell populations; and / or inhibition of metastasis of selected cell populations (eg, cancer cells). The growth of selected cell populations can be modulated in vitro, in vivo or ex vivo.

In the methods of the present invention, immunoconjugates and chemotherapeutic agents can be administered in vitro, in vivo, or ex vivo separately or as components of a single composition. Combined administration includes concomitant administration using separate preparations or a single pharmaceutical preparation and sequential administration in any order, where a period of time is preferably present during which both (or all) active agents simultaneously exhibit their biological activity. Preferably, such combination therapy leads to a synergistic therapeutic effect. Anticancer drugs that can be administered include DNA alkylating agents, such as melphalan; proteasome inhibitor such as bortezomib (Velcade); and immunomodulatory or antiangiogenic agents such as thalidomide and lenalidomide (Revlimid), along with the corticosteroid dexamethasone. Thus, for example, an antibody-maytansinoid conjugate can be combined with only one of the chemotherapeutic agents listed above, or with a combination of two or more chemotherapeutic agents listed above. For example, an antibody-maytansinoid conjugate may be combined with bortezomib and lenalidomide or thalidomide with or without dexamethasone. Also, the antibody-maytansinoid conjugate can be combined with melphalan and bortezomib or lenalidomide with or without dexamethasone. The order of administration and dose for each agent is readily apparent to those skilled in the art using an approved administration schedule for individual agents (see, for example, Physicians Desk Reference, (PDR) 2006 for preferred doses and treatment regimens for thalidomide (pp. 979-983 ), Velcade (pp. 2102-2106) and melphalanum (pp. 976-979)).

Immunoconjugates and chemotherapeutic agents can be used with suitable pharmaceutically acceptable carriers, diluents and / or excipients, which are well known and can be determined by a person skilled in the art in accordance with the requirements of the clinical situation. Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco's phosphate-buffered saline, approximately 6.5 pH, which contains about 1-25 mg / ml human serum albumin, (2) 0.9% w / v. a solution of sodium chloride (NaCl) and (3) 5% wt./about. glucose solution.

The compounds and compositions described herein may be administered in a suitable form, preferably parenterally, more preferably intravenously. For parenteral administration, the compounds or compositions may be aqueous or non-aqueous sterile solutions, suspensions or emulsions. Propylene glycol, oils and injectable organic esters such as ethyl oleate can be used as a solvent or carrier. Compositions may also contain adjuvants, emulsifiers or dispersants.

The compositions may also exist in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or any other sterile injectable medium.

The “therapeutically effective amount” of chemotherapeutic agents and immunoconjugates described herein refers to a treatment regimen for suppressing the proliferation of selected cell populations and / or treating a patient’s disease, and is selected according to a variety of factors, including age, body weight, gender , diet and medical condition of the patient, severity of the disease, route of administration and pharmacological considerations, such as activity, effectiveness, pharmacokinetic and toxicological profile of nodule Nogo compound employed. A “therapeutically effective amount” can also be defined with reference to standard medical texts, for example the Physicians Desk Reference 2004. The patient is preferably an animal, more preferably a mammal, most preferably a human. The gender of the patient may be male or female, and the patient may be an infant, child or adult.

Examples of suitable immunoconjugate administration protocols are provided below. Immunoconjugates can be administered daily for approximately 5 days, or as an IV bolus daily for approximately 5 days, or as a continuous infusion for approximately 5 days.

Alternatively, immunoconjugates may be administered once a week for 6 weeks or longer. As another alternative, immunoconjugates may be administered once every two or three weeks. Dosages in the form of a bolus are administered in a volume of from about 50 ml to about 400 ml of normal saline, to which from about 5 ml to about 10 ml of human serum albumin can be added. By continuous infusion, the drug is administered in a volume of from about 250 ml to about 500 ml of normal saline, from about 25 ml to about 50 ml of human serum albumin can be added over 24 hours. Doses will be from about 10 pg to about 1000 mg / kg per person, in / in (range from about 100 ng to about 10 mg / kg).

1-4 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols regarding the route of administration, excipients, diluents, doses and time of administration can be determined by a specialist in accordance with the requirements of the clinical situation.

The present invention also provides pharmaceutical kits comprising one or more containers filled with one or more ingredients of the pharmaceutical compounds and / or compositions of the present invention, including one or more immunoconjugates and one or more. chemotherapeutic agents. Such kits may also include, for example, other compounds and / or compositions, a device (a) for administering the compounds and / or compositions, and written instructions in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceuticals or biological products.

Anticancer therapeutic agents and their dosages, routes of administration, and recommended uses are known in the art and are described in such literature as the Physician's Desk Reference (PDR). The PDR discloses doses of drugs that are used in the treatment of various types of cancer. The therapeutically effective treatment regimens and dosages of the aforementioned chemotherapeutic drugs will depend on the particular type of cancer to be treated, the severity of the disease, and other factors familiar to the medical practitioner in the art and may be determined by the physician. The content of PDR is expressly incorporated herein by reference in its entirety. The 2006 Physician's Desk Reference (PDR) disclosed a mechanism of action, preferred treatment regimens, and dosages for thalidomide (pp. 979-983), Velcade (pp. 2102-2106), and melphalan (pp. 976-979). The content of PDR is expressly incorporated herein by reference in its entirety. One of skill in the art can review PDR data using one or more of the following parameters to determine the treatment regimen and dosage of chemotherapeutic agents and conjugates that may be used in accordance with the disclosure of this invention. Such parameters include:

1. Comprehensive index:

a) by manufacturer;

b) by product (company name or trade name of the drug);

c) the category index (for example, “proteasome inhibitors”, “DNA alkylating agents”, “melphalan”, etc .;

e) an index of generic / chemical names (commonly known generic names of drugs).

2. Color images of medicines.

3. Product information consistent with FDA marking:

a) chemical information;

b) function / action;

c) indications and contraindications;

e) clinical studies, side effects, warnings.

Analogs and derivatives

One skilled in the art from therapeutic agents, such as cytotoxic agents or chemotherapeutic agents, will readily understand that each of the agents described herein can be modified so that the resulting compound retains the specificity and / or activity of the parent compound. It will also be apparent to those skilled in the art that many of these compounds may be used in place of the therapeutic agents described herein. Thus, therapeutic agents of the present invention include analogs and derivatives of the compounds described herein.

All references cited in this description and the examples below are expressly incorporated by reference in their entirety.

Examples

The invention will now be described with reference to non-limiting examples. Unless otherwise specified, all percentages and ratios are given by volume.

Mice were inoculated with tumor cell lines of multiple human myeloma; the tumors were allowed to take root (average tumor size of approximately 100 mm ³ ) before treatment. Doses of conjugates have been described based on the concentration of DM1. Efficiency was expressed as% growth of the treated tumor against the control (% T / C) and the logarithm of cell death (LCK), determined on the basis of the period of tumor growth by half and delayed tumor growth as a result of treatment. It was believed that percent T / C values of less than 42% and / or LCK values of 0.5 or more indicate activity; percent T / C values of less than 10% were considered to indicate high activity (Bissery et al., Cancer Res, 51: 4845-4852 (1991).

Example 1. The antitumor effect of the combination therapy of xenografts of human multiple myeloma (MOLP-8) huN901-DM1 and melphalan

The antitumor effect of the combination of huN901-DMl and melphalan was evaluated on a model of an implanted subcutaneous xenograft of multiple myeloma. Naked Balb / c mice (20 animals) were grafted with human multiple myeloma MOLP-8 cells (1 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the size of the tumors reached approximately 150 mm ³ (21 days after the introduction of tumor cells), the mice were randomly divided into four groups (5 animals per group). The first group of mice received huN901-DM1 intravenously (DM1 dose of 200 μg / kg, single injection, 22nd day after the introduction of tumor cells). The second group of animals received melphalan intraperitoneally (12 mg / kg, single injection, 23rd day after the introduction of tumor cells). The third group of mice received a combination of huN901-DM1 and melphalan using the same doses, regimens and routes of administration as in groups 1 and 2. The control group of animals received phosphate buffered saline (PBS) using such regimens and routes of administration as in the groups 1 and 2. Tumor growth was monitored by measuring tumor size twice a week. Tumor size was calculated by the formula

length × width × height × ½

Tumor growth data is shown in FIG. 1A. In the control group of animals, the size of the tumors reached 1000 mm in approximately 33 days. Treatment with huN901-DM1 or melphalan alone resulted in a delay in tumor growth of 11 days and 14 days, respectively. Conversely, treatment with a combination of melphalan and huN901-DM1 led to a delay in tumor growth of 35 days, and the combination showed high activity according to NCI standards (T / C = 4%, see table 1 (fig.1B)).

The logarithm of cell death (LCK) as a result of combination therapy was 2.1, which exceeds the sum of the LCK values for individual drugs, indicating a synergistic activity.

Example 2. The effect of antitumor combination therapy of huN901-DM1 and thalidomide on xenografts of multiple human myeloma (MOLP-8)

The antitumor effect of the combination of huN901-DM1 and thalidomide was evaluated on a model of an implanted subcutaneous xenograft of multiple myeloma. SCID mice (36 animals) were inoculated with MOLP-8 human multiple myeloma cells (1 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the size of the tumors reached about 150 mm ³ (15 days after the introduction of tumor cells), the mice were randomly distributed into 6 groups (6 animals per group). Two groups received huN901-DM1 monotherapy intravenously at doses of DM1 of 100 mg / kg and 250 mg / kg, respectively (1 time per week × 2, 16th and 23rd days after the introduction of tumor cells). The third group of mice received monotherapy with thalidomide intraperitoneally at a dose of 200 mg / kg (a total of 11 doses, days 16, 18-22 and 25-29 after the introduction of tumor cells) in the form of a suspension in a 1% solution of carboxymethyl cellulose in phosphate buffer solution. Two groups received combinations of huN901-DM1 (100 mg / kg or 250 mg / kg) plus thalidomide, using the same doses, regimens and methods of administration as in the monotherapy groups. The control group of animals received a phosphate buffer solution intravenously (1 time per week × 2, 16th and 23rd days after the introduction of tumor cells). Tumor growth was monitored by measuring tumor size twice a week. Tumor size was calculated by the formula

length × width × height × ½

Tumor growth data is shown in FIG. 2A. The combination of huN901-DM1 plus thalidomide was active against MOLP-8 xenografts, which additionally led to synergistic activity (Table 2 (Fig. 2B)). The combination of huN901-DM1 and thalidomide in doses in which monotherapy was inactive (100 mg / kg huN901-DM1, 200 mg / kg thalidomide) was active according to NCI standards (T / C = 26%; Table 2 (Fig. 2B )).

The combination of huN901-DM1 (250 mg / kg) and thalidomide (200 mg / kg) gives a highly effective combination (T / C = 7%) with a logarithm of cell death of 1.0, which is higher than the sum of the LCK values for individual drugs.

Example 3. The antitumor effect of the combination therapy of huN901-DM1 and bortezomib on xenografts of multiple human myeloma (ORM2)

The antitumor effect of the combination of huN901-DM1 and bortezomib (Velcade, Millennium Pharmaceuticals) was evaluated on the model of the implanted subcutaneous xenografts of multiple myeloma. SCID mice (36 animals) were inoculated with human ORM2 multiple myeloma cells (1 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the tumor size reached about 70 mm ³ (12 days after the introduction of tumor cells), the mice were randomly divided into 6 groups (6 animals per group). Two groups received huN901-DM1 monotherapy intravenously at doses of DM1 of 100 mg / kg and 200 mg / kg, respectively (12th day after the introduction of tumor cells). The third group of mice received monotherapy with bortezomib intravenously at a dose of 1 mg / kg (13th and 16th days after the introduction of tumor cells). Two groups received combinations of huN901-DM1 (100 mg / kg; or 200 mg / kg) plus bortezomib, using the same doses, regimens, and modes of administration as in the monotherapy groups. The control group of animals received a phosphate buffer solution intravenously (12th day after the introduction of tumor cells). Tumor growth was monitored by measuring tumor size twice a week. Tumor size was calculated by the formula

length × width × height × ½

The combination of huN901-DM1 plus bortezomib showed high activity against ORM2 xenografts, which led to synergistic activity for both combinations of both doses. HuN901-DM1 monotherapy huN901-DM1 led to the fact that in 1 out of 6 and 3 out of 6 mice tumors were absent on the 91st day in dose groups of 100 and 200 mg / kg, respectively (see table 3 (fig.3B) ) At the same time, in the bortezomib monotherapy group, tumors were present in all mice. Both huN901-DM1 plus bortezomib combinations (1 mg / kg) resulted in complete regression of tumors in 6 out of 6 mice, with no tumors in mice on day 91, i.e. Date of last measurement of tumor size. Tumor growth curves are shown in FIG. 3A.

The combination of huN901-DM1 plus bortezomib showed high activity, which was synergistic.

Example 4. The antitumor effect of the combination therapy of huN901-DM1 and bortezomib on large tumor xenografts of multiple human myeloma (H929)

The antitumor effect of the combination of huN901-DM1 and bortezomib was evaluated using a model of the implanted subcutaneous xenografts of multiple myeloma. SCID mice (54 animals) were inoculated with human H929 multiple myeloma cells (1 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the tumor size reached about 300 mm ³ (34 days after the introduction of tumor cells), the mice were randomly distributed into 11 groups (6 animals per group). Two groups of mice received huN901-DM1 monotherapy intravenously at doses of DM1 of 50 mg / kg and 100 mg / kg, respectively (34th day after injection of tumor cells). Two groups of mice received intravenous bortezomib monotherapy at a low dose of 0.5 mg / kg and a high dose of 1 mg / kg (35th and 38th days after the introduction of tumor cells). Four groups of combination therapy were evaluated, receiving combinations of each of the doses of huN901-DM1 plus a high or low dose of bortezomib, using the same doses, regimens, and methods of administration as in the monotherapy groups. The control group of animals received a phosphate buffer solution intravenously (34th day after the introduction of tumor cells). Tumor growth was monitored by measuring tumor size twice a week. Tumor size was calculated by the formula

length × width × height × ½

The combination of low dose huN901-DM1, bortezomib, showed a synergistic effect against H929 tumors. The combination of huN901-DM1 and bortezomib in doses at which they were ineffective as monotherapy (50 μg / kg huN901-DM1,

0.5 μg / kg bortezomib), showed activity in accordance with NCI standards (T / C = 38%; see Table 4a (Fig. 4B)). The combination of huN901-DM1 (100 μg / kg) plus bortezomib at a dose of 0.5 μg / kg also proved to be an active composition (T / C = 17%,

1 out of 6 mice had no tumors at the end of the study on day 119; see figa).

The combination of huN901-DM1 with a high dose of bortezomib also showed a synergistic effect against H929 tumors. A high dose of bortezomib (1.0 mg / kg) was active as monotherapy in the model of large H929 tumors (T / C = 11%, 1 out of 6 mice had no tumors on day 119; see Table 4b (Fig. 4D)). The combined treatment with a high dose of bortezomib and huN901-DM1 at a dose of 50 and 100 μg / kg was highly active, as indicated by low T / C values (7% and 0%, respectively); moreover, in the group of the last combination in 5 out of 6 mice there were no tumors on the 119th day (see figs). The combination of huN901-DM1 plus bortezomib was highly active and showed a synergistic effect.

Example 5. The antitumor effect of the combination therapy of huN901-DM1 and lenalidomide on xenografts of multiple human myeloma (ORM2)

The antitumor effect of the combination of huN901-DM1 and lenalidomide was evaluated in a model of an implanted subcutaneous xenograft of multiple myeloma. SCID mice (20 animals) were grafted with human MOLP-8 multiple myeloma cells (1 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the size of the tumors reached about 130 mm ³ (16 days after the introduction of the tumor cells), the mice were randomly divided into 4 groups (5 animals per group). One group of mice received huN901-DM1 monotherapy intravenously (200 mg / kg, day 16 after tumor cell administration). The second group of mice received monotherapy with lenalidomide intraperitoneally (100 mg / kg, days 16-20 and 22-26 after the introduction of tumor cells) in the form of a suspension in 1% solution. carboxymethyl cellulose in phosphate buffered saline. The third group received a combination of huN901-DM1 plus lenalidomide, using the same doses, regimens and methods of administration as in the monotherapy groups. The control group of animals received a phosphate buffer solution intravenously (16th day after the introduction of tumor cells). Tumor growth was monitored by measuring tumor size twice a week. Tumor size was calculated by the formula

length × width × height × ½

Treatment with a single injection of huN901-DM1 (200 μg / kg, day 16) showed activity against multiple myeloma ORM2 xenografts (T / C = 26%, LCK = 0.9; see Table 5 (Fig. 5B)); the activity of lenalidomide as a monotherapy (100 mg / kg, days 16-20, 23-27) was comparable (T / C = 28%, LCK = 0.9). The combination “huN901-DM1 plus lenalidomide" was highly active (T / C = 1.4%, LCK = 1.7), with 1 of 5 mice having no tumors at the end of the study; tumors were present in all animals in the huN901-DM1 treatment groups or lenalidomide / dexamethasone. The results of this study demonstrate that combination treatment is synergistic, and the combination exhibits more pronounced activity than drugs as monotherapy. Tumor growth data are shown in Table 5 (Figure 5B) and Figure 5A.

Example 6. The dependence of the antitumor effect of the combination "huN901-DM1 plus bortezomib" on the scheme of administration

The antitumor effect of the combination of huN901-DMl and bortezomib (Velcade, Millennium Pharmaceuticals) was evaluated on the model of an implanted subcutaneous xenograft of multiple myeloma. The objective of this study was to determine the optimal administration regimen for combination therapy. SCID mice (18 animals) were grafted with human OPM2 multiple myeloma cells (1 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the size of the tumors reached approximately 70 mm ³ (12 days after the introduction of tumor cells), the mice were randomly divided into three groups (6 animals per group). The first group of mice received first bortezomib at a dose of 1 mg / kg on days 0 and 3, and then huN901-DM1 at a dose of 13 mg / kg on day 3. In an alternative administration regimen, the second group of mice received first huN901-DM1 at a dose of 13 mg / kg on days 0, 3, and 3 days later (3rd and 6th days) bortezomib at a dose of 1 mg / kg. The control group of animals received: phosphate buffered saline intravenously (12th day after the introduction of tumor cells). Tumor growth was monitored by measuring the size of the tumor twice a week. Tumor size was calculated by the formula

length × width × height × ½

Surprisingly, the combination of huN901-DM1 plus bortezomib showed high activity against ORM2 xenografts only if bortezomib was administered first (see FIG. 6). Thus, this scheme led to a complete regression of tumors, where 5 out of 6 mice had no tumors on the 120th day. If bortezomib was administered second (i.e., after administration of huN901-DM1), the treatment only led to a slight delay in tumor growth, and even on the 30th day, all animals had tumors. The data obtained indicate that the administration schedule is critical for combination therapy with bortezomib and an immunoconjugate. It is possible that preliminary treatment with bortezomib makes tumor cells susceptible to huN901-DM1-induced immunoconjugate death.

Example 7. Antitumor effect of treatment with the triple combination of "huN901-DM1 and lenalidomide plus a low dose of dexamethasone" xenografts of human multiple myeloma (MOLP-8)

The antitumor effect of the triple combination of “huN901-DM1 and lenalidomide plus a low dose of dexamethasone” was evaluated on the model of an implanted subcutaneous xenograft of multiple myeloma. SCID mice were inoculated with human MOLP-8 multiple myeloma cells (1.5 × 10 ⁷ cells / animal) by subcutaneous injection into the right shoulder of the mouse. When the size of the tumors reached about 100 mm ³ (13 days after the introduction of the tumor cells), 24 mice were randomly divided into 4 groups (6 animals per group). One group of mice received huN901-DM1 monotherapy intravenously (150 mg / kg, days 1 and 8 after administration of tumor cells). The second group of mice received a combination of lenalidomide / low dose of dexamethasone (100 mg / kg lenalidomide, intraperitoneal suspension in a 1% solution of carboxymethyl cellulose in phosphate buffer solution on days 1-5 and 8-12 after the introduction of tumor cells; dexamethasone at a dose of 1, 5 mg / kg, administration by subcutaneous injection on the 1st and 8th days). The third group received a triple combination of “huN901-DM1 plus lenalidomide / dexamethasone", using the same doses, regimens and methods of administration as in the monotherapy groups. The control group of animals received phosphate buffered saline intravenously (1st and 8th days). Tumor growth was monitored by measuring tumor size twice a week. Tumor size was calculated by the formula

length × width × height × ½

Treatment with huN901-DM1 (150 μg / kg, 1 time per week × 2) or lenalidomide and dexamethasone showed equal activity against MOLP-8 tumors (T / C = 33%, LCK = 0.5; see table 7 (Fig. 7 .7b)). The triple combination huN901-DM1 / lenalidomide / dexamethasone was highly active (T / C = 0%, LCK = 1.4), with partial regression in all mice (6/6) and complete regression of tumors in 4 of 6 mice, compared with no regression in the huN901-DM1 group and in the lenalidomide / dexamethasone group. Tumor growth data are shown in Table 7 (Fig. 7b) and in Fig. 7A.

Satellite treatment groups of tumor-bearing MOLP-8 mice (3 animals per group) received treatment in parallel with a performance study; animals: they were killed on the 3rd day (after 48 hours of treatment), and the tumors were excised for immunohistochemical analysis using antibodies to cleaved caspase-3, to assess apoptosis. Tumors of mice treated with the triple combination, huN901-DM1 / lenalidomide / dexamethasone, showed a significant increase in caspase-3 staining relative to controls and monotherapy groups where apoptosis levels corresponded or approximately corresponded to the baseline value (data shown in Fig. 8A). Such a sharp increase in apoptosis when using the triple combination is evident at an early stage of the treatment phase before any changes in tumor volume were detected in the study of antitumor activity.

The results of the studies demonstrate that treatment with the triple combination of huN901-DM1 / lenalidomide / dexamethasone is synergistic, demonstrating higher antitumor activity and a significant increase in tumor cell apoptosis compared to single-agent monotherapy.

Claims (53)

1. A method of treating or modulating the growth of a population of malignant tumor cells, comprising administering to an object with a malignant tumor an effective amount of lenalidomide, at least one corticosteroid, and at least one immunoconjugate, where the immunoconjugate contains at least one binding to cells an agent that binds to CD56, and at least one antimitotic agent, wherein the cell-binding agent is an antibody, a single chain antibody, or an antigen-binding fragment of an antibody. 2. The method according to claim 1, in which the population of malignant tumor cells contains one or more breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, multiple myeloma cells, neuroblastoma cells, neuroendocrine cancer cells, stomach cancer cells squamous cell cancer, small cell lung cancer or testicular cancer cells. 3. The method according to claim 1, in which separate formulations are administered simultaneously, sequentially or separately. 4. The method according to claim 3, in which the combination is administered every second day, on alternating days, on a weekly basis or during a period of time between day 0 and day 7, or between 0 and 4 weeks. 5. The method according to claim 1, in which the combination is administered parenterally. 6. The method according to claim 1, wherein the cell-binding agent is a monoclonal antibody, a single chain monoclonal antibody, or an antigen-binding fragment of a monoclonal antibody. 7. The method of claim 1, wherein the cell-binding agent is a chimeric antibody or antigen-binding fragment of a chimeric antibody. 8. The method of claim 1, wherein the cell-binding agent is a domain antibody or antigen-binding fragment of a domain antibody. 9. The method according to claim 1, wherein the cell-binding agent is a restored antibody, a single-chain restored antibody, or an antigen-binding fragment of a restored antibody. 10. The method of claim 1, wherein the cell-binding agent is a humanized antibody, a single chain humanized antibody, or an antigen-binding fragment of a humanized antibody. 11. The method of claim 1, wherein the cell-binding agent is a human antibody, a single chain human antibody, or an antigen binding fragment of a human antibody. 12. The method of claim 1, wherein the cell-binding agent is an N901 antibody, an N901 single chain antibody, or an antigen binding fragment of an N901 antibody. 13. The method according to claim 1, wherein the cell-binding agent is a humanized or restored N901 antibody, a single-chain humanized or restored N901 antibody, or an antigen-binding fragment of a humanized or restored N901 antibody. 14. The method according to claim 1, in which the antimitotic agent is an agent selected from the group consisting of maytansinoids, vinca alkoloids, dolastatins, auristatins, cryptophycins, tubulizins and taxanes. 15. The method according to 14, in which the antimitotic agent is maytanside. 16. The method according to clause 15, in which maytanside has a component of N-methyl-alanine-containing C-3 thiol. 17. The method according to clause 16, in which maytanoside is a compound of the formula:

where l is an integer from 1 to 10; a May is maytanside. 18. The method according to clause 16, in which maytanoside is a compound of the formula:

where n is an integer from 3 to 8; a May is maytanside. 19. The method according to clause 16, in which maytanoside is a compound of the formula:

where R ₁ , R ₂ , R ₃ , R ₄ are H, CH ₃ or CH ₂ CH ₃ and are the same or different; m is 0, 1, 2 or 3; a May is maytanside. 20. The method according to 17, in which R ₁ , R ₂ , R ₃ , R ₄ are H, and m is 1. 21. The method according to clause 16, in which maytanoside is a compound of the formula:

where l is 1, 2 or 3; Y ₀ is Cl or H; and X ₃ is H or CH ₃ . 22. The method according to item 21, in which 1 is 2. 23. The method according to claim 1, in which the maytanoside has a component N-methyl-alanine-containing C-3 thiol. 24. The method according to item 23, in which maytanoside is a compound of the formula:

where o is 1, 2 or 3; p is an integer from 0 to 10; a May is maytanside. 25. The method according to item 23, in which maytanoside is a compound of the formula:

where o is 1, 2 or 3; q is an integer from 0 to 10; Y ₀ is Cl or H; X ₃ is H or CH ₃ . 26. The method according to claim 1, in which the immunoconjugate contains maytanoside and a humanized monoclonal antibody or fragment thereof that binds the CD56 antigen expressed by neuroendocrine cancer cells or small cell lung cancer cells. 27. The method according to p, in which the maytanoside has a component of N-methyl-alanine-containing C-3 thiol. 28. The method according to item 27, in which maytanside is a compound of the formula:

where R ₁ , R ₂ , R ₃ , R ₄ are H, CH ₃ or CH ₂ CH ₃ , and are the same or different; m is 0, 1, 2 or 3; a May is maytanside. 29. The method according to p, in which R ₁ , R ₂ , R ₃ , R ₄ are H, and m is 0. 30. The method according to item 27, in which maytanoside is a compound of the formula:

where l is 1, 2 or 3; Y ₀ is Cl or H; and X ₃ is H or CH ₃ . 31. The method according to claim 30, wherein 1 is 2. 32. The method according to any one of claims 1 or 26, in which the corticosteroid is selected from the group consisting of dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone. 33. The method according to p, in which the corticosteroid is dexamethasone. 34. The method according to claim 1, in which the antimitotic agent is associated with a cell-binding agent through a linker molecule selected from the group consisting of N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (2- pyridyldithito) butanoate (SPDB) and N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 35. The method according to clause 34, in which the linker molecule is N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 36. The method according to claim 2, in which the population of malignant tumor cells are multiple myeloma cells. 37. The method according to claim 1, in which the corticosteroid is dexamethasone, and the immunoconjugate contains DM1 associated with a humanized or restored antibody N901. 38. The method according to clause 37, in which DM1 and the antibody are linked by N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 39. The method according to claim 1, wherein the cell-bound agent is a humanized or restored N901 antibody, the antimitotic agent is DM1, and the cell-binding agent is coupled to the antimitotic agent via the linker molecule N-succinimidyl 4- (2-pyridyldithio) pentanoate ( SPP). 40. A method of treating a malignant tumor, comprising administering to a subject with a malignant tumor a combination comprising a synergistically effective amount of lenalidomide, at least one corticosteroid, and at least one immunoconjugate, and a pharmaceutically acceptable carrier for each;
where the immunoconjugate contains at least one cell-binding agent that binds to CD56, and at least one antimitotic agent; and
wherein the cell-binding agent is an antibody, a single chain antibody, or an antigen binding fragment of an antibody. 41. The method of claim 40, wherein the cell-binding agent is a humanized or restored N901 antibody, a single chain humanized or restored N901 antibody, or an antigen-binding fragment of a humanized or restored N901 antibody. 42. The method of claim 40, wherein the corticosteroid is dexamethasone and the immunoconjugate contains DM1 coupled to a humanized or restored antibody N901. 43. The method of claim 40, wherein the antimitotic agent is coupled to the cell-binding agent through a linker molecule selected from the group consisting of N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (2- pyridyldithito) butanoate (SPDB) and N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 44. The method according to item 43, in which the linker molecule is 4- (2-pyridyldithio) pentanoate (SPP). 45. The method according to p, in which the malignant tumor is multiple myeloma. 46. The method according to paragraph 41, in which DM1 and the antibody are coupled with N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 47. The method according to paragraph 41, wherein the cell-bound agent is a humanized or restored N901 antibody, the antimitotic agent is DM1, and the cell-binding agent is coupled to the antimitotic agent via the linker molecule N-succinimidyl 4- (2-pyridyldithio) pentanoate ( SPP). 48. The method according to claim 1, comprising administering to a subject with a malignant tumor a combination containing a synergistically effective amount of (i) lenalidomide, (ii) a corticosteroid, and (iii) an immunoconjugate containing maytanside and a humanized or restored monoclonal antibody, or a fragment thereof, which binds CD56 antigen expressed by multiple myeloma malignant tumor cells and a pharmaceutically acceptable carrier for each. 49. The method of claim 48, wherein the corticosteroid is dexamethasone and the immunoconjugate contains DM1 coupled to a humanized or restored antibody N901. 50. The method of claim 48, wherein the antimitotic agent is coupled to the cell-binding agent via a linker molecule selected from the group consisting of N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (2- pyridyldithito) butanoate (SPDB) and N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 51. The method of claim 50, wherein the linker molecule is N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 52. The method of claim 49, wherein DM 1 and the antibody are coupled by N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP). 53. The method of claim 48, wherein the cell-bound agent is a humanized or restored N901 antibody, the antimitotic agent is DM1, and the cell-binding agent is coupled to the antimitotic agent via the linker molecule N-succinimidyl 4- (2-pyridyldithio) pentanoate ( SPP).

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