Cutaneously administered antibody-drug conjugates for use in cancer treatment - Patent Application 20070122997

Abstract

The present application relates to a pharmaceutical composition for use in the treatment of cancer, which is administered subcutaneously, and which comprises at least one conjugate comprising an antibody that specifically binds to a cell surface antigen on the surface of a cancer cell and at least one amatoxin-linker payload. The present invention further relates to a method for treating a patient suffering from cancer using the pharmaceutical composition of the present invention.

Description

[Technical Field]

[0001] The present invention relates to the subcutaneous administration of pharmaceutical compositions of antibody-drug conjugates (ADCs) comprising one or more cytotoxic amatoxin moieties attached to an antibody or antigen-binding antibody fragment, and their use in the treatment of cancer. In a further aspect, the present invention relates to methods of treating cancer in a patient using the pharmaceutical compositions of the invention. [Background technology]

[0002] Conventional cancer treatments often involve the low therapeutic range and nonspecificity of chemotherapy drugs, resulting in the high mitotic rate of normal cells and causing many adverse effects. Monoclonal antibodies have shown great therapeutic potential for the treatment of several diseases, especially cancer. Prior to the development of antibody-drug conjugates (ADCs) for cancer treatment, monoclonal antibodies attracted attention due to their target specificity, therapeutic index, and generally fewer side effects than conventional treatments such as chemotherapy or chemoradiotherapy.

[0003] The design and development of ADCs presents many challenges, including a concentration-dependent tendency for degradation or aggregation caused by the hydrophobicity of each ADC payload. Attempts to alleviate these constraints by lowering the concentration of the ADC in each formulation have resulted in limited bioavailability and tissue penetration.

[0004] Commonly used ADC toxins such as auristatins, maytansinoids, and calicheamicins typically have IC in the subnanomolar range. 50 These drugs have in vitro potencies 100-1000 times higher than those of traditional chemotherapy drugs. Such increased potency is necessary for effective tumor cell killing by ADCs, and their delivery to intracellular drug targets is related to the amount of antigen expression on the tumor cell surface and the efficiency of internalization. However, improved in vitro potency is often associated with increased hydrophobicity of each payload.

[0005] Hydrophobicity represents an important factor contributing to the overall physicochemical properties of an ADC payload: increasing hydrophobicity often improves in vitro potency, but also comes with poor solubility, risk of metabolic instability, and an increased probability of nonspecific off-target effects.

[0006] Subcutaneous delivery of biotherapeutics has become a valuable alternative to intravenous administration across many disease areas. The choice of administration route depends on several factors, including patient convenience and the pharmacokinetic properties of the drug. Each route has its advantages and disadvantages. Intravenous (iv) administration has a rapid onset of action and near 100% bioavailability, but can be painful, requires hospitalization, and can result in high peak serum levels (e.g., high C) that can induce toxicity. max ) As used herein, "C max " refers to the maximum observed plasma concentration of a given drug, such as the conjugates of the present invention. On the other hand, subcutaneous (sc) administration, which is often used for antibody drugs, has the advantage that it is very easy to administer and can even be done by the patient themselves.

[0007] Although the pharmacokinetic profiles of subcutaneous and intravenous formulations differ, subcutaneous administration of antibodies has proven effective, safe, and well-tolerated, and is generally preferred by patients and healthcare providers because it reduces healthcare costs and resource utilization associated with drug delivery.

[0008] However, subcutaneous administration of antibody-based therapeutics, including ADCs, is generally limited by the small injectable volume. As a result, high formulation concentrations of ADCs are required to administer a therapeutically effective dose. However, higher-concentration ADC formulations containing hydrophilic payloads risk increasing the tendency of the ADC to aggregate, which is correlated with toxicity. ADC aggregation is also correlated with a reduced half-life and narrow therapeutic index. Using low-concentration formulations of ADCs reduces their bioavailability and ability to penetrate tissues and exert their pharmacological effects.

[0009] Potential drawbacks of the subcutaneous route of administration include difficulty in controlling absorption rates and local irritation or skin toxicity. Significant skin toxicity has been reported with intravenous (iv) administration of trastuzumab-emtansine with a maytansine derivative (DM1) payload. Skin toxicity is likely caused by extravasation, where the intravenously administered drug accidentally leaks into the tissues surrounding the vein.

[0010] Another concern regarding subcutaneous administration of antibodies and ADCs is the isoelectric point (pI) of the antibody or ADC. A pI of 7–9 makes the antibody positively charged at physiological pH. Positively charged antibodies exhibit reduced bioavailability of approximately 30%, while their negatively charged counterparts exhibit increased bioavailability of up to 70% after subcutaneous administration (Yadav, et al. J. Biol. Chem. 2015, 290, 29732–29741). While these findings relate to antibodies, there is little information regarding the effect of charge on the bioavailability of ADCs. However, positively charged ADCs are likely to exhibit similarly reduced bioavailability.

[0011] Strategies have been developed to overcome the limitations of small subcutaneous injectable volumes (typically 1–2 ml) and poor bioavailability. Recombinant human hyaluronidase PH20 (rHuPH20) combined with antibodies allows for larger subcutaneous injection volumes, up to 5 ml or more. rHuPH20 acts by locally degrading hyaluronan (HA), a large glycosaminoglycan and component of the extracellular, pericellular, and intracellular matrix. Hyaluronan is a major component of skin that forms a gel-like substance with water, creating resistance to bulk fluid flow and limiting subcutaneous drug delivery, dispersion, and absorption of large volumes. Combining antibodies with hyaluronidase promotes bulk fluid flow and improves the pharmacokinetic profile after subcutaneous administration. Antibodies approved for subcutaneous administration in combination with rHuPH20 include rituximab (Rituxan Hycela / mAbThera sc), trastuzumab (Herceptin Hylecta), and daratumumab (Darzalex Faspro).

[0012] WO2018 / 187074A1 discloses a method for treating cancer using subcutaneous administration of an antibody-drug conjugate containing an SN38 payload, however the corresponding ADC has not been approved for subcutaneous administration.

[0013] Thus, there is an unmet medical need for highly effective ADCs that are administered subcutaneously to maximize efficacy and minimize toxicity. Summary of the Invention [Means for solving the problem]

[0014] Surprisingly and unexpectedly, the inventors have found that subcutaneous administration of a pharmaceutical composition for use in the treatment of cancer according to the present invention, comprising a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) at least one linker linking said target binding moiety to said at least one amatoxin, results in reduced peak serum levels (C max They found that the toxicity of the compound was reduced.

[0015] It was therefore an object of the present invention to provide a pharmaceutical composition for subcutaneous administration, comprising (i) a target-binding moiety, (ii) at least one amatoxin, and (iii) at least one linker connecting said target-binding moiety to said at least one amatoxin.

[0016] Preferably, the pharmaceutical composition of the invention is for subcutaneous administration and comprises an antibody, preferably a monoclonal antibody or antigen-binding fragment thereof, as the target-binding moiety. Preferably, the target-binding moiety of the invention is an IgG isotype antibody.

[0017] In a particular object of the present invention, the pharmaceutical composition comprises a conjugate linked to at least one amatoxin via a non-cleavable or cleavable linker. Preferably, the cleavable linker of the conjugate according to the present invention is an enzymatically cleavable linker, preferably a self-immolative linker.

[0018] According to a further object of the present invention, the antibody of the conjugate of the present invention does not induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) and comprises at least one amino acid substitution at positions D265, L234, L235, or G236 (according to the EU numbering system). In particular, the antibody of the conjugate of the present invention comprises the amino acid substitutions L234A, L235A, and D265C (according to the EU numbering system).

[0019] According to a further object of the invention, at least one linker of the conjugates of the invention linking at least one amatoxin to the antibody portion of the ADC is covalently attached to the antibody portion via any of the naturally occurring cysteine ​​residues of the antibody, preferably via any of the naturally occurring cysteine ​​residues that form the interchain disulfide bonds of the antibody and / or via a disulfide bond, or to the engineered cysteine ​​residue D265C (according to the EU numbering system) in the Fc region of the antibody.

[0020] According to some embodiments, the pharmaceutical compositions of the invention comprise a conjugate comprising an amatoxin-linker moiety according to any of Formulas XII-XXII disclosed herein, wherein the conjugate comprises 1 to about 8, preferably 1.5, 2 to about 3 or 3.5, and particularly preferably 2 amatoxin-linker moieties.

[0021] A further object of the present invention is to provide pharmaceutical compositions for use in methods for treating cancer.

[0022] These and further objects are met by the methods and means of the present invention according to the independent claims. The dependent claims relate to particular embodiments.

[0023] The general advantages of the present invention and its features are described in detail below. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the Markush structures of various amatoxins. The bold numbers (1-8) indicate the standard numbering of the eight amino acids that form the amatoxins. Also shown are the standard designations for the atoms in amino acids 1, 3, and 4 (Greek letters α-γ, Greek letters α-δ, and numbers 1'-7', respectively). [Figure 2-1] Figure 2 shows a comparison of the efficacy of subcutaneously administered conjugates with intravenously administered conjugates. (A) Mouse prostate cancer model using C4-2 tumor cells. [Figure 2-2] Figure 2 shows a comparison of the efficacy of subcutaneously and intravenously administered conjugates. (B) Maximum tolerated dose of anti-PSMA ADC conjugated to amatoxin linker (XII) or (XIV) disclosed herein. "DIG-LALA-D265C-(XIV)" refers to an isotype control antibody conjugated to amatoxin-linker payload (XIV). (C) Comparison of half-life, Cmax, and AUC of anti-PSMA ADC and anti-CD37 upon subcutaneous and intravenous administration. Subcutaneous administration of the pharmaceutical composition of the present invention is shown to increase half-life, decrease Cmax, and increase AUC. [Figure 3] Figure 3 shows an NHP tolerability study of intravenous and subcutaneous administration of anti-PSMA-(XII) conjugates. Cynomolgus monkeys were treated with markers of liver damage following subcutaneous and intravenous administration of anti-PSMA-(XII). Upper panel: 7.5 mg / kg intravenous administration of anti-PSMA-(XII) as indicated. Lower panel: 7.5 mg / kg and 10 mg / kg subcutaneous administration of anti-PSMA-(XII) as indicated. AST (aspartate aminotransferase), ALT (alanine aminotransferase), and LDH (lactate dehydrogenase) levels in serum of cynomolgus monkeys after intravenous or subcutaneous treatment with amatoxin-conjugates as indicated. Subcutaneous administration of anti-PSMA-(XII) conjugates was better tolerated than the corresponding intravenous route, as indicated by increases in the liver enzymes ALT and AST following intravenous administration of the conjugates. Animals that were euthanized for ethical reasons or died were marked with a cross. [Figure 4] Figure 4 shows a comparison of the efficacy of anti-CD37-(XII) conjugates administered intravenously versus subcutaneously. The efficacy of the conjugate anti-CD37-(XII) disclosed herein was compared when administered by intravenous or subcutaneous routes. Probability of survival plots for (A) the Raji-Luc model and (B) the murine MEC2 tumor model. [Figure 5-1]Figure 5 shows pharmacokinetic and biodistribution studies of anti-PSMA-(XII) in mice. (A) Concentration of conjugated anti-PSMA-(XII) in mouse serum following subcutaneous administration as indicated. (B) Concentration of conjugated anti-PSMA-(XII) in mouse serum following intravenous administration as indicated. [Figure 5-2] Figure 5 shows pharmacokinetic and biodistribution studies of anti-PSMA-(XII) in mice. (C) PK data for subcutaneous and intravenous administration of conjugated anti-PSMA-(XII). The data show that subcutaneous administration reduces Cmax, but AUC remains nearly unchanged. [Figure 6-1] Figure 6 shows pharmacokinetic and biodistribution studies of anti-CD37-(XII) in mice. (A) Concentration of conjugated anti-CD37-(XII) in mouse serum following subcutaneous administration of the conjugated anti-CD37-(XII) as indicated. (B) Concentration of conjugated anti-CD37-(XII) in mouse serum following intravenous administration as indicated. [Figure 6-2] Figure 6 shows pharmacokinetic and biodistribution studies of anti-CD37-(XII) in mice. (C) PK data for subcutaneous and intravenous administration of conjugated anti-CD37-(XII). The data show that subcutaneous administration reduces Cmax, but the AUC remains nearly unchanged. [Figure 7-1] Figure 7 shows a pharmacokinetic study of the concentration of αPSMA-(XII) conjugate in serum of cynomolgus monkeys. (A) Serum concentration of the conjugate anti-PSMA-(XII) following intravenous and subcutaneous administration as indicated. Intravenous administration results in a higher and more rapid Cmax compared to subcutaneous administration of the conjugate. [Figure 7-2] Figure 7 shows a pharmacokinetic study of the concentration of αPSMA-(XII) conjugate in serum of cynomolgus monkeys. (B) Detailed results of the pharmacokinetic study showing that subcutaneous administration of the conjugate reduces Cmax (Cmax(iv) ≈219 μg / ml vs. Cmax(sc) ≈96 μg / ml at a dose of 7.5 mg / kg). [Figure 8]Figure 8 shows a comparison of the antitumor efficacy of intravenously and subcutaneously administered anti-GCC conjugates. (A) Antitumor efficacy of anti-GCC-LALA-D265C-(XIV) administered subcutaneously at 2.5 mg / kg (open squares) and intravenously at 2.5 mg / kg (filled squares). (B) Antitumor efficacy of anti-GCC-LALA-D265C-(XII) administered subcutaneously at 6 mg / kg (open triangles) and intravenously at 6 mg / kg (filled triangles). [Figure 9-1] Figure 9 shows the pharmacokinetics of amanitin-based anti-PSMA-(XIV) ADCs. Serum concentrations of anti-PSMA ADC h3 / F11-LALA-D265C Var16-(XIV) over time following a single subcutaneous dose of 10 mg / kg or 5 mg / kg or a single intravenous dose of 5 mg / kg in male CB17-SCID mice. [Figure 9-2] Figure 9 shows the pharmacokinetics of amanitin-based anti-PSMA-(XIV) ADCs. Serum concentrations of anti-PSMA ADC h3 / F11-LALA-D265C Var16-(XIV) over time following a single subcutaneous dose of 10 mg / kg or 5 mg / kg or a single intravenous dose of 5 mg / kg in male CB17-SCID mice. DETAILED DESCRIPTION OF THE INVENTION

[0025] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific components of the described apparatus or the specific process steps of the described methods, as such apparatus and methods may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. As used in the specification and appended claims, the singular forms "a," "an," and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It should also be understood that when a parameter range delimited by numerical values ​​is given, the range is deemed to include those limits. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring to each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated herein as if it were individually recited herein.

[0026] Throughout this specification and the claims that follow, unless the context requires otherwise, the terms "comprises," and variations thereof, such as "comprises," "including," and "comprising," should be understood to mean the inclusion of stated elements, integers, or steps, but not the exclusion of other unstated elements, integers, or steps. The term "consisting of" is a specific embodiment of the term "comprising," which excludes other unstated elements, integers, or steps.

[0027] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate, unrelated embodiments. Features discussed in one embodiment are meant to be disclosed in conjunction with other embodiments set forth herein. In some cases, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, this does not necessarily mean that the feature is not disclosed in the other embodiments, as one skilled in the art would understand. Those skilled in the art will understand that it is an intent of the present application to disclose the feature for the other embodiments as well, but that this has not been done merely for the sake of clarity and to keep the specification manageable.

[0028] Furthermore, the contents of the prior art documents mentioned herein are incorporated by reference. This refers particularly to prior art documents that disclose standard or conventional methods. In this case, incorporation by reference is primarily for the purpose of providing a fully enabling disclosure and avoiding lengthy repetition. Chemical terminology used throughout this application shall be interpreted in accordance with the "Glossary of Chemical Terms" published by the International Union of Pure and Applied Chemistry (IUPAC) (ISBN: 0-9678550-9-8).

[0029] Throughout this application, the term "about" is used to refer to + / - 10% of the numerical value with which it is used.

[0030] According to a first aspect of the present invention, the present invention relates to a pharmaceutical composition for use in the treatment of cancer, the composition comprising a conjugate comprising (i) a target-binding moiety, (ii) at least one amatoxin, and (iii) at least one linker linking the target-binding moiety to the at least one amatoxin, the composition being administered subcutaneously.

[0031] As used herein, the term "pharmaceutical composition" means a product containing pharmaceutical excipients, such as buffers, preservatives, and tonicity adjusting agents, together with an active compound or its salt, which is useful for treating or preventing or lessening the severity of a disease or disorder by administration to a mammal, preferably a human.

[0032] The term "amatoxin" or "amatoxins" as used in conjunction with the pharmaceutical compositions of the present invention refers to a bicyclic peptide consisting of eight amino acids found in Amanita phalloides (see Figure 1). Amatoxin specifically inhibits DNA-dependent RNA polymerase II in mammalian cells, thereby inhibiting transcription and protein biosynthesis in affected cells. When transcription is inhibited in cells, growth and proliferation are arrested. Although not covalently bound, the complex between amanitin and RNA polymerase II is tightly packed (K D =3 nM). Dissociation of amanitin from the enzyme is a very slow process, and therefore recovery of affected cells is unlikely. If the inhibition of transcription continues long enough, the cell will undergo programmed cell death (apoptosis).

[0033] In the context of the present invention, the term "amatoxin" includes any bicyclic peptide consisting of eight amino acids isolated from the genus Amanita, as described by Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260), as well as any chemical derivatives thereof, as well as any semisynthetic analogues thereof, including any synthetic analogues constructed from building blocks related to the master structure of the natural compound (cyclic, eight amino acids), as well as any synthetic or semisynthetic analogues containing non-hydroxylated amino acids instead of hydroxylated amino acids, and any synthetic or semisynthetic analogues in which the sulfoxide moiety is replaced by a sulfone, a thioether, or an atom other than sulfur (e.g., a carbon atom, as in the carbon analogue of amanitin).

[0034] As used herein, a "derivative" of a compound refers to a species that has a chemical structure similar to that of the compound, but that contains at least one chemical group not present in the compound from which it is derived and / or that lacks at least one chemical group present in the compound from which it is derived. The compound to which a derivative is compared is known as the "parent" compound. Typically, a "derivative" may be produced from the parent compound in one or more chemical reaction steps.

[0035] As used herein, an "analog" of a compound is structurally related to, but not identical to, the compound and exhibits at least one activity of the compound. The compound to which the analog is compared is known as the "parent" compound. Such activities include, but are not limited to, binding activity to another compound, inhibitory activity, such as enzyme inhibitory activity, toxic effect, and activating activity, such as enzyme activation activity. An analog is not required to exhibit such activity to the same extent as the parent compound. A compound is considered an analog within the context of this application if it exhibits an activity to the extent of at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, and more preferably at least 50%) of the activity of the parent compound. Thus, as used herein, "analog of amatoxin" refers to a compound that is structurally related to any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid, and that exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, 50%, 60%, more preferably at least 70%, 80%, 90%) inhibitory activity against mammalian RNA polymerase II compared to at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid. An "analog of amatoxin" suitable for use in the present invention may even exhibit greater inhibitory activity against mammalian RNA polymerase II than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, or amanuric acid. Inhibitory activity is measured as the concentration at which 50% inhibition occurs (IC 50The inhibitory activity against mammalian RNA polymerase II can be determined indirectly by measuring the inhibitory activity against cell proliferation, or the inhibitory activity of the amatoxins and their derivatives disclosed herein may be evaluated using, for example, the RNA polymerase II activity assay disclosed in Voss et al. BMC Molecular Biology 2014, 15:7.

[0036] "Semisynthetic analogs" refer to analogs obtained by chemical synthesis using compounds derived from natural sources (e.g., plant materials, bacterial cultures, fungal cultures, or cell cultures) as starting materials. Typically, "semisynthetic analogs" of the present invention are synthesized starting from compounds isolated from Amanita mushrooms. In contrast, "synthetic analogs" refer to analogs synthesized by so-called total synthesis from small (typically petrochemical) building blocks. Usually, this total synthesis is carried out without the aid of biological processes.

[0037] According to some embodiments of the present invention, the amatoxin may be selected from the group consisting of α-amanitin, β-amanitin, amanin, amaninamide, analogs thereof, derivatives thereof, and salts thereof.

[0038] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those that have a functional group (e.g., a carboxyl group, an amino group, a hydroxyl group, a thiol, or a thiol capture group) that can react with a linker molecule or target binding moiety, as defined below.

[0039] In the context of the present invention, the term "amanitin" refers in particular to an aspartic acid or asparagine residue at position 1, a proline residue, particularly a hydroxyproline residue, at position 2, an isoleucine, hydroxyisoleucine, or dihydroxyisoleucine (or aspartic acid in the case of amanurinic acid) at position 3, a tryptophan or hydroxytryptophan residue (or proline in the case of proamanurinic acid) at position 4, a glycine residue (or an isoleucine residue in the case of amanurinic acid and proamanurinic acid) at positions 5 and 7, an isoleucine residue at position 6, and a cysteine ​​residue, particularly oxidized to sulfoxide or sulfo, at position 8. The term "amanitin" refers to a bicyclic structure based on derivatives of cysteine ​​that have been converted into amanitin derivatives (see Figure 1 for numbering and a representative example of amanitin), and also includes any chemical derivatives thereof, any semi-synthetic analogues thereof, any synthetic analogues thereof that are constructed from building blocks related to the master structure of the natural compound (cyclic, 8 amino acids), any synthetic or semi-synthetic analogues that contain non-hydroxylated amino acids in place of hydroxylated amino acids, and any synthetic or semi-synthetic analogues, provided that in each case, any such derivative or analogue is functionally active by inhibiting mammalian RNA polymerase II.

[0040] The indented conjugates contained in the pharmaceutical compositions disclosed herein comprise (i) at least one amatoxin and (ii) at least one linker connecting the target-binding moiety to the at least one amatoxin. Thus, the conjugates may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amatoxin moieties. Preferably, the conjugates comprise about 2 to about 3 or 4 amatoxin moieties connected to the target-binding moiety by at least one linker, e.g., each of the amatoxin moieties is connected to the target-binding moiety by a linker, such that the number of linkers corresponds to the number of amatoxin moieties. In some embodiments, the pharmaceutical compositions of the invention comprise a conjugate disclosed herein comprising about 2 (e.g., about 1.5 to about 2.5) amatoxin-linker moieties connected to a target-binding moiety, e.g., an antibody or antigen-binding fragment thereof, preferably a monoclonal antibody.

[0041] As used herein, the term "target-binding moiety" refers to any molecule or portion of a molecule that can specifically bind to a target molecule or target epitope. Preferred target-binding moieties in the context of this application are (i) antibodies or antigen-binding fragments thereof, (ii) antibody-like proteins, and (iii) nucleic acid aptamers, (iv) anticalins, or (v) "target-binding moieties" suitable for use in the present invention that typically have a molecular mass of 40,000 Da (40 kDa) or greater.

[0042] In the context of this application, a "linker" refers to a molecule that increases the distance between the target-binding moiety and the amatoxin, such as to reduce steric interference between the two components (which may otherwise reduce the amatoxin's ability to interact with RNA polymerase II). The linker may also serve other purposes, such as facilitating the release of the amatoxin specifically in cells targeted by the target-binding moiety. The linker, preferably the bond between the linker and the amatoxin on one side and the bond between the linker and the target-binding moiety or antibody on the other side, is preferably not cleaved, degraded, or hydrolyzed under extracellular physiological conditions, such as in blood, but is preferably cleavable intracellularly, particularly within target cells (e.g., cancer cells), more specifically within the lysosomes of cancer cells. To provide selective stability, the linker may contain a functional group that is preferably pH-sensitive or protease-sensitive. Alternatively, the bond connecting the linker to the target-binding moiety may provide selective stability. Preferably, the linker has a length of at least 1, preferably 1 to 30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), one side of the linker is reacted with the amatoxin and the other side is reacted with the target binding moiety. In the context of the present invention, the linker is preferably a C, optionally bearing a substituent. 1-30 -Alkyl, C 1-30 -heteroalkyl, C 2-30 -Alkenyl, C 2-30 -heteroalkenyl, C 2-30 -alkynyl, C 2-30-heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group. The linker may contain one or more structural elements such as amide, ester, ether, thioether, disulfide, or hydrocarbon moiety. The linker may also contain a combination of two or more of these structural elements. Each of these structural elements may be present multiple times in the linker, for example, two, three, four, five, or six times.

[0043] In some embodiments, the linker may comprise a disulfide bond. It is understood that the linker must be coupled to the amatoxin and target-binding moiety either in one step or in two or more subsequent steps. To this end, the linker will have two groups, preferably at the proximal and distal ends, that are either (i) capable of forming a covalent bond with a group, preferably an activated group, on the amatoxin or target-binding peptide, or (ii) activated or capable of being activated to form a covalent bond with a group on the amatoxin. Thus, chemical groups, such as ester, ether, urethane, or peptide bonds, are preferably present at the distal and proximal ends of the linker as a result of the coupling reaction.

[0044] As used herein, the term "subcutaneous administration," which may also be referred to as "sc administration" or "subQ administration" and grammatical variations of any of these, refers to injection of a pharmaceutical composition of the present invention into the subcutaneous tissue (the layer of skin immediately below the dermis and epidermis (collectively referred to as skin)). Subcutaneous administration may be performed, for example, by a hypodermic needle and syringe, or by other means such as an auto-injector or injection pen (such as those described in WO12085029A1 or WO20154170A1, which are incorporated herein by reference).

[0045] According to some embodiments, the target-binding moiety of the present invention is one of (i) an antibody, preferably a monoclonal antibody, (ii) an antigen-binding fragment thereof, preferably a variable region (Fv), Fab fragment, or F(ab)2 fragment, (iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv), and (iv) an antibody-like protein. According to preferred embodiments, the target-binding moiety is an antibody, preferably a monoclonal antibody.

[0046] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes, or fragments of immunoglobulin genes, or cDNAs derived therefrom, including the kappa and lambda light chain constant region genes and the alpha, delta, epsilon, gamma, and mu heavy chain constant region genes, as well as any of a number of different variable region genes.

[0047] A basic immunoglobulin (antibody) unit is typically a tetramer consisting of two identical pairs of polypeptide chains: light chains (L, having a molecular weight of approximately 25 kDa) and heavy chains (H, having a molecular weight of approximately 50-70 kDa). Each heavy chain consists of a heavy chain variable region (VH or VH) and a heavy chain constant region (CH or CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL or VL) and a light chain constant region (CL or CL). The VH and VL regions can be further divided into hypervariable regions, also called complementarity-determining regions (CDRs), interspersed with more conserved regions than framework regions (FRs). The VH and VL regions each consist of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen.

[0048] CDR is the most important for the binding of an antibody or its antigen-binding portion. FR can be replaced with other sequences as long as the three-dimensional structure required for antigen binding is maintained. Structural changes in the construct will most likely result in the loss of sufficient binding to the antigen.

[0049] The term "antigen-binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to a given antigen in its native form. Examples of antigen-binding portions of antibodies include a Fab fragment; a monovalent fragment consisting of the VL, VH, CL, and CH1 regions; a F(ab')2 fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 regions; a Fv fragment consisting of the VL and VH regions of a single arm of an antibody; and a dAb fragment consisting of a VH region and isolated complementarity-determining regions (CDRs).

[0050] The antibody, antibody fragment, or antibody derivative thereof according to the present invention may be a monoclonal antibody. As used herein, the term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules of a single binding specificity and affinity for a particular epitope, representing a homogeneous antibody population, i.e., a homogeneous population of whole immunoglobulins or fragments or derivatives thereof. Preferably, such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA, and / or IgM, fragments or derivatives thereof. Preferably, the monoclonal antibody of the present invention is of the IgG isotype, such as IgG1 or IgG4, more preferably the IgG1 isotype. As used herein, the term "antibody" also includes and refers to antibody moieties or antibody portions contained in the conjugates according to the present invention.

[0051] As used herein, the term "fragment" or "antigen-binding fragment" refers to a fragment of the above-mentioned antibody that retains target binding ability, such as the CDRs (complementarity-determining regions), hypervariable region, variable region (Fv), IgG heavy chain (consisting of the VH region, CH1 region, hinge region, CH2 region, and CH3 region), IgG light chain (consisting of the VL region and CL region), and / or Fab and / or F(ab)2.

[0052] As used herein, the term "derivative" or "antigen-binding derivative" refers to protein constructs that are structurally distinct from, but have some structural relationship to, the common antibody concept, such as scFv, Fab, and / or F(ab)2, as well as bi-, tri-, or higher specificity antibody constructs, all of which have approximately the same target binding specificity as the monoclonal antibodies of the invention, all of which are described below.

[0053] Other antibody derivatives known to those skilled in the art are diabodies (e.g., as disclosed in Proc Natl Acad Sci US A. 1993 Jul 15;90(14):6444-8), camelid antibodies, domain antibodies, scFvs, bivalent homodimers having two chains consisting of IgA (two IgG structures linked by a J chain and a secretory component), shark antibodies (IgNAR), antibodies consisting of a New World primate framework and non-New World primate CDRs, dimerization constructs comprising CH3+VL+VH, other scaffold protein formats comprising CDRs, and antibody conjugates (e.g., antibodies conjugated to drugs, toxins, cytokines, aptamers, nucleic acids (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), therapeutic polypeptides, radioisotopes, or labels, or fragments or derivatives thereof).

[0054] As used herein, the term "antibody-like protein" refers to a protein engineered to specifically bind to a target molecule (e.g., by mutagenesis of an Ig loop). Typically, such antibody-like proteins contain at least one variable peptide loop attached to both ends of a protein scaffold. This dual structural constraint significantly enhances the binding affinity of antibody-like proteins, to a level comparable to that of antibodies. The variable peptide loop is typically 10 to 20 amino acids in length. The scaffold protein may be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affilin proteins, affibodies, anti-calins, and engineered ankyrin repeat proteins (see, e.g., Binz et al., 2005, or WO2012135345A1). Antibody-like proteins can be obtained from large libraries of mutants, such as by panning from large phage display libraries, and can be isolated similarly to conventional antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

[0055] The term "Fab" as used herein relates to an IgG fragment containing the antigen-binding region, this fragment consisting of one constant region and one variable region from each of the heavy and light chains of the antibody.

[0056] The term "F(ab)2" as used herein relates to an IgG fragment consisting of two Fab fragments linked together by disulfide bonds.

[0057] The term "scFv" as used herein refers to a single-chain variable fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked together by a short linker that usually contains serine (S) and / or glycine (G) residues. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide.

[0058] Engineered antibody formats are, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, and the like.

[0059] IgG, scFv, Fab, and / or F(ab)2 are antibody formats well known to those skilled in the art, and the relevant enabling techniques are available from the respective textbooks.

[0060] According to a preferred embodiment of the invention, the antibody, antigen-binding fragment thereof, or antigen-binding derivative thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment or antigen-binding derivative thereof, respectively; more preferably, the antibody or antigen-binding fragment thereof is a humanized antibody or a human antibody.

[0061] Monoclonal antibodies (mAbs) derived from mice can produce undesirable immunological side effects due to the fact that they contain proteins from other species that may raise antibodies. To overcome this problem, antibody humanization and maturation methods have been designed to generate antibody molecules with minimal immunogenicity when applied to humans, while ideally retaining the specificity and affinity of the non-human parent antibody (for a review, see Almagro and Fransson 2008, Front Biosci. 2008 Jan 1;13:1619-33). Using these methods, for example, the framework regions of a mouse mAb are replaced with corresponding human framework regions (so-called CDR grafting). WO200907861 discloses the generation of humanized forms of mouse antibodies by combining the CDR regions of a non-human antibody with human constant regions using recombinant DNA technology. US6548640 to the UK Medical Research Council describes CDR grafting techniques and US5859205 to Celltech describes the production of humanized antibodies.

[0062] As used herein, the term "chimeric antibody" refers to an antibody that consists of the original antigen-binding variable region of an antibody with a constant region derived from a different species. Because antibodies, especially monoclonal antibodies, were originally mostly derived from mice, chimeric antibodies typically contain human constant regions and mouse variable regions to reduce immunogenicity in humans. Examples of chimeric antibodies used in clinical therapy include infliximab, rituximab, and abciximab.

[0063] As used herein, the term "humanized antibody" refers to an antibody, fragment, or derivative thereof, in which at least a portion of the antibody's constant and / or framework regions, and optionally a portion of the CDR regions, are derived from or adapted to human immunoglobulin sequences. Methods for antibody humanization are known in the art and are described, for example, in Riechmann et al., Nature 332:323-327, 1988; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370.

[0064] The antibodies, antibody fragments thereof, or antibody derivatives thereof disclosed herein may contain humanized sequences, particularly humanized sequences of antigen-binding regions based on preferred VH and VL sequences that maintain appropriate ligand affinity. The amino acid sequence modifications to obtain the humanized sequences may occur in the CDR and / or framework regions of the original antibody, and / or in the antibody constant region sequences.

[0065] The antibody, antibody fragment, or antibody derivative can be glycosylated. The glycan can be an N-linked oligosaccharide chain of asparagine 297 of the heavy chain.

[0066] The antibodies, fragments, or derivatives of the present invention may be produced by transfecting host cells with an expression vector containing the coding sequence for an antibody of the present invention. The expression vector or recombinant plasmid is obtained by placing the coding antibody sequence under the control of suitable regulatory genetic elements, such as a promoter and enhancer sequence (e.g., a CMV promoter). The heavy and light chain sequences may be expressed from individual co-transfected expression vectors or from a dual expression vector. The transfection may be transient or stable. The transfected cells are then cultured to produce the transfected antibody construct. After stable transfection, stable clones secreting antibodies with properly linked heavy and light chains are selected by screening using a suitable assay, such as ELISA, subcloned, and expanded for future production.

[0067] According to one preferred embodiment, the antibody, or antibody portion or antigen-binding fragment thereof, of the conjugate of the pharmaceutical composition according to the invention is an IgG isotype antibody, such as an IgG1 isotype antibody, an IgG2 isotype antibody, an IgG3 isotype antibody, or an IgG4 isotype antibody.

[0068] The use of antibodies characterized by reduced or eliminated effector functions in conjugates of pharmaceutical compositions for use according to the present invention may be desirable, for example, to prevent unwanted cytokine secretion or to kill cells expressing Fcγ receptors, such as macrophages. Accordingly, the antibodies or antigen-binding fragments of the present invention may contain modifications and / or mutations that alter the properties of the antibody and / or fragment, such as reducing ADCC, ADCP, or complement-dependent cytotoxicity (CDC), as known in the art. Preferably, ADCC, ADCP, and CDC are reduced by at least 90%, more preferably at least 95%, more preferably at least 97.5%, and even more preferably at least 98% or 99%, compared to antibodies comprising a wild-type Fc region. According to a preferred embodiment, the pharmaceutical composition for use according to the present invention comprises a conjugate containing an antibody that does not induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0069] Binding of IgG1 to activating and inhibitory Fcγ receptors (FcγR) or the first component of complement (C1q) depends on residues located in the hinge and CH2 regions. Two regions in the CH2 region are important for FcγR and complement C1q binding and have unique sequences. Substitution of human IgG1 and IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduced ADCC and CDC (Armour, et al., Eur. J. Immunol. 29(8)(1999)2613-2624; Shields, et al., J. Biol. Chem. 276(9)(2001)6591-6604, WO2021 / 234402A2).

[0070] Thus, in one embodiment, a pharmaceutical composition for use according to the present invention comprises an antibody portion or antigen-binding fragment as part of a conjugate disclosed herein comprising a mutant Fc region, the mutant Fc region comprising at least one amino acid modification relative to a wild-type Fc region such that the molecule has reduced affinity for the IgG1 Fc receptors FcγRI, FcγRII, and FcγRIII, and complement component C1q, compared to the wild-type Fc region.

[0071] Affinity to an Fc region, such as IgG1 binding to FcγR, can be determined using a variety of techniques known in the art, including, but not limited to, equilibrium assays (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or surface plasmon resonance assays such as those disclosed in Wilkinson et al. PLoS One. 2021;16(12):e0260954, or other reaction mechanisms of kinetic-based assays (e.g., BIACORE™ analysis or Octet™ analysis (forteBIO)), as well as other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration).

[0072] Thus, pharmaceutical compositions for use according to the present invention include conjugates comprising the antibodies described herein that have been genetically modified to contain a mutant Fc region that contains a modification of at least one amino acid residue that directly contacts FcγR based on structural and crystallographic analysis. The term "genetically modified" or "genetic modification" as used herein refers to modifying the amino acid sequence of a given or naturally occurring polypeptide or protein, such as the Fc region of an antibody, or a portion thereof, by genetic engineering methods such as site-directed mutagenesis, as described in Biochem. J. (1986) Vol. 237:1-7 or J Biol Chem. (2015) Vol. 290(5):2577-2592, in the sense of nucleotide and / or amino acid substitution, insertion, deletion, or reversion, or any combination thereof. The term "amino acid substitution" or "mutation" as used herein refers to a modification of the amino acid sequence of a protein in which one or more amino acids are replaced with the same number of different amino acids, resulting in a protein containing an amino acid sequence that differs from that of the original protein. Conservative amino acid substitutions are understood to refer to substitutions that do not significantly affect the structure and function of proteins due to similar size, charge, polarity, and / or conformation. Conservative amino acid groups in this sense include, for example, nonpolar amino acids Gly, Ala, Val, Ile, and Leu; aromatic amino acids Phe, Trp, and Tyr; positively charged amino acids Lys, Arg, and His; and negatively charged amino acids Asp and Glu.

[0073] The Fc region of the antibody may further comprise at least one cysteine ​​amino acid substitution at a site where the engineered cysteine ​​is available for binding but does not disrupt the folding and assembly of the immunoglobulin. Corresponding cysteine-substituted or cysteine-engineered antibodies are disclosed in WO2016040856A2, or Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; WO2009 / 052249, and WO2016 / 142049. A preferred cysteine ​​substitution in the Fc region of the antibody of the present invention disclosed herein is D265C (according to the EU numbering system), as disclosed in WO2016142049A1.

[0074] According to one embodiment, the antibody portion comprised in the pharmaceutical composition for use according to the invention comprises at least one amino acid substitution at positions D265, L234, L235, or G236 (according to the EU numbering system), and preferably comprises two or three amino acid substitutions at the above positions.

[0075] According to a preferred embodiment, the conjugate of the invention for use in a pharmaceutical composition according to the invention comprises an antibody or antibody portion comprising an Fc region containing at least one amino acid substitution selected from L234A, L234S, L234G, L235A, L235G, L235S, L235T, G236R, and D265C, wherein the amino acid numbering is according to the EU numbering system. The EU numbering system, also sometimes referred to as the "Kabat-like EU index," refers to the numbering of human IgG1 EU antibodies, which refers to the EU antibody numbering of Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85.

[0076] According to a preferred embodiment, the antibody or antibody portion of the conjugate of the present invention disclosed herein comprises the amino acid substitution D265C (according to the EU numbering system). It is particularly preferred that the Fc region of the antibody comprises the amino acid substitutions L234A, L235A, and D265C (according to the EU numbering system). Corresponding antibodies comprising these mutations are particularly suitable for ADCs containing highly toxic payloads, because they lack Fc effector functions ("Fc silenced"), thereby avoiding killing of macrophages and other cells expressing Fcγ receptors on their cell surface.

[0077] According to one embodiment, the linker of the conjugate is attached to the antibody or antibody portion via any of the naturally occurring cysteine ​​residues of the antibody, preferably via any of the naturally occurring cysteine ​​residues that form the interchain disulfide bonds of the antibody and / or via a disulfide bond. Corresponding methods for attaching the linkers disclosed herein to the antibody portion of the conjugates of the invention are disclosed, for example, in WO2005 / 084390A2.

[0078] According to one embodiment, the amatoxin of the conjugate of the pharmaceutical composition of the present invention for use in treating cancer disclosed herein is attached to the antibody via a cleavable or non-cleavable linker.

[0079] A "cleavable linker" according to the present invention is understood to include at least one cleavage site. As used herein, the term "cleavage site" refers to a moiety that is susceptible to specific cleavage at a predetermined location under certain conditions, such as a specific enzymatic or reducing environment in a particular body or cellular compartment. For example, a cleavable linker is designed to exploit differences in local environments, such as the extracellular and intracellular environments (e.g., including pH, reduction potential, or enzyme concentration), to trigger the release of an amatoxin within a target cell. Generally, cleavable linkers are relatively stable in the circulatory system but are particularly susceptible to cleavage in the intracellular environment by one or more reaction mechanisms (e.g., including, but not limited to, the activity of proteases, peptidases, and glucuronidases). As used herein, a cleavable linker is substantially stable in circulating plasma and / or outside target cells (e.g., cancer cells) and may be cleaved at any effective rate within target cells or in close proximity to target cells (e.g., in the tumor microenvironment).

[0080] Suitable cleavable linkers according to the present invention may include, for example, linkers that can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference). Suitable cleavable linkers may include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

[0081] For example, linkers that are hydrolyzable under acidic conditions may include hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc. (see, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the entire disclosure of which is incorporated herein by reference).Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH below 5.5 or 5.0, which is close to the pH of lysosomes.

[0082] For example, the linker that may be used in the conjugates of the present invention can be cleaved under reducing conditions, such as disulfides. Various disulfide linkers are known in the art, including those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; U.S. Patent No. 4,880,935, the entire disclosure of which is incorporated herein by reference).

[0083] According to some embodiments, the cleavage site may be cleavable by at least one protease selected from the group consisting of cysteine ​​proteases, metalloproteases, serine proteases, threonine proteases, and aspartic proteases.

[0084] Cysteine ​​proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism involving a nucleophilic cysteine ​​thiol in the catalytic triad or diad.

[0085] Metalloproteases are proteases whose catalytic mechanism involves a metal. Most metalloproteases require zinc, but some use cobalt. The metal ion coordinates to the protein through three ligands. The ligands that coordinate to the metal ion can vary depending on the ligand: histidine, glutamate, aspartate, lysine, and arginine. The fourth coordination position is occupied by a labile water molecule.

[0086] Serine proteases are enzymes that cleave peptide bonds in proteins, with serine acting as the nucleophilic amino acid in the active site of the enzyme. Serine proteases are broadly classified into two categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0087] Threonine proteases are a family of proteolytic enzymes that contain a threonine (Thr) residue in their active site. The prototypic members of this class of enzymes are the catalytic subunits of the proteasome, while acyltransferases have convergently evolved the same active site geometry and mechanism.

[0088] Aspartic proteases are catalytic protease enzymes that use activated water molecules bound to one or more aspartate residues to catalyze their peptide substrates. Generally, they have two highly conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.

[0089] In some embodiments, the cleavable site is cleavable by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase, and β-galactosidase, preferably cathepsin B.

[0090] In some embodiments, the cleavage site is a disulfide bond, and specific cleavage is achieved by a reducing environment, such as an intracellular reducing environment (e.g., acidic pH conditions). For example, the corresponding linker may have the following structure: (Amatoxin)-(CH2)2-SS-(CH2)2-XS-(antibody) (Amatoxin)-(CH2)3-SS-(CH2)2-XS-(antibody) (Amatoxin)-(CH2)2-SS-(CH2)3-XS-(antibody) (Amatoxin)-(CH2)3-SS-(CH2)3-XS-(antibody) wherein X is as disclosed above.

[0091] In some embodiments, the linker is a pH-sensitive linker, which is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are cleavable under acidic conditions. This cleavage strategy generally takes advantage of the lower pH of endosomal (pH about 5-6) and lysosomal (pH about 4.8) intracellular compartments compared to the cytosol (pH about 7.4), causing hydrolysis of an acid-labile group in the linker, such as a hydrazone (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-labile and / or hydrolyzable linker. For example, an acid-labile linker that is hydrolyzable in the lysosome and contains an acid-labile group (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) can be used. See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123; Neville et al. (1989) Biol. Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which approximates the pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to a therapeutic agent via an acylhydrazone bond). See, for example, U.S. Patent No. 5,622,929.

[0092] According to some embodiments, the cleavable linker of the present invention is an enzymatically cleavable linker. An enzymatically cleavable linker comprises a cleavage site, which is an enzymatically cleavable moiety comprising two or more amino acids. Preferably, the enzymatically cleavable moiety is a phenylalanine-lysine (Phe-Lys), valine-lysine (Val-Lys), phenylalanine-alanine (Phe-Ala), valine-alanine (Val-Ala), phenylalanine-citrulline (Phe-Cit), or valine-citrulline (Val-Cit) dipeptide, or, for example, a valine-alanine-valine (Val-Ala-Val), leucine-alanine-leucine (Leu-Ala-Leu), glycine-phenylalanine-lysine (Val-Lys), or a valine-citrulline (Val-Cit) dipeptide. and the like. Examples of such tripeptides include, for example, Asp-cBu-Cit, iGlu-cBu-Ala, iGlu-cBu-Cit, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Ala-Ala-Asn, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, or for example, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or alanine-alanine-proline-valine (Ala Ala Pro Val) peptides, or β-glucuronides or β-galactosides.

[0093] In some embodiments, the enzymatically cleavable linker of the present invention is a β-glucuronic acid-based linker. Easy drug release can be achieved by cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and is overexpressed in some tumor types, but its extracellular enzymatic activity is low. The use of a β-glucuronic acid-based linker may circumvent the tendency of the conjugates of the present invention to aggregate due to the hydrophilicity of β-glucuronides. Corresponding linkers are disclosed, for example, in WO2007011968A2, and β-galactoside-cleavable linkers are disclosed in WO19192979A1, the contents of which are incorporated herein by reference.

[0094] In some embodiments, the cleavable linker of the conjugates of the present invention disclosed herein is a self-immolative linker. The term "self-immolative linker" or "self-immolative spacer" refers to a bifunctional chemical moiety that can covalently link two chemical moieties into a generally stable tripartite molecule. A self-immolative spacer can spontaneously separate from a second moiety when the bond to the first moiety is cleaved.

[0095] The linkers of the conjugates of the present invention disclosed herein contain "self-immolative" groups such as PAB or PABC (para-aminobenzyloxycarbonyl), as described, for example, in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al. al (1983) J. Med. Chem. 26:638-644; US6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; WO98 / 13059; US20040052793; US6677435; US5621002; US20040121940; WO2004 / 032828, or WO2005 / 112919. Other such chemical moieties capable of this process ("self-immolative linkers") include methylene carbamates and heteroaryl groups, such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in U.S. Patent Publication Nos. 20160303254 and 20150079114, and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and US 7,223,837. In some embodiments, dipeptides are used in combination with self-immolative linkers.

[0096] In a preferred embodiment, the enzymatically cleavable linker according to the invention is a linker cleavable by cathepsin B and comprises a dipeptide selected from Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit, and Val-Cit, or a tripeptide selected from valine-alanine-valine (Val-Ala-Val), leucine-alanine-leucine (Leu-Ala-Leu), glycine-phenylalanine-lysine (Gly-Phe-Lys), isoleucine-alanine-leucine (Ile-Ala-Leu) tripeptides, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly), or alanine-alanine-proline-valine (Ala Ala Pro Val) peptides.

[0097] In a particularly preferred embodiment, the enzymatically cleavable linker according to the present invention is a cathepsin B cleavable linker and comprises a dipeptide selected from Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit, and Val-Cit, and in particular, the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and the amatoxin disclosed herein below, where the wavy line indicates the binding site between the amatoxin and the antibody of the conjugate disclosed herein. [ka] [ka] [ka] [ka] [ka] [ka]

[0098] Thus, the conjugates disclosed herein that are included in the pharmaceutical compositions of the present invention include, for example, an enzymatically cleavable moiety that includes any one of the dipeptide-PAB moieties disclosed above: Phe-Lys-PAB, Val-LysPAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit-PAB, or Val-Cit-PAB.

[0099] Preferably, the cleavable moiety of the conjugate of the invention comprises the dipeptide-PAB moiety Val-Ala-PAB. [ka] Here, the PAB moiety is attached to an amatoxin.

[0100] According to some embodiments, the cleavable linkers of the invention disclosed herein comprise a thiol reactive group selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazin-2-ylamino group, methyl-sulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate, or maleimide.

[0101] According to one preferred embodiment, the thiol reactive group is a maleimide (meleimidyl moiety) as shown below. [ka]

[0102] Linkers (e.g., cleavable linkers and / or non-cleavable linkers) containing the above-described thiol-reactive groups are particularly useful for covalent coupling of the linker-amatoxin conjugates disclosed herein to antibodies containing a reactive thiol (e.g., cysteine-engineered antibodies containing at least one reactive cysteine ​​residue for coupling).

[0103] According to a particularly preferred embodiment, the linker of the present invention comprises the following: (i) the structure prior to coupling to a target binding moiety, such as an antibody, as disclosed herein; or (ii) the structure after said coupling. [ka] [ka]

[0104] According to some embodiments, the linker of the conjugate of the present invention is a non-cleavable linker. A "non-cleavable linker" is understood to be not subject to enzymatic cleavage, such as by cathepsin B, and is released from the conjugate of the present invention during degradation of the antibody portion of the conjugate of the present invention within the target cell (e.g., lysosomal degradation). Non-cleavable linkers suitable for use in the present invention may include, for example, one or more groups selected from a bond, -(C=O)-, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be substituted and / or contain one or more heteroatoms (e.g., S, N, or O) in place of one or more carbon atoms. Non-limiting examples of such groups include (CH2) p , (C=O)(CH2) p , and polyethylene glycol (PEG; (CH2CH2O) p ) units (wherein p is an integer of 1 to 6 selected independently in each case).

[0105] In some embodiments, a non-cleavable linker according to the present invention may be a bond, —(C═O)—, a —C(O)NH— group, a —OC(O)NH— group, C-C alkylene, C-C heteroalkylene, C-C alkenylene, C-C heteroalkenylene, C-C alkynylene, C-C heteroalkynylene, C-C cycloalkylene, heterocycloalkylene, arylene, heteroarylene, —(CHCHO) p - groups (wherein p is an integer from 1 to 6), wherein each C-C alkylene, C-C heteroalkylene, C-C alkenylene, C-C heteroalkenylene, C-C alkynylene, C-C heteroalkynylene, C-C cycloalkylene, heterocycloalkylene, arylene, or heteroarylene is optionally substituted by 1 to 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro, as appropriate.

[0106] For example, each C-C alkylene, C-C heteroalkylene, C-C alkenylene, C-C heteroalkenylene, C-C alkynylene, C-C heteroalkynylene, C-C cycloalkylene, heterocycloalkylene, arylene, or heteroarylene in a non-cleavable linker disclosed herein may be optionally interrupted by one or more heteroatoms selected from O, S, and N, and may be substituted with, for example, from 1 to 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0107] According to a preferred embodiment, the non-cleavable linker of the conjugate of the present invention is -(CH2) n -units (wherein n is an integer of 2 to 12, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably n is an integer of 2 to 6, for example, 1, 2, 3, 4, 5, or 6).

[0108] In a preferred embodiment, the non-cleavable linker of the conjugate of the invention is -(CH2) n - (wherein n is 6), and the linker is represented by the following formula: [ka]

[0109] In some embodiments, the non-cleavable linker of the present invention disclosed herein further comprises a thiol-reactive group, which may be selected from, for example, bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazin-2-ylamino, methyl-sulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate, or maleimide.

[0110] According to a preferred embodiment, the thiol-reactive group is a maleimide (a meleimidyl moiety) as disclosed above. For example, a non-cleavable linker comprising the maleimide may have the following structure, where the wavy line at the end of the linker indicates the point of attachment to the amatoxin: [ka] In the formula, n is an integer of 2 to 12, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably, n is an integer of 2 to 6, for example, 1, 2, 3, 4, 5, or 6, and more preferably, n is 6.

[0111] After attachment to a reactive sulfhydryl of an antibody, such as a naturally occurring or engineered cysteine ​​residue of the antibody, the mereimidyl moiety of a cleavable or non-cleavable linker disclosed herein comprises the following structure: [ka] Here, the wavy line represents the attachment site of a cleavable or non-cleavable linker (L) disclosed herein, and the sulfur atom is part of the reactive cysteine ​​of the antibody.

[0112] According to a preferred embodiment, conjugates of the present invention comprising a cleavable or non-cleavable linker disclosed herein and further comprising a thiol-reactive group may be linked to a naturally occurring sulfhydryl moiety in the antibody of the conjugate, or the cleavable or non-cleavable linker of the conjugate of the present invention comprising a thiol-reactive group may be linked to a sulfhydryl moiety introduced into the antibody by genetic modification, such as that described in Nat Biotechnol. 2008 Aug;26(8):925-32 or WO2006 / 034488A2. Preferably, the cleavable or non-cleavable linker disclosed herein comprising a thio-reactive group is linked to a sulfhydryl moiety introduced into the Fc region of each antibody of the conjugate of the present invention by genetic modification, such as D265C (EU numbering).

[0113] In some embodiments, the at least one amatoxin and at least one linker of the conjugates of the pharmaceutical compositions of the invention disclosed herein are represented by Formula (Ia): [ka] During the ceremony, R1 is H, OH, OR A , or OR C and R2 is H, OH, OR B , or OR C and R A and R B When present, they combine together with the oxygen atom to which they are attached to form a 5-membered heterocycloalkyl group; R3 is H, R C or R4, R5, R6, and R7 are each independently H, OH, OR C , R C and R8 is OH, NH2, OR C , or NHR C and Q is -S-, -S(O)-, or -SO2-; R C teeth [ka] wherein the sulfur atom is part of a reactive cysteine ​​of the antibody, and L is a linker (e.g., a cleavable linker or a non-cleavable linker as defined herein) and is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl; or -((CH2) m O) n (CH2) m -wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0114] In some embodiments, R A and R B are joined together with the oxygen atom to which they are attached to form a 5-membered heterocycloalkyl of the formula: [ka] In the formula, Y is -(C=O)-, -(C=S)-, -(C=NR E )-, or -(CR E R E’ )- and R E and R E’ are each independently H, C1-C6 alkylene-R C , C1-C6 heteroalkylene-R C , C2-C6 alkenylene-R C , C2-C6 heteroalkenylene-R C , C2-C6 alkynylene-R C , C2-C6 heteroalkynylene-R C , cycloalkylene-RC , heterocycloalkylene-R C , Arylene-R C , or heteroarylene-R C or a combination thereof, wherein each C1-C6 alkylene-R C , C1-C6 heteroalkylene-R C , C2-C6 alkenylene-R C , C2-C6 heteroalkenylene-R C , C2-C6 alkynylene-R C , C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , Arylene-R C , or heteroarylene-R C is optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0115] In some embodiments, in the at least one amatoxin according to formula (Ia) disclosed above, R1, R2, and R9 are OH; R3 is H, R C , or R D and R4, R5, R6, and R7 are each independently H, OH, OR C , R C and R8 is OH, NH2, OR C , NHR C and Q is -S-, -S(O)-, or -SO2-; R Cis as disclosed above.

[0116] In some embodiments, the at least one amatoxin and at least one linker of the conjugates of the pharmaceutical compositions of the invention disclosed herein are represented by the following formula (IIa): [ka] wherein R1, R2, and R9 are OH; R5 is independently H, OH, OR C , R C and R8 is OH, NH2, OR C , NHR C , or NR C and Q is -S-, -S(O)-, or -SO2-; R C teeth [ka] wherein the sulfur atom is part of a reactive cysteine ​​of the antibody, and L is a linker (e.g., a non-cleavable linker or a cleavable linker as defined below) that is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or comprises a dipeptide, or -((CH2) m O) n (CH2) m - (wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10), or L is a cleavable linker, preferably an enzymatically cleavable linker, more preferably a self-immolative cathepsin B cleavable linker as disclosed herein.

[0117] According to some embodiments, the linker of the conjugates of the invention disclosed herein is attached to at least one amatoxin via (i) the γ C-atom of amatoxin amino acid 1, (ii) the δ C-atom of amatoxin amino acid 3, or (iii) the 6′-C-atom of amatoxin amino acid 4.

[0118] In one embodiment of the present invention, the pharmaceutical composition comprises a conjugate described herein, wherein the conjugate comprises an amatoxin comprising (i) amino acid 4 having a 6'-deoxy position, and (ii) amino acid 8 having an S-deoxy position.

[0119] According to a particularly preferred embodiment of the present invention, the pharmaceutical composition for use in treating cancer according to the present invention comprises a conjugate comprising as a linker-amatoxin moiety at least one of the compounds of formulae (I) to (XI) below: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0120] Therefore, the pharmaceutical composition of the present invention for use in treating cancer contains a conjugate comprising an antibody covalently bound to at least one of the amatoxin-linker conjugates (I) to (XI), for example, 1, 2, 3, 4, 5, 6, 7, or 8, preferably about 1 to about 4 or about 3 to about 6, preferably about 2 to about 3, and more preferably about 2, amatoxin-linker conjugates (I) to (XI).

[0121] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer according to the present invention comprises a conjugate comprising an antibody covalently linked to an amatoxin linker moiety via a thioether bond according to any one of Formulas XII-XXII below: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] wherein the amatoxin-linker moiety is attached to the thiol group of a cysteine ​​residue of the antibody or antibody portion of the conjugate of the present invention, and n is preferably about 1, 2, 3 to about 4, 5, 6, 7, or 8, and preferably about 1, 1.5, 2, 2.5 to about 3.5, 4.5, or 5.5, more preferably about 1.5 to about 3.5, and most preferably about 2. The thiol group of the cysteine ​​residue of the antibody or antibody portion may be a naturally occurring cysteine ​​residue of the antibody, e.g., one that forms an interchain disulfide bond after reduction of the cysteine ​​residue for the thiol-based bond, or the thiol group of the cysteine ​​residue of the antibody may be genetically modified, preferably at position D265 (D265C according to EU numbering).

[0122] According to a preferred embodiment, the pharmaceutical composition of the present invention for use in treating cancer disclosed herein can be used to treat, for example, solid tumor or non-solid tumor.For example, solid tumor includes gastric cancer, adenocarcinoma, melanoma, ovarian cancer, uterine cancer, cervical cancer, breast cancer (including triple-negative breast cancer), bronchial carcinoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, thymoma, testicular cancer, neuroblastoma, glioma, prostate cancer, castration-resistant prostate cancer, gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, esophageal cancer, laryngeal cancer, parotid cancer, bile duct cancer, rectal cancer, endometrial cancer, desmoid tumor, desmoplastic small round cell tumor, neuroectodermal tumor, retinoblastoma, Selected from the group including rhabdomyosarcoma, Wilms' tumor, osteosarcoma, chondrosarcoma, lung cancer, non-small cell lung cancer (NSCLC), alveolar rhabdomyosarcoma, rhabdomyosarcoma, Askin tumor, intraperitoneal desmoplastic small cell tumor, malignant papillary renal cell carcinoma, meningioma; small cell lung carcinoma, ependymoma, nasal neuroblastoma (olfactory neuroblastoma), fibromatosis, ganglioglioma, islet cell tumor, basal and squamous cell carcinoma, large cell neuroendocrine carcinoma (LCNEC), Leydig cell tumor, salivary gland carcinoma, pineoblastoma, pleomorphic low malignant grade adenocarcinoma, schwannoma, teratoma, thymoma. The non-solid tumor may be, for example, Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma (DBNHL), subtypes of non-Hodgkin's lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter's syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), or one of multiple myeloma, Burkitt's lymphoma, anaplastic large cell lymphoma, marginal zone B-cell lymphoma.

[0123] As used herein, the term "cancer" refers to a general term for diseases in which abnormal cells divide without control. Cancer cells can invade nearby tissues and spread to other parts of the body through the bloodstream and lymphatic system. There are several major types of cancer, for example, carcinoma is cancer that begins in the skin or in the tissues that cover or coat the internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that begins in blood-forming tissues, such as the bone marrow, and causes the production of many abnormal blood cells that enter the bloodstream. Lymphoma is cancer that begins in cells of the immune system.

[0124] Tumors form when normal cells lose their ability to behave as a defined, controlled, and coordinated unit. Generally, a solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. A single tumor may have different cell populations and even different processes within it. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcoma, carcinoma, and lymphoma. Leukemia (cancer of the blood) generally does not form solid tumors.

[0125] According to one embodiment, the cancer cells or tumors disclosed above are characterized by hemizygous deletion of TP53, POLR2A, or del(17p13).For example, cancer cells or tumors that can be treated with the pharmaceutical compositions of the present invention disclosed herein are characterized by hemizygous deletion of the POLR2A gene, or hemizygous deletion of the TP53 gene and the POLR2A gene.The term "hemizygous" used in the present invention refers to an individual or cell that has only one complete allele of a gene or chromosome segment, instead of the usual two.Hemizygote refers to a cell or organism whose genome contains only one complete allele at a given locus, whether the allele is wild-type or mutant, for example, any cell of the tumor or cancer disclosed above is hemizygous for the chromosome locus 17p13.Preferably, the cell of the cancer or tumor disclosed above is hemizygous for the genes TP53 and POLR2A. As used herein, "TP53" refers to the "tumor protein 53" gene, which encodes a tumor suppressor protein (P53) containing transcriptional activation, DNA binding, and oligomerization domains. The encoded protein regulates the expression of target genes in response to various cellular stresses, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes. Mutations in this gene are associated with various human cancers, including hereditary cancers such as Li-Fraumeni syndrome.

[0126] The tumor suppressor gene TP53 is often inactivated by mutation or deletion in most human tumors. As used herein, "POLR2A" refers to the POLR2A gene, which encodes the largest subunit of the human RNA polymerase II complex and is essential for polymerase activity in mRNA synthesis. Hemizygous deletions of chromosome 17p13, such as del(17p13.1), can be detected by fluorescence in situ hybridization (FISH), as disclosed in Merz et al. Am J Hematol. 2016 Nov;91(11):E473-E477.

[0127] The cancer cells or tumors disclosed herein may not be homogeneous cell populations, for example, with respect to the deletion of TP53 and / or POLR2A. For example, about 1%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% to about 70%, 75%, 80%, 85%, 90%, 95%, 100, or about 70%, 75%, 80%, 85% to about 90%, 92.5%, 95%, 97.5%, 100% of the cancer cells disclosed above may be del(17p13.1), TP53, and / or POLR2A. The tumors or cancer cells may be hemizygous for TP53 and / or POLR2A, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cancer cells disclosed herein are del(17p13.1) or hemizygous for TP53 and / or POLR2A. The use of the pharmaceutical compositions of the present invention disclosed herein in treating cancer according to the present invention may be particularly advantageous, for example, for any of the tumors or cancers disclosed above that are characterized by hemizygous deletion of chromosome 17p13.1, TP53, and / or POLR2A, because the tumors or cancer cells are at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold more sensitive to cancer treatment using the pharmaceutical compositions of the present invention disclosed herein. Therefore, for example, it may be beneficial to determine whether the cells of a cancer disclosed herein comprise or consist of cells hemizygous for deletion of TP53 and / or POLR2A, since the desired therapeutic effect may be achieved using at least 10, 25, 50, 100, 250, 500, or 1000 times less of the conjugates, pharmaceutical compositions, or compositions of the present invention disclosed herein. Assays for assessing the susceptibility of the cancer cells or tumors disclosed above to treatment with pharmaceutical compositions of the present invention, including the conjugates disclosed herein, can be performed, for example, as described in Nature. 2015 April 30; 520 (7549): 697-701.

[0128] The pharmaceutical compositions for use according to the present invention disclosed herein are preferably liquid, and preferably in a dosage form adapted for subcutaneous administration to a patient suffering from cancer in need thereof. According to some embodiments, the pharmaceutical compositions for use according to the present invention disclosed herein further comprise one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives.

[0129] For example, a pharmaceutical composition for use according to the present invention may comprise at least one buffering agent to achieve a pH of 4 to 8, preferably 5 to 7, more preferably about 5.5 to about 6.5, or, for example, a pH of about 5, 5.5, 6, 6.5. The corresponding buffer may preferably be one or more selected from the group consisting of malate, formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carboxylate, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), imidazole, BIS-TRIS propane, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)propanesulfonic acid) (MOPS), hydroxyethylpiperazineethanesulfonic acid (HEPES), pyrophosphate, and triethanolamine, and more preferably a histidine buffer, such as, but not limited to, L-histidine / HCl. The concentration of the at least one buffering agent may range from 0.1 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM to about 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, preferably 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 25 mM, 30 mM, 40 mM, 50 mM, more preferably about 5 mM, 10 mM, 25 mM to about 50 mM.

[0130] For example, the pharmaceutical compositions of the present invention disclosed herein may also contain, for example, one or more stabilizers. The stabilizers used in the pharmaceutical compositions of the present invention may be any stabilizer commonly used in the art for stabilizing proteins, without limitation. Preferably, the stabilizer may be, for example, one or more selected from the group consisting of carbohydrates, sugars or hydrates thereof, sugar alcohols or hydrates thereof, and amino acids. For example, the carbohydrates, sugars, or sugar alcohols used as stabilizers may be, but are not limited to, one or more selected from the group consisting of trehalose or hydrates thereof, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, polyglycitol, cyclodextrin, hydroxypropylcyclodextrin, and glucose. The sugar or sugar alcohol used as a stabilizer may be present in the pharmaceutical compositions of the present invention disclosed herein at a concentration of about 0.1 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 75 mM, 100 mM to about 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 500 mM, preferably about 10 mM, 25 mM, 50 mM, 100 mM, 125 mM to 150 mM, 200 mM, 250 mM, or, for example, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM. When present in a pharmaceutical composition of the present invention, the amino acid may be one or more selected from the group consisting of, but not limited to, glutamine, glutamic acid, glycine, lysine, lysilysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine, and alanine.The amino acids used as stabilizers in the pharmaceutical compositions according to the present invention may be at concentrations of, for example, about 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, 25 mM, 50 mM to about 75 mM, 100 mM, preferably about 5 mM, 7.5 mM, 10 mM, 25 mM to about 30 mM, 40 mM, 50 mM.

[0131] For example, pharmaceutical compositions according to the invention may contain polyoxyethylene-sorbitan fatty acid esters (polysorbates or Tweens), polyethylene-polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ethers [Triton-X], and polyoxyethylene-polyoxypropylene copolymers [poloxamers and Pluronics], and sodium dodecyl sulfate (SDS), polysorbate 20 or polysorbate 80, polyethylene glycol hexadecyl ether (Brij® 56); polyethylene glycol octadecyl ether (Brij® 72); polyoxyethylene 10 oleyl ether (Brij® 97); poloxamer 188, t-octyl It may further contain a non-ionic surfactant such as phenoxypolyethoxyethanol (TRITON® X100); polyethylene glycol sorbitan monolaurate (TWEEN® 20); polyoxyethylene sorbitan monopalmitate (TWEEN® 40); polyethylene glycol sorbitan monostearate (TWEEN® 60); polyoxyethylene sorbitan tristearate (TWEEN® 65); polyethylene glycol sorbitan monooleate (TWEEN® 80); polyoxyethylene sorbitan trioleate (TWEEN® 85); tris(hydroxymethyl)aminomethane lauryl sulfate (TRIZMA® dodecyl sulfate); block copolymer of polyethylene and polypropylene glycol (Pluronic® F68). The nonionic surfactants disclosed above may be present in the pharmaceutical compositions according to the present invention in an amount ranging from about 0.01 percent (w / v) to about 0.5% (w / v), preferably from about 0.1% (w / v), 0.2% (w / v), 0.3% (w / v) to about 0.4% (w / v), and more preferably from about 0.1% (w / v) to 0.25% (w / v), 0.35% (w / v), or 0.5% (w / v), where the expression "(w / v)" refers to weight per volume.

[0132] For example, pharmaceutical compositions for use according to the invention disclosed herein may also include a diluent selected from the group including, for example, mannitol, microcrystalline cellulose, lactose, starch, dibasic anhydrous calcium phosphate, tribasic calcium phosphate, kaolin, sucrose, precipitated calcium carbonate, sorbitol, maltodextrin, powdered cellulose, microcrystalline cellulose, and other materials known for such properties. The diluent may be present in a pharmaceutical composition for use according to the present invention at a concentration of about 0.1% (w / v), 0.25% (w / v), 0.5% (w / v), 1% (w / v), 2.5% (w / v), 5% (w / v), 7.5% (w / v), 10% (w / v), 12.5% ​​(w / v), 15% (w / v), 20% (w / v), 25% (w / v), 30% (w / v) to about 35% (w / v), 40% (w / v), It may be present at 45% (w / v), 50% (w / v), or about 35% (w / v), 40% (w / v), 50% to about 60% (w / v), 75% (w / v), or about 0.5% (w / v), 1% (w / v), 2.5% (w / v) to about 7.5% (w / v), 10% (w / v), 12.5% ​​(w / v), 15% (w / v), 20% (w / v), 25% (w / v), 30% (w / v).

[0133] For example, pharmaceutical compositions for use according to the invention disclosed herein may include a lubricant selected from the group including stearic acid, sodium stearyl fumarate, polyethylene glycol, magnesium stearate, calcium stearate, talc, zinc stearate, hydrogenated castor oil, silica, colloidal silica, corn starch, calcium silicate, magnesium silicate, silicon hydrogel, and other materials known for such properties. The lubricant may be present in a pharmaceutical composition for use according to the present invention at, for example, about 0% (w / v), 0.1% (w / v), 0.25% (w / v), 0.5% (w / v), 0.75% (w / v), 1% (w / v) to about 1.25% (w / v), 1.5% (w / v), 1.75% (w / v), 2% (w / v), 2.5% (w / v), 3.0% (w / v), or about 1.25% (w / v), 1.5% (w / v), 1.75% (w / v), 2% (w / v), 2.5% (w / v) to about 3% (w / v), 3.5% (w / v), 4% (w / v), 5% (w / v).

[0134] For example, pharmaceutical compositions for use according to the invention disclosed herein may include a binder selected from the group including polyvinylpyrrolidone, hydroxypropyl methylcellulose, gum arabic, alginic acid, hydroxypropyl cellulose, sodium carboxymethylcellulose, compressible sugar, ethyl cellulose, gelatin, liquid glucose, methyl cellulose, pregelatinized starch, and other materials known to those skilled in the art. The binder in the dosage form ranges from 0% to 5.0% by weight.

[0135] For example, pharmaceutical compositions for use according to the invention disclosed herein may include a glidant selected from the group including colloidal silicon dioxide, colloidal silica, corn starch, talc, calcium silicate, magnesium silicate, colloidal silicon, or silicon hydrogel. The glidant may be present in the pharmaceutical composition in an amount ranging from about 0% (w / v), 0.1% (w / v), 0.25% (w / v), 0.5% (w / v), 0.75% (w / v), 1% (w / v) to about 1.25% (w / v), 1.5% (w / v), 1.75% (w / v), 2.0% by weight, or in a range from about 1.25% (w / v), 1.5% (w / v), 1.75% (w / v) to about 2% (w / v).

[0136] The pharmaceutical compositions of the present invention may further comprise, for example, a preservative. A "preservative" according to the present invention is a compound that can be added to the pharmaceutical compositions disclosed herein to inhibit bacterial growth. For example, the addition of a preservative facilitates the preparation of multi-use or multi-dose pharmaceutical compositions of the present invention. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols (such as phenol), butyl and benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol at a concentration of about 0.9% (w / v) to about 2.0% (w / v), e.g., 0.9% (w / v), 1% (w / v), 1.25% (w / v), 1.5% (w / v), 1.75% (w / v), or 2% (w / v).

[0137] Typically, the subcutaneous injection volume of the pharmaceutical composition of the present invention is 3 ml or less, or 2 ml or less, because it is desirable to use a pharmaceutical composition in which the concentration of the conjugate of the present invention to be administered is high. Therefore, the conjugate of the present invention disclosed herein can be administered at a concentration of about 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml to about 50 mg / ml, 60 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 175 mg / ml, or 200 mg / ml, or at a concentration of about 50 mg / ml, 60 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml to about 160 mg / ml, 180 mg / ml, or 200 mg / ml. It may be desirable to use a pharmaceutical composition according to the present invention containing the compound at a concentration of about 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 350 mg / ml, 400 mg / ml, 450 mg / ml, 500 mg / ml, or at a concentration of about 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 75 mg / ml, or 100 mg / ml, preferably at a concentration of about 25 mg / ml.

[0138] According to some embodiments, the pharmaceutical compositions of the present invention for use in treating cancer disclosed herein are injected in a volume of 3 ml or less, 2.5 ml or less, 2.0 ml or less, or 1.5 ml or less, with an amount of the complex of the present invention selected from 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg (e.g., fixed-dose administration). The effective amount of the complex of the present invention to be injected, and accordingly, the amount and composition of the pharmaceutical composition of the present invention to be injected, may depend on the type of cancer or tumor, the stage of the cancer, the overall health of the patient being treated, and the patient's weight and size. Fixed-dose subcutaneous administration of the pharmaceutical compositions of the present invention may be desirable, for example, when intended as a combination treatment with standard therapy. Such combination therapy may be desirable, for example, to reduce the risk of adverse reactions to a higher dose of standard therapy or a corresponding monotherapy using subcutaneous administration of the pharmaceutical composition of the present invention, (iii) may, for example, lower overall costs, and (iv) may improve patient compliance.

[0139] In one embodiment, the viscosity of the pharmaceutical composition of the present invention is about 5 cP, 10 cP, 15 cP, 20 cP, 30 cP, 40 cP, 50 cP, 60 cP to about 70 cP, 80 cP, 90 cP, 100 cP, or about 10 cP, 15 cP, 20 cP to about 30 cP, 40 cP, 50 cP, 60 cP, preferably 5 cP, 10 cP to about 25 cP, 30 cP. The term "viscosity" as used herein may also refer to "kinematic viscosity" or "absolute viscosity." Kinematic viscosity is a measure of the resistance flow of a fluid under the influence of gravity. When two fluids of the same volume are placed in the same capillary viscometer and allowed to flow by gravity, the more viscous fluid will take longer to flow through the capillary than the less viscous fluid. If one fluid takes 100 seconds to complete its flow and the other takes 200 seconds, then the viscosity of the second fluid is twice that of the first on the kinematic viscosity scale. "Absolute viscosity", sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density: Absolute viscosity = kinematic viscosity x density

[0140] The dimension of kinematic viscosity is L 2 / T, where L is length and T is time. Kinematic viscosity is generally expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is millipascal-second (mPa-s), where 1 cP = 1 mPa-s. The viscosity of the pharmaceutical composition of the present invention can be determined, for example, using a commercially available glass capillary viscometer or by the method disclosed in WO2016027859A1 (the contents of which are incorporated herein by reference).

[0141] Pharmaceutical compositions comprising antibodies or immunoconjugates (such as conjugates of the invention) at concentrations high enough to allow subcutaneous administration of an effective amount of the conjugates of the invention may have a viscosity greater than 20 cP, 30 cP, 40 cP, 50 cP, etc. Thus, it may be advantageous to include one or more viscosity-lowering agents in the pharmaceutical compositions of the invention disclosed herein.

[0142] Therefore, the pharmaceutical compositions of the present invention disclosed herein may optionally further comprise a viscosity-lowering agent. For example, the pharmaceutical compositions of the present invention may comprise 1-butyl-3-methylimidazolium methanesulfonate (BMI Mes) and, optionally, one or more additional ionic liquids in an effective amount to significantly reduce viscosity. Representative ionic liquids include 4-(3-butyl-1-imidazolio)-1-butane methanesulfonate (BIM), 1-butyl-3-methylimidazolium methanesulfonate (BMI Mes), 4-ethyl-4-methylmorpholinium methylcarboxylate (EMMC), and 1-butyl-1-methylpyrrolidinium chloride (BMP Chloride), preferably at a concentration of about 0.10 to about 0.50 M, equivalent to about 20 to 150 mg / mL, as disclosed in EP 3043774 A1. Other viscosity-lowering agents, such as those disclosed in US Pat. No. 9,605,051 B2, may also be used.

[0143] In some embodiments, pharmaceutical compositions provided herein further comprise a recombinantly produced human hyaluronidase, such as ruHuPH20 (HYLENEX®). The use of such recombinant hyaluronidase in pharmaceutical compositions provided herein can be advantageous, for example, when the pharmaceutical compositions of the invention need to be administered subcutaneously to patients requiring larger injection volumes to administer a therapeutically effective amount of a conjugate of the invention, or when it is desirable to reduce the injection pressure required to achieve subcutaneous injection.

[0144] Thus, according to one embodiment, the pharmaceutical compositions of the invention for the uses disclosed herein may further comprise a soluble recombinant hyaluronidase, preferably a soluble recombinant human hyaluronidase, more preferably a soluble recombinant human PH20. As used herein, "soluble recombinant human PH20 (rHuPH20)" refers to a composition comprising a soluble form of human PH20 recombinantly expressed in and secreted from Chinese hamster ovary (CHO) cells, such as disclosed in WO 2004 / 078140 A2, or preferably a mutant form of rHuPH20, such as disclosed in WO 2013 / 102144 A2. The contents of these two applications are incorporated herein by reference.

[0145] As used herein, the term "hyaluronidase" or "hyaluronidase activity" refers to hyaluronidases, a family of enzymes that catalyze the degradation of hyaluronic acid (HA). There are three major types of hyaluronidases: two classes of eukaryotic endoglycosidase hydrolases and prokaryotic lyase-type glycosidases. In humans, there are five functional hyaluronidases: HYAL1, HYAL2, HYAL3, HYAL4, and HYAL5 (also known as SPAM1 or PH-20), as well as the pseudogene HYAL6 (also known as HYALP1). The genes for HYAL1-3 are clustered on chromosome 3, and HYAL4-6 are clustered on chromosome 7. HYAL1 and HYAL2 are the major hyaluronidases in most tissues. GPI-anchored HYAL2 is responsible for cleaving high-molecular-weight HA, most of which is bound to the CD44 receptor. The resulting HA fragments of various sizes are then internalized into endolysosomes and further hydrolyzed by HYAL1 to generate HA oligosaccharides.

[0146] According to their enzymatic reaction mechanism, hyaluronidases are hyaluronoglucosidases (EC 3.2.1.35). That is, they cleave the (1->4) bond between N-acetylglucosamine and glucuronate. The term "hyaluronidase" can also refer to hyaluronoglucuronidases (EC 3.2.1.36), which cleave the (1->3) bond. Bacterial hyaluronate lyases (EC 4.2.2.1) are also sometimes referred to as hyaluronidases, although this is uncommon.

[0147] Mammalian hyaluronidases (EC 3.2.1.35) are endo-β-N-acetylhexosaminidases that produce tetra- and hexasaccharides as their major end products. They possess both hydrolytic and transglycosidase activities and can degrade hyaluronan and chondroitin sulfates (CS), generally C4-S and C6-S.

[0148] Mammalian hyaluronidases can be further divided into two groups: neutral-active and acid-active enzymes. There are six hyaluronidase-like genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, and PH20 / SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown to have enzymatic activity toward any known substrates. HYAL4 is a chondroitinase and shows little activity toward hyaluronan. HYAL1 is the prototypical acid-active enzyme, and PH20 is the prototypical neutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 and HYAL2, generally lose catalytic activity at neutral pH (i.e., pH 7). For example, HYAL1 has little in vitro catalytic activity above pH 4.5 (Frost et al., Anal. Biochemistry, 1997). HYAL2 is an acid-active enzyme with very low in vitro specific activity. Recombinant human hyaluronidase PH20 (rHuPH20) has been found to have optimal reaction rates in the pH range of 4.5 to 5.5 for rHuPH20 and HA substrates ranging in size from 90 to 752 kDa according to Michaelis-Menten kinetics during the initial reaction time (see, e.g., Anal. Biochem. 2015 Jul. 1;480:74-81). Therefore, the pH of a pharmaceutical composition for use according to the present invention further comprising rHuPH20 should be selected so that the activity of rHuPH20 is essentially optimal without adversely affecting the stability of the complexes in the pharmaceutical compositions for use according to the present invention disclosed herein. Thus, the pH of a pharmaceutical composition for use according to the present invention may be, for example, about pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0 to about pH 5.5, pH 6.0, pH 6.2, pH 6.5, or, for example, about pH 5.5, pH 6.0 to about pH 6.5, pH 6.7, pH 7.0.If the pH of the pharmaceutical compositions of the invention requires a pH that results in a hyaluronidase activity that corresponds to less than 90%, 80%, 70%, 60%, 50%, or 40% of the activity of rHuPH20 at pH 4.5 to 5.5, the amount of rHuPH20 in the pharmaceutical compositions for use according to the invention may be increased to compensate for the decrease in activity. The hyaluronidase activity of rHuPH20 can be determined, for example, as disclosed in WO 2013 / 102144 or Anal Biochem. 2015 Jul 1;480:74-81.

[0149] Pharmaceutical compositions for use according to the present invention may contain, for example, about 0.1 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 75 μg / ml to about 80 μg / ml, 90 μg / ml to 100 μg / ml, or about 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml to about 30 μg / ml, 35 μg / ml, 40 μg / ml, or 50 μg / ml of rHuPH20.

[0150] For example, pharmaceutical compositions for use according to the present invention may contain, for example, about 50 U / ml, 100 U / ml, 150 U / ml, 200 U / ml, 250 U / ml, 300 U / ml, 400 U / ml, 500 U / ml, 600 U / ml, 750 U / ml, 800 U / ml, 900 U / ml, 100 U / ml to about 1250 U / ml, 1500 U / ml, 1750 U / ml, 2000 U / ml, 2500 U / ml, 3000 U / ml, 35 The hyaluronidase activity may be from about 1250 U / ml, 1500 U / ml, 1750 U / ml, 2000 U / ml, 2500 U / ml, 3000 U / ml, 3500 U / ml, 3750 U / ml, 4000 U / ml, 4500 U / ml, 5000 U / ml, or from about 1250 U / ml, 1500 U / ml, 1750 U / ml, 2000 U / ml, 2500 U / ml, 3000 U / ml, 3500 U / ml, 3750 U / ml, 4000 U / ml to about 5000 U / ml, 7500 U / ml, 10,000 U / ml.

[0151] In some embodiments, pharmaceutical compositions for use according to the present invention may comprise a mixture of a conjugate of the present invention in an amount disclosed herein, preferably a therapeutically effective amount, and rHuPH20 in an amount disclosed above. Alternatively, pharmaceutical compositions for use according to the present invention may comprise a first component and a second component, wherein the first component comprises hyaluronidase rHuPH20 in an amount disclosed above, and the second component comprises a conjugate of the present invention as disclosed herein. The first and second components of the pharmaceutical composition may be administered, for example, simultaneously or sequentially. However, it is preferred that the first and second components of the pharmaceutical composition be administered sequentially, with the first component administered before the second component. Thus, the second component may be administered within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 40 hours, or 48 hours after administration of the first component, and preferably within 5 minutes, 15 minutes, 30 minutes to 1 hour after administration of the first component. The first and second components of the pharmaceutical composition of the present invention may be subcutaneously administered at the same injection site, or may be subcutaneously administered at injection sites 0.5 cm, 1 cm, 1.5 cm, 2 cm, or 2.5 cm apart from each other.

[0152] In one embodiment, the subcutaneous injection volume of a pharmaceutical composition for use according to the invention disclosed herein comprising rHuPH20 disclosed herein may be, for example, about 0.5 ml, 1 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml, 4.5 ml, 5 ml to about 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, or about 6 ml, 7 ml, 8 ml, 9 ml to about 10 ml, or, for example, 0.75 ml, 1 ml, 1.25 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml, 4.5 ml, 5 ml, 5.5 ml, 6 ml, 6.5 ml, 7 ml, 7.5 ml, or 10 ml.

[0153] In some embodiments, the pharmaceutical composition for use according to the present invention is co-administered with an immune checkpoint inhibitor. In the context of the present invention, the term "immune checkpoint inhibitor," or simply "checkpoint inhibitor" or "ICI," refers to any agent or compound that directly or indirectly reduces the amount or inhibits the function of an immune checkpoint receptor protein or molecule found on the surface of immune cells (e.g., T cells), or any agent or compound that directly or indirectly reduces the amount or inhibits the function of a ligand that binds to the immune checkpoint receptor protein or molecule as a soluble compound or on the surface of an immune cell inhibitory cell. Such inhibitory cells may be, for example, cancer cells, regulatory T cells, tolerogenic antigen-presenting cells, myeloid-derived suppressor cells, tumor-associated macrophages, or cancer-associated fibroblasts. The ligand typically binds to an immune checkpoint receptor protein or molecule on an immune cell. Non-limiting examples of immune checkpoint receptor protein-ligand pairs include PD-1 and PD-L1. PD-1 is an immune checkpoint receptor protein found on T cells. PD-L1, which can be overexpressed by cancer cells, binds to PD-1 and helps the cancer cells evade attack by the host immune system. Therefore, immune checkpoint inhibitors prevent PD-1 / PD-L1 interaction by blocking PD-1 on T cells (i.e., acting as PD-I inhibitors) or PD-L1 on cancer cells (i.e., acting as PD-L1 inhibitors), thereby maintaining or restoring anti-tumor T cell activity or blocking inhibitory cancer cell activity.

[0154] Thus, immune checkpoint inhibitors are antagonists of immune inhibitory receptors such as PD-1 (in this case, they inhibit PD-1 or PD-L1 in the PD-1 / PD-L1 pathway). Examples of PD-1 or PD-L1 inhibitors include pembrolizumab, pidilizumab, cemiplimab, JTX-4014, spartalizumab, sintilimab (IBI308), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, BCD-100, AGEN-2034, and toripalimab. These include, but are not limited to, humanized or human antibodies that antagonize or block human PD-1 function, such as nivolumab (TAB001, JS001), or AMP-514 (MEDI0680), or fully human antibodies that block PD-1, such as avelumab, durvalumab, cosibelimab (CK-301), WBP-3155 (CS1001), atezolizumab, embafolimab (KN035), or the recombinant anti-PD-L1 probody CX-072 (pakmilimab).

[0155] Pembrolizumab (formerly known as lambrolizumab, trade name Keytruda, also known as MK-3475), as disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, is a humanized IgG4 monoclonal antibody that binds to PD-1 and contains a C228P mutation designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US8,354,509 and WO2009 / 114335. Pembrolizumab has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0156] Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cellular cytotoxicity (ADCC). Nivolumab is disclosed, for example, in US8,008,449 and WO2006 / 121168. Nivolumab has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0157] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, for example, in WO2009 / 101611.

[0158] PD1-1 to PD1-5 refer to the anti-PD-1 antibodies disclosed in WO2018 / 220169.

[0159] Ipilimumab (CAS Registry Number: 477202-00-9, 10D1, or sometimes referred to as MDX010 or MDX-101) is a human IgG1 antibody that binds to cytotoxic T-lymphocyte antigen-4 (CTLA4). CTLA-4 is an inhibitory molecule that competes with stimulatory CD28 for binding to B7 on antigen-presenting cells. Both CTLA-4 and CD28 are presented on the surface of T cells. Ipilimumab is a human IgG1 that binds to CTLA-4 and prevents the inhibition of T cell-mediated immune responses against tumors. Ipilimumab is disclosed, for example, as antibody "10D1" in WO01 / 14424.

[0160] Embafolimab (CAS Registry Number: 2102192-68-5) is a subcutaneously (SC) administered single-domain anti-programmed death-ligand 1 (PD-L1) antibody disclosed in US 11,377,497 B2.

[0161] As used herein, the INN designations for antibodies disclosed herein are meant to encompass all biosimilar antibodies of the corresponding original antibody disclosed herein, including, but not limited to, biosimilar antibodies authorized under 42 USC §262(k) in the United States and equivalent legislation in other jurisdictions. As used herein, the term "biosimilar" refers to an antibody that contains an identical amino acid sequence compared to the antibody originally identified by the INN, although the biosimilar antibody may differ in glycosylation.

[0162] The pharmaceutical compositions for use disclosed herein may be co-administered with an immune checkpoint inhibitor, for example, in a patient suffering from a solid tumor, wherein the solid tumor is selected from the list of solid tumors disclosed herein.

[0163] The pharmaceutical compositions for use according to the present invention disclosed herein may be co-administered with, for example, the pharmaceutical compositions of the present invention administered subcutaneously. For example, the pharmaceutical compositions for use according to the present invention may be administered, for example, before, simultaneously with, or after the administration of an immune checkpoint inhibitor. When administered before the administration of an immune checkpoint inhibitor, the pharmaceutical compositions disclosed herein may be subcutaneously administered 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, or 48 hours before the administration of the immune checkpoint inhibitor. Alternatively, the immune checkpoint inhibitor disclosed herein may be administered, for example, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, or 48 hours before the administration of the pharmaceutical compositions of the present invention. The pharmaceutical composition for use according to the invention and the immune checkpoint inhibitor disclosed herein may also, in one alternative, be administered simultaneously, e.g., 1, 2, 3, 5, 10, 15 minutes apart from each other. Corresponding combinations of conjugates according to the invention and immune checkpoint inhibitors are disclosed, for example, in WO2022 / 096604A1, the entire contents of which are incorporated herein by reference.

[0164] In one embodiment, the pharmaceutical composition for use in treating cancer according to the present invention is administered subcutaneously for the treatment of any of the above cancers and tumor types. For example, the pharmaceutical composition may be subcutaneously injected using a suitable means for subcutaneous injection, such as the syringe or auto-injector disclosed above. The pharmaceutical composition may be subcutaneously injected, for example, into the side or back of the upper arm, the abdomen, or the front of the patient's thigh, depending on the volume to be injected and taking into account the overall health of the patient requiring cancer treatment.

[0165] According to one embodiment, the pharmaceutical composition for use in treating cancer is administered subcutaneously at least once (e.g., in at least one dose) to a patient diagnosed with at least one type of cancer disclosed herein.

[0166] According to one embodiment, the antibody of the conjugate of the invention specifically binds to a cell surface antigen (such as a tumor-specific antigen) on a tumor cell or a tumor-associated antigen. As used herein, the term "specifically binds" or any grammatical variation thereof means that the antibody specifically binds to an antigen with at least about 10 -6 M, 10 -7 M, 10 -8 M, or about 10 -8 M ~ about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or approximately 5 x 10 -9 M, 5 x 10 -10 M ~ approx. 2.5×10 -11 M, 5 x 10 -11 M, 2.5 x 10 -12 M, 5 x 10 -12 K of M d The term "tumor-specific antigen" refers to the binding of a targeting moiety of the present invention (such as an antibody or antibody portion of the conjugates disclosed herein) with a targeting moiety of the present invention (such as an antibody or antibody portion of the conjugates disclosed herein). As used herein, a tumor-specific antigen is an antigenic substance produced in tumor cells, i.e., that induces an immune response in the host. A tumor antigen is a tumor marker that is useful in identifying tumor cells in diagnostic tests. A tumor antigen can be, for example, a glycan, protein, or peptide that is expressed on the surface of cancer cells but not on healthy tissues or cells. As used herein, the term "tumor-specific antigen" (TSA) can be, for example, a glycan, protein, or peptide that is present only on tumor cells and not on any other cells. The term "tumor-associated antigen" (TAA) refers to a glycan, protein, or peptide that is present on some tumor cells and also on some normal, non-cancerous cells.

[0167] According to one embodiment, the conjugate of the present invention contained in the pharmaceutical composition for use in treating cancer disclosed herein may target any suitable epitope on a target cell, such as an epitope on a cancer cell, where the cancer may be, for example, a cancer cell of any of the cancers disclosed herein. As used herein, the term "epitope" refers to a portion of a macromolecule, preferably a polypeptide recognized by an antigen-binding molecule, such as an antibody of the present invention disclosed herein, or an antigen-binding fragment or derivative thereof, particularly by the antigen-binding site of the molecule. An epitope defines the minimal binding site for an antibody molecule and thus represents the specific target of the antibody molecule. An epitope can be further defined as a structural epitope or a functional epitope. A "structural epitope" consists of amino acids or other molecules in the region that is in close contact with the antibody, usually as revealed by its structure. A "functional epitope" is defined as a portion of a molecule that energetically contributes to binding such that alterations reduce binding affinity. Therefore, whether proximal or not, residues that contact the paratope and which residues contribute to affinity are important considerations when defining an epitope. A structural epitope may be, for example, a linear contiguous sequence of about 5 to about 50 or 100 amino acids in length, or may be a conformational epitope formed by the three-dimensional structure of a polypeptide and may include discontinuous amino acids of the polypeptide. Epitopes that can be specifically bound by antibodies of the complexes of the present invention also include, for example, non-proteinaceous structures on cancer cells (e.g., Lewis antigens (sialyl Lewis x (SLe))). x ) and sialyl Lewis a (SLe a )) and the like).

[0168] According to some embodiments, the antibody of the conjugate of the invention is selected from the group consisting of EpCAM, HER2 / neu, EGFR (HER1, ErbB1), TROP-2, BCMA, CD37, STEAP1, FXYD3, CA125, CD30, NCAM (CD56), MUC1, CEA (CD66e), VEGF, AFP, AXL, TYRO3, MER, CD20, CD19, CD52, CD268, CD28, CD80, CD22, CD4, CD2, CD33, CD30, CD38, CD52, CD80, CD140b, PSMA, TYR, FCRL2, MUC17, GPR143, NMNAT2, MAGE, MAGEC2, MAG E-A3, MART-1, WT-1, EPHA2, KRT19, CLDN7, DKK1, FGF19, SCN3A, SCN2A, GAS1, S100Z, GAPT, GPR35, NY-ESO, cadherin 24, DLK1, GPR173, ALK, GFRA3, GUCY2C, DLL3, PSMA, PROX1, PSCA, glypican-1, mesothelin (prostate stem cell antigen), GAGE-1 (G antigen 1), ganglioside / GD2, GnT-V, β1,6-N (acetylglucosaminyltransferase-V), UPAR (urokinase-type plasminogen activator receptor), sialyl Lewis x (SLe x ), and sialyl Lewis a (SLe a The antibody specifically binds to a cell surface antigen (such as a tumor-specific antigen) or a tumor-associated antigen on a tumor cell selected from the group consisting of:

[0169] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate according to Formula (I) as disclosed in WO2018 / 115466A1, wherein the antibody "J22.9-ISY-D265C" refers to an anti-BCMA antibody comprising a heavy chain amino acid sequence according to SEQ ID NO: 1 and a light chain amino acid sequence according to SEQ ID NO: 2 (SEQ ID NOs: 11, 12 as disclosed herein) as disclosed in WO2018 / 115466 (the contents of which are incorporated herein by reference). [ka] wherein said cancer is selected from the group comprising multiple myeloma, diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL), in particular multiple myeloma.

[0170] According to one preferred embodiment, the pharmaceutical compositions for use in treating cancer disclosed herein are administered subcutaneously to a patient suffering from relapsed / refractory multiple myeloma, wherein the patient is characterized by the following criteria: · Male or female, aged 18 or older. Life expectancy >12 weeks. Eastern Cooperative Oncology Group performance status (PS) of 0-2. ·Confirmed diagnosis of active MM according to the diagnostic criteria established by the International Myeloma Working Group (IMWG). · Must have undergone SCT or be considered transplant ineligible. Patients must have received prior anti-myeloma therapy, which must include immunomodulatory drugs, proteasome inhibitors, and anti-CD38 therapy, alone or in combination. Patients must also be resistant or intolerant to any established standard of care that provides meaningful clinical benefit to the patient as assessed by the investigator. · Measurable disease by IMWG criteria.

[0171] Thus, the pharmaceutical compositions of the present invention for use in treating relapsed / refractory multiple myeloma are administered to patients as disclosed herein.

[0172] Accordingly, the present invention relates to a method of treating a patient suffering from relapsed / refractory multiple myeloma, said patient being characterized by the above criteria.

[0173] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C, which comprises a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12, as disclosed in WO2022 / 194988A2 (the contents of which are incorporated herein by reference), wherein the conjugate is a compound represented by formula (XII), (X III), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII), preferably (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XX), or (XXII), more preferably (XII), (XIII), (XIV), (XVII), or (XX), particularly preferably (XII), (XIII), or (XIV).

[0174] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C, which comprises a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12 disclosed in WO2022 / 194988A2 (SEQ ID NOs: 13 and 14 disclosed herein), wherein the conjugate comprises an amatoxin-linker (XII).

[0175] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C, which comprises a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12 disclosed in WO2022 / 194988A2 (SEQ ID NOs: 13 and 14 disclosed herein), wherein the conjugate comprises an amatoxin-linker (XIII).

[0176] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C, which comprises a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12 disclosed in WO2022 / 194988A2 (SEQ ID NOs: 13 and 14 disclosed herein), wherein the conjugate comprises an amatoxin-linker (XIV).

[0177] According to a more preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C, which comprises a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12 (SEQ ID NOs: 13 and 14 disclosed herein) as disclosed in WO2022 / 194988A2, wherein the conjugate comprises an amatoxin-linker (XII), and the cancer is non-Hodgkin's lymphoma (NHL), filtrate, or the like. and the cancer is selected from the group comprising follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma (DBNHL), subtypes of non-Hodgkin's lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter's syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis, and pemphigus vulgaris, wherein the pharmaceutical composition is administered subcutaneously and may, for example, be formulated as disclosed herein.

[0178] According to a more preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C comprising a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12 disclosed in WO2022 / 194988A2 (SEQ ID NOs: 13 and 14 disclosed herein), wherein the conjugate comprises an amatoxin-linker (XIII), The cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma (DBNHL), subtypes of non-Hodgkin's lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter's syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis, and pemphigus vulgaris.

[0179] According to a more preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-CD37 antibody chHH1-HDPLALA-D266C, which comprises a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12 disclosed in WO2022 / 194988A2 (SEQ ID NOs: 13 and 14 disclosed herein), wherein the conjugate comprises an amatoxin-linker (XIV); The cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma (DBNHL), subtypes of non-Hodgkin's lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter's syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis, and pemphigus vulgaris.

[0180] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises 3-F11-var1, 3-F11-var2, 3-F11-var3, 3-F11-var4, 3-F11-var5, 3-F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11-var14, 3-F11-var15, 3-F11-var16, 3-F11-var17, 3-F11-var18, 3-F11-var19 ... and conjugates comprising an anti-PSMA antibody disclosed in WO2020 / 025564 (the contents of which are incorporated herein by reference) selected from the group including 3-F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11-var14, 3-F11-var115, or 3-F11-var16, but most preferably the antibody is 3-F11-var1, 3-F11-var13, or 3-F11-var16 linked to an amatoxin-linker moiety according to any of formulas (XII), (XIII), (XIV), (XVII), or (XX).

[0181] [Table 1]

[0182] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var1 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XII), wherein the anti-PSMA antibody 3-F11-var1 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 8.

[0183] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var1 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XIII), wherein the anti-PSMA antibody 3-F11-var1 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 8.

[0184] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var1 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XIV), wherein the anti-PSMA antibody 3-F11-var1 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 8.

[0185] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var13 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XII), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 10.

[0186] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var13 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XIII), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 10.

[0187] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var16 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XIV), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 10.

[0188] According to a preferred embodiment, the pharmaceutical composition for treating cancer disclosed herein is administered subcutaneously and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var16 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XII), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NOs: 7 and 10.

[0189] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var16 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XIII), wherein the anti-PSMA antibody 3-F11-var16 comprises the VH and VL sequences according to SEQ ID NOs: 9 and 10.

[0190] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is for subcutaneous administration and comprises a conjugate comprising the anti-PSMA antibody 3-F11-var16 disclosed in WO2020 / 025564 linked to an amatoxin linker moiety (XIV), wherein the anti-PSMA antibody 3-F11-var16 comprises the VH and VL sequences according to SEQ ID NOs: 9 and 10.

[0191] According to a preferred embodiment, the pharmaceutical composition for use in treating cancer disclosed herein is administered subcutaneously and comprises a compound represented by formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII) disclosed herein, preferably (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XX), or (XXII), more preferably ( (XII), (XIII), (XIV), (XVII), (XX), most preferably antibodies mAb1, mAb8, mAb41 containing the mutations L234A, L235A, and D265C (numbering according to the EU numbering system) linked to an amatoxin-linker moiety according to (XII) or (XIV).

[0192] [Table 2]

[0193] The term "GCC" refers to guanylyl cyclase C (GUCY2C, EC: 4.6.1.2), preferably human GCC, a member of the receptor enzyme protein family that synthesizes guanosine 3',5'-cyclic monophosphate (cyclic GMP; cGMP). GUCY2C is a transmembrane receptor for the endogenous hormone ligands guanylin and uroguanylin. Ligand binding to the extracellular receptor catalyzes the conversion of GTP to cyclic GMP (cGMP), initiating downstream cGMP-related signaling pathways involved in regulating intestinal homeostatic functions such as epithelial cell proliferation, differentiation, and apoptosis.

[0194] According to one embodiment, the pharmaceutical compositions for use in treating cancer according to the invention disclosed herein are administered to the patient multiple times, for example, two or three times, or periodically, for example, every 30, 60, 90, 120, 180, or 360 days, or about every 21, 28 to about 35, 36, 37, 38, 39, 40, 41, or 42 days, or every 22, 23, 24, 25, 26, or 27 days.

[0195] According to one embodiment, the pharmaceutical composition for use in treating cancer according to the present invention disclosed herein increases the maximum tolerated dose (MTD) of the pharmaceutical composition when administered intravenously by at least 30%, 40%, or 50% compared to the MTD. As used herein, the term "maximum tolerated dose" (MTD) refers to the highest tolerated dose of a drug (such as the conjugate of the present invention disclosed herein) that can be administered to an animal without causing significant toxicity or mortality. Thus, the pharmaceutical composition for use in treating cancer according to the present invention is characterized by a therapeutic index (TI) greater than that of a corresponding pharmaceutical composition for use in treating cancer administered to a mammal (e.g., a patient suffering from cancer) via an intravenous (iv) route. As used herein, the term "therapeutic index" refers to the ratio of the blood concentration at which a drug becomes toxic to the concentration at which the drug is effective. The higher the therapeutic index (TI), the safer the drug. TI can be calculated, for example, as TI = MTD / MED, where "MED" refers to the minimum effective dose, which is the lower limit of the therapeutically effective dose.

[0196] According to one embodiment, the present invention relates to the use of a conjugate comprising an amatoxin linker moiety according to any of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), or (XI), or a conjugate according to any of formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII) in the manufacture of a pharmaceutical composition for use in the treatment of cancer according to the methods disclosed herein, to be administered subcutaneously.

[0197] According to one embodiment, the present invention relates to an immune checkpoint inhibitor selected from the group of avelumab, nivolumab, pembrolizumab, ipilimumab, or durvalumab, embafolimab, for use in the manufacture of a pharmaceutical composition according to the invention disclosed herein.

[0198] In one aspect, the present invention relates to a method of treating a patient suffering from cancer, comprising subcutaneously administering to the patient an effective amount of the pharmaceutical composition disclosed herein. As used herein, the term "effective amount" refers to the amount of the pharmaceutical composition disclosed herein or the conjugate disclosed herein that results in one or more desired responses in a subject, such as killing of cancer cells that express a cell surface antigen, tumor-associated antigen, or tumor-specific antigen that is specifically bound by the antibody of the conjugate.

[0199] According to some embodiments, the method for treating a patient disclosed herein comprises administering the pharmaceutical composition of the present invention to one or more injection sites of the patient.The multiple injection sites may include two or more injection sites into which the pharmaceutical composition of the present invention disclosed herein is subcutaneously administered.Depending on the composition of the pharmaceutical composition of the present invention and the overall health condition of the patient (for example, the amount of subcutaneous fat of each patient), the pharmaceutical composition of the present invention may be administered to one or more injection sites.

[0200] Pharmaceutical compositions of the present invention comprising two components, such as a conjugate according to the present invention disclosed herein and a second component (e.g., rHuPh20), may require the first and second components to be injected in close spatial proximity, e.g., 0.5 cm to about 3 cm apart. The first and second components may be administered subcutaneously to the patient, e.g., as disclosed herein, where the first component may be administered before, simultaneously with, or after the administration of the second component. Alternatively, the second component (rHuPH20) may be administered before, simultaneously with, or after the administration of the first component disclosed herein. However, the second component is preferably administered before the administration of the first component, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to about 15, 20, 25, 30, 35, 40, 45, or 60 minutes, or, for example, about 15, 20, 25, 30 to about 90 minutes before the administration of the first component. For example, the first or second component of the pharmaceutical composition of the present invention may be administered subcutaneously within 1, 2, 3, 4, 5, 15, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 40 hours, or 48 hours after the subcutaneous administration of the first or second component. Corresponding treatments may be repeated periodically as medically indicated, for example, every 21, 28, 35, 42, 60, 90, 120, 180, 360 days, etc., twice, three times, or four times.

[0201] In some embodiments, a method of treating a patient according to the present invention comprises subcutaneously administering to said patient about 0.5 ml, 0.75 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml to about 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml, or, for example, about 1.25 ml, 1.75 ml, 2.25 ml to about 2.5 ml, 3 ml, 3.5 ml, of a pharmaceutical composition of the invention, wherein the volume injected may depend, for example, on the use of rHuPH20 to facilitate subcutaneous injection of a pharmaceutical composition of the invention as disclosed herein.

[0202] The effective amount of the pharmaceutical composition of the present invention to be subcutaneously administered to the patient may vary depending on several factors, such as the type of cancer, the stage of cancer, the age or weight of the patient, or the body surface of the patient. In some embodiments, the effective amount of the pharmaceutical composition of the present invention to be subcutaneously administered to the patient may be from about 60 μg / kg, 75 μg / kg, 100 μg / kg, 125 μg / kg, 150 μg / kg, 200 μg / kg body weight (bw) to about 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 900 μg / kg, 1 mg / kg, 1 mg / kg, or 1 mg / kg. The effective dose is 25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg body weight, or about 175 μg / kg, 250 μg / kg, 275 μg / kg, 300 μg / kg, 350 μg / kg, 475 μg / kg, 550 μg / kg, 75 μg / kg, 850 μg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg body weight to about 375 μg / kg, 425 μg / kg, 475 μg / kg, 950 μg / kg, 2.5 mg / kg, 3 mg / kg body weight. When determining the effective dose using the patient's body surface area (BSA), the body surface area (BSA) can be calculated using the DuPont formula. BSA=0.007184×W 0.425 ×H 0.725 where W is the weight (kg) and H is the patient's height (cm).

[0203] According to one embodiment, the present invention relates to a method of treating a patient suffering from cancer, said method comprising subcutaneously administering to said patient a pharmaceutical composition of the present invention as disclosed herein, wherein said patient has failed first-line, second-line, or third-line cancer treatments according to the conventional standard of care for the respective cancer.

[0204] In one embodiment, the present invention relates to a method of delivering an amatoxin-linker payload disclosed herein (e.g., according to formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), or (XI) disclosed herein) to a cell, said method comprising subcutaneously administering to a patient in need thereof a pharmaceutical composition comprising a conjugate of the present invention that specifically binds to an epitope on said cell.

[0205] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Example]

[0206] While the present invention has been illustrated and described in detail in the drawings and the foregoing description, this illustration and description is to be considered illustrative and not restrictive, and the invention is not limited to the disclosed embodiments. Those skilled in the art will understand and effect other modifications to the disclosed embodiments in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope of the invention.

[0207] Example 1: Amatoxin-Linker Payload Conjugation The antibody was conjugated to the amatoxin-linker conjugate using the so-called THIOMAB® technology, which utilizes a cysteine-engineered antibody. In this method, the conjugation is mediated by the coupling of a maleimide residue in the toxin-linker construct to the free SH group of an engineered cysteine ​​residue in the antibody, such as that introduced by site-directed mutagenesis. The conjugation is shown in the following reaction scheme: [ka]

[0208] The principles of this conjugation method are disclosed in Junutula JR et al. (2008). Nat Biotechnology Vol. 26:925-932, the contents of which are incorporated herein by reference. Briefly, prior to conjugation of a cysteine-engineered antibody (sometimes referred to as THIOMAB®) to the amatoxin linker payload disclosed herein, which includes a maleimide-containing linker, any blocked cysteines or glutathione present at the introduced cysteines are removed by gentle reduction, e.g., with a 10-40-fold molar excess of a reducing agent, TCEP, or dithiothreitol (DTT), in PBS at 25°C, followed by diafiltration. To reform interchain disulfide bonds, the cysteine-engineered antibody is incubated for 3 hours at 25°C with, e.g., a 2-20-fold molar excess of CuSO4 or dhAA (Sigma-Aldrich) over the reducing agent concentration. Interchain disulfide bond formation is monitored, for example, by non-reducing SDS-PAGE or denaturing reverse-phase high-performance liquid chromatography (HPLC) PLRP column chromatography. Conjugation of the maleimide-linker amatoxin payload to the cysteine-modified antibody is performed, for example, using 2-4 molar equivalents of the amatoxin linker payload. Quenching is performed, for example, using 20-25 molar equivalents of N-acetyl-L-cysteine, followed by incubation at 25°C for approximately 1 hour. The antibody complex is then purified, for example, on a HiTrap® S column (GE Healthcare Bio-Sciences) to remove excess reagent, optionally preceded by a 15-minute quenching step with N-acetyl-L-cysteine.

[0209] The antibody used in this experiment contains a D265C substitution in both Fc regions to provide a cysteine ​​residue with such a free SH group. The respective technique is disclosed in WO2016 / 142049A1 (the contents of which are incorporated herein by reference), and results in a homogenous product with a consistent drug-to-antibody ratio ("DAR") of 2 and site-specific conjugation.

[0210] Example 2: Mouse prostate cancer model 2.5×10 6 C4-2 tumor cells in 200 μL of RPMI (without PR and with 50% GFR Matrigel) were inoculated subcutaneously into the right flank of male CB17 Scid animals. The mean tumor volume was approximately 140 mm. 3 (Preferably 80 to 200 mm 3 Once tumor volume reached 1600 mm, animals were randomized according to tumor size (n=10). Treatment began on day 1 after group assignment. Mice were treated intravenously or subcutaneously (between the shoulders) (2.5 ml / kg) with a single dose of PBS or an anti-PSMA conjugate comprising an anti-PSMA antibody disclosed in WO2020 / 025564A1 conjugated to an amatoxin-linker moiety (XII) at 20 mg / kg. Tumor volume was measured twice weekly with a caliper and body weight was measured simultaneously. Tumors with a tumor volume >1600 mm were treated intravenously or subcutaneously (2.5 ml / kg) with an anti-PSMA conjugate comprising an anti-PSMA antibody disclosed in WO2020 / 025564A1 conjugated to an amatoxin-linker moiety (XII). 3 If the mice reached a critical mass or needed to be sacrificed for ethical reasons, the animals were sacrificed and autopsies were performed. The results are shown in Figure 2A, which shows that a single subcutaneous injection of the anti-PSMA conjugate (open triangles) was equally effective as a single intravenous dose (filled triangles).

[0211] Example 3: Murine chronic B-cell leukemia cancer model On day -3, 2.5 x 10 6MEC-2 (human CLL) tumor cells in 200 μL of PBS were inoculated intravenously into female CB17 Scid animals. Treatment began 3 days after tumor cell inoculation (n=10). Mice were treated intravenously or subcutaneously (10 ml / kg) with PBS or the anti-CD37 conjugate chHH1-HDPLALA-D266C conjugated to an amatoxin-linker moiety (XII). Body weight was measured twice weekly. Clinical signs and survival were monitored and recorded daily. If ethical reasons required the sacrifice of a mouse, the animal was sacrificed and a necropsy was performed. The results are shown in Figure 2B. The figure shows that a single dose of the conjugate administered subcutaneously at a dose of 1 mg / kg (open triangle) or 3 mg / kg (open square) was equally effective in extending survival compared to the corresponding treatment with the conjugate administered intravenously at the same doses of 1 mg / kg (closed triangle) and 3 mg / kg (closed square).

[0212] Example 4: Antitumor efficacy of anti-CD37-(XII) conjugates in a Raji-Luc (human Burkitt's lymphoma) disseminated xenograft model in female CB17-SCID mice The objective of this study was to evaluate the antitumor efficacy of anti-CD37-(XII) conjugates after a single subcutaneous administration in a Raji-Luc (human Burkitt's lymphoma) disseminated xenograft model in female CB17-SCID mice.

[0213] Test Design Twenty female CB17-SCID mice were assigned to two groups of 10 mice each, and on day -3, 2.5 × 10 mice per group were administered. 6 Raji-Luc tumor cells (in 200 μl of phenol red-free RPMI) were inoculated intravenously. On day 0, the animals received a final subcutaneous injection of anti-CD37-(XII) conjugate (dose: 0.5 or 1.0 mg / kg).

[0214] Concurrently, as part of Study A, 30 female CB17-SCID mice bearing Raji-Luc tumor cells were treated intravenously with a single dose of anti-CD37-(XII) conjugate (dose: 0.5 or 1.0 mg / kg) or PBS. These data served as the baseline.

[0215] Starting on day 1 after treatment, tumor cell-dependent luciferase activity was measured weekly by noninvasive whole-body bioimaging (Caliper IVIS). Body weight was measured at the same time. Clinical signs and survival were monitored daily. Animals were sacrificed and necropsied if one or more termination criteria were met or at the end of the study (day 97).

[0216] Results and Conclusions Intravenous inoculation of female CB17-SCID (PBS control group 1) mice with luciferase-expressing human Burkitt's lymphoma (Raji Luc) resulted in a gradual increase in luciferase-dependent whole-body luminescence, weight loss after 11 days, and 100% mortality within 18 days of treatment. Mortality was primarily associated with significant hind limb paralysis.

[0217] Anti-CD37-(XII) conjugates controlled Raji-Luc tumor-dependent mean bioluminescence to levels close to those of day 1 in 10 of 10 mice (100%, 0.5 mg / kg iv and 0.5 or 1.0 mg / kg sc) or 8 of 10 mice (80%, 1.0 mg / kg iv). Body weight gain was nearly continuous until the end of the study. Most clinical and necropsy findings may represent effects caused by the Raji_Luc tumors but are not considered related to the test item.

[0218] In conclusion, a single subcutaneous administration of 0.5 or 1.0 mg / kg anti-CD37-(XII) on day 0 (tumor inoculation on day -3) resulted in a highly potent inhibitory effect (10 of 10 mice) of intravenous human Burkitt's lymphoma growth compared with PBS (iv), normal weight gain, and 100% survival at the end of the study (day 97). There were no statistically significant differences in bioluminescence between animals treated with the same dose (0.5 or 1.0 mg / kg) but different (sc or iv) routes of administration. The results are shown in Figure 4A and Figure 4C.

[0219] Example 5: Efficacy of anti-CD37-(XII) conjugates in a disseminated MEC-2 tumor xenograft model in female CB-17 SCID mice The study consisted of two experimental groups containing 10 animals each. On day -3, 2.5 x 10 6 Twenty animals were intravenously inoculated with 200 μL of MEC-2 (human CLL) tumor cells in PBS. Treatment began on day 0 (3 days after tumor cell inoculation). Mice were treated with anti-CD37-(XII) as shown in Figure 4B. Body weights were measured twice weekly. Clinical signs and survival were recorded daily. Animals were sacrificed and autopsied upon the occurrence of termination criteria or for ethical reasons. The results of the study are shown in Figure 4B. The figure demonstrates that there are no statistically significant differences between animals treated with the same dose but via different (sc or iv) administration routes.

[0220] Example 6: Quantification of αPSMA-(XII) complex concentrations in serum A sandwich ELISA assay was used to quantify serum concentrations of αPSMA-(XII) conjugates derived from pharmacokinetic studies of αPSMA-(XII) conjugates following a single intravenous infusion or subcutaneous administration to cynomolgus monkeys. In the sandwich ELISA, polyclonal anti-amanitin antibodies were used to capture the conjugates from cynomolgus monkey serum samples. Rabbit anti-human IgG H&L HRP antibodies were used for detection. Test samples were analyzed in duplicate, with a calibration range of 1.6–200 pM. Optical density (OD) was measured at wavelengths of 450 nm and 570 nm and calculated using the OD difference. Serum concentrations of αPSMA-(XII) conjugates were calculated from the calibration curve and reconverted to the linear range. The results are shown in Figures 7A and 7B. Following intravenous administration of the αPSMA-(XII) conjugate disclosed herein at a concentration of 7.5 mg / kg, C max The C was approximately 219 μg / ml (average of 3 animals), but when administered subcutaneously, the C was 0.01 at a dose of 7.5 mg / kg. max was approximately 96 μg / ml, and the dose of complex αPSMA-(XII) was 10 mg / kg. max The αPSMA-(XII) conjugate was also well tolerated, with no animals dying when administered subcutaneously at a dose of 7.5 mg / kg, whereas two of three animals died when administered intravenously (see Figure 7). The increased tolerability of the conjugated αPSMA-(XII) is also reflected in the concentrations of the liver enzymes ALT and AST, as shown in Figure 3. Here, intravenous administration of the conjugated αPSMA-(XII) conjugate resulted in an increase in both markers, peaking between 5 and 10 days after intravenous administration of the conjugate, whereas subcutaneous administration of the same dose of the conjugate only resulted in a small increase in both liver enzymes. Thus, subcutaneous administration of the conjugated αPSMA-(XII) conjugate significantly increased C max Furthermore, C max The reduction in β-glucan increases tolerance as shown in Figure 3.

[0221] Example 6: Subcutaneous administration (sc) with anti-GCC (Guanylyl Cyclase C (GUCY2C)) conjugate MTD by anti-GCC ATAC The maximum tolerated dose (MTD) of αGCC-antibody conjugates containing amatoxin-linker moieties (XIV) and (XII) was determined after intravenous or subcutaneous administration of DAR2 ADCs (αGCC-LALA-D265C-(XIV) and αGCC-LALA-D265C-(XII)) to female NOD / SCID mice. Here, each anti-GCC antibody contained heavy and light chain sequences according to SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Conjugates were administered on day 0, body weights were measured twice weekly, and clinical observations and mortality were recorded daily. The initial dose was increased or decreased in subsequent groups to define the maximum tolerated dose (MTD), as shown in Table 1 below.

[0222] [Table 4]

[0223] Antitumor efficacy of amanitin-based anti-GCC ADCs The anti-tumor efficacy of the anti-GCC ADCs anti-GCC-LALA-D265C-(XII) and anti-GCC-LALA-D265C-(XIV) was evaluated in a female NOD / SCID HEK293-GUCY2C-(HDP)-2B3 subcutaneous xenograft model after a single subcutaneous administration. 5.0 × 10 per mouse 6 HEK293-GUCY2C-(HDP)-2B3 tumor cells (in 200 μL of RPMI medium (without PR and containing 50% GFR Matrigel, Corning 356231)) were inoculated subcutaneously into the right flank of female NOD / SCID mice. The mean tumor volume was 150–160 mm. 3Once tumor size reached 1000, 50 animals were selected and assigned to five groups of 10 animals each based on the anti-GCC ATAC efficacy relative to tumor size. On the same or following day (day 1), the animals received a single subcutaneous or intravenous dose of anti-GCC-LALA-D265C-(XII) ADC (2.5 mg / kg) or anti-GCC-LALA-D265C-(XIV) ADC (6.0 mg / kg), or PBS as a control. Tumor volume was measured three times weekly using calipers, and body weights were also measured. Clinical signs and survival were monitored daily. Animals were sacrificed and necropsied when one or more termination criteria were met or at the end of the study (day 61).

[0224] result A single subcutaneous or intravenous dose of 2.5 mg / kg anti-GCC-LALA-D265C-(XIV) ADC or 6.0 mg / kg anti-GCC-LALA-D265C-(XII) ADC, regardless of toxin-linker or route of administration, resulted in a statistically significant reduction in mean tumor volume (vs. PBS control) within approximately 12 days of treatment, with (almost) permanent complete tumor reduction (0 mm ) in 3 and 7 (anti-GCC-LALA-D265C-(XIV), sc and iv, respectively) or 5 and 8 (anti-GCC-LALA-D265C-(XII) ADC, sc and iv, respectively) of 10 mice. 3 The results are shown in Figure 8.

[0225] In conclusion, a single subcutaneous administration of 2.5 mg / kg anti-GCC-LALA-D265C-(XIV) or 6.0 mg / kg anti-GCC-LALA-D265C-(XII) ADCs resulted in a strong inhibitory effect on subcutaneous human embryonic kidney carcinoma growth, with slightly delayed weight gain and approximately 30% and 60% survival, respectively, compared to PBS controls (day 61). There were no statistically significant differences in tumor inhibition, weight gain, and survival between animals treated with the same test item and dose but via different (sc or iv) routes of administration.

[0226] The HEK293-GUCY2C-HDP-2B3 cells used in the xenograft model described above express human GUCY2C and are described in PCT / EP2023 / 080350. Briefly, HEK293-GUCY2C-HDP-2B3 were obtained by transient transfection of HEK293 wild-type cells with an overexpression plasmid encoding human guanylate cyclase 2C (GUCY2C or GCC) with the amino acid sequence according to Uniprot no. P25092 (version 13-SEP-2023) and resistance to geneticin (G418). Selection of stably transfected cells was performed after 4 days by placing the cells in culture medium containing G418 to select for a stable cell pool expressing GCC and G418 resistance. Stable single-cell clones were isolated from this cell pool using limiting dilution. For the stable cell clone HEK293-GUCY2C-HDP-2B3, GCC surface expression was confirmed by flow cytometry and cytotoxicity assay (BrdU ELISA).

[0227] Example 7 Pharmacokinetics of Subcutaneously Administered Anti-PSMA-(XIV) ADC The serum pharmacokinetics of the amanitin-based anti-PSMA ADC h3 / F11-LALA-D265C Var16-(XIV) was evaluated after a single subcutaneous administration of 10 or 5 mg / kg or a single intravenous administration of 5 mg / kg to male CB17-SCID mice. Male CB17-SCID mice were assigned to cohorts (3 mice per group, based on the sampling time point) with 18 mice per group. Animals received a single subcutaneous (5 or 10 mg / kg) or intravenous (5 mg / kg) dose of h3 / F11-LALA-D265C Var16-30.2347. Body weights were measured twice weekly for up to 14 days. Clinical signs and mortality were recorded daily. Blood samples were collected at 12 time points from 5 minutes to 336 hours (14 days) post-dose. The pharmacokinetic parameters of h3 / F11-LALA-D265C Var16-(XIV) in serum were calculated and compared.

[0228] result: Subcutaneous administration of 5.0 mg / kg of h3 / F11-LALA-D265C Var16-(XIV) resulted in a slight increase in the dose-normalized AUC and half-life, and a significant increase in the dose-normalized C max A favorable PK profile was demonstrated, with significant reductions in

[0229] After subcutaneous administration, C of h3 / F11-LALA-D265C Var16-(XIV) at doses of 5 and 10 mg / kg max are proportional to the dose, and C max _ D The values ​​were 7.54 and 7.33 kg·μg / mL / mg. The half-life (13.8 and 11.0 days), dose-normalized AUC (161 days·kg·μg / mL / mg and 119 days·kg·μg / mL / mg), and clearance (6.2 mL / day / kg and 8.4 mL / day / kg) were in similar ranges.

[0230] Taken together with the pharmacokinetic results of subcutaneously administered anti-PSMA-(XII) ADCs (Figure 7), these results support the finding that the effect of subcutaneous administration on the pharmacokinetics of amanitin-based ADCs is independent of the amanitin derivative used as the payload.

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[0232] Refer to the patent documents EP3043774A1 WOO98 / 13059 WO01 / 14424 W02004 / 032828, WO2005 / 084390A² WO2005 / 112919 WO2006 / 121168 WO2007011968A² WO2009 / 101611 WO2009 / 114335 WO2009 / 052249 WO2016 / 142049 WO2016027859A¹ WO2018 / 220169. WO2018 / 115466: WO19192979A1 WO2012135345A1 WO2016040856A2 WO 2021 / 234402A2 WO2022 / 096604A1, US20030130189; US20030096743; US20040052793;; US20040018194 US20040052793 US20040121940;. U.S. Patent No. 8,008,449 U.S. Patent No. 8,354,509 and U.S. Patent No. 6,835,807 U.S. Patent No. 6,268,488 U.S. Patent No. 5,621,002 U.S. Patent No. 6,677,435; U.S. Patent No. 6,218,519 U.S. Patent No. 6,759,509; U.S. Patent No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 6,180,370. US Patent No. 7,521,541; U.S. Patent No. 7,723,485 U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929 U.S. Patent No. 4,880,935 U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; U.S. Patent No. 9,605,051B2 U.S. Patent No. 11,377,497

Claims

1. 1. A pharmaceutical composition for use in the treatment of cancer, the pharmaceutical composition comprising a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) at least one linker linking the target binding moiety to the at least one amatoxin, the pharmaceutical composition being administered subcutaneously.

2. The target binding portion of the complex comprises: (i) an antibody, preferably a monoclonal antibody; (ii) an antigen-binding fragment thereof, preferably a variable region (Fv), a Fab fragment, or a F(ab)2 fragment; (iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv), and (iv) antibody-like proteins The pharmaceutical composition of claim 1, wherein the compound is selected from the group consisting of:

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the antibody of said conjugate is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody or a human antibody.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody of the complex is an IgG isotype antibody.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the IgG type antibody of the complex is an IgG1 isotype antibody, an IgG2 isotype antibody, an IgG3 isotype antibody, or an IgG4 isotype antibody.

6. 6. The pharmaceutical composition of claim 2, wherein the antibody portion of the conjugate does not induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

7. 7. The pharmaceutical composition of claim 2, wherein the antibody portion of the conjugate comprises an Fc region comprising at least one amino acid substitution at position D265, L234, L235, or G236 (according to the EU numbering system).

8. 8. The pharmaceutical composition of claim 7, wherein the Fc region of the antibody portion of the conjugate comprises at least one amino acid substitution selected from L234A, L234S, L234G, L235A, L235G, L235S, L235T, G236R, and D265C (according to the EU numbering system).

9. 9. The conjugate of claim 8, wherein the Fc region of the antibody comprises the amino acid substitution D265C (according to the EU numbering system).

10. 10. The pharmaceutical composition of claim 6, wherein the Fc region of the antibody comprises the amino acid substitutions L234A, L235A, and D265C (according to the EU numbering system).

11. 11. The pharmaceutical composition according to claim 1, wherein the linker is attached to the antibody moiety via any of the naturally occurring cysteine ​​residues of the antibody, preferably any of the naturally occurring cysteine ​​residues that form the interchain disulfide bonds of the antibody, and / or via a disulfide bond.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the amatoxin of the conjugate is attached to the antibody via a cleavable or non-cleavable linker.

13. 13. The pharmaceutical composition of claim 12, wherein the cleavable linker of the conjugate is selected from the group consisting of enzymatically cleavable linkers, preferably linkers cleavable by proteases, chemically cleavable linkers, preferably linkers containing disulfide bridges.

14. 14. The pharmaceutical composition of claim 13, wherein the enzymatically cleavable linker of the conjugate comprises a valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg) dipeptide, a phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or an alanine-alanine-proline-valine (Ala Ala Pro Vai) peptide, or a β-glucuronide or a β-galactoside.

15. 15. The pharmaceutical composition of claim 14, wherein the enzymatically cleavable linker of the conjugate is a linker cleavable by cathepsin B.

16. 16. The pharmaceutical composition of any one of claims 13 to 15, wherein the cleavable linker is a self-immolative linker, and the self-immolative linker comprises a para-aminobenzyloxycarbonyl (PAB or PABC) moiety.

17. 17. The pharmaceutical composition of claim 1, wherein the linker of the conjugate is attached to the amatoxin via (i) the γ C-atom of amatoxin amino acid 1, or (ii) the δ C-atom of amatoxin amino acid 3, or (iii) the 6'-C-atom of amatoxin amino acid 4.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the conjugate comprises at least one amatoxin linker moiety selected from any of the following compounds (I) to (XI): 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】

19. The antibody of the conjugate is linked to at least one amatoxin linker moiety via a thioether bond according to any of Formulas XII-XXII below: 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 19. The pharmaceutical composition of claim 1, wherein the amatoxin-linker moiety is attached to a thiol group of a cysteine ​​residue in the antibody portion of the conjugate, and n is from about 1, 2, 3 to about 4, 5, 6, 7, or 8.

20. 20. The pharmaceutical composition of claim 19, wherein n is from about 1.5 or 2 to about 3 or 3.5, preferably about 2.

21. The above cancers include gastric cancer, adenocarcinoma, melanoma, ovarian cancer, uterine cancer, cervical cancer, breast cancer, including triple-negative breast cancer, bronchial carcinoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, thymoma, testicular cancer, neuroblastoma, glioma, prostate cancer, castration-resistant prostate cancer, gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), stomach cancer, esophageal cancer, laryngeal cancer, parotid cancer, bile duct cancer, rectal cancer, endometrial cancer, desmoid tumor, desmoplastic small round cell tumor, neuroectodermal tumor, retinoblastoma, rhabdomyosarcoma, and sarcoma.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the tumor is a solid or non-solid tumor selected from the group comprising myosinoma, Wilms' tumor, osteosarcoma, Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma (DBNHL), subtypes of non-Hodgkin's lymphoma such as mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), Richter's syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), or multiple myeloma.

22. 22. The pharmaceutical composition of claim 21, wherein the cancer cells or tumor are characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13), or at least 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 40%, or 50% or more of the tumor cells are characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13).

23. 23. The pharmaceutical composition of any one of claims 1 to 22, further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the pharmaceutical composition is co-administered with an immune checkpoint inhibitor.

25. 25. The pharmaceutical composition of claim 24, further comprising a recombinant hyaluronidase.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the antibody of the complex specifically binds to a cell surface antigen on a tumor cell or a tumor-associated antigen.

27. The cell surface antigens on the tumor cells include EpCAM, HER2 / neu, EGFR (HER1, ErbB1), TROP-2, BCMA, CD37, STEAP1, FXYD3, CA125, CD30, NCAM (CD56), MUC1, CEA (CD66e), VEGF, AFP, AXL, TYR03, MER, CD20, CD19, CD52, CD268, CD28, CD80, CD22, CD4, CD2, CD33, CD30, CD38, CD52, CD80, CD140b, PSMA, TYR, FCRL2, MUC17, GPR143, NMNAT2, MAGE, MAGEC2, MAGE-A3, MART-1, WT-1, EPHA2, KRT19, CLDN7, DKK1, FGF19, SCN3A, SCN2A, GAS1, S100Z, GAPT, GPR35, NY-ESO, cadherin 24, DLK1, GPR173, ALK, GFRA3, GUCY2C, DLL3, PSMA, PROX1, PSCA, glypican-1, mesothelin (prostate stem cell antigen), GAGE-1 (G antigen 1), ganglioside / GD2, GnT-V, β1,6-N (acetylglucosaminyltransferase-V), UPAR (urokinase-type plasminogen activator receptor), sialyl Lewis x (SLe ｘ ), and sialyl Lewis a (SLe ａ 27. The pharmaceutical composition according to any one of claims 1 to 26, wherein the compound is selected from the group consisting of:

28. 28. The pharmaceutical composition of any one of claims 1 to 27, wherein the pharmaceutical composition is administered to a patient diagnosed with at least one type of cancer, and wherein the treatment comprises subcutaneous administration of at least one dose of the pharmaceutical composition.

29. 29. The pharmaceutical composition of any one of claims 1 to 28, wherein the pharmaceutical composition is administered to the patient multiple times, preferably about every 21, 28 to about 35, 36, 37, 38, 39, 40, 41, 42 days, or every 22, 23, 24, 25, 26, 27 days.

30. 30. The pharmaceutical composition of any one of claims 1 to 29, wherein subcutaneous administration of the pharmaceutical composition increases the maximum tolerated dose (MTD) by at least 30%, 40%, 50% compared to the MTD of the pharmaceutical composition when administered intravenously.

31. Use of a conjugate according to any one of claims 1 to 20 in the manufacture of a pharmaceutical composition for subcutaneous administration.

32. A method for treating a patient suffering from cancer, comprising subcutaneously administering to said patient an effective amount of the pharmaceutical composition of any one of claims 1 to 28.

33. 33. The method of claim 32, wherein the pharmaceutical composition or complex is administered at a single injection site.

34. 33. The method of claim 32, wherein the pharmaceutical composition or complex is administered at multiple injection sites.

35. 36. The method of any one of claims 33 to 35, wherein the pharmaceutical composition is administered subcutaneously in a volume of about 0.5 ml, 0.75 ml, 1 ml to about 1.5 ml, 2 ml, 3 ml, 4 ml.

36. An effective amount of the conjugate administered in the pharmaceutical composition is from about 100 μg / kg, 125 μg / kg, 150 μg / kg, 200 μg / kg body weight (b.w.) to about 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 900 μg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg 35. The method of any one of claims 32 to 34, wherein the dose is from about 175 μg / kg, 250 μg / kg, 275 μg / kg, 300 μg / kg, 350 μg / kg, 475 μg / kg, 550 μg / kg, 75 μg / kg, 850 μg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg body weight to about 375 μg / kg, 425 μg / kg, 475 μg / kg, 950 μg / kg, 2.5 mg / kg, 3 mg / kg body weight.

37. 37. The method of claim 36, wherein the patient has failed a conventional standard first-line, second-line, or third-line cancer treatment for their respective cancer.

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