Antibody-drug conjugates for subcutaneous administration for use in treatment of cancer

Abstract

The present invention relates to a pharmaceutical composition for administration in the treatment of cancer wherein the pharmaceutical composition is administered subcutaneously and wherein the pharmaceutical composition of the invention comprises at least one conjugate comprising an antibody that specifically binds to a cell surface antigen on a cancer cell and at least one amatoxin-linker load. The invention further relates to methods of treating patients with cancer using the pharmaceutical compositions of the invention.

Description

Technical Field

[0001] This invention relates to the subcutaneous administration of antibody-drug conjugates (ADCs) and their respective uses in cancer treatment, said ADCs comprising one or more cytotoxic amatoxin moieties conjugated to an antibody or antigen-binding antibody fragment. In another aspect, this invention relates to a method of treating a patient with cancer using the pharmaceutical compositions of the invention. Background Technology

[0002] Traditional cancer therapies often suffer from a narrow therapeutic window and the nonspecificity of chemotherapy drugs, which can affect normal cells with high mitotic rates and lead to numerous adverse reactions. Monoclonal antibodies have shown great therapeutic potential in the treatment of several diseases, particularly cancer. Prior to the development of antibody-drug conjugates (ADCs) for cancer treatment, monoclonal antibodies had already attracted attention due to their targeting specificity, therapeutic index, and generally fewer side effects compared to traditional therapies such as chemotherapy or chemoradiotherapy.

[0003] The design and development of ADCs face several challenges, including easy degradation or concentration-dependent aggregation tendencies due to the hydrophobicity of the corresponding ADC loading. Attempts to mitigate these limitations have led to reduced ADC concentrations in their respective formulations, resulting in limited bioavailability and tissue penetration.

[0004] Commonly used ADC toxins (such as auristatins, maytansinoids, and calicheamicins) typically have 100-1000 times higher in vitro potency than traditional chemotherapy agents, with an IC50 value of [missing information]. 50 At the sub-nanomolar level, this enhanced potency is crucial for ADCs to effectively kill tumor cells, where delivery to intracellular drug targets is related to tumor cell surface antigen expression levels and internalization efficiency. However, improvements in in vitro potency are generally associated with increased hydrophobicity of the corresponding payload.

[0005] Hydrophobicity is a significant factor influencing the overall physicochemical properties of ADC payloads. While increased hydrophobicity typically leads to improved in vitro efficacy, it also introduces risks of poor solubility, metabolic instability, and a higher probability of nonspecific off-target effects.

[0006] In many disease areas, subcutaneous delivery of biologics has emerged as a valuable alternative to intravenous administration. 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 administration results in rapid onset of action and near-100% bioavailability, but it can also cause pain, require hospitalization, and lead to peak serum levels (e.g., high C60) that may trigger toxicities. max As used in this article, "C" max "" refers to the highest observed plasma concentration of a given drug (such as, for example, the conjugates of the present invention). On the other hand, subcutaneous (sc) administration, commonly used for antibody-based drugs, has the advantage of being very easy to administer and can even be done by the patient themselves.

[0007] Despite the differences in pharmacokinetic characteristics between subcutaneous and intravenous formulations, subcutaneous administration of antibodies has proven to be safe, effective, and well-tolerated, and is generally preferred by patients and healthcare professionals due to its reduction in medical costs and resource usage associated with drug delivery.

[0008] However, subcutaneous administration of antibody-based therapies (including ADCs) is generally limited by small injectable volumes. Therefore, high formulation concentrations of ADCs are required to deliver an effective therapeutic dose. However, higher concentrations of ADC formulations containing hydrophobic loadings carry the risk of increased ADC aggregation, which in turn is associated with toxicity. ADC aggregation is also associated with reduced half-life and a narrow therapeutic index. The use of low-concentration ADC formulations reduces bioavailability and the ability to penetrate tissues and exert pharmacological effects.

[0009] Potential drawbacks of subcutaneous administration include uncontrollable absorption rates and local irritation or skin toxicity. Serious skin toxicity has been reported with intravenous (iv) administration of trastuzumab-emtansine loaded with a maytansine derivative (DM1). This skin toxicity may be due to extravasation, i.e., accidental leakage of the intravenously administered drug into the perivenous tissue.

[0010] Another concern regarding the subcutaneous administration of antibodies and ADCs is their isoelectric point (pI). Antibodies with an isoelectric point between 7 and 9 are positively charged at physiological pH. After subcutaneous administration, positively charged antibodies exhibit approximately a 30% decrease in bioavailability; while their negatively charged counterparts show an increase in bioavailability of up to 70% (Yadav et al. J. Biol. Chem. 2015, 290, 29732–29741.). Although these findings pertain to antibodies, little information is available regarding the effect of charge on the bioavailability of ADCs; however, positively charged ADCs are likely to face a similarly reduced bioavailability.

[0011] Strategies have been developed to overcome the low subcutaneous injection volume (typically between 1–2 ml) and poor bioavailability. The combined use of recombinant human hyaluronidase PH20 (rHuPH20) with antibodies allows for larger subcutaneous injection volumes of 5 ml or more. rHuPH20 works by locally degrading hyaluronic acid (HA), a large mucopolysaccharide and a component of the extracellular, pericellular, and intracellular matrix. Hyaluronic acid is a key component of the skin, forming a gel-like substance with water that resists the flow of large volumes of fluids and limits the delivery, diffusion, and absorption of large subcutaneous drugs. Combining antibodies with hyaluronidase promotes the flow of large volumes of fluids and improves pharmacokinetic characteristics after subcutaneous administration. Several antibodies have been approved for subcutaneous administration in combination with rHuPH20, such as rituximab (Rituxan Hycela / mAbThera subcutaneously), trastuzumab (Herceptin Hylecta), and daratumumab (Darzalex Faspro).

[0012] WO 2018 / 187074 A1 discloses a method for treating cancer using subcutaneous administration of an antibody-drug conjugate containing an SN38 payload. However, no corresponding ADC has yet been approved for subcutaneous administration.

[0013] Therefore, there remains an unmet medical need for highly efficient ADCs that can be administered subcutaneously to maximize efficacy and minimize toxicity. Summary of the Invention

[0014] The inventors surprisingly and unexpectedly discovered that subcutaneous administration of the pharmaceutical composition according to the invention for use in the treatment of cancer resulted in a reduced peak serum level (reduced C4). max The pharmaceutical composition comprises a conjugate containing (i) a target-binding moiety, (ii) at least one amatoxin, and (iii) at least one linker connecting the target-binding moiety to the at least one amatoxin.

[0015] Therefore, one object of the present invention is to provide a pharmaceutical composition that is administered subcutaneously and comprises (i) a target-binding portion, (ii) at least one amatoxin, and (iii) at least one linker connecting said target-binding portion to said at least one amatoxin.

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

[0017] In one specific aspect of the invention, the pharmaceutical composition comprises a conjugate conjugated to at least one amatoxin via an uncleavable or cleavable linker. Preferably, the cleavable linker of the conjugate according to the invention is an enzyme-cleavable linker, and preferably a self-degrading linker.

[0018] According to another object of the invention, the antibodies of the conjugates of the invention do not cause antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) and include at least one amino acid substitution at position D265, L234, L235, or G236 (according to the EU numbering system). In particular, the antibodies of the conjugates of the invention contain amino acid substitutions L234A, L235A, and D265C (according to the EU numbering system).

[0019] According to another object of the invention, at least one linker of the conjugate of the invention, which links at least one amatoxin and an ADC, is covalently bound to the antibody portion via any of the naturally occurring cysteine ​​residues of the antibody, preferably via any of the naturally occurring cysteine ​​residues forming the interchain disulfide bond of the antibody and / or via disulfide linkage, or covalently bound to engineered cysteine ​​residue D265C (according to the EU numbering system) of the Fc region of the antibody.

[0020] According to some aspects, the pharmaceutical composition of the present invention comprises a conjugate containing an amatoxin linker portion according to any one of formulas XII to XXII as disclosed herein, wherein the conjugate comprises between 1 and 8, preferably between 1.5, 2 and 3 or 3.5, and particularly preferably 2 amatoxin linker portions.

[0021] Another object of the present invention is to provide a pharmaceutical composition for use in methods of treating cancer.

[0022] These and other objectives are achieved by the methods and means according to the independent claims of the invention. Dependent claims relate to specific embodiments.

[0023] The general advantages of the invention and its features will now be described in detail. Attached Figure Description

[0024] Figure 1 Markush structures of various amatoxins. The bolded numbers (1 to 8) specify the standard numbers of the eight amino acids that form amatoxins. The standard names of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters α to γ, Greek letters α to δ, and numbers 1' to 7', respectively).

[0025] Figure 2 Comparison of the efficacy of conjugates administered subcutaneously and intravenously. (A) Mouse prostate cancer model established using C4-2 tumor cells; (B) Maximum tolerated dose of anti-PSMAADC conjugated to the amatoxin linker (XII) or (XIV) as disclosed herein, “DIG-LALA-D265C-(XIV)” refers to an isotype control antibody conjugated to the amatoxin-linker load (XIV); (C) Comparison of half-life, Cmax, and AUC of anti-PSMAADC and anti-CD37 administered subcutaneously and intravenously, showing that subcutaneous administration of the pharmaceutical compositions of the present invention results in an increased half-life and a decreased Cmax. max And the increased AUC.

[0026] Figure 3 NHP Tolerance Study of Intravenous and Subcutaneous Administration of Anti-PSMA-(XII) Conjugate. The NHP study used cynomolgus monkeys to record liver injury markers after subcutaneous and intravenous administration of anti-PSMA-(XII). Top: Intravenous administration of anti-PSMA-(XII) at 7.5 mg / kg as directed; Bottom: Subcutaneous administration of anti-PSMA-(XII) at 7.5 mg / kg and 10 mg / kg as directed. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LDH: Serum lactate dehydrogenase concentration in cynomolgus monkeys after treatment with the amatoxin-conjugate intravenously or subcutaneously as directed. As indicated by the elevated liver enzymes ALT and AST after intravenous administration of the conjugate, subcutaneous administration of the anti-PSMA-(XII) conjugate was better tolerated than the corresponding intravenous administration. Animals marked with a cross were euthanized or died for ethical reasons.

[0027] Figure 4 Comparison of the efficacy of intravenous versus subcutaneous administration of anti-CD37-(XII) conjugates. The efficacy of the conjugates against anti-CD37-(XII) as disclosed in this application was compared when administered intravenously or subcutaneously. (A) Raji-Luc model (B) Survival probability graph of mouse MEC2 tumor model.

[0028] Figure 5 Pharmacokinetics and biodistribution of anti-PSMA-(XII) in mice. (A) Serum concentration of the conjugate anti-PSMA-(XII) after subcutaneous administration as directed; (B) Serum concentration of the conjugate anti-PSMA-(XII) after intravenous administration as directed. (C) PK data for subcutaneous and intravenous administration of the conjugate anti-PSMA-(XII). The data indicate that subcutaneous administration resulted in a decrease in Cmax, while AUC remained essentially unchanged.

[0029] Figure 6Pharmacokinetics and biodistribution of anti-CD37-(XII) in mice. (A) Serum concentration of the conjugate anti-CD37-(XII) after subcutaneous administration as directed; (B) Serum concentration of the conjugate anti-CD37-(XII) after intravenous administration as directed; (C) PK data for subcutaneous and intravenous administration of the conjugate anti-CD37-(XII). The data indicate that subcutaneous administration resulted in a decrease in Cmax, while AUC remained essentially unchanged.

[0030] Figure 7 Pharmacokinetic study of serum concentrations of αPSMA-(XII) conjugate in cynomolgus monkeys. (A) Serum concentrations of the conjugate anti-PSMA-(XII) after intravenous and subcutaneous administration as directed. Intravenous administration resulted in higher and faster C60 concentrations compared to subcutaneous administration. max (B) Detailed pharmacokinetic results indicate that subcutaneous administration of the conjugate resulted in lower C. max (At a dose of 7.5 mg / kg, approximately 219 μg / ml of C) max(静脉内) With approximately 96 μg / ml of C max(皮下) ).

[0031] Figure 8 Comparison of the antitumor efficacy of intravenous and subcutaneous administration of anti-GCC conjugates. (A) Antitumor efficacy of anti-GCC-LALA-D265C-(XIV) administered subcutaneously (hollow square) and intravenously (solid square) at 2.5 mg / kg; (B) Antitumor efficacy of anti-GCC-LALA-D265C-(XII) administered subcutaneously (hollow triangle) and intravenously (solid triangle) at 6 mg / kg.

[0032] Figure 9 Pharmacokinetics of anti-PSMA-(XIV)ADC based on muscarinic acid. Changes in serum concentrations of anti-PSMAADC h3 / F11-LALA-D265C Var16-(XIV) over time following a single subcutaneous administration of 10 mg / kg or 5 mg / kg, or an intravenous administration of 5 mg / kg, to male CB17-SCID mice. Detailed Implementation

[0033] Before describing the invention in detail, it should be understood that the invention is not limited to the specific components of the described apparatus or the process steps of the described method, as these apparatuses and methods can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and appended claims, the singular forms "a," "an," and "the" include singular and / or plural referents unless the context explicitly states otherwise. Furthermore, it should be understood that where a parameter range is given by numerical value, that range is considered to include those limits. The expression of numerical ranges herein is intended only as a way of abbreviating each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were separately stated herein.

[0034] Throughout this specification and the following claims, unless the context otherwise requires, the term "comprise" and its variations such as "comprises" and "comprising" should be understood to imply inclusion of the stated member, integer, or step, but not to exclude any other member, integer, or step not stated. The term "consisting of" is a specific embodiment of the term "comprise," in which any other member, integer, or step not stated is excluded.

[0035] It should also be understood that the embodiments disclosed herein are not intended to be construed as isolated embodiments unrelated to each other. Features discussed with respect to one embodiment are also intended to be disclosed when referring to other embodiments shown herein. If, in a case, a particular feature is not disclosed in one embodiment but is disclosed in another, those skilled in the art will understand that this does not necessarily mean that the feature was not intended to be disclosed in the other embodiment. Those skilled in the art will understand that it is in the spirit of the invention to disclose the feature with respect to other embodiments, but this has not been done only for clarity and to keep the specification to a manageable length.

[0036] Furthermore, the contents of prior art documents cited herein are incorporated by reference, especially those concerning public standards or conventional methods. In such cases, the primary purpose of inclusion by reference is to provide sufficient implementable disclosure and to avoid lengthy repetition. Chemical terminology used throughout this invention should be interpreted in accordance with the "Compendium of Chemical Terminology" published by the International Union of Pure and Applied Chemistry, ISBN: 0-9678550-9-8.

[0037] Throughout this invention, the term “about” is used, which shall refer to + / - 10% of the numerical value used.

[0038] According to a first aspect of the invention, the present invention relates to a pharmaceutical composition for use in the treatment of cancer, wherein the pharmaceutical composition comprises a conjugate, wherein the conjugate comprises (i) a target-binding portion, (ii) at least one amatoxin, and (iii) at least one linker connecting the target-binding portion to the at least one amatoxin, wherein the pharmaceutical composition is administered subcutaneously.

[0039] As used herein, the term "pharmaceutical composition" means comprising pharmaceutical excipients such as buffers, preservatives, and tension modifiers, as well as the active compound or a salt thereof, which, by administration to a mammal, preferably a human, can be used to treat or prevent a disease or condition or to reduce its severity.

[0040] As used with respect to the pharmaceutical compositions of the present invention, the terms "amatoxin" or "amatoxin class" refer to a dicyclic peptide composed of eight amino acids found in the poison amanita mushroom (see [link to original text]). Figure 1 Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby also inhibiting transcription and protein biosynthesis in invaded cells. Repression of transcription in cells leads to the cessation of growth and proliferation. Although there is no covalent binding, the covalent bond between amatoxins and RNA polymerase II is very tight (K...). D =3nM). The dissociation of muscarinine from the enzyme is a very slow process, making it unlikely that the attacked cells will recover. When transcriptional repression persists long enough, the cells will undergo programmed cell death (apoptosis).

[0041] In the context of this invention, the term "amanita toxin" includes all bicyclic peptides consisting of eight amino acids as isolated from the genus *Amanita* and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260), all other chemical derivatives thereof, all other chemical derivatives thereof, all other semi-synthetic analogs thereof; all synthetic analogs thereof constructed from synthetic building blocks based on the main structure (cyclic, eight amino acids) of the natural compound; all other synthetic or semi-synthetic analogs thereof containing non-hydroxylated amino acids in place of hydroxylated amino acids; and all other synthetic and semi-synthetic analogs wherein the sulfoxide portion is replaced by a sulfone, a thioether, or by an atom other than sulfur (e.g., the carbon atom in the carbanalog of amanita phalloids).

[0042] As used herein, a "derivative" of a compound refers to a substance that has a similar chemical structure to the compound but also contains at least one chemical group absent in the compound from which it is derived and / or lacks at least one chemical group present in the compound from which it is derived. The compound compared to the derivative is called the "parent" compound. Typically, a "derivative" can be generated from a parent compound in one or more chemical reaction steps.

[0043] As used herein, an “analogy” of a compound is structurally related to but not identical to the compound and exhibits at least one of the compound’s activities. The compound to which the analog is compared is referred to as the “parent” compound. The activities mentioned above include, but are not limited to: binding activity with another compound; inhibitory activity, such as enzyme inhibitory activity; toxic effects; and activating activity, such as enzyme activating activity. It is not required that the analog exhibits the same degree of such activity as the parent compound. A compound is considered an analog within the context of this invention if it 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%, and more preferably at least 50%) of the activity of the parent compound. Therefore, as used herein, “analogs of amatoxins” refers to compounds that are structurally related to α-amaminine, β-amaminine, γ-amaminine, ε-amaminine, amanin, amaninamide, amanullin, and amanullinic acid and exhibit 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%, and more preferably at least 70%, 80%, 90%) of the inhibitory activity against mammalian RNA polymerase II compared to at least one of α-amaminine, β-amaminine, γ-amaminine, ε-amaminine, amanin, amaninamide, amanullin, and amanullinic acid. The "analytes of amatoxins" suitable for use in this invention may even exhibit higher inhibitory activity against mammalian RNA polymerase II than any of α-amaminine, β-amaminine, γ-amaminine, ε-amaminine, amatoxin, amatoxin amide, amatoxin cyclic peptide, and amatoxin cyclic peptide carboxylic acid. Inhibitory activity can be determined by determining the concentration at which 50% inhibition occurs (IC50). 50The inhibitory activity against mammalian RNA polymerase II can be measured indirectly by measuring its inhibitory activity against cell proliferation, or alternatively, for amatoxins and their respective derivatives disclosed herein, an RNA polymerase II activity assay can be used as disclosed by Voss et al., BMC Molecular Biology 2014, 15:7.

[0044] "Semi-synthetic analogs" refer to analogs obtained through chemical synthesis using compounds from natural sources (e.g., plant materials, bacterial cultures, fungal cultures, or cell cultures) as starting materials. Typically, the "semi-synthetic analogs" of this invention are synthesized starting from compounds isolated from mushrooms of the Amanita family. Conversely, "synthetic analogs" refer to analogs synthesized through the so-called total synthesis of small (usually petrochemical) synthetic building blocks. This total synthesis is typically carried out without the aid of biological methods.

[0045] According to some embodiments of the present invention, the amatoxin may be selected from α-amaminine, β-amaminine, amatoxin, amatoxin amide and its analogues, derivatives and salts.

[0046] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those having functional groups (e.g., carboxyl, amino, hydroxyl, thiol, or thiol scavenging groups) capable of reacting with linker molecules or target binding sites as defined below.

[0047] In the context of this invention, the term "amaminine" specifically refers to a bicyclic structure based on an aspartic or asparagine residue at position 1, a proline residue at position 2 (particularly a hydroxyproline residue), an isoleucine, hydroxyisoleucine, or dihydroxyisoleucine (or aspartic, for amanullic acid), a tryptophan or hydroxytryptophan residue at position 4 (or proline, for proamanullin), glycine residues at positions 5 and 7 (or isoleucine residues, in the case of amanullic acid and proamanullin), an isoleucine residue at position 6, and a cysteine ​​residue at position 8, particularly a derivative of cysteine ​​oxidized to a sulfoxide or sulfone derivative (see Amanuline numbering and representative examples for details). Figure 1), and additionally include all its chemical derivatives; all its other semi-synthetic analogs; all its synthetic analogs derived from synthetic building blocks based on the main structure (cyclic, 8 amino acids) of the natural compound; all other synthetic or semi-synthetic analogs containing non-hydroxylated amino acids in place of hydroxylated amino acids; and all other synthetic and semi-synthetic analogs, in each case of any such derivative or analog having functional activity by inhibiting mammalian RNA polymerase II.

[0048] The conjugates of the present invention, as included in the pharmaceutical compositions disclosed herein, comprise (i) at least one amatoxin and (ii) at least one linker connecting the target-binding portion to the at least one amatoxin. Thus, the conjugates comprise one, two, three, four, five, six, seven, eight, nine, or ten amatoxin moieties, preferably about two to about three or four amatoxin moieties connected to the target-binding portion via at least one linker, for example, each of the amatoxin moieties connected to the target-binding portion via a linker, such that the number of linkers corresponds to the number of amatoxin moieties. In some embodiments, the pharmaceutical compositions of the present invention comprise the conjugates disclosed herein, comprising about two (e.g., about 1.5 to about 2.5) amatoxin-linker moieties bound to the target-binding portion, wherein the target-binding portion is, for example, an antibody or an antibody-binding fragment thereof, preferably a monoclonal antibody.

[0049] 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. In the context of this invention, preferred target-binding moieties are (i) antibodies or antigen-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers, (iv) anticalcinin, or (v) “target-binding moieties” suitable for use in this invention, typically having a molecular weight of 40,000 Da (40 kDa) or higher.

[0050] In the context of this invention, a "linker" refers to a molecule that increases the distance between two components, such as a molecule that alleviates steric hindrance between the target-binding moiety and amatoxins, which may otherwise reduce the ability of amatoxins to interact with RNA polymerase II. The linker can be used for another purpose, as it can specifically promote the release of amatoxins in cells targeted by the target-binding moiety. Preferably, the linker, and the bond between the linker on one side and amatoxins, and the bond between the linker on the other side and the target-binding moiety or antibody, are not cleaved, degraded, or hydrolyzed under physiological conditions outside the cell (e.g., in blood), but can be cleaved intracellularly, particularly within target cells (e.g., cancer cells), and more particularly within the lysosomes of cancer cells. To provide this selective stability, the linker may contain preferably pH-sensitive or protease-sensitive functional groups. Alternatively, the bond connecting the linker to the target-binding moiety may provide selective stability. The linker preferably has a length of at least one atom, more preferably 1-30 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), wherein one side of the linker has reacted with amatoxins and the other side has reacted with the target binding portion. In the context of this invention, the linker is preferably C. 1-30 -alkyl, C 1-30 -heteroalkyl, C 2-30 -Alkenyl, C 2-30 -heterene group, C 2-30 -Alynyl group, C 2-30 - A cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaryl group, optionally substituted. The linker may contain one or more structural elements, such as amides, esters, ethers, thioethers, disulfides, hydrocarbon moieties, etc. The linker may also contain combinations of two or more of these structural elements. Each of these structural elements may be present in the linker more than once, for example, twice, three times, four times, five times, or six times.

[0051] In some embodiments, the linker may contain a disulfide bond. It should be understood that the linker must be attached to the amatoxin and the target-binding moiety in a single step or two or more subsequent steps. For this purpose, the group intended to be the linker carries two groups (preferably at the proximal and distal ends), which may (i) form a covalent bond with a group on the amatoxin or target-binding peptide, preferably an activated group, or (ii) be activated or be able to be activated to form a covalent bond with a group on the amatoxin. Therefore, it is preferred that the chemical groups are located at the distal and proximal ends of the linker, as a result of such coupling reactions, such as esters, ethers, carbamates, peptide bonds, etc.

[0052] As used herein, the term “subcutaneous administration” (which may also be referred to as “sc administration” or “subQ administration” and any grammatical variations thereof) means the injection of the pharmaceutical composition of the present invention into the subcutaneous layer of skin, that is, the layer of skin just beneath the dermis and epidermis, collectively referred to as the cutis. For example, subcutaneous administration can be performed by a subcutaneous injection needle and syringe or by alternative means such as an autoinjector or injection pen, such as those described in WO12085029 A1 or WO20154170 A1, which are incorporated herein by reference.

[0053] According to some embodiments, the target-binding portion of the present invention is one of the following: (i) an antibody, preferably a monoclonal antibody; (ii) an antigen-binding fragment thereof, preferably a variable domain (Fv), a Fab fragment, or an F(ab)2 fragment; (iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv); and (iv) an antibody-like protein. According to a preferred embodiment, the target-binding portion is an antibody, preferably a monoclonal antibody.

[0054] As used herein, the term "antibody" refers to a protein composed of one or more polypeptide chains encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene or cDNA derived from an immunoglobulin gene or a fragment thereof. The immunoglobulin gene includes any one of the light chain κ, λ and heavy chain α, δ, ε, γ and μ constant region genes, as well as many different variable region genes.

[0055] The basic structural unit of immunoglobulins (antibodies) is typically a tetramer composed of two pairs of identical polypeptide chains: a light chain (L, with a molecular weight of approximately 25 kDa) and a heavy chain (H, with a molecular weight of approximately 50-70 kDa). Each heavy chain includes a heavy chain variable region (abbreviated as VH or VH) and a heavy chain constant region (abbreviated as CH or CH). The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL or VL) and a light chain constant region (abbreviated as CL or CL). The VH and VL regions can be further subdivided into hypervariable regions, also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region consists of three CDRs (FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4) arranged sequentially from the amino terminus to the carboxyl terminus, and four FRs. The variable regions of the heavy and light chains form binding domains that interact with the antigen.

[0056] The CDR (Containing Derivative) is crucial for the binding of the antibody or its antigen-binding portion. The FR (Front-Ending Sequence) can be replaced by other sequences if the three-dimensional structure required for antigen binding is preserved. Structural changes to the construct typically result in insufficient binding to the antigen.

[0057] The term "antigen-binding moiety" in (monoclonal) antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to a given antigen in its natural form. Examples of antigen-binding moieties include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains, F(ab')2 fragments, bivalent fragments containing two Fab fragments connected by disulfide bridges in the hinge region, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of VL and VH domains of the antibody's single arm, and dAb fragments consisting of a VH domain and a separated complementarity-determining region (CDR).

[0058] The antibody or 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 an article of an antibody molecule having a single binding specificity and affinity for a particular epitope, representing a homogeneous antibody group, i.e., a homogeneous group composed of a complete immunoglobulin or its fragments or derivatives. Preferably, the antibody is selected from IgG, IgD, IgE, IgA and / or IgM or its fragments or derivatives, and preferably the monoclonal antibody of the present invention is an IgG isotype, such as IgG1 or IgG4, more preferably an IgG1 isotype. The term "antibody" as used herein shall also include and refer to antibody portions or antibody moieties contained in the conjugates of the present invention.

[0059] As used herein, the term “fragment” or “antigen-binding fragment” should refer to fragments of such antibodies that retain the ability to bind to the target, such as CDR (complementarity-determining region), hypervariable region, variable domain (Fv), IgG heavy chain (composed of VH, CH1, hinge region, CH2 and CH3 regions), IgG light chain (composed of VL and CL regions) and / or Fab and / or F(ab)2.

[0060] As used herein, the term "derivative" should refer to protein constructs that differ structurally from the conventional antibody concept but still share some structural relationships, such as scFv, Fab, and / or F(ab)2, as well as bispecific, trispecific, or highly specific antibody constructs, all of which have substantially the same target binding specificity as the monoclonal antibodies of this invention. All of these terms will be explained below.

[0061] Other antibody derivatives known to those skilled in the art are bispecific antibodies (such as those disclosed, for example, in Proc Natl Acad SciU S A. 1993 Jul 15; 90(14): 6444-8), arabinotropic antibodies, domain antibodies, divalent homodimers having two chains consisting of scFv, IgA (two IgG structures linked by a J chain and a secretory component), shark-derived antibodies (IgNAR), antibodies consisting of a New World primate framework region plus a non-New World primate CDR, dimer constructs containing CH3+VL+VH, other scaffold protein forms containing CDRs, and antibody conjugates (e.g., antibodies or fragments or derivatives thereof linked to drugs, toxins, cytokines, aptamers, nucleic acids (such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), therapeutic peptides, radioisotopes, or tags).

[0062] As used herein, the term "antibody-like protein" refers to a protein engineered (e.g., through mutation of an Ig loop) to specifically bind to a target molecule. Typically, such antibody-like proteins contain at least one variable peptide ring connected at both ends to a protein backbone. This dual structural constraint significantly increases the binding affinity of the antibody-like protein to levels comparable to antibodies. The length of the variable peptide ring typically consists of 10 to 20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globulin. Antibody-like proteins include, but are not limited to, affinities, anticalcitonins, and engineered ankylosing spicules (see, for example, Binz et al. 2005 or WO2012135345A1). Antibody-like proteins can be derived from large libraries of mutants, for example, by panning from large phage display libraries, and can be isolated similarly to conventional antibodies. Similarly, antibody-like binding proteins can be obtained through combined mutations of surface-exposed residues in globulins.

[0063] As used herein, the term "Fab" refers to an IgG fragment containing an antigen-binding region, the fragment consisting of a constant domain and a variable domain from each heavy and light chain of the antibody.

[0064] As used herein, the term “F(ab)2” refers to an IgG fragment consisting of two Fab fragments linked together by disulfide bonds.

[0065] As used herein, the term "scFv" refers to a single-chain variable fragment, which is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked together by short linkers, typically containing serine (S) and / or glycine (G) residues. Despite the removal of the constant region and the introduction of linker peptides, the chimeric molecule retains the specificity of the original immunoglobulin.

[0066] Modified antibody forms include, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, and immunoconjugates.

[0067] IgG, scFv, Fab, and / or F(ab)2 are antibody forms well known to those skilled in the art. Related enabling techniques are available from relevant textbooks.

[0068] According to a preferred embodiment of the present invention, the antibody or its antigen-binding fragment or antigen-binding derivative is a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody, or its antigen-binding fragment or antigen-binding derivative. More preferably, the antibody or its antigen-binding fragment is a humanized antibody or a human antibody.

[0069] Mouse-derived monoclonal antibodies (mAbs) can cause unwanted immune side effects because they actually contain proteins from another species that may trigger antibodies. To overcome this problem, antibody humanization and maturation methods have been designed to produce antibody molecules with minimal immunogenicity when administered to humans, while ideally retaining the specificity and affinity of the non-human parent antibody (see review by Almagro and Fransson 2008, Front Biosci. 2008 Jan 1; 13:1619-33.). Using these methods, for example, the frame region of a mouse mAb is replaced with the corresponding human frame region (so-called CDR transplantation). WO200907861 discloses the generation of humanized forms of mouse antibodies by linking the CDR region of a non-human antibody to a human constant region using recombinant DNA technology. US6548640 of the Medical Research Council describes CDR transplantation techniques, and US5859205 of Celltech describes the generation of humanized antibodies.

[0070] As used herein, the term "chimeric antibody" refers to an antibody consisting of an original antigen-binding variable domain and a constant domain from a different species. Since most antibodies (especially monoclonal antibodies) are originally derived from mice, typical chimeric antibodies include a human constant domain and a mouse variable domain to reduce immunogenicity in humans. Examples of chimeric antibodies used in clinical treatment include infliximab, rituximab, and abciximab.

[0071] As used herein, the term "humanized antibody" refers to an antibody, fragment thereof, or derivative thereof, wherein at least a portion of the constant region and / or frame region of the antibody, and optionally a portion of the CDR region, is derived from or modulated from a human immunoglobulin sequence. Methods for antibody humanization are known in the art and have been described, for example, in Riechmann et al., Nature 332:323-327, 1988; Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762 and 6,180,370.

[0072] The antibodies, antibody fragments thereof, or antibody derivatives disclosed herein may contain humanized sequences, particularly preferred VH and VL-based antigen-binding regions that maintain appropriate ligand affinity. Amino acid sequence modifications performed to obtain said humanized sequences may occur in the CDR region and / or frame region of the original antibody and / or in the antibody constant region sequence.

[0073] The antibody, its antibody fragment, or its antibody derivative may be glycosylated. The polysaccharide may be an N-linked oligosaccharide chain at the asparagine 297 position of the heavy chain.

[0074] The antibodies, fragments, or derivatives of the present invention can be produced by transfecting host cells with an expression vector containing the coding sequence of the antibody of the present invention. The expression vector or recombinant plasmid is produced by placing the antibody-coding sequence under the control of suitable regulatory genetic elements, including promoter and enhancer sequences, such as the CMV promoter. The heavy and light chain sequences can be expressed by a single co-transfected expression vector or by a dual-expression vector. The transfection can be transient or stable. Transfected cells are then cultured to produce transfected antibody constructs. When stable transfection is performed, stable clones secreting antibodies with appropriately related heavy and light chains are selected by screening using appropriate assays (e.g., ELISA), subcloning is performed, and the clones are propagated for future production.

[0075] According to a preferred embodiment, the antibody or antibody portion or antigen-binding fragment of the conjugate of the pharmaceutical composition according to the present invention is an IgG isotype antibody, for example, an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype antibody.

[0076] In conjugates of pharmaceutical compositions for use according to the invention, the use of antibodies characterized by reduced or eliminated effector functions may be expected to prevent, for example, unwanted cytokine secretion or killing of cells expressing Fcγ receptors (such as macrophages). Therefore, the antibodies or antigen-binding fragments of the invention may also include modifications and / or mutations that alter the properties of the antibody and / or fragment, such as those known in the art that reduce ADCC, ADCP, or complement-dependent cytotoxicity (CDC). Preferably, ADCC, ADCP, and CDC are reduced by at least 90% or more, more preferably at least 95%, more preferably at least 97.5%, and even more preferably at least 98% or 99% compared to antibodies containing a wild-type Fc region. According to a preferred embodiment, pharmaceutical compositions for use according to the invention comprise conjugates containing antibodies, wherein said antibodies do not cause antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0077] The binding of IgG1 to the activating and inhibitory Fcγ receptor (FcγR) or the first component (C1q) of complement depends on residues located in the hinge region and the CH2 domain. Both regions of the CH2 domain are crucial for the binding of FcγR and complement C1q and have unique sequences. Human IgG1 and IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduce 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, WO 2021 / 234402 A2).

[0078] Therefore, in one embodiment, the pharmaceutical composition for use according to the invention comprises an antibody portion or antigen-binding fragment as part of a conjugate as disclosed herein, which includes a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region, such that the molecule has a reduced affinity for IgG1 Fc receptors FcγRI, FcγRII, and FcγRIII and complement component C1q compared to the wild-type Fc region.

[0079] Affinity to the Fc region (such as binding of IgG1 to FcγR) can be determined using a variety of techniques known in the art, such as, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by surface plasmon resonance assays, as disclosed, for example, Wilkinson et al. PLoS One. 2021; 16(12):e0260954, or other mechanisms based on kinetic assays (e.g., BIACORE). TM Analysis or Octet TM 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).

[0080] Therefore, the compositions for use according to the invention comprise conjugates containing antibodies as described herein, which have been genetically engineered to include a variant Fc region containing at least one amino acid residue directly contacting the FcγR based on structural and crystallographic analysis. As used herein, the terms “genetically engineered” or “genetically engineered” refer to the modification of the amino acid sequence or a portion thereof (such as, for example, the Fc region of an antibody) of a given or native polypeptide or protein, in terms of nucleotide and / or amino acid substitutions, insertions, deletions, or inversions, or any combination thereof, by genetic technology methods (such as, for example, site-directed mutagenesis, as described in Biochem. J. (1986) Vol. 237:1-7 or J Biol Chem. (2015) Vol. 290(5):2577-2592). As used herein, the terms “amino acid substitution” or “mutation” refer to the modification of the amino acid sequence of a protein, wherein one or more amino acids are substituted with the same number of different amino acids to produce a protein containing an amino acid sequence different from that of the original protein. Conservative amino acid substitutions are understood to involve substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity, and / or conformation. In this sense, representatives of conserved amino acid groups 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.

[0081] The Fc region of the antibody may further include at least one cysteine ​​amino acid substitution at a position where engineered cysteine ​​can be used for conjugation without interfering with immunoglobulin folding and assembly. Corresponding cysteine-substituted or cysteine-engineered antibodies have been disclosed in WO2016040856A2 or Junutula et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US 7521541; US ​​7723485; WO2009 / 052249 and WO2016 / 142049. The 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.

[0082] According to one embodiment, the antibody portion contained in the pharmaceutical composition for use according to the invention contains at least one amino acid substitution at position D265, L234, L235 or G236 (according to the EU numbering system), preferably containing two or three amino acid substitutions at said position.

[0083] According to a preferred embodiment, the conjugate of the pharmaceutical composition for use according to the present invention comprises an antibody, or an 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 may also be referred to as "e.g., the Kabat EU index" and refers to the numbering method for human IgG1 EU antibodies, specifically the numbering method for EU antibodies as described in Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85.

[0084] According to preferred embodiments, the antibody or antibody moiety of the conjugate disclosed herein comprises the amino acid substitution D265C (according to the EU numbering system). Particularly preferred, the Fc region of the antibody comprises the amino acid substitutions L234A, L235A, and D265C (according to the EU numbering system). Corresponding antibodies containing the aforementioned mutations are particularly suitable for ADCs containing highly toxic payloads because they lack Fc effector function (“Fc silencing”), thereby avoiding killing, for example, macrophages expressing Fcγ receptors on their cell surface.

[0085] According to one embodiment, the linker of the conjugate is linked to the antibody or antibody moiety via any of the naturally occurring cysteine ​​residues of the antibody, preferably via any of the cysteine ​​residues forming the interchain disulfide bond of the antibody and / or via disulfide linkage. For example, WO2005 / 084390A2 discloses a corresponding method for conjugating the linker as disclosed herein to the antibody moiety of the conjugate of the present invention. According to one embodiment, as disclosed herein, the amatoxin of the conjugate of the pharmaceutical composition of the present invention for use in the treatment of cancer is linked to the antibody via a cleavable linker or a non-cleavable linker.

[0086] The “cleavable linker” according to the present invention is understood to include at least one cleavable site. As used herein, the term “cleavable site” should refer to a portion that is susceptible to specific cleavage at a defined location under specific conditions. These conditions are, for example, specific enzyme or reduction environments within a specific in vivo or cellular compartment. For example, a cleavable linker is designed to utilize differences in local environments, such as extracellular and intracellular environments, including, for example, pH, reduction potential, or enzyme concentration, to trigger the release of amatoxins from target cells. Typically, cleavable linkers are relatively stable in circulation but are particularly readily cleaved in the intracellular environment by one or more mechanisms, including but not limited to, the activity of proteases, peptidases, and glucuronidases. The cleavable linkers used herein are substantially stable in circulating plasma and / or outside target cells (e.g., cancer cells) and can be cleaved at some efficient rate within or near target cells (e.g., in the tumor microenvironment).

[0087] Suitable cleavable linkers according to the invention may include, for example, those that can be cleaved (e.g., by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under alkaline 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 hydrazine, disulfides, thioethers, or dipeptides.

[0088] For example, hydrolyzable linkers under acidic conditions may include hydrazones, ureas, thioureas, cis-aconitic amides, orthoesters, acetals, ketals, etc. (see, for example, U.S. Patents 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 disclosure of each of which is incorporated herein by reference in its entirety). Such linkers are relatively stable under neutral pH conditions (such as those in blood), but unstable below pH 5.5 or 5.0 (approximately the pH of lysosomes).

[0089] For example, the linker group of the conjugates used in this invention is cleavable under reducing conditions such as disulfides. Various disulfide linkers known in the art include, for example, those 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-pyridyl-dithio)toluene), SPDB, and SMPT, see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; U.S. Patent No. 4,880,935, the disclosure of each of which is incorporated herein by reference in its entirety.

[0090] According to some embodiments, the cleavable site can be cleaved by at least one protease selected from the following: cysteine ​​protease, metalloproteinase, serine protease, threonine protease, and aspartic protease.

[0091] Cysteine ​​proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism involving the catalysis of nucleophilic cysteine ​​thiols in a triplet or dummy.

[0092] Metalloproteinases are proteases whose catalytic mechanisms involve metals. Most metalloproteinases require zinc, but some use cobalt. The metal ion coordinates to the protein via three ligands. The ligands for coordinating the metal ion can vary depending on histidine, glutamate, aspartic acid, lysine, and arginine. The fourth coordination site is occupied by a variable water molecule.

[0093] Serine proteases are enzymes that cleave peptide bonds in proteins; serine acts as a nucleophilic amino acid at the enzyme's active site. Based on their structure, serine proteases can be divided into two main categories: chymotrypsin-like proteases or subtilisin-like proteases.

[0094] Threonine proteases are a family of proteases whose active sites contain a threonine (Thr) residue. Typical members of this class are the catalytic subunits of the proteasome; however, acyltransferases have convergently evolved to share the same active site geometry and mechanism.

[0095] Aspartic proteases are catalytic proteases that use activated water molecules bound to one or more aspartic residues to catalyze their peptide substrates. Generally, they have two highly conserved aspartic residues at their active site and exhibit optimal activity at acidic pH. Almost all known aspartic proteases are inhibited by gasstatin.

[0096] In some embodiments, the cleavable site is cleaved by at least one agent selected from: cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase and β-galactosidase, preferably cathepsin B.

[0097] In some embodiments, the cleavable site is a disulfide bond and specific cleavage occurs via a reducing environment, such as an intracellular reducing environment, like acidic pH conditions. For example, the corresponding linker has the following structure:

[0098] (Amanita toxin)-(CH2)2-SS-(CH2)2-XS-(antibody)

[0099] (Amatoxin)-(CH2)3-SS-(CH2)2-XS-(antibody);

[0100] (Amanita toxin)-(CH2)2-SS-(CH2)3-XS-(antibody);

[0101] (Amatoxin)-(CH2)3-SS-(CH2)3-XS-(antibody),

[0102] Where X is as disclosed above.

[0103] In some embodiments, the linker is a pH-sensitive linker and is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are cleavable under acidic conditions. This cleavage strategy typically utilizes the lower pH of the intracellular compartments of endosomes (pH 5–6) and lysosomes (pH 4.8) compared to the cytoplasm (pH ~7.4) to trigger the hydrolysis of acid-labile groups in the linker, such as hydrazones (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-labile and / or hydrolyzable linker. For example, acid-labile linkers that are hydrolyzable in lysosomes and contain acid-labile groups (e.g., hydrazones, thioureas, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used. See, for example, U.S. Patents 5,122,368; 5,824,805; and 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 those in blood), but unstable below pH 5.5 or 5.0 (approximately the pH of lysosomes). In some embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to a therapeutic agent via an acylhydrazone bond). See, for example, U.S. Patent 5,622,929.

[0104] According to some embodiments, the cleavable linker of the present invention is an enzymatically cleavable linker. The enzymatically cleavable linker includes a cleavable site, which is an enzymatically cleavable moiety comprising two or more amino acids. Preferably, the enzymatically cleavable moiety includes 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, valine-alanine-valine (Val-Ala-Val), leucine-alanine-leucine (Leu-Ala-Leu), glycine-phenylalanine-lysine (Gly-Phe -Lys), isoleucine-alanine-leucine (Ile-Ala-Leu) tripeptide, or 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) peptide or β-glucuronide or β-galactoside.

[0105] In some embodiments, the enzymatically cleavable linker according to the invention is a β-glucuronide-based linker. The easy release of the drug can be achieved by cleaving the β-glucuronide bond by a lysosomal enzyme, β-glucuronidease. This enzyme is abundant in lysosomes and overexpressed in some tumor types, while its activity is low extracellularly. The β-glucuronide-based linker can be used to circumvent the tendency of the conjugates of the invention to aggregate due to the hydrophilicity of β-glucuronide. Relevant linkers are disclosed, for example, in WO2007011968A2, or β-galactoside-cleavable linkers in WO19192979A1, the contents of which are incorporated herein by reference.

[0106] In some embodiments, the cleavable linker of the conjugates of the present invention, as disclosed herein, is a self-degradable linker. The term "self-degradable linker" or "self-degradable spacer" refers to a bifunctional chemical moiety capable of covalently linking two chemical parts into a generally stable tripartate molecule. If the bond with the first part is cleaved, the self-degradable spacer is capable of spontaneously separating from the second part. The linker of the conjugates of the present invention, as disclosed herein, comprises a "self-degradable" group, such as the aforementioned PAB or PABC (p-aminobenzyloxycarbonyl), which is disclosed, for example, in: Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al. (1983) J. Med. Chem. 26:638-644; US6214345; US20030130189; US US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; WO098 / 13059; US20040052793; US6677435; US5621002; US20040121940; WO02004 / 032828 or WO2005 / 112919. Other such chemical components (“self-degrading linkers”) capable of carrying out this process include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoliums, and aminopyrimidines. Linking groups containing such heterocyclic self-degrading groups are disclosed, for example, in: U.S. Patent Nos. 20160303254 and 20150079114, and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and US 7223837. In some embodiments, the dipeptide is used in combination with the self-degrading linking group.

[0107] In a preferred embodiment, the enzymatically cleavable linker according to the present invention is a cathepsin B-cleavable linker and comprises a dipeptide selected from the following: Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit, and Val-Cit, or a tripeptide selected from the following: valine-alanine-valine (Val-Ala-Val), leucine-alanine-leucine (Leu-Ala-Leu), glycine-phenylalanine-lysine (Gly-Phe-Lys), isoleucine-alanine-leucine (Ile-Ala-Leu) tripeptide, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly), or alanine-alanine-proline-valine (Ala Ala Pro Val) peptide.

[0108] In a particularly preferred embodiment, the enzymatically cleavable linker according to the 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, wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and amatoxin as disclosed below, wherein the wavy line indicates the linking site of the amatoxin and antibody in the conjugate as disclosed herein:

[0109]

[0110]

[0111] Therefore, the conjugates as disclosed herein included in the pharmaceutical compositions of the present invention comprise, for example, an enzymatically cleavable portion comprising any of the following dipeptide-PAB portions disclosed above: Phe-Lys-PAB, Val-Lys-PAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit-PAB, or Val-Cit-PAB.

[0112] Preferably, the cleavable portion of the conjugate of the present invention comprises the dipeptide-PAB moiety Val-Ala-PAB.

[0113]

[0114] The PAB portion is connected to amatoxins.

[0115] According to some embodiments, the cleavable linker of the present invention disclosed herein comprises a thiol reactive group selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazine-2-ylamino group, methyl-sulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methyl thiosulfonate or maleimide.

[0116] According to a preferred embodiment, the thiol reactive group is a maleimide (maleimide group moiety) as described below:

[0117]

[0118] Linkers containing the reactive group (e.g., cleavable and / or non-cleavable linkers) are particularly useful for covalently linking linker-amatoxin conjugates, as disclosed herein, to antibodies containing reactive thiols (such as, for example, cysteine-engineered antibodies containing at least one reactive cysteine ​​residue for coupling).

[0119] According to a particularly preferred embodiment, the linker of the present invention comprises structure (i) before coupling to the target-binding portion, or comprises structure (ii) after coupling to the target-binding portion, the target-binding portion being, for example, an antibody as disclosed herein:

[0120]

[0121] According to some embodiments, the linker of the conjugates of the present invention is an uncleavable linker. "Uncleavable linker" is understood to be resistant to enzymatic cleavage by, for example, cathepsin B and released from the conjugate of the present invention during degradation (e.g., lysosomal degradation) within target cells. Suitable uncleavable linkers according to the present invention may, for example, comprise one or more groups selected from: bond, -(C=O)-, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkenylene, C2-C6 alkyne, C2-C6 alkyne, C3-C6 cycloalkylene, heterocycloalkylene, aryl, heteroaryl, and combinations thereof, each of which may optionally be substituted, and / or may contain one or more heteroatoms (e.g., S, N, or O) on one or more carbon atoms. Non-limiting examples of such groups include (CH2). p (C=O)(CH2) p And polyethylene glycol (PEG; (CH2CH2O) p (a) unit, where each occurrence of p is an integer selected independently from 1 to 6.

[0122] In some embodiments, the non-cleavable linker according to the invention comprises one or more of the following: bond, -(C=O)-, -C(O)NH- group, -OC(O)NH- group, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkyneyl, C2-C6 heteroalkynyl, C3-C6 cycloalkylene, heterocycloalkyl, aryl, heteroaryl, -(CH2CH2O)p- group, wherein p is an integer from 1 to 6, wherein each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 The alkenyl, C2-C6 heteroalkyl, C2-C6 ynylene, C2-C6 heteroalkylene, C3-C6 cycloalkylene, heteroalkylene, aryl or heteroaryl may optionally be substituted by 1 to 5 groups, each time independently selected from the following groups: alkyl, alkenyl, ynylene, cycloalkyl, heteroalkylene, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate group, aryl, heteroaryl, thionyl, sulfonyl, hydroxyl, alkoxy, thiol, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, mercapto and nitro.

[0123] For example, each of the C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkyneylene, C2-C6 heterokyneylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene groups as disclosed herein may optionally be interrupted by one or more heteroatoms selected from O, S, and N and may optionally be substituted, for example, by one to five groups that, each time appearing independently, are selected from: alkyl, alkenyl, alkyne, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate group, aryl, heteroaryl, thionyl, sulfonyl, hydroxyl, alkoxy, thiol, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, mercapto, and nitro.

[0124] According to a preferred embodiment, the insulatory linker of the conjugate of the present invention comprises -(CH2). n - unit, where n is an integer from 2 to 12, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or preferably where n is an integer from 2 to 6, for example, n is 1, 2, 3, 4, 5 or 6.

[0125] In a preferred embodiment, the uncleavable group of the conjugate of the present invention comprises -(CH2). n - where n is 6, and the connection basis is represented by the following formula:

[0126]

[0127] In some embodiments, the non-cleavable linker as disclosed herein further comprises a thiol reactive group. The thiol reactive group of the non-cleavable linker as disclosed above may be selected, for example, from the following: bromoacetamide, iodoacetamide, methanesulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazine-2-ylamino group, methyl-sulfonylphenyltetrazole or methanesulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methyl thiosulfonate or maleimide.

[0128] According to a preferred embodiment, the thiol reactive group is a maleimide (maleimide group portion) as disclosed above. For example, the insoluble linker containing the maleimide may have, for example, the following structure, wherein the wavy line at the end of the linker indicates the connection point of the amatoxin:

[0129]

[0130] Where n is an integer from 2 to 12, for example, n is 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably where n is an integer from 2 to 6, for example, n is 1, 2, 3, 4, 5 or 6, more preferably, n is 6.

[0131] Following a reactive thiol group (e.g., a naturally occurring or engineered cysteine ​​residue on the antibody) conjugated to the antibody, the maleimide moiety of a cleavable or incleavable linker, as disclosed herein, comprises the following structure:

[0132]

[0133] The wavy line indicates the linking site of the cleavable or non-cleavable linker (L) as disclosed herein, and the sulfur atom is part of the reactive cysteine ​​of the antibody.

[0134] According to a preferred embodiment, the conjugate of the present invention comprises a cleavable or non-cleavable linker as described herein, and further comprises a thiol-reactive group that can be coupled to a naturally occurring thiol moiety of an antibody of the conjugate, or the cleavable or non-cleavable linker of the conjugate of the present invention comprising a thiol-reactive group can be coupled to a thiol moiety of an antibody introduced by genetic engineering as described, for example, in Nat Biotechnol. 2008 Aug; 26(8):925-32 or WO2006 / 034488A2. Preferably, the cleavable or non-cleavable linker comprising a thiol-reactive group as disclosed herein is coupled to a thiol moiety of the Fc region of the corresponding antibody introduced into the conjugate according to the present invention by genetic engineering, such as, for example, D265C (according to EU number).

[0135] In some embodiments, at least one amatoxin and at least one linker of the pharmaceutical composition conjugate of the present invention disclosed herein are represented by formula (Ia):

[0136]

[0137] in:

[0138] R1 represents H, OH, or OR. A OR C ;

[0139] R2 represents H, OH, or OR. B OR C ;

[0140] R A and R B (When present) they combine with the oxygen atoms to which they are attached to form 5-membered heterocyclic alkyl groups;

[0141] R3 represents H and R. C ,or

[0142] Each of R4, R5, R6, and R7 is independently H, OH, OR. C R C ,

[0143] R8 represents OH, NH2, or OR. C or NHR C ,

[0144] Q is -S-, -S(O)-, or -SO2-;

[0145] R C for-

[0146]

[0147] The sulfur atom is part of the reactive cysteine ​​residue of the antibody, and L is a linker (e.g., a cleavable or uncleavable linker as defined herein) and is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 ynyl, optionally substituted C2-C6 heteroynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or –((CH2) m O) n (CH2) m – where m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0148] In some implementation schemes, R A and RB Together with the oxygen atoms attached to them, they form 5-membered heterocyclic alkyl groups of the following formula:

[0149]

[0150] Where Y is -(C=O)-, -(C=S)-, -(C=NR)-, E) -or-(CR) E R E’ )-;and

[0151] Where R E and R E Each is independently H, C1-C6 alkylene-R C C1-C6 heteroalkylene-R C C2-C6 imenoyl-R C C2-C6 hemienyl-R C C2-C6 ynethynyl-R C C2-C6 hypoyne-R C , cycloalkylene-R C heterocyclic alkyl-R C , aryl-R C or heteroaryl-R C Or combinations thereof, wherein each C1-C6 alkylene-R C C1-C6 heteroalkylene-R C C2-C6 imenoyl-R C C2-C6 hemienyl-R C C2-C6 ynethynyl-R C C2-C6 hypoyne-R C , cycloalkylene-R C heterocyclic alkyl-R C , aryl-R C or heteroaryl-R C The substituent may be selected independently from the following groups each time it appears: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, thionyl, sulfonyl, hydroxyl, alkoxy, thiol, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, mercapto, and nitro.

[0152] In some embodiments, R1, R2 and R9 are OH in at least one amatoxin according to formula (Ia) disclosed above;

[0153] R3 represents H and R. C Or R D ;

[0154] Each of R4, R5, R6, and R7 is independently H, OH, OR. C R C ,

[0155] R8 represents OH, NH2, or OR. C NHR C ,

[0156] Q is -S-, -S(O)-, or -SO2-;

[0157] R C As disclosed above.

[0158] In some embodiments, at least one amatoxin and at least one linker of the pharmaceutical composition conjugate of the present invention disclosed herein are represented by formula (IIa):

[0159]

[0160] Where R1, R2, and R9 are OH;

[0161] R5 can be H, OH, or OR independently. C R C ,

[0162] R8 represents OH, NH2, or OR. C NHR C or NR C

[0163] Q is -S-, -S(O)-, or -SO2-;

[0164] R C for-

[0165]

[0166] The sulfur atom is part of the reactive cysteine ​​residue of the antibody, and L is a linker, such as an uncleavable or cleavable linker as defined below, and is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 ynynyl, optionally substituted C2-C6 heteroynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or comprises a dipeptide; or –((CH2) m O) n (CH2) m– where m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and; or L is a cleavable linker, preferably an enzyme-cleavable linker, more preferably a self-degrading cathepsin B-cleavable linker as disclosed herein.

[0167] According to some embodiments, the linker of the conjugate of the present invention, as disclosed herein, is linked to at least one 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.

[0168] In one embodiment of the invention, the pharmaceutical composition of the invention comprises a conjugate as described herein, which comprises amatoxin, the amatoxin comprising (i) amino acid 4 having a 6'-deoxy position and (ii) amino acid 8 having an S-deoxy position.

[0169] According to a particularly preferred embodiment of the invention, the pharmaceutical composition according to the invention for use in treating cancer comprises a conjugate comprising at least one of the following compounds of formula (I) to (XI) as a linker-amatoxin moiety:

[0170]

[0171]

[0172]

[0173]

[0174] Therefore, the pharmaceutical composition of the present invention for use in treating cancer comprises a conjugate comprising an antibody covalently bound to at least one, for example, one, two, three, four, five, six, seven or eight amatoxin-linker conjugates (I)-(XI), preferably about one to about four, or about three to about six, preferably about two to about three, more preferably about two amatoxin-linker conjugates (I)-(XI).

[0175] According to a preferred embodiment, the pharmaceutical composition according to the invention for use in treating cancer comprises a conjugate containing an antibody covalently conjugated to the amatoxin linker moiety via a thioether bond according to any one of formulas XII to XXII.

[0176]

[0177]

[0178]

[0179]

[0180]

[0181] The amatoxin-linking base is coupled to a thiol group of a cysteine ​​residue of the antibody or antibody moiety according to the invention, and wherein n is preferably about 1, 2, 3 to about 4, 5, 6, 7, 8, preferably, wherein n is about 1, 1.5, 2, 2.5 to about 3.5, 4.5, 5.5, more preferably, wherein n is about 1.5 to about 3.5, and most preferably, wherein n is about 2. The thiol group of the antibody or antibody moiety may, for example, be a naturally occurring cysteine ​​residue of the antibody, such as those forming an interchain disulfide bond upon reduction of the cysteine ​​residue based on the thiol conjugation, or the thiol group of the cysteine ​​residue of the antibody may be genetically engineered, preferably at position D265 (according to EU number, D265C).

[0182] According to preferred embodiments, the pharmaceutical compositions of the present invention, as disclosed herein, for use in cancer treatment, may be used, for example, in the treatment of solid tumors or non-solid tumors. For example, the solid tumor is selected from the group including: gastric cancer, adenocarcinoma, melanoma, ovarian cancer, uterine cancer, cervical cancer, breast cancer (including triple-negative breast cancer), bronchial cancer, 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 gland cancer, biliary tract cancer, rectal cancer, endometrial cancer, desmoidoma, fibroproliferative small round cell tumor, neuroectodermal tumor, retinoblastoma, rhabdomyosarcoma, Wilms' tumors, osteosarcoma, chondrosarcoma, lung cancer, non-small cell lung cancer (NSCLC), alveolar rhabdomyosarcoma, rhabdomyosarcoma, Askin's tumor. Tumor), intra-abdominal fibroproliferative small cell tumor, malignant papillary renal cell carcinoma, meningioma, small cell lung cancer, ependymoma, nasal glioma (olfactory neuroblastoma), fibromatosis, ganglion glioma, islet cell tumor, basal cell carcinoma and squamous cell carcinoma, large cell neuroendocrine carcinoma (LCNEC), Leydig cell tumor, salivary gland carcinoma, pineal blastoma, pleomorphic low-grade adenocarcinoma, schwannoma, teratoma, thymoma. Non-solid tumors may include, for example, one of the following: Hodgkin-lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma (DBNHL), subtypes of non-Hodgkin lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), or multiple myeloma, Burkitt's lymphoma, anaplastic large cell lymphoma, or marginal zone B-cell lymphoma.

[0183] As used herein, the term "cancer" should be a general term for a disease in which abnormal cells divide uncontrollably. Cancer cells can invade nearby tissues and can spread to other parts of the body via the bloodstream and lymphatic system. There are several main types of cancer; for example, carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is cancer that begins in hematopoietic tissues (such as bone marrow) and causes a large number of abnormal blood cells to be produced and enter the bloodstream. Lymphoma is cancer that begins in cells of the immune system.

[0184] Tumors form when normal cells lose their ability to function as specific, controlled, and coordinated units. Typically, solid tumors are abnormal masses of tissue that do not usually contain cysts or fluid-filled areas. A single tumor may even contain different cell populations with different abnormal processes. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named according to the types of cells that form them; examples of solid tumors include sarcomas, carcinomas, and lymphomas. Leukemia (blood cancers) typically does not form solid tumors.

[0185] According to one embodiment, cancer cells or tumors disclosed above are characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13). For example, cancer cells or tumors that can be treated, for instance, with the pharmaceutical compositions of the present invention disclosed herein are characterized by a hemizygous deletion of the POLR2A gene, or a hemizygous deletion of both the TP53 and POLR2A genes. As used in the present invention, the term "hemizygous" refers to an individual or cell having only one complete gene or chromosomal segment allele, rather than the usual two. A hemizygote is a cell or organism whose genome contains only one complete allele at a given locus, regardless of whether the allele is wild-type or mutant. For example, cells of any of the tumors or cancers described above are hemizygous at chromosomal locus 17p13, and preferably, cells of cancers or tumors disclosed above are hemizygous for both the TP53 and POLR2A genes. As used herein, "TP53" refers to the "tumor protein 53" gene, which encodes a tumor suppressor protein (P53) comprising a transcriptional activation domain, a DNA-binding domain, and an oligomerization domain. The encoded protein responds to various cellular stresses to regulate the expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes. Mutations in this gene are associated with a variety of human cancers, including hereditary cancers such as Li-Fraumeni syndrome.

[0186] In most human tumors, the tumor suppressor gene TP53 is typically inactivated through mutation or deletion. As used herein, “POLR2A” refers to the POLR2A gene encoding the largest subunit of the human RNA polymerase II complex and is essential for polymerase activity in mRNA synthesis. Hemizygous deletions of chromosome 17p13 (e.g., del(17p13.1)) can be detected by fluorescence in situ hybridization (FISH) as disclosed by Merz et al., Am J Hematol. 2016 Nov; 91(11): E473-E477.

[0187] For example, with regard to the absence of TP53 and / or POLR2A, the cells of the cancer types or tumors disclosed herein may not be a homogeneous cell population. For example, approximately 1%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% to approximately 70%, 75%, 80%, 85%, 90%, 95%, 100% of the cancer cells disclosed above, or approximately 70%, 75%, 80%, 85% to approximately 90%, 92.5%, 95%, 97.5%, 100%, may be... Hemizygotes of del(17p13.1), TP53, and / or POLR2A, or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of cancer cells as disclosed above are del(17p13) hemizygotes, or TP53 and / or POLR2A hemizygotes. The use of the pharmaceutical compositions disclosed herein in the treatment of cancers according to the invention may be particularly advantageous, for example, for any of the tumors or cancers as disclosed above characterized by the absence of chromatin 17p13.1, TP53, and / or POLR2A hemizygotes, because said tumors or cancers are more sensitive to the use of the pharmaceutical compositions of the invention as disclosed herein by at least 10, 25, 50, 100, 250, 500, or 1000 times. Therefore, determining whether cancer cells as disclosed herein contain or consist of cells that are hemizygous as lacking TP53 and / or POLR2A may be advantageous, for example, because as little as 10, 25, 50, 100, 250, 500, or 1000 times the conjugates, pharmaceutical compositions, or compositions disclosed herein can be used to achieve the desired therapeutic effect. Assays assessing the sensitivity of cancer cells or tumors as disclosed above to treatment with pharmaceutical compositions of the present invention containing conjugates as disclosed herein can be performed, for example, as described in Nature. 2015 April 30; 520(7549):697–701.

[0188] The pharmaceutical compositions for use according to the invention disclosed herein are preferably in liquid form, and preferably preparations for subcutaneous administration to patients in need of cancer. According to some embodiments, the pharmaceutical compositions for use according to the invention disclosed herein further comprise one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, binders, lubricants, flow aids, disintegrants, absorbents, and / or preservatives.

[0189] For example, the pharmaceutical compositions available according to the invention may contain at least one buffer to achieve a pH of 4 to 8, preferably 5 to 7, more preferably about 5.5 to about 6.5, or, for example, about 5, 5.5, 6, 6.5. The appropriate buffer may preferably be one or more selected from the following: malate, fumarate, citrate, acetate, propionate, pyridine, piperazine, carcodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carbonate, 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), hydroxyethylpiperazine ethanesulfonic acid (HEPES), pyrophosphate, and triethanolamine, with histidine buffers being more preferred, for example, L-histidine / HCl, but not limited thereto. The concentration range of the at least one buffer is 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, and more preferably about 5 mM, 10 mM, 25 mM to about 50 mM.

[0190] For example, the pharmaceutical compositions of the present invention disclosed herein may further comprise one or more stabilizers. In the pharmaceutical compositions according to the invention, stabilizers may be used without limitation, provided they are commonly used in the art for the purpose of stabilizing proteins, and preferably, said stabilizers may be selected, for example, from one or more of the following: carbohydrates, sugars or their hydrates, sugar alcohols or their hydrates, and amino acids. For example, carbohydrates, sugars, or sugar alcohols used as stabilizers may be selected from one or more of the following: trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabinitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idoteol, inositol, heptaheptaol, isomaltose, maltitol, polyhydroxyol, cyclodextrin, hydroxypropyl cyclodextrin, and glucose, but are not limited thereto. The sugar or sugar alcohol used as a stabilizer may be present in the pharmaceutical compositions of the present invention as disclosed herein at concentrations 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, at concentrations of 10 mM, 25 mM, 50 mM, 100 mM, 150 mM. The amino acids (if present) in the pharmaceutical compositions of the present invention may be selected from one or more of the following: glutamine, glutamic acid, glycine, lysine, dilysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolidone, histidine, and alanine, but are not limited thereto. The amino acids used as stabilizers in the pharmaceutical compositions according to the present invention may, for example, have concentrations from about 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, 25 mM, 50 mM to about 75 mM, 100 mM, preferably from about 5 mM, 7.5 mM, 10 mM, 25 mM to about 30 mM, 40 mM, 50 mM.

[0191] For example, the pharmaceutical composition according to the invention may further comprise a nonionic surfactant, such as, for example, polyoxyethylene sorbitan fatty acid ester (polysorbate or Tween), polyoxyethylene-polypropylene glycol, polyoxyethylene stearate, polyoxyethylene alkyl ether, for example, polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether [Triton-X], and polyoxyethylene-polypropylene copolymer [poloxam and pranic], and sodium dodecyl sulfate (SDS), polysorbate 20 or polysorbate 80; polyethylene glycol hexadecyl ether (… 56); Polyethylene glycol octadecyl ether ( 72); Polyoxyethylene 10 oil ether ( 97); Poloxamer 188, tert-octylphenoxypolyethoxyethanol ( (R)X 100); Polyethylene glycol dehydrated sorbitan monolaurate ( 20); Polyoxyethylene dehydrated sorbitan monopalmitate ( 40); Polyethylene glycol dehydrated sorbitan monostearate ( (R)60); Polyoxyethylene dehydrated sorbitan tristearate ( 65); Polyethylene glycol dehydrated sorbitan monooleate (TWEEN 80); Polyoxyethylene dehydrated sorbitan trioleate ( 85); Tris(hydroxymethyl)aminomethane lauryl sulfate ( Dodecyl sulfate); block copolymers of polyethylene glycol and polypropylene glycol ( F68). The nonionic surfactant disclosed above may be present in the pharmaceutical composition according to the invention, for example, in the range of about 0.01% (w / v) to about 0.5% (w / v), preferably about 0.1% (w / v), 0.2% (w / v), 0.3% (w / v) to about 0.4% (w / v), and more preferably about 0.1% (w / v) to 0.25% (w / v), 0.35% (w / v), 0.5% (w / v), wherein the expression "(w / v)" refers to weight-volume ratio.

[0192] For example, the pharmaceutical compositions for use according to the invention disclosed herein may further comprise, for example, a diluent selected from the following: mannitol, microcrystalline cellulose, lactose, starch, anhydrous calcium hydrogen phosphate, tricalcium phosphate, kaolin, sucrose, precipitated calcium carbonate, sorbitol, maltodextrin, powdered cellulose, microcrystalline cellulose, and other known substances having such properties. The diluent may be in amounts from 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), 45% (w / v), 50% (w / v), etc. (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) are present in the pharmaceutical compositions available for use according to the invention.

[0193] For example, the pharmaceutical compositions for use according to the present invention, as disclosed herein, may contain lubricants selected from: stearic acid, sodium stearoyl fumarate, polyethylene glycol, magnesium stearate, calcium stearate, talc, zinc stearate, hydrogenated castor oil, silica, colloidal silica, corn starch, calcium silicate, magnesium silicate, silica hydrogel, and other known substances having such properties. The lubricant may be present in the pharmaceutical composition for use according to the invention, for example, from about 0%, 0.1%, 0.25%, 0.5%, 0.75%, 1% to about 1.25%, 1.5%, 1.75%, 2%, 2.5%, 3.0%, or from about 1.25%, 1.5%, 1.75%, 2%, 2.5% to about 3%, 3.5%, 4%, 5% (w / v).

[0194] For example, pharmaceutical compositions for use according to the present invention as disclosed herein may contain a binder selected from the group consisting of: polyvinylpyrrolidone, hydroxypropyl methylcellulose, gum arabic, alginate, hydroxypropyl cellulose, sodium carboxymethyl cellulose, compressible sugar, ethyl cellulose, gelatin, liquid glucose, methylcellulose, pregelatinized starch, and other substances known to those skilled in the art. The binder in the dosage form ranges from 0% to 5.0% by weight.

[0195] For example, a pharmaceutical composition for use according to the invention disclosed herein may comprise a flow aid selected from the group consisting of colloidal silica, colloidal silica, corn starch, talc, calcium silicate, magnesium silicate, colloidal silica, or silicone hydrogel. The flow aid may be present in the pharmaceutical composition, for example, in amounts 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 from about 1.25% (w / v), 1.5% (w / v), 1.75% (w / v) to about 2% (w / v) by weight.

[0196] The pharmaceutical compositions of the present invention may, for example, further comprise a preservative, wherein the "preservative" according to the invention is a compound that can be added to the pharmaceutical compositions disclosed herein to reduce bacterial growth. Adding a preservative may, for example, facilitate the multi-purpose or multi-dosing production of the pharmaceutical compositions of the present invention. Examples of potential preservatives include octadecyl dimethylbenzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride (a mixture of alkylbenzyl dimethyl ammonium chloride, wherein the alkyl group is a long-chain compound), and benzyl chloride. Other types of preservatives include aromatic alcohols (such as phenol, butanol, and benzyl alcohol), parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The preferred preservative described herein is benzyl alcohol at a concentration of about 0.9% (w / v) to about 2.0% (w / v), such as 0.9% (w / v), 1% (w / v), 1.25% (w / v), 1.5% (w / v), 1.75% (w / v) or 2% (w / v).

[0197] Typically, the injection volume of pharmaceutical compositions used in this invention via the subcutaneous route is 3 ml or less, 2 ml or less, because it is desirable to use pharmaceutical compositions having high concentrations of the conjugates of this invention for administration. Therefore, it is desirable to use pharmaceutical compositions according to the invention comprising the conjugates of this invention as disclosed herein, at concentrations 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, 200 mg / ml, or about 50 mg / ml, 60 mg / ml, 75 mg / ml, 100 mg / ml, ... 125 mg / ml to about 160 mg / ml, 180 mg / ml, 200 mg / ml, 250 mg / ml, 300 mg / ml, 350 mg / ml, 400 mg / ml, 450 mg / ml, 500 mg / ml or 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 about 25 mg / ml.

[0198] According to some embodiments, the amount of the conjugate of the invention disclosed herein for use in the treatment of cancer, injected in volumes of 3 ml or less, 2.5 ml or less, 2.0 ml or less, 1.5 ml or less, is 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 conjugate of the invention to be injected, and correspondingly the amount and composition of the pharmaceutical composition of the invention to be injected, may depend on the type of cancer or tumor, the stage of cancer, the overall health condition of the patient to be treated, and the person's weight and body type. Fixed-dose subcutaneous administration of the pharmaceutical composition of the invention may be desirable, for example, if combination therapy (e.g., in combination with standard care treatment) is planned. The combination therapy may be desirable, for example, to reduce the risk of adverse reactions relative to a single therapy of standard care or a corresponding single therapy of subcutaneous administration of the pharmaceutical composition of the present invention, (iii) may reduce overall costs and (iv) improve patient medication adherence.

[0199] In one embodiment, the viscosity of the pharmaceutical composition of the present invention is from 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 from about 10 cP, 15 cP, 20 cP to about 30 cP, 40 cP, 50 cP, 60 cP, preferably between 5 cP, 10 cP to about 25 cP, 30 cP. As used herein, the term “viscosity” may be “kinematic viscosity” or “absolute viscosity.” “Kinematic viscosity” is a measure of a fluid’s resistance to flow under gravity. When equal volumes of two fluids are placed in the same capillary viscometer and allowed to flow under gravity, the viscous fluid takes longer to pass through the capillary than the less viscous fluid. If one fluid completes its flow in 100 seconds and another fluid completes its flow in 200 seconds, then on the kinematic viscosity scale, the viscosity of the second fluid is twice that of the first fluid. "Absolute viscosity" (sometimes also called dynamic viscosity or simple viscosity) is the product of kinematic viscosity and fluid density: Absolute viscosity = kinematic viscosity × density.

[0200] The dimension of kinematic viscosity is L. 2 / T, where L is length and T is time. Kinematic viscosity is typically expressed in centistokees (cSt). The SI unit for kinematic viscosity is mm. 2 / s, which is 1 cSt. Absolute viscosity is expressed in 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, for example, according to the method disclosed in WO2016027859A1 (the contents of which are incorporated herein by reference).

[0201] Pharmaceutical compositions containing antibodies or immunoconjugates (such as the conjugates of the present invention) may have a viscosity higher than 20 cP, 30 cP, 40 cP, or 50 cP at concentrations sufficient for subcutaneous administration of an effective amount of the conjugates of the present invention. Therefore, incorporating one or more viscosity-reducing agents into the pharmaceutical compositions of the present invention as disclosed herein may be advantageous.

[0202] Therefore, the pharmaceutical compositions of the present invention as disclosed herein may optionally further comprise a viscosity reducer. For example, the pharmaceutical compositions of the present invention may comprise an effective amount of 1-butyl-3-methylimidazolium methanesulfonate (BMIMes) and optionally one or more other ionic liquids to significantly reduce viscosity. Representative ionic liquids include 4-(3-butyl-1-imidazolium)-1-butanesulfonate (BIM), 1-butyl-3-methylimidazolium methanesulfonate (BMIMes), 4-ethyl-4-methylmorpholinium methyl carbonate (EMMC), and 1-butyl-1-methylpyrrolidone chloride (BMP chloride), preferably at a concentration between about 0.10 and about 0.50 M, equivalent to about 20-150 mg / mL as disclosed in EP3043774A1. Other viscosity reducers, such as those disclosed in US9,605,051B2, may be used.

[0203] In some embodiments, the pharmaceutical composition according to the invention further comprises recombinant human hyaluronidase, such as, for example, ruHuPH20. The use of such recombinant hyaluronidase in pharmaceutical compositions according to the invention may be advantageous, for example, if it is necessary to administer a larger injection volume of the pharmaceutical composition of the invention to a patient in need subcutaneously to administer a therapeutically effective dose of the conjugate of the invention, or in cases where it is desirable to reduce the injection pressure that needs to be applied to affect the subcutaneous injection.

[0204] Therefore, according to one embodiment, the pharmaceutical composition for use as disclosed herein may further comprise soluble recombinant hyaluronidase, preferably soluble recombinant human hyaluronidase, more preferably soluble recombinant human PH20. As used herein, "soluble recombinant human PH20 (rHuPH20)" means a composition containing a soluble form of human PH20 recombinantly expressed and secreted in Chinese hamster ovary (CHO) cells, such as disclosed in, for example, WO2004 / 078140A2, or preferably a variant of rHuPH20 disclosed in, for example, WO2013 / 102144A2, the contents of which are incorporated herein by reference.

[0205] As used herein, the term "hyaluronidase" or "hyaluronidase activity" refers to hyaluronidase, a family of enzymes that catalyze the degradation of hyaluronic acid (HA). There are three main classes of hyaluronidases: two eukaryotic endoglucosidases and one prokaryotic lysin-type glycosidase. In the human body, there are five functional hyaluronidases: HYAL1, HYAL2, HYAL3, HYAL4, and HYAL5 (also known as SPAM1 or PH-20); and the pseudogene HYAL6 (also known as HYALP1). The gene clusters of HYAL1-3 are located on chromosome 3, while the clusters of HYAL4-6 are located on chromosome 7. HYAL1 and HYAL2 are the major hyaluronidases in most tissues. GPI-anchored HYAL2 is responsible for cleaving high molecular weight HA, which mostly binds to the CD44 receptor. The resulting HA fragments of varying sizes are then further internalized into endosomes and lysosomes, where they are hydrolyzed by HYAL1 to generate HA oligosaccharides.

[0206] Based on their enzymatic mechanism, hyaluronidases are hyaluronoglucosidase (EC 3.2.1.35), meaning they cleave the (1->4)-bond between N-acetylglucosamine and glucuronide. The term hyaluronidase can also refer to hyaluronoglucuronidase (EC 3.2.1.36), which cleaves the (1->3)-bond. Additionally, bacterial hyaluronide lyase (EC 4.2.2.1) can be called hyaluronidase, although this is less common.

[0207] Mammalian hyaluronidase (EC 3.2.1.35) is an endo-β-N-acetylhexosaminease that produces tetrasaccharides and hexasaccharides as its main end products. It possesses both hydrolytic and transglycosidic activities and can degrade hyaluronic acid and chondroitin sulfate (CS), typically C4-S and C6-S.

[0208] Mammalian hyaluronidases can be further divided into two categories: neutral-active enzymes and acidic-active enzymes. There are six classes of hyaluronidase genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, and PH20 / SPAM1. HYALP1 is a pseudogene, HYAL3 has not been shown to have enzymatic activity against any known substrate, and HYAL4 is a chondroitinase with low activity towards hyaluronic acid. HYAL1 is a typical acidic-active enzyme, and PH20 is a typical neutral-active enzyme. Acidic-active hyaluronidases (such as HYAL1 and HYAL2) generally lack catalytic activity at neutral pH (i.e., pH 7). For example, HYAL1 has almost no in vitro catalytic activity above pH 4.5 (Frost et al., Anal Biochemistry, 1997). HYAL2 is an acidic-active enzyme with extremely low in vitro specific activity. Studies have found that recombinant human hyaluronidase PH20 (rHuPH20) exhibits optimal reaction rates on HA substrates with sizes ranging from 90 to 752 kDa at pH ranges of 4.5–5.5, where rHuPH20 follows Michaelis-Menten kinetics in the initial reaction time (see, for example, Anal Biochem. 2015 Jul 1; 480:74–81). Therefore, if a pharmaceutical composition according to the invention comprises rHuPH20, the pH of the pharmaceutical composition for use according to the invention, which further comprises rHuPH20, should be selected such that the activity of rHuPH20 is substantially optimal without negatively impacting the stability of the conjugate of the pharmaceutical composition for use according to the invention as disclosed herein. Therefore, the pH of the pharmaceutical composition supplied according to the 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 required for the pharmaceutical composition of the invention results in hyaluronidase activity corresponding to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, or less than 40% of rHuPH20 activity at pH 4.5-pH 5.5, the amount of rHuPH20 in the pharmaceutical composition supplied according to the invention may be increased to offset the decrease in activity. The hyaluronidase activity of rHuPH20 may be determined, for example, as disclosed in WO2013 / 102144, or, for example, as disclosed in Anal Biochem.2015 Jul 1;480:74-81.

[0209] The pharmaceutical compositions available according to the invention may, for example, comprise 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, 50 μg / ml of rHuPH20.

[0210] For example, the pharmaceutical compositions for use according to the present invention may contain, for example, from 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. Hyaluronidase activities of approximately 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 approximately 5000 U / ml, 7500 U / ml, 10000 U / ml, with values ​​of approximately 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 approximately 5000 U / ml, 7500 U / ml, 10000 U / ml.

[0211] In some embodiments, the pharmaceutical composition for use according to the invention may comprise a mixture of a conjugate in the amount disclosed herein (preferably a therapeutically effective amount) and rHuPH20 in the amount disclosed above. Alternatively, the pharmaceutical composition for use according to the invention may comprise first and second components, wherein the first component comprises hyaluronidase rHuPH20 in the amount disclosed above and the second component comprises a conjugate as disclosed herein. The first and second components of the pharmaceutical composition may be administered, for example, simultaneously or sequentially. However, preferably, the first and second components of the pharmaceutical composition are administered sequentially, with the first component administered before the second component. Thus, the second component may be administered at 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 the administration of the first component; preferably, it may be administered at 5 minutes, 15 minutes, 30 minutes, or up to 1 hour after the administration of the first component. The first and second components of the pharmaceutical composition of the present invention can preferably be administered subcutaneously at the same injection site or at injection sites that are 0.5 cm, 1 cm, 1.5 cm, 2 cm, or 2.5 cm apart.

[0212] In one embodiment, the subcutaneous injection volume comprising the pharmaceutical composition of the invention as 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.

[0213] In some embodiments, the pharmaceutical compositions available according to the invention are administered in combination with immune checkpoint inhibitors. In the context of this invention, the term "immune checkpoint inhibitor," or simply "checkpoint inhibitor" or "ICI," refers to any agent or compound that directly or indirectly reduces the level of immune checkpoint receptor proteins or molecules present on the surface of immune cells (e.g., T cells) or inhibits their function, or directly or indirectly reduces the level of ligands that bind to said immune checkpoint receptor proteins or molecules (as soluble compounds or on the surface of immunosuppressive cells). These immunosuppressive cells can be, for example, cancer cells, regulatory T cells, tolerant antigen-presenting cells, myeloid-derived suppressor cells, tumor-associated macrophages, or cancer-associated fibroblasts. The ligands are typically capable of binding immune checkpoint receptor proteins or molecules on immune cells. Non-limiting examples of immune checkpoint receptor protein-ligand pairs are PD-1 and PD-L1. PD-1 is an immune checkpoint receptor protein present on T cells. PD-L1 (which can be overexpressed by cancer cells) binds to PD-1 and helps cancer cells evade host immune system attacks. Therefore, immune checkpoint inhibitors prevent PD-1 / PD-L1 interaction by blocking PD-1 on T cells (i.e., acting as PD-1 inhibitors) or PD-L1 on cancer cells (i.e., acting as PD-L1 inhibitors), thereby maintaining or restoring the activity of anti-tumor T-cells or blocking the activity of suppressor cancer cells.

[0214] Therefore, immune checkpoint inhibitors are antagonists of immunosuppressive receptors (such as PD-1), in which case the antagonist inhibits PD-1 or PD-L1 in the PD-1 / PD-L1 pathway. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, humanized or human antibodies that antagonize or block human PD-1 function, such as 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, and AGN-20. 34. Toripalimab (TAB001, JS001) or AMP-514 (MEDI0680), and fully human antibodies such as nivolumab (blocking PD-1) or avelumab, durvalumab, cosibelimab (CK-301), WBP-3155 (CS1001), atezolizumab, envafolimab (KN035) or recombinant anti-PD-L1 precursor CX-072 (pacmilimab).

[0215] Pembrolizumab (formerly known as lambolizumab; trade name Keytruda; also known as MK-3475), disclosed for example 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; it contains a mutation at C228P designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed for example in US 8,354,509 and WO2009 / 114335. It is FDA approved for the treatment of patients with unresectable or metastatic melanoma and metastatic NSCLC.

[0216] Nivolumab (CAS Registry No.: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cytotoxicity (ADCC). Nivolumab is disclosed, for example, in US 8,008,449 and WO2006 / 121168. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0217] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, for example, in WO2009 / 101611.

[0218] PD1-1 to PD1-5 refer to anti-PD-1 antibodies as disclosed in WO2018 / 220169.

[0219] Ipilumumab (CAS Registry No.: 477202-00-9, also known as 10D1, 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 competitively binds to B7 on antigen-presenting cells against stimulating CD28. Both CTLA-4 and CD28 are expressed on the surface of T cells. Ipilumumab is a human IgG1 antibody that binds to CTLA-4, preventing the suppression of T-cell-mediated antitumor immune responses. Ipilumumab is disclosed, for example, as antibody "10D1" in WO 01 / 14424.

[0220] Envafolimab (CAS Registry No.: 2102192-68-5) is a subcutaneous (SC) single-domain anti-programmed death-ligand 1 (PD-L1) antibody, as disclosed in US11,377,497B2.

[0221] As used herein, the INN designation for the antibodies disclosed herein is intended to also encompass all biosimilar antibodies of the corresponding originator antibodies disclosed herein, including, but not limited to, biosimilar antibodies approved in the United States under 42:262(k) of the United States Code and in other jurisdictions under equivalent regulations. As used herein, the term "biosimilar" means an antibody that contains the same amino acid sequence as the antibody originally identified by the INN; however, biosimilar antibodies may differ in their glycosylation.

[0222] The pharmaceutical compositions disclosed herein may be administered, for example, in patients with solid tumors selected from the list of solid tumors disclosed herein, in combination with immune checkpoint inhibitors.

[0223] The pharmaceutical compositions for use according to the invention disclosed herein may be administered, for example, in conjunction with the subcutaneous pharmaceutical compositions of the invention. For example, the pharmaceutical compositions for use according to the invention may be administered, for example, before, simultaneously with, or after the administration of an immune checkpoint inhibitor. If administered before the administration of an immune checkpoint inhibitor, the pharmaceutical compositions disclosed herein may be administered subcutaneously 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 inhibitors disclosed herein may also 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 invention. In an alternative embodiment, the pharmaceutical composition according to the invention may also be administered simultaneously with an immune checkpoint inhibitor as disclosed herein, for example, at intervals of 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, or 15 minutes. Corresponding combinations of the conjugates according to the invention with immune checkpoint inhibitors are disclosed, for example, in WO2022 / 096604A1, the entire contents of which are incorporated herein by reference.

[0224] In one embodiment, the pharmaceutical composition according to the invention for use in treating cancer is administered subcutaneously for treating any of the aforementioned cancers and tumor types. For example, the pharmaceutical composition can be administered subcutaneously using a suitable means for subcutaneous injection (such as a syringe or auto-injector as disclosed above). For example, depending on the volume to be injected and taking into account the overall health of the patient requiring cancer treatment, the pharmaceutical composition can be injected subcutaneously into the side or back of the upper arm, the abdomen, or the front of the patient's thigh.

[0225] According to one embodiment, a pharmaceutical composition for use in the treatment of cancer is administered subcutaneously at least once (e.g., at least one dose) to a patient diagnosed with at least one type of cancer as disclosed herein.

[0226] According to one embodiment, the antibody of the conjugate of the present invention specifically binds to cell surface antigens on tumor cells, such as tumor-specific antigens or tumor-associated antigens. As used herein, the term "specific binding" or any grammatical variation thereof means that the binding of the targeting portion of the present invention (such as, for example, the antibody or antibody portion of the conjugate disclosed herein) to its antigen has a binding strength of at least about 10. -6M, 10 -7 M, 10 -8 M, or about 10 -8 M to approximately 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or approximately 5x10 -9 M, 5x10 -10 M to approximately 2.5x10 -11 M, 5x10 -11 M, 2.5x10 -12 M, 5x10 -12 M of K d As used herein, tumor-specific antigens are antigenic substances produced in tumor cells that trigger an immune response in the host. Tumor antigens are useful tumor markers for identifying tumor cells using diagnostic tests. Tumor antigens may be, for example, one of the glycans, proteins, or peptides expressed on the surface of cancer cells and not expressed in healthy tissues or cells. As used herein, the term "tumor-specific antigen" (TSA) may be, for example, one of the glycans, proteins, or peptides and 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-cancer cells.

[0227] According to one embodiment, the conjugates of the present invention contained in pharmaceutical compositions disclosed herein for use in the treatment of cancer can target any suitable epitope on target cells (such as, for example, epitopes on cancer cells), said cancer being, for example, any of the cancers disclosed herein. As used herein, the term “epitaph” refers to a portion of a macromolecule (preferably a polypeptide) that is recognized by an antigen-recognizing molecule (such as an antibody or its antigen-binding fragment or its antigen-binding derivative as disclosed herein), and more particularly by the antigen-binding site of said molecule. An epitope defines the minimal binding site of an antibody molecule and thus represents a 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 a region in close contact with an antibody and is generally revealed by its structure. A “functional epitope” is defined as those molecular portions that contribute energy to binding such that when they are altered, the binding affinity decreases. Therefore, when defining an epitope, it is important to consider which residues bind to the paratope and which residues contribute affinity (regardless of whether they are adjacent to the paratope). Structural epitopes can be, for example, linear continuous sequences of about 5 to about 50 or 100 amino acids in length, or conformational epitopes formed by the three-dimensional structure of a polypeptide. Epitopes that can be specifically bound by antibodies to the conjugates of the present invention can also be, for example, contained on non-protein structures of cancer cells, such as, for example, glycans, such as Lewis antigens (sialic acid Lewis x (SLe)). x ) and sialic acid Lewis a (SLe a )).

[0228] According to some embodiments, the antibodies of the conjugates of the present invention specifically bind to surface antigens on tumor cells, such as tumor-specific antigens, or selected from tumor-associated antigens including: 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, MUC1. 17. 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, phosphatidylinositol polysaccharide-1, mesothelin, (prostate stem cell antigen), GAGE-1 (G antigen 1), gangliosides / GD2, GnT-V, β1,6-N (acetylglucosamine transferase-V), UPAR (urokinase type plasminogen activator receptor), sialic acid Lewis x (SLe x ) and sialic acid Lewis a (SLe a ).

[0229] According to a preferred embodiment, the pharmaceutical composition disclosed herein for use in the treatment of cancer is administered subcutaneously and comprises a conjugate of formula (I) as disclosed in WO2018 / 115466A1, wherein the antibody “J22.9-ISY-D265C” refers to an anti-BCMA antibody comprising the heavy chain amino acid sequence of SEQ ID NO:1 disclosed in WO2018 / 115466 (the contents of which are incorporated herein by reference) and the light chain amino acid sequence of SEQ ID NO:2 (as disclosed herein in SEQ ID NO:11,12).

[0230]

[0231] The cancers mentioned are selected from the group comprising: multiple myeloma, diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL), particularly multiple myeloma.

[0232] According to a preferred embodiment, a pharmaceutical composition disclosed herein for use in the treatment of cancer is administered to a patient suffering from relapsed / refractory multiple myeloma, wherein the patient is characterized by the following criteria:

[0233] • Male or female aged ≥18 years.

[0234] • Life expectancy > 12 weeks.

[0235] The Eastern Oncology Collaboration Group proposed a performance status (PS) of 0 to 2.

[0236] • Diagnosed with active MM according to the diagnostic criteria established by the International Myeloma Working Group (IMWG).

[0237] • Must have undergone SCT or be deemed unsuitable for transplantation.

[0238] • Prior treatment with antimyeloma therapy is required, which must include immunomodulatory drugs, proteasome inhibitors, and anti-CD38 therapy, either alone or in combination. Furthermore, the patient should be refractory to or intolerant of any established standard of care that, as evaluated by the investigator, provides meaningful clinical benefit to the patient.

[0239] • Diseases that are measurable according to IMWG standards.

[0240] Therefore, the pharmaceutical composition of the present invention for use in the treatment of relapsed or refractory multiple myeloma is administered to patients as disclosed herein.

[0241] Therefore, the present invention relates to a method for treating patients with refractory relapsed multiple myeloma, wherein the patients are characterized by the above-mentioned criteria.

[0242] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-CD37 antibody chHH1-HDPLALA-D266C, comprising the heavy chain amino acid sequence according to SEQ ID NO:11 and the light chain amino acid sequence according to SEQ ID NO:12 as disclosed in WO 2022 / 194988 A2 (the contents of which are incorporated herein by reference), wherein the conjugate comprises one of the amatoxin-linking moiety of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII) as disclosed herein, preferably (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XX), or (XXII), more preferably (XII), (XIII), (XIV), (XVII), or (XX), particularly preferably (XII), (XIII), or (XIV).

[0243] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-CD37 antibody chHH1-HDPLALA-D266C, comprising the heavy chain amino acid sequence according to SEQ ID NO:11 as disclosed in WO 2022 / 194988 A2 and the light chain amino acid sequence according to SEQ ID NO:12 (as disclosed herein in SEQ ID NO:13,14), wherein the conjugate comprises an amatoxin-linker (XII).

[0244] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-CD37 antibody chHH1-HDPLALA-D266C, comprising the heavy chain amino acid sequence according to SEQ ID NO:11 as disclosed in WO 2022 / 194988 A2 and the light chain amino acid sequence according to SEQ ID NO:12 (as disclosed herein in SEQ ID NO:13,14), wherein the conjugate comprises an amatoxin-linker (XIII).

[0245] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-CD37 antibody chHH1-HDPLALA-D266C, comprising the heavy chain amino acid sequence according to SEQ ID NO:11 as disclosed in WO 2022 / 194988 A2 and the light chain amino acid sequence according to SEQ ID NO:12 (as disclosed herein in SEQ ID NO:13,14), wherein the conjugate comprises an amatoxin-linker (XIV).

[0246] According to a preferred embodiment, the pharmaceutical composition disclosed herein for use in the treatment of cancer is administered subcutaneously and comprises a conjugate containing an anti-CD37 antibody chHH1-HDPLALA-D266C, which contains the heavy chain amino acid sequence according to SEQ ID NO:11 as disclosed in WO 2022 / 194988 A2 and the light chain amino acid sequence according to SEQ ID NO:12 (as disclosed herein in SEQ ID NO:12). NO:13,14), wherein the conjugate comprises an amatoxin-linker (XII) and wherein the cancer is selected from: non-Hodgkin lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma (DBNHL), subtypes of non-Hodgkin lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatous polyangiitis and microscopic polyangiitis, and pemphigus vulgaris, wherein the pharmaceutical composition is administered subcutaneously and can be formulated as disclosed herein.

[0247] According to a preferred embodiment, the pharmaceutical composition disclosed herein for use in the treatment of cancer is administered subcutaneously and comprises a conjugate containing an anti-CD37 antibody chHH1-HDPLALA-D266C, which contains the heavy chain amino acid sequence according to SEQ ID NO:11 as disclosed in WO 2022 / 194988 A2 and the light chain amino acid sequence according to SEQ ID NO:12 (as disclosed herein in SEQ ID NO:12). NO:13,14), wherein the conjugate contains an amatoxin-linker (XIII) and wherein the cancer is selected from: non-Hodgkin lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma (DBNHL), subtypes of non-Hodgkin lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatous polyangiitis and microscopic polyangiitis, and pemphigus vulgaris.

[0248] According to a preferred embodiment, the pharmaceutical composition disclosed herein for use in the treatment of cancer is administered subcutaneously and comprises a conjugate containing an anti-CD37 antibody chHH1-HDPLALA-D266C, which contains the heavy chain amino acid sequence according to SEQ ID NO:11 as disclosed in WO 2022 / 194988 A2 and the light chain amino acid sequence according to SEQ ID NO:12 (as disclosed herein in SEQ ID NO:12). NO:13,14), wherein the conjugate contains an amatoxin-linker (XIV) and wherein the cancer is selected from: non-Hodgkin lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma (DBNHL), subtypes of non-Hodgkin lymphoma (including mantle cell lymphoma (MCL)), chronic lymphocytic leukemia (CLL), Richter syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatous polyangiitis and microscopic polyangiitis, and pemphigus vulgaris.

[0249] According to a preferred embodiment, the pharmaceutical composition disclosed herein for use in the treatment of cancer is administered subcutaneously and comprises a conjugate containing an anti-PSMA antibody selected from the group disclosed in WO 2020 / 025564 (the contents of which are incorporated herein by reference): 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, or 3-F11-var16, conjugated to the formulas (XII) and (XIII) disclosed herein. (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), (XX), the amatoxin-linker portion, and most preferably the antibody is one of 3-F11-var1, 3-F11-var13 or 3-F11-var16, conjugated to the amatoxin-linker portion according to any one of formula (XII), (XIII), (XIV), (XVII) or (XX):

[0250] Antibodies (as disclosed in WO 2020 / 025564) Amatoxins - Linker 3-F11-var1 (containing SEQ ID NO:7,8) (XII), (XIII), (XIV), (XVII) or (XX) 3-F11-var13 (containing SEQ ID NO:7,10) (XII), (XIII), (XIV), (XVII) or (XX) 3-F11-var16 (containing SEQ ID NO: 9, 10) (XII), (XIII), (XIV), (XVII) or (XX)

[0251] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var1 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XII), wherein the anti-PSMA antibody 3-F11-var1 comprises the VH and VL sequences according to SEQ ID NO:7,8.

[0252] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var1 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XIII), wherein the anti-PSMA antibody 3-F11-var1 comprises the VH and VL sequences according to SEQ ID NO:7,8.

[0253] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var1 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XIV), wherein the anti-PSMA antibody 3-F11-var1 comprises the VH and VL sequences according to SEQ ID NO:7,8.

[0254] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var13 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XII), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NO:7,10.

[0255] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var13 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XIII), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NO:7,10.

[0256] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var13 as disclosed in WO 2020 / 025564 conjugated to an amatoxin linker moiety (XIV), wherein the anti-PSMA antibody 3-F11-var13 comprises the VH and VL sequences according to SEQ ID NO:7,10.

[0257] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var16 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XII), wherein the anti-PSMA antibody 3-F11-var16 comprises the VH and VL sequences according to SEQ ID NO: 9, 10.

[0258] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var16 as disclosed in WO 2020 / 025564 conjugated to the amatoxin linker portion (XIII), wherein the anti-PSMA antibody 3-F11-var16 comprises the VH and VL sequences according to SEQ ID NO: 9, 10.

[0259] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-PSMA antibody 3-F11-var16 as disclosed in WO 2020 / 025564 conjugated to an amatoxin linker portion (XIV), wherein the anti-PSMA antibody 3-F11-var16 comprises the VH and VL sequences according to SEQ ID NO: 9, 10.

[0260] According to a preferred embodiment, a pharmaceutical composition for use in the treatment of cancer, as disclosed herein, is administered subcutaneously and comprises a conjugate comprising an anti-GCC antibody selected from the group disclosed in PCT / EP2023 / 080350 (the contents of which are incorporated herein by reference): antibody mAb1, mAb8, mAb, mAb41, each comprising mutants L234A, L235A, and D265C (numbered according to the EU numbering system), conjugated to an amatoxin-linker portion of a formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII) as disclosed herein, preferably (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XX), or (XXII), more preferably (XII), (XIII), (XIV), (XVII), (XX), and most preferably (XII) or (XIV).

[0261] Antibodies (such as those disclosed in PCT / EP2023 / 080350) Amatoxins - Linker mAb1 contains SEQ ID NO:1,2 (XII), (XIII), (XIV), (XVII) or (XX) mAb8 contains SEQ ID NO:4,3 (XII), (XIII), (XIV), (XVII) or (XX) mAb41 contains SEQ ID NO:5,6 (XII), (XIII), (XIV), (XVII) or (XX)

[0262] The term "GCC" refers to guanylate cyclase C (GUCY2C, EC: 4.6.1.2), preferably human GCC, which is a member of the receptor enzyme protein family that synthesizes guanosine 3′,5′-cyclic monophosphate (cyclic GMP; cGMP). GUCY2C is a transmembrane receptor for endogenous hormone ligands: guanylin and uroguanylin. Ligands binding to this extracellular receptor catalyze the conversion of GTP to cyclic GMP (cGMP) and initiate downstream cGMP-related signaling pathways, which are involved in the regulation of intestinal homeostasis processes such as epithelial cell proliferation, differentiation, and apoptosis.

[0263] According to one embodiment, the pharmaceutical composition of the invention disclosed herein for use in cancer treatment is administered to the patient multiple times, for example, two or three times, or periodically, for example, every 30 days, 60 days, 90 days, 120 days, 180 days, 360 days, or about every 21 days, 28 days to about 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, or every 22 days, 23 days, 24 days, 25 days, 26 days, 27 days.

[0264] According to one embodiment, the pharmaceutical composition of the invention disclosed herein for use in treating cancer increases the MTD by at least 30%, 40%, or 50% compared to the maximum tolerated dose (MTD) of the pharmaceutical composition when administered intravenously. As used herein, the term "maximum tolerated dose" (MTD) refers to the highest tolerated dose of a drug (such as the conjugates of the invention disclosed herein) that can be administered to an animal without causing serious toxicity or death. Therefore, the pharmaceutical composition of the invention for use in treating cancer is characterized by a higher therapeutic index (TI) compared to a corresponding pharmaceutical composition for use in treating cancer administered intravenously (iv) to, for example, a mammal (e.g., a patient with cancer). As used herein, the term "therapeutic index" refers to the ratio of the concentration of a drug in the blood at which it is toxic to that at which it is effective. A higher therapeutic index (TI) indicates greater safety of the drug. TI can be calculated, for example, as: TI = MTD / MED, where the term "MED" refers to the minimum effective dose, which is the lower limit of the therapeutically effective dose.

[0265] According to one embodiment, the present invention relates to the use of a conjugate comprising an amatoxin linker portion according to any one of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), or (XI), or a conjugate according to any one of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII) in the preparation of a pharmaceutical composition according to the present invention for use in the treatment of cancer, as disclosed herein, wherein the pharmaceutical composition is administered subcutaneously.

[0266] According to one embodiment, the present invention relates to immune checkpoint inhibitors selected from the group consisting of avelumumab, nivolumab, pembrolizumab, ipilimumab or durvalumab, and envorimab, for use in preparing pharmaceutical compositions according to the invention as disclosed herein.

[0267] According to one aspect, the present invention relates to a method of treating a patient suffering from cancer, wherein the method comprises subcutaneously administering an effective dose of a pharmaceutical composition as disclosed herein to the patient. As used herein, the term "effective dose" refers to the amount of a pharmaceutical composition or conjugate as disclosed herein that produces one or more expected responses in a subject (e.g., killing cancer cells expressing cell surface antigens, tumor-associated antigens, or tumor-specific antigens specifically bound by antibodies to the conjugate).

[0268] According to some embodiments, the methods of treating a patient disclosed herein include administering the pharmaceutical composition of the present invention to the patient at a single injection site or to multiple injection sites. Multiple injection sites may include two or more injection sites where the pharmaceutical composition of the present invention, as disclosed herein, is injected subcutaneously. Depending on the composition of the pharmaceutical composition of the present invention and the patient's overall health condition (such as the amount of subcutaneous fat in the corresponding patient), the pharmaceutical composition of the present invention may be administered at a single injection site or multiple injection sites.

[0269] Pharmaceutical compositions of the present invention comprising two components (e.g., conjugates according to the invention as disclosed herein and a second component such as, for example, rHuPH20) may require spatial proximity injection of the first and second components, for example, within 0.5 cm to about 3 cm of each other. The first and second components may be injected subcutaneously into the patient as disclosed herein, for example, 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, for example, before, simultaneously with, or after the administration of the first component as disclosed herein; however, it is preferred to administer the second component from about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes to about 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 60 minutes before the administration of the first component, or, for example, from about 15 minutes, 20 minutes, 25 minutes, 30 minutes 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 can be applied subcutaneously at 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 the subcutaneous application of the first or second component. Depending on medical needs, the corresponding treatment can be repeated periodically, for example, every 21 days, 28 days, 35 days, 42 days, 60 days, 90 days, 120 days, 180 days, or 360 days, for example, twice, three times, or four times.

[0270] In some embodiments, a method of treating a patient according to the invention comprises subcutaneously administering to the 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 the pharmaceutical composition of the invention, wherein the volume to be injected may, for example, depend on the use of rHuPH20 to facilitate subcutaneous injection of the pharmaceutical composition of the invention as disclosed herein.

[0271] The effective dose of the pharmaceutical composition according to the invention to be administered subcutaneously to the subject may vary depending on various factors, such as cancer type, cancer stage, patient age, or patient weight or body surface area. In some embodiments, the effective dose of the pharmaceutical composition according to the invention administered subcutaneously to the patient is 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 0.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg body weight, or approximately 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, or approximately 375 μg / kg, 425 μg / kg, 475 μg / kg, 950 μg / kg, 2.5 mg / kg, 3 mg / kg body weight. If the effective dose is determined using the patient's body surface area, the body surface area (BSA) can be calculated according to the Du Bois formula:

[0272] BSA = 0.007184 × W 0.425 ×H 0.725 Where W is the patient's weight in kg and H is the patient's height in cm.

[0273] According to one embodiment, the present invention relates to a method for treating a patient suffering from cancer, wherein the method comprises subcutaneously administering a pharmaceutical composition of the present invention as disclosed herein to the patient, wherein the patient has previously failed standard care first-line, second-line, or third-line cancer treatment for their respective cancer.

[0274] In one embodiment, the present invention relates to a method of delivering an amatoxin-linker payload (e.g., according to one of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), or (XI)) as disclosed herein) to cells, wherein the method comprises subcutaneously administering a pharmaceutical composition comprising the conjugate of the present invention to a patient in need, the conjugate specifically binding to an epitope on the cell.

[0275] sequence list

[0276]

[0277]

[0278]

[0279]

[0280] Example

[0281] Although the invention has been described and illustrated in detail in the accompanying drawings and the foregoing description, such descriptions and illustrations should be considered illustrative or exemplary rather than restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and implemented by those skilled in the art in carrying out the claimed invention by studying 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 plural. The fact that certain embodiments are recited in dissimilar dependent claims does not imply that combinations of these embodiments cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope.

[0282] Example 1: Amatoxin-linker loading conjugation

[0283] By using so-called cysteine-engineered antibodies The technique enables the conjugation of antibodies to amatoxin linker conjugates. In this method, conjugation is performed by conjugating a maleimide residue of the toxin linker construct with a free SH group of an engineered cysteine ​​residue in an antibody, introduced, for example, through site-directed mutagenesis, as shown in the following reaction protocol:

[0284]

[0285] The principle of this conjugation method is disclosed in Junutula JR et al. (2008), Nat Biotechnology Vol. 26:925-932, the contents of which are incorporated herein by reference. In short: [The text abruptly ends here, likely due to an incomplete translation or a missing section.] Prior to conjugation with a maleimide-linked amatoxin-based loading as disclosed herein, the blocking cysteine ​​or glutathione present on the introduced cysteine ​​may be removed, for example, by mild reduction in PBS at 25°C with the addition of a 10-40 molar excess of reducing agent TCEP or dithiothreitol (DTT), followed by dialysis filtration. To reform the interchain disulfide bond, the cysteine-engineered antibody may be incubated, for example, with a 2-20 molar excess of CuSO4 relative to the reducing agent concentration or with dhAA (Sigma-Aldrich) at 25°C for three hours. Interchain disulfide bond formation may be monitored, for example, by non-reducing SDS-PAGE or by denaturing reversed-phase high-performance liquid chromatography (HPLC) PLRP column chromatography. Conjugation of the maleimide-linked amatoxin loading with the cysteine-engineered antibody may be accomplished, for example, using a 2-4 molar equivalent of the amatoxin-linked loading. Quenching can be performed, for example, using 20-25 molar equivalents of N-acetyl-L-cysteine, followed by incubation at 25°C for approximately 1 hour. Subsequently, the antibody conjugate can be, for example, in… Purification on an S-column (GE Healthcare Bio-Sciences) to remove excess reactants, optionally followed by treatment with N-acetyl-L-cysteine ​​for 15 minutes prior to the quenching step.

[0286] To provide cysteine ​​residues having the free SH group, the antibody used in this experiment contains a D265C substitution in both Fc domains. The corresponding technique is disclosed in WO2016 / 142049A1, the contents of which are incorporated herein by reference, and it results in a homogeneous product having a fixed drug-antibody ratio (“DAR”) of 2 and site-specific conjugation.

[0287] Example 2: Mouse prostate cancer model

[0288] Using 2.5×10 containing 50% GFR Matrigel 6 One C4-2 tumor cell / 200 μL RPMI (excluding PR) was subcutaneously injected into the right flank of male CB17 Scid animals. The tumor volume was approximately 140 mm². 3 (Preferred range is 80-200mm) 3Animals were randomized to group (n=10) according to tumor size. Treatment began one day after grouping. Mice were treated with a single dose of PBS or 20 mg / kg of an anti-PSMA conjugate (2.5 ml / kg) containing an anti-PSMA antibody conjugated to the amatoxin-linker moiety (XII) as disclosed in WO2020 / 025564A1, either intravenously or subcutaneously (between the shoulders). Tumor volume was measured twice weekly by calipers, and body weight was determined in parallel. When tumor volume >1600 mm², treatment was initiated. 3 When it is necessary to euthanize a mouse for ethical reasons, the animal is euthanized and an autopsy is performed. The results are as follows: Figure 2 As shown in Figure A, a single subcutaneous injection (hollow triangle) of the anti-PSMA conjugate is as effective as a single intravenous injection (solid triangle).

[0289] Example 3: Mouse chronic B-cell leukemia cancer model

[0290] Female CB17 Scid animals were intravenously inoculated on day 3 with 2.5 × 10⁻⁶ ppm of PBS in 200 μL of water. 6 MEC-2 (human CLL) tumor cells were inoculated. Treatment began three days after cell seeding (n=10). Mice were treated intravenously or subcutaneously (10 ml / kg) with PBS or a chHH1-HDPLALA-D266C conjugate to the amatoxin-linker moiety (XII). Body weight was determined twice weekly. Clinical symptoms and survival were monitored and recorded daily. When euthanasia was necessary for ethical reasons, the animals were euthanized and autopsies were performed. Results are as follows: Figure 2 As shown in B, a single dose of the conjugate administered subcutaneously at doses of 1 mg / kg (hollow triangle) or 3 mg / kg (hollow square) is equally effective in prolonging survival probability compared to corresponding treatments administered intravenously at the same doses of 1 mg / kg (solid triangle) and 3 mg / kg (solid square).

[0291] Example 4: Antitumor efficacy of anti-CD37-(XII) conjugate in a Raji-Luc (human Burkitt lymphoma) diffuse xenograft model in female CB17-SCID mice.

[0292] The aim of this study was to evaluate the antitumor efficacy of the anti-CD37-(XII) conjugate in a female CB17-SCID mouse model of Raji-Luc (human Burkitt lymphoma) diffuse xenograft after a single subcutaneous dose.

[0293] Research Design

[0294] Twenty female CB17-SCID mice were divided into two groups of 10 each, and on day 3, each animal was given 2.5 × 10⁻⁶ mg / L of medication. 6 Raji-Luc tumor cells (in 200 μl of RPMI without phenol red) were intravenously inoculated. On day 0, animals were subcutaneously administered the final anti-CD37-(XII) conjugate (dose: 0.5 or 1.0 mg / kg).

[0295] In parallel, as part of Study A, 30 female CB17-SCID mice loaded with Raji-Luc tumor cells received a single intravenous (iv) dose of either anti-CD37-(XII) conjugate (dose: 0.5 or 1.0 mg / kg) or PBS. These data are for reference only.

[0296] Tumor cell-dependent luciferase activity was determined weekly by non-invasive whole-body bioimaging (Caliper IVIS) starting on day 1 post-treatment. Body weight was determined in parallel. Clinical signs and survival were monitored daily. Animals were euthanized and autopsied when one or more termination criteria were met or at the end of the study (day 97).

[0297] Results and Conclusions

[0298] Intravenous inoculation of female CB17-SCID (PBS control 1) mice with human Burkitt lymphoma expressing luciferase (Raji Luc) resulted in progressively increasing luciferase-dependent whole-body fluorescence, weight loss after day 11, and 100% mortality within 18 days post-treatment. Mortality was primarily associated with significant hindlimb paralysis.

[0299] The anti-CD37-(XII) conjugate controlled Raji-Luc tumor-dependent mean bioluminescence levels close to day 1, in 10 out of 10 animals (100%, 0.5 mg / kg intravenously and 0.5 or 1.0 mg / kg subcutaneously) or 8 out of 10 animals (80%, 1.0 mg / kg intravenously). Body weight increased continuously in most animals until the end of the study. Most common clinical manifestations and autopsy findings were representative of the effects caused by the Raji-Luc tumor and were not considered relevant to the tested items.

[0300] In summary, compared with PBS (intravenous), a single subcutaneous dose of 0.5 or 1.0 mg / kg anti-CD37-(XII) on day 0 (tumor inoculation on day -3) resulted in a very strong inhibitory effect on intravenous human Burkitt lymphoma growth (10 out of 10 animals), 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 via different routes of administration (subcutaneous or intravenous). Results are as follows... Figure 4 As shown in A and 4C.

[0301] Example 5: Antitumor efficacy of anti-CD37-(XII) in a MEC-2 tumor xenograft model in female CB17-SCID mice

[0302] The study consisted of two experimental groups, with 10 animals in each group. The 20 animals were given 2.5 × 10⁻⁶ ppm of PBS in 200 μL on day -3. 6 Mice were intravenously inoculated with MEC-2 (human CLL) tumor cells. Treatment began on day 0 (three days after tumor cell inoculation). Mice were treated with anti-CD37-(XII), as follows: Figure 4 As shown in B. Body weight was determined twice weekly. Clinical signs and survival rates were recorded daily. Animals were euthanized and autopsied when termination criteria were met or for ethical reasons. The results of this study are as follows: Figure 4 As shown in B, there were no statistically significant differences between animals treated with the same dose but via different routes of administration (subcutaneous or intravenous).

[0303] Example 6: Quantification of serum αPSMA-(XII) conjugate concentration

[0304] Serum concentrations of the αPSMA-(XII) conjugate, derived from the pharmacokinetics of the conjugate, were determined using a sandwich ELISA to quantify the pharmacokinetics of the conjugate in cynomolgus monkeys following a single intravenous infusion or subcutaneous administration. For the sandwich ELISA, the conjugate was captured from cynomolgus monkey serum samples using a polyclonal anti-muscarinic antibody. Detection was performed using rabbit anti-human IgGH&L HRP antibody. Two copies of the study samples were analyzed, and the standard range was measured from 1.6 to 200 pM. Optical density (OD) was measured at wavelengths of 450 nm and 570 nm, and the difference in OD was used for calculation. Serum concentrations of the αPSMA-(XII) conjugate were calculated from the standard curve and then converted to a linear range. Results are as follows: Figure 7 As shown in A and B. Intravenous administration of the αPSMA-(XII) conjugate as disclosed herein at a concentration of 7.5 mg / kg resulted in approximately 219 μg / ml of C. max(Average of 3 animals), while subcutaneous administration of the conjugate at a dose of 7.5 mg / kg resulted in approximately 96 μg / ml of C. max And subcutaneous administration of the conjugate αPSMA-(XII) at a dose of 10 mg / kg resulted in approximately 142 μg / ml of C. max Tolerance to the αPSMA-(XII) conjugate was also increased; no animal deaths were observed when administered subcutaneously at a dose of 7.5 mg / kg, compared to intravenous administration (which resulted in the death of 2 out of 3 animals). (See also: [link to relevant information]) Figure 7 (As indicated). Increased tolerance to the conjugate αPSMA-(XII) is also reflected in the concentrations of liver enzymes ALT and AST, such as... Figure 3 As shown. In this study, intravenous administration of the conjugate αPSMA-(XII) resulted in an increase in both markers, peaking 5 to 10 days after administration, while subcutaneous administration of the same dose resulted in only a slight increase in both liver enzymes. Therefore, subcutaneous administration of the conjugate αPSMA-(XII) led to a decrease in C... max Decreased C max This, in turn, leads to increased tolerance, such as Figure 3 As shown.

[0305] Example 6: Subcutaneous administration (sc) using an anti-GCC (guanylate cyclase C (GUCY2C)) conjugate

[0306] MTD with anti-GCCATAC

[0307] Following intravenous or subcutaneous administration of DAR2 ADCs (αGCC-LALA-D265C-(XIV) and αGCC-LALA-D265C-(XII)) to female NOD / SCID mice, the maximum tolerated dose (MTD) of the αGCC antibody conjugates comprising the amatoxin-linker moieties (XIV) and (XII), wherein the corresponding anti-GCC antibodies comprise the heavy and light chain sequences according to SEQ ID NO:3 and SEQ ID NO:4, was determined. The conjugates were administered on day 0, body weight was determined twice weekly, and clinical observations and mortality were recorded daily. The initial dose was increased or decreased in subsequent groups to determine the maximum tolerated dose (MTD), as shown in Table 1 below.

[0308] Table 1

[0309]

[0310] Antitumor efficacy of amatoxins based on anti-GCC ADC

[0311] The antitumor efficacy of the anti-GCC ADCs anti-GCC-LALA-D265C-(XII) and anti-GCC-LALA-D265C-(XIV) was evaluated after single subcutaneous administration to a HEK293-GUCY2C-(HDP)-2B3 subcutaneous xenograft model in female NOD / SCID mice. Female NOD / SCID mice were administered 5.0 × 10⁶ mmol / L of the ADCs per animal. 6 HEK293-GUCY2C-(HDP)-2B3 tumor cells (in 200 μL RPMI medium (without PR) containing 50% GFR Matrigel, Corning 356231) were seeded into their right flank. When the average tumor volume reached 150-160 mm, 3 Fifty animals were selected and divided into five groups of ten animals each, based on tumor size. On the same day or the following day (Day 1), 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 weight was determined in parallel. Clinical signs and survival were monitored daily. Animals were euthanized and autopsied when one or more termination criteria were met or when the study was terminated on day 61.

[0312] result

[0313] 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 reduced mean tumor volume (statistically significant relative to PBS control) within approximately 12 days post-treatment, regardless of the toxin-linker or route of administration, and in 3 and 7 out of 10 animals (anti-GCC-LALA-D265C-(XIV), subcutaneously and intravenously, respectively) or 5 and 8 out of 10 animals (anti-GCC-LALA-D265C-(XII) ADC, subcutaneously and intravenously, respectively) resulted in (almost) permanent complete reduction (0 mm). 3 Tumor remission. Results as follows: Figure 8 As described.

[0314] In summary, a single subcutaneous dose of 2.5 mg / kg anti-GCC-LALA-D265C-(XIV) or 6.0 mg / kg anti-GCC-LALA-D265C-(XII) ADC resulted in strong inhibition of subcutaneous human embryonic renal cell carcinoma growth, slightly delayed weight gain (relative to PBS control), and approximately 30% and 60% survival (day 61), respectively. There were no statistically significant differences in tumor suppression, weight gain, and survival between animals treated with the same test items and doses but via different routes of administration (subcutaneous or intravenous).

[0315] HEK293-GUCY2C-HDP-2B3 cells used in the above-mentioned xenograft model express human GUCY2C and are described in PCT / EP2023 / 080350. In short, HEK293-GUCY2C-HDP-2B3 is obtained by transiently transfecting HEK293 wild-type cells with an overexpression plasmid encoding human guanylate cyclase 2C (GUCY2C or GCC) according to Uniprot P25092 (September 13, 2023 version) and resistance to G418. Stable cell pools expressing GCC and G418 resistance were selected after 4 days by placing cells in a medium containing G418. Stable single-cell clones were isolated from this cell pool using limiting dilutions. The expression of GCC on the surface of the stable cell clone HEK293-GUCY2C-HDP-2B3 was confirmed by flow cytometry and cytotoxicity assay (BrdU ELISA).

[0316] Example 7: Pharmacokinetics of subcutaneous administration of anti-PSMA-(XIV)ADC

[0317] Serum pharmacokinetics of the amanita-based anti-PSMA ADC h3 / F11-LALA-D265CVar16-(XIV) were evaluated in male CB17-SCID mice following a single subcutaneous administration of 10 or 5 mg / kg or an intravenous administration of 5 mg / kg. Male CB17-SCID mice were randomly assigned to cohorts of 18 animals each (3 animals per cohort based on sample collection time point). 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 weight was determined 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) after administration. Pharmacokinetic parameters of h3 / F11-LALA-D265CVar16-(XIV) in plasma were calculated and compared.

[0318] result:

[0319] Compared with intravenous administration of the same dose, subcutaneous administration of 5.0 mg / kg h3 / F11-LALA-D265C Var16-(XIV) showed better pharmacokinetic characteristics, with a slightly increased normalized AUC and half-life and a significantly reduced dose-normalized C. max .

[0320] Following subcutaneous administration, the C of h3 / F11-LALA-D265C Var16-(XIV) at doses of 5 and 10 mg / kg max The concentrations were 7.54 and 7.33 kg·μg / mL / mg, respectively, compared with C. max_D The values ​​were dose-proportional. The dose-normalized AUC (161 and 119 days·kg·μg / mL / mg) and clearance (6.2 and 8.4 mL / day / kg) were in similar ranges.

[0321] Considering the pharmacokinetic results of subcutaneously administered anti-PSMA-(XII)ADC ( Figure 7 These results support the view that the effect of subcutaneous administration on the pharmacokinetics of amanitan-based ADCs is independent of the amanitan derivative used as the loading.

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Claims

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

2. The pharmaceutical composition for use according to claim 1, wherein the target-binding portion of the conjugate is selected from: (i) Antibody, preferably a monoclonal antibody. (ii) its antigen-binding fragment, preferably a variable domain (Fv), a Fab fragment, or an F(ab)2 fragment. (iii) its antigen-binding derivative, preferably single-chain Fv (scFv), and (iv) Antibody-like proteins.

3. The pharmaceutical composition for use according to claim 1 or claim 2, wherein the antibody of the conjugate is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody or a human antibody.

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

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

6. The pharmaceutical composition for use according to any one of claims 2 to 5, wherein the antibody portion of the conjugate does not cause antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

7. The pharmaceutical composition for use according to any one of claims 2 to 6, 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. The pharmaceutical composition for use according to 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. The pharmaceutical composition for use according to claim 8, wherein the Fc region of the antibody comprises amino acid substitution D265C (according to the EU numbering system).

10. The pharmaceutical composition for use according to any one of claims 6 to 9, wherein the Fc region of said antibody comprises amino acid substitutions L234A, L235A and D265C (according to the EU numbering system).

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the linker is connected to the antibody moiety via any one of the naturally occurring cysteine ​​residues of the antibody, preferably via any one of the naturally occurring cysteine ​​residues forming the interchain disulfide bond of the antibody and / or via a disulfide linkage.

12. The pharmaceutical composition for use according to any one of claims 1 to 11, wherein the amatoxin of the conjugate is linked to the antibody via a cleavable linker or an uncleavable linker.

13. The pharmaceutical composition for use according to claim 12, wherein the cleavable linker of the conjugate is selected from enzymatically cleavable linkers, preferably protease-cleavable linkers; and chemically cleavable linkers, preferably linkers comprising disulfide bridges.

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

15. The pharmaceutical composition for use according to claim 14, wherein the enzymatically cleavable linker of the conjugate is a cathepsin B-cleavable linker.

16. The pharmaceutical composition for use according to any one of claims 13 to 15, wherein the cleavable linker is a self-degradable linker, wherein the self-degradable linker comprises a p-aminobenzyloxycarbonyl (PAB or PABC) moiety.

17. The pharmaceutical composition for use according to any one of claims 1 to 16, wherein the linker of the conjugate is linked to the amatoxin via (i) a γC atom of amatoxin amino acid 1, (ii) a δC atom of amatoxin amino acid 3, or (iii) a 6'-C atom of amatoxin amino acid 4.

18. The pharmaceutical composition for use according to any one of claims 1 to 17, wherein the conjugate comprises at least one amatoxin linker portion selected from any one of compounds (I)-(XI):

19. The pharmaceutical composition for use according to any one of claims 1 to 18, wherein the antibody of the conjugate is conjugated to at least one amatoxin linker portion via a thioether bond according to any one of formulas XII to XXII: The amatoxin-linking base portion is coupled to the thiol group of the cysteine ​​residue of the antibody portion of the conjugate, wherein n is about 1, 2, 3 to about 4, 5, 6, 7 or 8.

20. The pharmaceutical composition for use according to claim 19, wherein n is about 1.5 or 2 to about 3 or 3.5, preferably wherein n is about 2.

21. The pharmaceutical composition for use according to any one of claims 1 to 20, wherein the cancer is a solid tumor or non-solid tumor selected from: gastric cancer, adenocarcinoma, melanoma, ovarian cancer, uterine cancer, cervical cancer, breast cancer (including triple-negative breast cancer), bronchial cancer, Ewing 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 gland cancer, and biliary tract cancer. Rectal cancer, endometrial cancer, desmoidoma, fibroproliferative small round cell tumor, neuroectodermal tumor, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, osteosarcoma, 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 multiple myeloma.

22. The pharmaceutical composition for use according to claim 21, wherein the cancer cells of the tumor are characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13), or wherein 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 said TP53, POLR2A, or del(17p13).

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

24. The pharmaceutical composition for use according to any one of claims 1 to 23, wherein the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor.

25. The pharmaceutical composition for use according to claim 24, wherein the pharmaceutical composition further comprises recombinant hyaluronidase.

26. The pharmaceutical composition for use according to any one of claims 1 to 25, wherein the antibody of the conjugate specifically binds to cell surface antigens on tumor cells or to tumor-associated antigens.

27. The pharmaceutical composition for use according to any one of claims 1 to 26, wherein the cell surface antigen on the tumor cells is selected from: 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, GPR1 43. NMNAT2, MAGE, MAGEC2, MAGE-A3, MART-1, WT-1, EPHA2, KRT19, CLDN7, DKK1, FGF19, SCN3A, SCN2A, GAS1, S100Z, GAPT, GPR35, NY-ESO, Cathelin 24, DLK1, GPR173, ALK, GFRA3, GUCY2C, DLL3, PSMA, PROX1, PSCA, Phosphatidylinositol Glycan-1, Mesothelin (Prostate Stem Cell Antigen), GAGE-1 (G Antigen 1), Ganglioside / GD2, GnT-V, β1,6-N (Acetylglucosamine aminotransferase-V), UPAR (Urokinase-type plasminogen activator receptor), Sialyl Lewis X (SLe x ) and sialylated Lewis a (SLe a ).

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

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

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

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

32. A method of treating a patient suffering from cancer, wherein the method comprises administering to the subject an effective dose of the pharmaceutical composition according to any one of claims 1 to 28.

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

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

35. The method according to 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. The method according to any one of claims 32 to 34, wherein the effective amount of the conjugate administered with the pharmaceutical composition is about 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, 1.2 mg / kg, etc. 5 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg body weight or approximately 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 approximately 375 μg / kg, 425 μg / kg, 475 μg / kg, 950 μg / kg, 2.5 mg / kg, 3 mg / kg body weight.

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

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