EA040940B1 - HYDROPHILIC ANTIBODY-DRUG CONJUGATES - Google Patents

Description

This application asserts the benefit of U.S. Provisional Application No. 61/940,759 filed February 17, 2014 and U.S. Provisional Application No. 61/947368 filed March 3

2014, each of which is included in this description in its entirety.

Prerequisites for the creation of the invention

Great interest surrounds the use of monoclonal antibodies (mAb) for targeted delivery of cytotoxic agents to malignant cells. Designing antibody drug conjugates by attaching cytotoxic agents to an antibody, usually via a linker, involves consideration of a number of factors. These factors include the identity and location of the chemical group(s) for conjugation with the cytotoxic agent, the release mechanism of the cytotoxic agent, the structural element(s) (if any) that release the agent, and the structural modification of the released free agent, if any. In addition, if the cytotoxic agent is to be released after internalization of the antibody, the building blocks for the release of the agent and the mechanism for its release must be consonant with the intracellular migration of the conjugate.

Although a number of different classes of drugs have been evaluated for antibody delivery, only a few classes of drugs have proven to be sufficiently active as antibody-drug conjugates while having an appropriate toxicity profile to warrant clinical development. One such class is the auristatins related to the natural product dolastatin 10. Exemplary auristatins include MMAE (N-methylvaline-valin-dolaisoleuin-dolaproin-norephedrine) and MMAF (N-methylvaline-valindolaisoleuin-dolaproin-phenylalanine).

MMAE is an example of a cytotoxic agent that is active as a free drug and is potent after conjugation with monoclonal antibodies (mAbs) and released after internalization into cells. MMAE was successfully conjugated to mAb at the N-terminal amino acid MMAE via a cathepsin-cleavable peptide linker containing maleimidocaproyl-valine-citrulline (mc-vc-) and an independent immolative group, p-aminobenzylcarbamoyl (PABC), to obtain antibody-drug conjugates. following structure, mAb-(mc-vc-PABC-MMAE)p. (In the formula above, p refers to the number of units (mc-vc-PABCMMAE) per mAb or antibody). After cleavage of the bond between the vc peptide and the independent immolative PABC group, the PABC group frees itself from MMAE, releasing free MMAE.

Another auristatin, MMAF, is less active as a free drug (compared to MMAE), but is potent after antibody conjugation, internalization, and release into cells. MMAF was successfully conjugated to a monoclonal antibody (mAb) at its N-terminal amino acid through a cathepsin-cleavable peptide linker containing maleimidocaproyl-valine-citrulline (mc-vc-) and an independent immolative group, raminobenzylcarbamoyl (PABC), to obtaining antibody-drug conjugates of the following structure, mAb-(mc-vc-PABC-MMAE) _p . (p refers to the number of units (mc-vc-PABC-MMAF) per mAb or antibody). Upon cleavage of the bond between the peptide and the PABC subunit, the independent immolative PABC group frees itself from MMAF, releasing free MMAF.

MMAF is also active as a non-cleavable maleimidocaproil MMAF (mcMMAF) linker drug conjugate. When this conjugate, mAb-(mcMMAF)p, is internalized into cells, the active particles released are cys-mcMMAF. Because the linker is non-cleavable, the maleimidocaproyl and cysteine residue of the antibody remain attached to the N-terminus of MMAF. MMAF has also been reported to be active as a C-terminal conjugate attached at its C-terminal amino acid, phenylalanine, to a peptide-maleimidocaproyl linker. When this conjugate, (MMAF-peptide-mc) _p -mAb is internalized into cells, the active moiety, MMAF, is released after cleavage of the MMAF(phenylalanine)-peptide bond.

In animal models, these MMAE and MMAF conjugates generally exhibited a drug loading dependent decrease in pharmacokinetic properties. In particular, as the number of drug-linker units attached to each antibody increased, the pharmacokinetics (PK) of the conjugates decreased.

As a consequence, another important consideration in the development of antibody drug conjugates is the amount of drug that can be delivered to the target (i.e., the number of cytotoxic agents attached to each target (e.g., antibody), referred to as the drug load or drug loading). Historically, it has been assumed that higher drug loads were superior to lower drug loads (eg, 8x loads vs 4x loads). The point is that more highly loaded conjugates would deliver more drug (cytotoxic agents) to target cells. This consideration was supported by observations that conjugates with higher drug loadings were more active against cell lines in vitro. Some more recent studies have shown, however, that this assumption has not been confirmed in animal models.

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Conjugates having a drug load of 4 or 8 specific auristatins have been observed to have similar activity in mouse models. See, for example, Hamblett et al., Clinical Cancer Res. 10:7063-70 (2004). In Hamblett et al. it has been reported that more loaded ADCs were cleared more rapidly from the circulation in animal models. This faster hatching suggested a higher PK susceptibility for more stressed species compared to less stressed species. See Hamblett et al. In addition, more loaded conjugates had lower MTDs in mice and, as a result, had the lowest published therapeutic indices. Id. In contrast, the 2-drug-engineered ADCs in the monoclonal antibody were reported to have the same or better PK properties and therapeutic indices as compared to some 4-fold loaded ADCs. For example, see Junutula et al., Clinical Cancer Res. 16:4769 (2010). Thus, the latest trend is to develop ADCs with low drug loads.

Alternative approaches to overcome PK-susceptibility to higher loading ADCs are the addition of solubilizing groups to ADCs. For example, in an attempt to overcome PK susceptibility, polyethylene glycol polymers or other water-soluble polymers have been incorporated into linkers (eg, between the drug and the antibody attachment site). Another approach is to add drug-polymers to the antibody, where each polymer contains a large number of drugs. These alternatives, however, did not necessarily achieve the desired result. In addition, the addition of solubilizing groups can increase the complexity of making such conjugates.

Thus, there remains a need for formats for antibody drug conjugates (and more generally formats for other conjugates) that provide higher drug loadings while retaining other desirable characteristics of less loaded conjugates, such as favorable PK properties. Surprisingly, the present invention addresses these needs.

Brief summary of the invention

The present invention provides, inter alia, hydrophilic ligand-linker drug conjugates. By designing conjugates having hydrophilicity similar to that of an unconjugated target drug (eg, a ligand such as an antibody), the conjugates retain the ability to provide pharmacokinetic (PK) properties similar to those of the unconjugated target drug in vivo. Conjugates can also have higher drug loading (i.e., higher numbers of hydrophilic drug linkers per target) than less loaded conjugates, while maintaining the same desirable PK properties and having the same or higher activity in vivo. (For example, 4-fold loaded or 8-fold loaded conjugates may have the same or better PK properties than their 2-fold or 4-fold loaded counterparts, respectively; such 4-fold loaded or 8-fold loaded conjugates may have the same or better activity than their 2-fold or 4-fold loaded counterparts, respectively). Thus, targeting drugs selected on the basis of certain desirable properties can be conjugated to drug linkers without significantly adversely affecting desirable properties such as the PK properties of the targeting drug itself. A method for the preparation and use of such conjugates is also provided.

Linker-drug compounds for conjugation with targeting agents (ligands) and methods for making and using such compounds are also provided. Also provided are linker-drug compounds to which drug compounds can be attached, as well as methods for making and using such compounds.

Brief description of the drawings

In FIG. 1 shows a typical synthesis scheme for assembling the ligand-linker-drug conjugates described herein.

In FIG. 2 shows the results of a mouse study comparing the pharmacokinetic stability of an unconjugated antibody, two hydrophilic ADCs, and three control ADCs. ADCs are shown in order from top to bottom.

In FIG. 3 shows the results of a study in mice comparing the pharmacokinetic stability of five hydrophilic ADCs and a control ADC.

In FIG. 4 shows the results of HIC chromatography of antibody drug conjugates.

In FIG. 5 shows the results of HIC chromatography of antibody drug conjugates.

In FIG. 6 shows the results of mouse xenograft studies comparing the activity of 4-fold loaded and 8-fold loaded ADCs.

In FIG. 7 shows the results of mouse xenograft studies comparing the activity of 4-fold loaded and 8-fold loaded ADCs.

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In FIG. 8 shows the results of mouse xenograft studies comparing the activity of 4-fold loaded and 8-fold loaded ADCs.

In FIG. 9 shows the results of studies in mouse xenografts comparing the activity of 4-fold loaded and 8-fold loaded ADCs.

Abbreviations and definitions

Unless otherwise indicated, the following terms and expressions used in this specification are intended to have the following meanings. When a trade name is used in this specification, the trade name includes the product formulation, generic drug and active pharmaceutical ingredient(s) of the trade name product, unless the context otherwise indicates.

The term hydrophilicity index refers to the degree of hydrophilicity of the conjugate relative to the hydrophilicity of the targeting agent itself (ie, the ligand, typically an antibody). The hydrophilic index is measured as the retention time of the conjugate to that of the corresponding unconjugated targeting agent (by itself) under high performance liquid chromatography (HPLC) conditions, as further described herein. For example, the retention time of a ligand-linker-drug conjugate can be determined relative to the retention time of the unconjugated ligand (usually an antibody). In certain embodiments of the present invention, the retention time of the conjugate is no more than two minutes slower than the retention time of the unconjugated ligand, as determined as described in the examples (referred to as hydrophilic index 2). In some embodiments, the retention time of the conjugate is no more than one minute slower than the retention time of the unconjugated ligand, as determined as described in the examples (referred to as hydrophilic index 1). In some embodiments, the retention time of the conjugate is no more than half a minute slower than the retention time of the unconjugated ligand, as determined as described in the examples (referred to as a hydrophilic index of 0.5). If a different hydrophobic column and/or interaction method is used, it can be calibrated using the conjugates from Table 1. 2 as a reference to determine the reference mobilities of the conjugates (elution time) with the chosen column and/or method. Certain reference mobilities on the chosen hydrophobic column and/or interaction method can be used to calculate the hydrophilicity index of the test sample (as will be determined below in Example 3). For example, the auristatin drug linkers T-Glu-DprMA, MC-MMAF and mc-vc-PABC-MMAE can be used to form conjugates for use as reference. In another example, auristatin T-Glu-Dpr-MA-h1F6 ADC, h1F6mc-MMAF and h1F6-mc-vc-PABC-MMAE ADC can be used as reference.

The term alkyl, by itself or as part of another substituent, means, unless otherwise indicated, a straight or branched hydrocarbon radical having the specified number of designated carbon atoms (i.e., C1-8 means one to eight carbon atoms). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. The term alkenyl refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term alkynyl refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers. The term cycloalkyl refers to hydrocarbon rings having the specified number of ring atoms (eg, C ₃ - ₆ cycloalkyl) and which are fully saturated or have no more than one double bond between the ring vertices. Cycloalkyl is also intended to refer to bicyclic and polycyclic hydrocarbon rings such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term heterocycloalkane or heterocycloalkyl refers to a cycloalkyl group containing one to five heteroatoms selected from N, O and S, the nitrogen and sulfur atoms being optionally oxidized and the nitrogen atom(s) being optionally quaternized. The heterocycloalkane may be a monocyclic, bicyclic or polycyclic ring system. Non-limiting examples of heterocycloalkane groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine and the like. The heterocycloalkane group may be attached to the rest of the molecule via a ring carbon or a heteroatom.

The term alkylene, by itself or as part of another substituent, means a divalent radical derived from an alkane, eg -CH ₂ CH ₂ CH ₂ CH ₂ -. Typically, an alkyl (or alkylene) group contains from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A lower alkyl or lower alkylene is a shorter chain alkyl or alkylene group, typically having four or fewer carbon atoms. Similarly, alkenylene and alkynylene refer to unsaturated forms of alkylene having double or triple bonds, respectively.

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As used herein, the wavy line ,_, that crosses a single, double, or triple bond in any chemical structure depicted herein, represents the point attachment of a single, double, or triple bond to the rest of the molecule.

The terms alkoxy, alkylamino, and alkylthio (or thioalkoxy) are used in their usual sense and refer to such alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. In addition, for dialkylamino groups, the alkyl moieties may be the same or different and may be combined to form a 3-7 membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as dialkylamino or -NR ^a R ^b is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

The terms halo or halogen, by themselves or as part of another substituent, mean, unless otherwise indicated, a fluorine, chlorine, bromine or iodine atom. In addition, terms such as haloalkyl are meant to include monohaloalkyl and polyhaloalkyl. For example, the term C ₁ . ₄ haloalkyl is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term aryl means, unless otherwise indicated, a polyunsaturated, typically aromatic, hydrocarbon group, which may be a single ring or multiple rings (up to three rings) that are fused together or covalently bonded. The term heteroaryl refers to aryl groups (or rings) that contain one to five heteroatoms selected from N, O and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule via a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl, and diphenyl, and non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyridinedinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidabenzolyl, benzopyrazolyl, benzothriazolyl, benzothriazolyl , indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, thilazolyl, isoxazolyl, , thienil and the like. Substituents for each of the above aryl and heteroaryl ring systems, when described as substituted, are selected from the group of acceptable substituents described below.

The term arylalkyl is intended to include those radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, and the like). Similarly, the term heteroaryl-alkyl means the inclusion of those radicals in which a heteroaryl group is attached to an alkyl group (eg, pyridylmethyl, thiazolylethyl, and the like).

The above terms (eg, alkyl, aryl, and heteroaryl) in some embodiments include both substituted and unsubstituted forms of the specified radical. Preferred substituents for each type of radical are shown below.

Unless otherwise indicated by the context, substituents for alkyl radicals (including groups often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) may be various groups selected from: -halo, -OR', -NR'R'', -SR' , SiR'RR', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R'', -OC(O)NR'R, NR''C(O )R', -NR'-C(O)NRR', -NR''C(O) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, - NHC(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -NR'S(O) ₂ R'', -CN and -NO ₂ in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such a radical. Each of R', R'' and R''' independently refers to hydrogen, unsubstituted C ₁ . ₈ alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C1_ ₈ alkyl, C ₁ . ₈ alkoxy or C ₁ . ₈ thioalkoxy groups, or unsubstituted aryl- _C1-4 alkyl groups. When R' and R are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' means the inclusion of 1-pyrrolidinyl and 4-morpholinyl.

Similarly, substituents for aryl and heteroaryl groups are varied and are generally selected from: -halo, -OR', -OC(O)R', -NR'R'', -SR', -R', - CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R',-NR'-C(O)NR''R''', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2) =NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -NR'S(O) ₂ R'', -N ₃ , perfluoro(C ₁ -C ₄ )alkoxy, and perfluoro(C ₁ C ₄ )alkyl in a number ranging from zero to the total number of open valences on the aromatic ring system, and where R', R'' and R''' are independently selected from hydrogen , C ₁ . ₈ αkyl, C ₁ . ₈ haloalkyl, C ₃ . ₆ cycloalkyl, C ₂ _ ₈ alkenyl, C ₂ - ₈ alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C ₁ -C ₄ alkyl and unsubstituted aryloxy-C 1 - ₄ alkyl. Other suitable substituents include each of the above aryl substituents attached to the ring atom with an alkylene bond of 1-4 carbon atoms.

The term base refers to a functional group that deprotonates water to produce a hydroxide ion. Typical bases are amines and nitrogen-containing heterocycles. Typical bases include -N(R ³ )(R ⁴ ), where R ³ and R ⁴ are independently selected from H or C ₁ . ₆ α-kyl, preferably H or methyl,

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where R ⁵ , R ⁶ , R ⁷ and R ⁸ , in each case, independently selected from hydrogen or C _1-6 alkyl, preferably H or methyl, and e represents 0-4. In some aspects, the base is a nitrogenous base.

The term antibody is used herein in its broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity (i.e., specific binding to a target antigen) . An intact antibody has mainly two regions: a variable region and a constant region. The variable region specifically binds and interacts with the target antigen. The variable region includes hypervariable regions (CDRs) that recognize and bind to a particular binding site on a particular antigen. The constant region can be recognized and interacted with by the immune system (see, for example, Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody can be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The antibody can be obtained from any suitable species. In some embodiments, the antibody is of human or murine origin. The monoclonal antibody may be, for example, human, humanized or chimeric.

The term monoclonal antibody as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical except for possible natural mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. The term Monoclonal refers to the nature of an antibody derived primarily from a homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method.

An intact antibody is one that includes an antigen-binding variable region as well as a light chain constant domain (CL) and a heavy chain constant domain, _CH 1 , _CH 2 , _CH 3 and _CH 4 , according to the class of the antibody. Constant domains can be natural sequence constant domains (eg, human natural sequence constant domains) or an amino acid sequence variant thereof.

An antibody fragment includes a portion of an intact antibody containing an antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, tribodies, tetrabodies, linear antibodies, single chain antibody molecules, ScFv, ScFv-Fc, multispecific antibody fragments derived from antibody fragment(s), fragment(s) produced by an expression Fab library, or epitope-binding fragments of any of the above, which specifically bind to a target antigen (eg, cancer cell antigen, viral antigen, or microbial antigen).

An antigen is an entity to which an antibody specifically binds. Antigen

- 5 040940 can be, for example, proteinaceous (eg protein, polypeptide or peptide), non-proteinaceous (eg carbohydrate) or a combination of the two.

The terms specific binding and specifically binds mean that a targeting agent or ligand, such as an antibody or antigen-binding fragment thereof, binds with high selectivity to its corresponding target antigen, and not to a variety of other antigens. For an antibody, the antibody or antibody fragment typically binds with an affinity of at least about 1 x 10' ⁷ M and preferably 10 ^-8 to 10 ^-9 , 10' ¹⁰ , 10 ^-11 or 10 ^-12 M and binds with a given antigen with an affinity that is at least twice its binding affinity for a non-specific antigen (eg BSA, casein) other than a predefined antigen or a closely related antigen having the same epitope.

The terms "inhibit" or "inhibit" means to reduce by a measurable amount or to prevent completely.

The term therapeutically effective amount refers to an amount of a conjugate (eg, an antibody drug conjugate) that is effective for treating a disease or disorder in a mammal. In the case of malignancy, a therapeutically effective amount of the conjugate may reduce the number of malignant cells; reduce the size of the tumor; inhibit (ie, slowly to some extent and preferably stop) the infiltration of malignant cells into peripheral organs; inhibit (ie, slowly to some extent and preferably stop) tumor metastasis; inhibit tumor growth; and/or alleviate one or more of the symptoms associated with the cancer. To the extent that a drug can inhibit growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by evaluating time to disease progression (TTP) and/or determining the rate of response (RR).

The term essential or essentially refers to the majority, i.e. >50% of the population, mixture or sample, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the population.

The terms intracellularly cleavable and intracellular cleavage refer to the metabolic process or reaction within a cell to a ligand-linker-drug conjugate (e.g., an antibody drug conjugate (ADC) or the like) resulting in a covalent bond (linker unit) between DE- the ligand (eg, antibody (Ab)) moiety is disrupted, resulting in the release of the DE moiety. The cleaved constituents of the ligand-linker-drug conjugate are thus intracellular metabolites.

The term cytotoxic activity refers to the cell-killing or cytotoxic effect of a ligand-linker-drug conjugate compound, typically via a released drug unit, on target cells. Cytotoxic activity can be expressed as an IC ₅₀ value (also referred to as half of the maximum inhibitory concentration), which is the concentration (molar or mass) per unit volume at which half of the cells survive when exposed to the conjugate.

The term cytotoxic agent, as used herein, refers to a substance that kills cells or otherwise causes destruction of cells.

The terms malignancy and malignant refer to or describe a physiological condition or disorder in mammals that is typically characterized by uncontrolled cell growth. The term tumor includes malignant cells.

An autoimmune disease is a disease or disorder arising from and directed against an individual's own tissues or proteins.

Non-limiting examples of a patient include human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird, and poultry. In a typical embodiment of the invention, the patient is a human.

The terms treat or treatment, unless the context otherwise indicates, refer to therapeutic treatment and preventive measures to prevent recurrence, in which the subject is to suppress or slow down (reduce) an undesirable physiological change or disorder, such as the development or spread of a malignant neoplasm. Beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization (i.e., not worsening) of the disease state, delay or retardation of disease progression, improvement or temporary relief of the disease state, and remission (partial or complete) , either detectable or undetectable. Treatment may also mean prolonging survival compared to expected survival if no treatment is given. Those in need of treatment include those who already have the condition or disorder.

In the context of cancer, the term treatment includes some of the

- 6 040940 or each of: inhibition of growth of tumor cells, malignant cells or tumors; inhibiting the replication of tumor cells or malignant cells, reducing the total tumor mass or reducing the number of malignant cells and alleviating one or more symptoms associated with this disease.

In the context of an autoimmune disease, the term treatment includes any or each of: inhibiting the replication of cells associated with the autoimmune disease condition, including, but not limited to, cells that produce an autoimmune antibody, reducing the autoimmune-like antibody load, and alleviating one or more symptoms. autoimmune disease.

The term pharmaceutically acceptable salt as used herein refers to pharmaceutically acceptable organic or inorganic salts of a compound (eg, drug, drug-linker, or ligand-linker-drug conjugate). The compound may contain at least one amino group, and accordingly acid addition salts may be formed with the amino group. Examples of salts include, but are not limited to sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate , ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, rtoluenesulfonate and pamoate (i.e., 1,1'-methylene-bis-(2- hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may include the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, its pharmaceutically acceptable salt may have more than one charged atom in its structure. Cases where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

Detailed description

General.

The present invention is based, in part, on the discovery that certain combinations of linking groups and cytotoxic agents can be used to prepare ligand-linker-drug conjugates, such as antibody-drug conjugates (ADC), which have hydrophilicity similar to that of unconjugated a ligand (ie, a targeting agent, such as an antibody or antigen-binding fragment thereof). By maintaining a similar hydrophilicity of the conjugate to that of the unconjugated ligand, the resulting conjugates can have higher drug loadings (e.g., at least 4 or 8 drug linkers per ligand) while retaining certain desirable characteristics of the ligand itself, such as decreased clearance in vivo, increased pharmacokinetic profile in vivo, higher exposure of the conjugates to the target cell(s), etc. Preferably, hydrophilic conjugates can be designed having hydrophilicity similar to that of the ligand without the need to include additional solubilizing groups such as polyethylene glycol or other water soluble polymers.

(Ligand-linker-drug conjugates are also referred to herein as drug-ligand conjugates, ligand-drug conjugates, or ligand-drug conjugates).

The hydrophilic linking groups (also referred to as linkers or linker units) of the conjugates are designed for increased hydrophilicity. The linking group provides for efficient release of the cytotoxic agent (also referred to as a drug moiety or drug) in the target cell, sufficient to induce cytotoxicity or a cytostatic effect in the case of a cytotoxic agent. Typically, hydrophilic linkers are designed to efficiently release the drug moiety once the conjugate is internalized into the target cell. Suitable recognition sites for releasing the drug moiety by cleavage are those that effectively separate the moiety from the hydrophilic linking group. Typically, the recognition site is a peptide cleavage site (eg, formed in combination with a cytotoxic agent that has an amino acid or peptide signature at the linking group attachment site). Examples of peptide cleavage sites include those recognized by intracellular proteases, such as those present in lysosomes. In embodiments where the linking group is attached to an effector moiety (DE) amino acid, AA1 forms a cleavable peptide bond with the DE unit. The cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches its target site. In other embodiments, AA1 forms an amide bond to an effector moiety attachment site ( _DE ) that is susceptible to cleavage (eg, by proteases) when the conjugate reaches the target site.

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In some embodiments, the drug units are auristatins, which are designed to increase hydrophilicity in combination with a hydrophilic linker unit. Advantageously, drug units can be designed to have hydrophilic substituents while maintaining strong cytotoxic activity. The auristatin drug units are attached at their C-terminus to a linker unit, as described in more detail herein.

Accordingly, in some embodiments, L ^H is a hydrophilic linker containing one, two, three, or four hydrophilic amino acids, wherein the first amino acid forms a cleavage site with the portion of the drug unit to which it is attached. In some embodiments, L ^H is a hydrophilic linker containing one or two hydrophilic amino acids, wherein the first amino acid forms a cleavage site with the portion of the drug unit to which it is attached. In some embodiments, L ^H is a hydrophilic linker containing an amino acid that forms a cleavage site with the portion of the drug unit to which it is attached. In some embodiments, the second, third and/or fourth hydrophilic amino acid is replaced with an optionally substituted alkylene or heteroalkylene group.

The hydrophilicity of a conjugate can be determined by comparing the hydrophilicity of the conjugate with that of the unconjugated targeting agent (ie, ligand or ligand unit), referred to as the hydrophilicity index. In some embodiments of the present invention, the retention time of the conjugate is no more than two minutes slower than the retention time of the unconjugated ligand, as determined as described in the examples. In some other embodiments of the present invention, the conjugate retention time is not more than one minute slower than the retention time of the unconjugated ligand, as determined as described in the examples. In some other embodiments of the present invention, the retention time of the conjugate is no more than half a minute slower than the retention time of the unconjugated ligand, as determined as described in the examples. With reference to the examples, example 3 discloses a preferred method for determining the hydrophilic index of a conjugate. Alternatively, another hydrophobic interaction column and/or method can be calibrated using the conjugates from Table. 2 as a reference for determining the reference mobilities of the conjugates (elution times) of the references using the selected column and/or method. The determined reference mobilities using the selected hydrophobic interaction column and/or method can be used to calculate the hydrophilicity index of the test sample (as will be determined below in Example 3). For example, auristatin T-Glu-Dpr-MA, mc-MMAF and mc-vc-PABC-MMAE can be used to form conjugates for use as reference. In another example, auristatin T-Glu-Dpr-MA-h1F6 ADC, h1F6-mc-MMAF and h1F6-mc-vc-PABC-MMAE ADC can be used as reference.

In view of the foregoing, in one group of embodiments, ligand-linker-drug conjugates are provided comprising a ligand unit and multiple linker drug units attached to the ligand unit. The linker unit contains a plurality of hydrophilic linkers (L ^H ), including a ligand attachment component, such as via a thioether bond. The drug unit contains a cytotoxic agent having an attachment component for connection to the linker unit. In another group of embodiments, ligand-linker-drug conjugates are provided wherein the linker moiety contains a plurality of hydrophilic linkers and the drug moiety contains a cytotoxic agent.

In related embodiments, methods are provided for administering ligandlinker-drug conjugates to a patient for the treatment of a disease. The disease may be, for example, a malignant neoplasm or an autoimmune disease. The ligand-linker drug conjugates are administered in a therapeutically effective amount and according to a therapeutically effective schedule. In some aspects, the dose of the conjugate is the same as or less than that of a similar doubly loaded conjugate (administered in a similar manner). In some aspects, the dose of the conjugate is the same as or less than the same quadruple loaded conjugate (administered in a similar manner). In some aspects, the dose of the conjugate is the same as or less than the same doubly loaded conjugate while the dosing schedule is the same or less frequent. In some aspects, the dose of the conjugate is the same as or less than the equivalent quadruple loaded conjugate while the dosing schedule is the same or less frequent. In some other aspects, the dose of the conjugate is less and the dosing schedule is the same or less frequent than a comparable doubly loaded conjugate. In some other aspects, the dose of the conjugate is less and the dosing schedule is the same or less frequent than a comparable four-fold loaded conjugate. The comparator conjugate can be, for example, the same ligand-drug-linker conjugate having a drug load of 2 or 4.

In another group of embodiments, methods are provided for making conjugates whether

- 8 040940 gand linker drug. In some aspects, the linker portion contains a collection of hydrophilic linkers (L ^H ) having a ligand attachment component (eg, an attached maleimide moiety) suitable for interaction with the ligand. In another group of embodiments, ligand-linker conjugates are provided wherein the linker portion contains a plurality of hydrophilic linkers (L ^H ) having features suitable for attachment and release of a drug moiety.

In another group of embodiments, methods are provided for preparing ligandlinker-drug conjugates. In some aspects, the linker portion contains a plurality of hydrophilic linkers (L ^H ) having an attached maleimide moiety suitable for reaction with a ligand. In another aspect, the linker portion comprises a plurality of hydrophilic linkers (L ^H ) having features suitable for drug release (if attached).

Ligand-linker-drug conjugates.

In one aspect, ligand-linker-drug conjugates are provided having the following formula

D----L ^H --L ^A —]---L

P' / P (I) or a pharmaceutically acceptable salt or solvate thereof, where:

L is a ligand that specifically binds to a target;

L ^a is a ligand attachment component;

L ^H is an optionally branched hydrophilic linker, each L ^H branch has the formula

-AA1 - R ^L1 - R ^L2 - R ^L3 in which

AA ₁ is a hydrophilic amino acid that forms a cleavable peptide bond with the C-terminus of the drug unit to which it is attached;

RL ¹ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ¹ and the absence of RL ² and RL ³ ;

RL ² is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ² and the absence of RL ³ ; And

RL ³ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ³ ;

index p is an integer in the range from 4 to about 20;

index p' is an integer from 1 to 4; And

D is having the formula

in which each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted-C1-C ₈ -alkyl; with the proviso that both R ¹ and R ² are not H, except that both of R ³ and R ³ are not H;

R ³ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -αkyl,

R ³ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl, and at least one of R ³ and R ³ is not H;

R ⁴ is selected from the group consisting of H and optionally substituted alkyl;

R ⁵ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl;

or R ⁴ and R ⁵ together form a carbocyclic ring and have the formula -(CR ^a R ^b )n- wherein R ^a and R ^b are independently selected from the group consisting of H and optionally substituted -C1-C ₈ -alkyl and n is selected from the group consisting of 2, 3, 4, 5 and 6;

R ⁶ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -αkyl;

R ⁷ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -αkyl;

each R ⁸ is independently selected from the group consisting of H, -OH, optionally substituted -C1-C8 alkyl, and optionally substituted -O-(C ₁ -C ₈ -alkyl);

R ¹² is selected from H, optionally substituted -C1-C8 alkyl, optionally substituted aryl, optionally substituted -X ¹ aryl, optionally substituted -C3- _C8 carbocycle, optionally substituted -X ¹ -(C3-C8 carbocycle), optionally substituted -C1-C8-alkylene-NH2, optionally substituted C3-C8 heterocycle and optionally substituted -X ¹ -(C3- _C8 heterocycle); and each X ¹ is independently -C ₁ -C ₁₀ -αkylene;

- 9 040940 where the ligand-linker-drug conjugate has a hydrophilic index less than or equal to 2;

where the left and right lines L ^H show covalent attachment to the unit of the drug and L ^A , respectively; and the wavy line of each D shows covalent attachment to L ^H

In a related aspect, ligand-linker-drug conjugates are provided having the following formula

or a pharmaceutically acceptable salt or solvate thereof, wherein:

L is a ligand that is specifically associated with the target;

L ^a is a ligand attachment component; L ^H is an optionally branched hydrophilic linker, each L ^H branch having the formula

-AA1 - R ^L1 - R ^L2 - R ^L3 in which

AA ₁ is a hydrophilic amino acid that forms a cleavable peptide bond with the C-terminus of the drug unit to which it is attached;

RL ¹ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the absence of RL ³ and RL ³ ;

RL ² is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ² and the absence of RL ³ ; And

RL ³ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ³ ;

index p is an integer in the range from 4 to about 20;

index p' is an integer from 1 to 4; And

D is having the formula

in which each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted-C1-C ₄ -alkyl; with the proviso that both R ¹ and R ² are not H, if both of R ³ and R ³ are not H;

R ³ is selected from the group consisting of H and optionally substituted -C ₁ -C ₄ -alkyl;

R ³ is selected from the group consisting of H and optionally substituted with -C ₁ -C ₄ -alkyl and at least one of R ³ and R ³ is not H;

R ⁴ is selected from the group consisting of H and optionally substituted alkyl;

R ⁵ is selected from the group consisting of H and optionally substituted -C ₁ -C ₄ -alkyl;

or R ⁴ and R ⁵ together form a carbocyclic ring and have the formula -(CR ^a R ^b )n-, in which R ^a and R ^b are independently selected from the group consisting of H and optionally substituted -C 1 -C ₄ -alkyl and n is selected from the group consisting of 2, 3, 4, 5 and 6;

R ⁶ is selected from the group consisting of H and optionally substituted -C ₁ -C ₄ -alkyl;

R ⁷ is selected from the group consisting of H and optionally substituted -C ₁ -C ₄ -alkyl;

each R ⁸ is independently selected from the group consisting of H, -OH, optionally substituted -C1-C4 alkyl, and optionally substituted -O-(C ₁ -C ₄ -alkyl);

R ¹² is selected from H, optionally substituted -C1-C8 alkyl, optionally substituted aryl, optionally substituted -X ¹ aryl, optionally substituted -C3- _C8 carbocycle, optionally substituted -X ¹ -(C3-C8 carbocycle), optionally substituted -C1-C8-alkylene-NH2, optionally substituted C3-C8 heterocycle and optionally substituted -X ¹ -(C3- _C8 heterocycle); and each X ¹ is independently -C ₁ -C ₁₀ -alkylene;

where the ligand-linker-drug conjugate has a hydrophilic index less than or equal to 2;

where the left and right lines L ^H show covalent attachment to the unit of the drug and L ^A , respectively; and the wavy line of each D shows covalent attachment to L ^H .

The various components of the ligand-linker-drug conjugates of formulas I and I' are presented in more detail below.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least four linker-drug units, wherein the ligand unit and each of the drug unit(s) are connected by a linker unit(s) comprising a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, each linker unit further comprises

- 10 040940 hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least six linker-drug units, wherein the ligand unit and each of the drug unit(s) are connected by a linker unit(s) comprising a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least eight linker-drug units, wherein the ligand unit and each of the drug unit(s) are connected by a linker unit(s) comprising a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least ten linker-drug units, wherein the ligand unit and each of the drug unit(s) are connected by a linker unit(s) comprising a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least sixteen linker-drug units, wherein the ligand unit and each of the drug unit(s) are connected by a linker unit(s) comprising a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

Drug unit.

Referring to the drug unit of formulas I and I', in some embodiments, R ¹² is selected from H, optionally substituted -C1-C8-alkyl, optionally substituted aryl, optionally substituted -X ¹ aryl, optionally substituted -C3- _C8 - a carbocycle, an optionally substituted -X ¹ -(C3-C8-carbocycle), an optionally substituted -CgCg-alkylene-KSh, an optionally substituted -C3-C8 heterocycle, and an optionally substituted -X ¹ -(C3- _C8 -heterocycle).

In some related embodiments, R ¹² is not a phenylalanine or proline side chain. In some additional embodiments, R ¹² is not a phenylalanine, methionine, tryptophan, or proline side chain.

In some embodiments, R ¹² is selected from natural L-amino acid side chains other than proline and glycine. In some other embodiments, R ¹² is selected from naturally occurring L-amino acid side chains other than proline, glycine, or phenylalanine. In some additional embodiments, R ¹² is selected from naturally occurring L-amino acid side chains other than proline, glycine, tryptophan, methionine, or phenylalanine.

In some other embodiments, R ¹² is selected from side chains of the hydrophilic amino acid group consisting of threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine, and thiazolylalanine .

In some embodiments, R ¹² is a threonine side chain.

Typical drug units have the following formula, or a pharmaceutically acceptable salt thereof, where the wavy line indicates the site of attachment to the linker unit. In some exemplary units, the drug unit is dimethyl- or monomethylauristatin F, as shown below:

- 11 040940

or a pharmaceutically acceptable salt or solvate thereof.

Other typical drug units are the dimethyl or monomethyl forms of auristatin T.

or a pharmaceutically acceptable salt or solvate thereof.

linker link.

Referring to the unit linker with formulas I and I', in some embodiments, the implementation of L ^A covalently linked to the sulfur atom of the ligand. In some aspects, the sulfur atom is a cysteine residue that can form an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is that of a cysteine residue that has been introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In other aspects, the sulfur atoms to which ^LA is attached are selected from cysteine residues forming an interchain disulfide bond between the antibody and the cysteine that has been introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction).

AA ₁ forms a cleavable bond with the drug unit. In embodiments where AA1 is attached to the amino acid of the drug unit, AA ₁ forms a cleavable peptide bond with the drug unit. The cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches the target site. In some embodiments, AA ₁ is a hydrophilic amino acid, typically an amino acid selected from the group consisting of glycine and L-form aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine. In some embodiments, AA ₁ is glutamic acid.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L- or D- forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NHCH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH ₂ CH ₂ CH ₂ C(O)-. In some other embodiments, R ^L1 is selected from the group consisting of the D-amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-GO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH ₂ CH ₂ C(O)-, -CH ₂ CH ₂ CH ₂ C(O)- and -CH ₂ CH ₂ CH ₂ CH ₂ C(O)-.

In embodiments where R ^L1 is present and is an optionally substituted alkylene, it may be C1-C6-alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl), -NH2 or -NH(C1-C3-alkyl). In some embodiments, R ^L1 is ethylenediamine, -NH-CH(COOH)-C2-NH- or -C(O)-CH(CH ₂ NH ₂ )-.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L- or D- forms of aspartic acid, glutamic acid, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-,

- 12 040940

-CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH^H^CCO) - and -CHSSCHSSCHSCCO)-. In some other embodiments, when R ^L2 is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L2 is present and is an optionally substituted alkylene, it may be a C1-C6 alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl), -NH2 or -NH(C1-C3-alkyl). In some embodiments, R ^L2 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH ₂ NH ₂ )-.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L- or D- forms of aspartic acid, glutamic acid, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH^HBDO)- and -CHSSCHSSCHCHCO)-.

In some other embodiments of the invention, where R ^L3 is present, it is selected from the group consisting of D-amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH^CBDO)- and -CHSSCHSSCHCHCO)-.

In embodiments where R ^L3 is present and is an optionally substituted alkylene, it may be a C1-C6 alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl), -NH2 or -NH(C1-C3-alkyl). In some embodiments, R ^L3 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH ₂ NH ₂ )-.

In some embodiments of the above, AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments of the above, AA1 is present and R ^L1 is present, and R ^l2 and R ^l3 are absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some embodiments of the above, AA ₁ is a hydrophilic amino acid and at least one of R ^L1 , R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is glutamic acid and at least one of R ^L1 , R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the foregoing, AA ₁ is glutamic acid, R ¹¹ is a hydrophilic amino acid, and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ and R ^L1 are hydrophilic amino acids and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as defined above.

In some embodiments of the above, L ^H does not contain a glycine dipeptide (Gly-Gly), tripeptide, or tetrapeptide. In some embodiments, L ^H does not include an Asn-(D)Lys peptide.

In some embodiments, L ^H comprises a modified peptide having two to four amino acids. The modified peptide has an amino acid at position 1 (AA ₁ ) that is selected to optimize the release of the drug moiety (eg, by protease cleavage of the amide peptide bond). At one or both positions, R ^L1 and R ^L2 is an amino acid that reorients the typical N bonds to C peptides (forming amide bonds) and facilitates the attachment of the last amino acid (e.g., R ^L2 or R ^L3 ) which, prior to attachment of the ligand unit, includes α-amino group protected as maleimide. An amino acid having an N-to-C bond is attached to the next group via its side chain. In some embodiments, this amino acid is an alpha amino acid.

- 13 040940

In other embodiments, it may be a beta or gamma amino acid. In some of these embodiments, the side chain is selected from -CH2NH2-,

-CH2CH2NH2-, -CH2CH2CH2NH2 and -CH2CH2CH2CH2NH2-.

In some L ^H embodiments, amino acids having an N-to-C reverse bond (RL ¹ ) are attached to RL2 or RL ³ , where RL2 or RL ³ is a hydrophilic amino acid or an optionally substituted alkylene, in accordance with any of the embodiments described by you. she.

In some L ^H embodiments, amino acids having an N-to-C reverse bond (RL ¹ ) are attached to RL2, where RL2 is a hydrophilic amino acid or an optionally substituted alkylene, in accordance with any of the embodiments described above.

In some other embodiments, L ^H is a hydrophilic, cleavable linker, each branch having the formula

in which R ²¹ is selected from CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ²² is selected from -CH2NH2, -CH2CH2NH2, -CH2OH and -CH2CH2OH. The left and right wavy lines indicate attachment to the drug unit and L ^a , or L ^H branch, respectively.

In other embodiments, L ^H or a branch thereof has the formula

in which R ²² is selected from -CH2NH2, -CH2CH2NH2, -CH2OH and -CH2CH2OH. In some embodiments, R ²² is selected from -CH2NH2 and -CH2CH2NH2. The left and right wavy lines indicate attachment to the drug unit and L ^A , or L ^H branch, respectively.

In some embodiments, L ^H or a branch thereof has the formula

The left and right wavy lines indicate drug attachment and L ^A , respectively.

In some embodiments, L ^H or a branch thereof has the formula

The left and right wavy lines indicate attachment to the drug unit and L ^A , or L ^H branch, respectively.

In some embodiments, L ^H or a branch thereof has the formula

- 14 040940

The left and right wavy lines indicate attachment to the drug unit and L ^A , or L ^H branch, respectively.

In some embodiments, L ^H or a branch thereof has the formula

The left and right wavy lines indicate attachment to the drug unit and L ^A , or L ^H branch, respectively.

In some embodiments, L ^H or a branch thereof has the formula

The left and right wavy lines indicate attachment to the drug unit and L ^A , or L ^H branch, respectively.

In some embodiments, L ^H or a branch thereof has the formula

The left and right wavy lines indicate drug attachment and L ^A , respectively.

In some embodiments, L ^H or a branch thereof has the formula

The left and right wavy lines indicate attachment to the drug unit and L ^A , or L ^H branch, respectively.

In some additional embodiments of the above, L ^H is a branched hydrophilic linker having the formula

where each R is independently selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and each of the bars next to R ³¹ indicates attachment to a DE unit, and the vertical dotted line indicates attachment to a ligand unit.

In some additional embodiments of the above, L ^H is a branched hydrophilic linker having the formula

- 15 040940

in which each of the bands indicates attachment to a drug unit, and a vertical dotted line indicates attachment to a ligand unit.

The L ^A subunit is described in more detail below.

Referring again to linker-ligand-drug conjugates of formula I and I', in some other embodiments, the ligand-linker-drug conjugates have the formula selected from

where S refers to the sulfur atom of the ligand.

Specific embodiments include the following:

- 16 040940 where S refers to the sulfur atom of the ligand, or a pharmaceutically acceptable salt or solvate thereof.

In yet another aspect, ligand-linker-drug conjugates are provided having the following formula

wherein

L is a ligand that specifically binds to a target;

L ^a is a ligand attachment component;

L ^H is an optional hydrophilic linker, each branch of L ^H having the formula:

-ΑΑχ - R ^L1 - R ^L2 - R ^L3 in which

AA ₁ is a hydrophilic amino acid that forms a cleavable bond with DE;

RL ¹ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ¹ in the absence of RL ³ and RL ³ ;

RL ² is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ² and the absence of RL ³ ; And

RL ³ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with L ^A in the presence of RL ³ ;

L ^A is a ligand attachment component;

index p is an integer in the range from 4 to about 20;

index p' is an integer from 1 to 4; and the ligand-linker-drug conjugate has a hydrophilic index less than or equal to 2; where the left and right lines L ^H indicate covalent attachments to the unit D _E and L ^A , respectively; And

D _E is an effector moiety, as further described herein.

The various components of the formula II ligand-linker-drug conjugates are presented below in more detail.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least four linker-DE units, wherein the ligand unit and each of the DE unit(s) are connected by a linker unit containing a plurality of hydrophilic linkers (L ^H ). In some other aspects, the inker units are attached to the ligand unit via a thioether bond. In some related aspects, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least six linker-DE units, wherein the ligand unit and each of the DE unit(s) are connected by a linker unit containing a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least eight linker-DE units, wherein the ligand unit and each of the DE unit(s) are connected by a linker unit containing a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates comprise a ligand unit and at least ten linker-DE units, wherein the ligand unit and each of the DE unit(s) are connected by a linker unit containing a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

In some aspects, ligand-linker-drug conjugates include a ligand unit and at least sixteen linker-DE units, where the ligand unit and each of the DE unit(s) are connected by a linker unit containing a plurality of hydrophilic linkers (L ^H ). In some other aspects, the linker units are attached to the ligand unit via a thioether bond. In some related aspects, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the ligand unit via a thioether bond.

With reference to a linker unit of Formula II, In some embodiments, L ^A is covalently bonded to the sulfur atom of the ligand. In some aspects, the sulfur atom is that of a cysteine residue that forms an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is that of a cysteine residue that has been introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In other aspects, the sulfur atom(s) to which L ^A is attached is selected from cysteine residues forming an interchain disulfide bond of the antibody and cysteine residues that have been introduced into the ligand unit (eg, by site-directed mutagenesis or chemical reaction).

AA ₁ forms a cleavable bond with the DE unit. In embodiments where AA ₁ is attached to an amino acid of the DE unit, AA ₁ forms a cleavable peptide bond with the DE unit. The cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches the target site. In other embodiments, AA ₁ forms an amide bond to an effector moiety (DE) attachment site that is susceptible to cleavage (eg, by proteases) when the conjugate reaches the target site. In some embodiments of Formula II, AA ₁ is a hydrophilic amino acid, typically a naturally occurring amino acid, selected from the group consisting of glycine and the L forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine. In some embodiments, AA ₁ is glutamate.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L- or D-forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NHCH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH ₂ CH ₂ CH ₂ CH ₂ C(O)-. In some other embodiments, R ^Li is selected from the group consisting of D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^Li is present and is an optionally substituted alkylene, it may be a C ₁ -C 6 -alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, - N(C ₁ -C ₃ -alkyl)-, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, R ^L1 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)CH(CH2NH2)-.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L- or D- forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NHCH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CHCHSCHCHCO)- and -CH2CH2CH2CH2C(O)-.

In some other embodiments, when R ^L2 is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NHCH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L2 is present and is an optionally substituted alkylene, it may be Q-C6-alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N( C1-C3-alkyl)-, -NH2 or -NH(C1-C3-alkyl). In some embodiments, RL ² is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH2NH ₂ )-.

In embodiments where RL ³ is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L- or D- forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NHCH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In some other embodiments, when RL ³ is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NHCH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where RL ³ is present and is an optionally substituted alkylene, it may be a C ₁ -C ₆ alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O) -, -COOH, -N(C ₁ -C ₃ -alkyl) -, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, RL ³ is ethylenediamine, -NH-CH(COOH)-CH ₂ -NH- or -C(O)CH(CH2NH2)-.

In some embodiments of the above, AA1 is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments of the above, AA ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some additional of the above, AA1 is a hydrophilic amino acid and at least one of R ^L1 , R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 is glutamic acid and at least one of R ^L1 , R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is glutamic acid, R ^L1 is a hydrophilic amino acid, and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some other embodiments of the above, AA ₁ and R ^L1 are hydrophilic amino acids and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 is an optionally substituted alkylene as defined above.

In some embodiments of the above, the LH does not contain a glycine dipeptide (Gly-Gly), tripeptide, or tetrapeptide. In some embodiments, the LH does not include peptidic Asn-(D)Lys.

In some embodiments, the LH includes a modified peptide having two to four amino acids. The modified peptide contains an amino acid at position 1 (AA1) which is chosen to optimize the yield of the DE unit (eg, protease cleavage via an amide peptide bond). At one or both positions of R ^L1 and R ^L2 is an amino acid that reverses the typical N to C orientation of the peptide bonds and facilitates the attachment of the last amino acid (e.g., R ^L2 or R ^L3 ) which, prior to attachment of the ligand unit, includes a protected α-amino group. like maleimide. An amino acid having an N-to-C bond is attached to the next group through its side chain. In some embodiments, this amino acid is an alpha amino acid. In other embodiments, it may be a beta or gamma amino acid. In some embodiments, the side chain is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2- and -CH2CH2CH2CH2NH2.

In some embodiments, the L ^H amino acid having an N-to-C bond (R ^L1 ) is attached to R ^L2 or R ^L3 , where R ^L2 or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene, in accordance with any of the embodiments, described above.

In some embodiments, L ^H , an amino acid having an N-to-C reverse bond (R ^L1 ), is attached to R ^L2 , where R ^L2 is an optionally substituted alkylene, in accordance with any of the embodiments described above.

In some additional embodiments, the implementation of L ^H is a hydrophilic, cleavable linker, each branch has the formula

where R ²¹ is selected from CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ²² is selected from -CH2NH2, -CH2CH2NH2, -CH2OH and -CH2CH2OH. The wavy lines on the left and right indicate link attachments DE and LA, or branches L ^H , respectively.

In additional embodiments, L ^H or a branch thereof has the formula

- 19 040940 o co ₂ n and ii I n .n. X X^

N' N

O

he , where R ²² is selected from -CH2NH2, -CH2CH2NH2, -CH2OH and -CH2CH2OH. In some embodiments, R ² is selected from -CH2NH ₂ and -CH ₂ CH ₂ NH ₂ . The wavy lines on the left and right indicate link attachments DE and L ^a , respectively.

In some embodiments, L ^H or a branch thereof has the formula

The wavy lines on the left and on the right indicate attachments to the link D _E and directly.

In some embodiments, L ^H or a branch thereof has the formula l ^a , or branches L ^H , respectively

The wavy lines on the left and on the right indicate attachments to the link D _E and directly.

In some embodiments, L ^H or a branch thereof has the formula

L ^A , or branches L ^H , respectively

The wavy lines on the left and right indicate attachments to the link DE and directly.

In some embodiments, L ^H or a branch thereof has the formula l ^a , or branches L ^H , respectively

The wavy lines on the left and right indicate attachments to the DE link and In some embodiments, L ^H or a branch thereof has the formula

L ^a , respectively.

The wavy lines on the left and on the right indicate attachments to the link D _E and directly.

In some embodiments, L ^H or a branch thereof has the formula

L ^a , or branches L ^H , respectively 20 040940

Wavy lines on the left and right indicate attachments to the link DE and L ^A , or branches L ^H , respectively

actually.

In some embodiments, L ^H or a branch thereof has the formula o

The wavy lines to the left and to the right represent link attachments DE and L ^A , or branches L ^H , respectively.

In some additional embodiments, L ^H is a branched hydrophilic linker having the formula

HN V where each R ³¹ is independently selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and each of the bars adjacent to R ³¹ shows attachment to a DE unit, and the vertical dotted line shows attachment to a ligand unit.

In some additional embodiments, L ^H is a branched hydrophilic linker having the formula

each of the bands shows attachment to the DE unit, and the vertical dotted line shows attachment to the ligand unit.

In some additional embodiments of the above, the branched hydrophilic linker has the formula

- 21 040940

Referring again to formula II ligand-linker-drug conjugates, in some further embodiments, the ligand-linker-drug conjugates have the formula selected from

wherein S is the sulfur atom of the ligand.

L ^A - ligand attachment component.

Referring again to formulas I, I' and II (see above), L ^A is the ligand attachment component. In some embodiments, L ^A may be a maleimide or a hydrolyzed maleimide or a succinimide group (shown in the figure below as a constituent of succinic acid). In some embodiments, where L ^A is attached to a ligand unit, it is a hydrolyzed maleimide or a succinimide group (shown as a constituent of succinic acid). Accordingly, in some embodiments, L ^A has the formula

where the wavy line indicates the point of attachment to L ^A and \\ indicates the point of attachment to L, the unit of the ligand.

In other embodiments, L ^A has the formula

where the wavy line indicates the point of attachment to L ^A and \\ indicates the point of attachment to L, the unit of the ligand.

- 22 040940

In the context of the present invention, in some embodiments (as shown above)

L ^a is the remainder of the maleimide group used to attach part of the ligand.

The design of L and L ^A allows for the easy addition of a ligand unit as well as the provision of an additional carboxyl group which increases the hydrophilicity of the ligand-drug conjugate.

The maleimidazot also becomes the a-amine of amino acid 3 (with reference to L ^H ).

L is a ligand.

Referring again to formulas I, I' and II, the ligand unit (L-) is a targeting agent that specifically binds to the target moiety. The ligand can specifically bind to a cell component or other target molecules of interest. The target component, or target, is usually located on the surface of the cell. In some aspects, the ligand moiety acts to deliver the Drug moiety to the specific target cell population with which the ligand moiety interacts. Ligands include, but are not limited to, proteins, polypeptides, and peptides, as well as non-protein substances such as carbohydrates. Suitable ligand units include, for example, antibodies, eg, full-length (intact) antibodies, as well as their antigen-binding fragments.

In embodiments where the ligand unit is a targetable agent other than an antibody, it may be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include an interferon, a lymphokine, a hormone, a growth factor and a colony stimulating factor, a vitamin, a nutrient transport molecule (such as, but not limited to, transferrin), or any other cell-binding molecule or substance.

In some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. In some aspects, the sulfur atom is that of a cysteine residue that forms an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is that of a cysteine residue that has been introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In additional aspects, the sulfur atoms attached to L ^A are selected from cysteine residues that form an interchain disulfide bond of the antibody and cysteine residues that have been introduced into the ligand unit (eg, site-directed mutagenesis or chemical reaction). In some embodiments, a cysteine residue is introduced into the Fc region at position 239 according to the EU index of the numbering system as in Kabat (Kabat E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91 3242).

In some aspects, the ligand unit forms a bond with the maleimide located on L ^H through the sulfhydryl group of the ligand, forming a thio-substituted succinimide. The sulfhydryl group may be present on the ligand in the natural state of the ligand, such as a natural antibody, or may be introduced into the ligand through chemical modification. Hydrolysis of the remaining succinimide produces the L ^A portion.

In one aspect, the ligand unit also contains one or more lysine residues, which can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

In another embodiment, the ligand unit may have one or more carbohydrate groups, which may be chemically modified to have one or more sulfhydryl groups.

In another embodiment, sulfhydryl groups may be formed by reduction of interchain disulfides. Accordingly, In some embodiments, the linker unit is conjugated to a cysteine residue of reduced interchain disulfides.

In another embodiment, the sulfhydryl group is chemically introduced into the antibody, for example by introducing a cysteine residue. Accordingly, in some embodiments, the linker unit is conjugated to an introduced cysteine residue.

Useful non-immunoreactive protein, polypeptide, or peptide ligands include, but are not limited to, transferrin, epidermal growth factor (EGF), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (TGF ), such as TGF-α and TGF-β, vaccinia growth factor (VGF), insulin and insulin-like growth factors I and II, somatostatin, lectins, and low-density lipoprotein apoprotein.

Particularly preferred ligands are antibodies. Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are a homogeneous population of antibodies to a specific antigenic determinant (e.g., cancer cell antigen, viral antigen, microbial antigen, protein, peptide, carbohydrate factor (VGF), chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen of interest can be obtained using any method known in the art, which involves the production of antibody molecules using continuous cell lines in culture.

- 23 040940

Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be obtained using any of the numerous methods known in the art (for example, Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983 , Immunology Today 4:72-79 and Olsson et al., 1982, Meth Enzymol 92:3-16).

The antibody may be an antigen-binding fragment, derivative or analogue of an antibody that immunospecifically binds to target cells (eg, cancer cell antigens, viral antigens, or microbial antigens), or another antibody associated with tumor cells or matrix. In this regard, antigen binding means that the fragment, derivative or analog is capable of specifically binding to the target moiety. In particular, in an exemplary embodiment, the antigenicity of the idiotype immunoglobulin molecule can be increased by deletion of the backbone and CDR sequences that are C-terminal of the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind an antigen, synthetic peptides containing CDR sequences can be used in antigen binding assays using any binding assay method known in the art (e.g., BIA core assay) (see, e.g., Kabat et al. al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md Kabat E et al., 1980, J. Immunology 125(3):961-969).

Other useful antibodies include antigen-binding antibody fragments such as, but not limited to, F(ab') ₂ fragments, Fab fragments, Fvs, single chain antibodies, diabodies, tribodies, tetrabodies, scFv, scFv-FV, or any other molecules derived from an antibody and having the same specificity as the antibody.

In addition, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human parts, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which the different parts are derived from different animal species, such as, for example, having a variable region derived from mouse monoclonal and human immunoglobulin constant regions. (See, for example, US Patent No. 4,816,567; and US Patent No. 4,816,397, which are incorporated herein by reference in their entirety). Humanized antibodies are non-human, animal antibody molecules having one or more non-human hypervariable regions (CDRs) and a framework region derived from a human immunoglobulin molecule. (See, for example, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA methods known in the art, for example, using the methods described in International Publication No. WO 87/02671; European Patent Publication No. 0184187; European Patent Publication No. 0171496; European Patent Publication No. 0173494; International Publication No. WO 86/01533; US Pat. No. 4,816,567; European Patent Publication No. 012 023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:12021207; Oi et al., 1986, BioTechniques 4:214; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534 and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.

Fully human antibodies are particularly desirable and can be generated using transgenic mice that are not capable of expressing endogenous immunoglobulin heavy and light chain variable region genes, but that can express human heavy and light chain variable region genes.

Antibodies may have modifications (eg, substitutions, deletions and/or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies may have amino acid residue modifications identified involved in the interaction between the anti-Fc domain and the FcRn receptor (see, for example, International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). Antibodies may also have modifications to amino acid residues identified as being involved in the interaction between the anti-Fc domain and the Fc gamma III receptor.

Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression methods.

The nucleotide sequence encoding antibodies immunospecific for a malignant cell antigen can be obtained, for example, from the GenBank database or a database like this, in literature publications, or by routine cloning and sequencing.

In a specific embodiment, the antibody can be used to treat cancer. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to those skilled in the art, such as recombinant expression methods. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or a database like this, in literature publications, or by routine cloning and sequencing.

In another specific embodiment, antibodies are used to treat autoimmune diseases in accordance with the compositions and methods of the invention. Antibodies immunospecific for the antigen of the cell that is responsible for the production of autoimmune antibodies can be obtained from an organization (for example, a researcher at a university or company) or produced by any method known to those skilled in the art, such as chemical synthesis methods. or recombinant expression.

In some embodiments, beneficial antibodies can bind to a receptor or receptor complex. The receptor or receptor complex may comprise, for example, a member of the immunoglobulin gene superfamily, a member of the TNF receptor superfamily, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.

In some embodiments, the antibody is a humanized CD70 antibody (see, for example, US 2009/0148942), a humanized CD19 antibody (see, for example, US 2009/0136526), a chimeric, or a humanized CD30 antibody (see, for example, US 2010 /0239571), a humanized CD33 antibody (US 2013/0309223), a humanized Beta6 antibody (see, for example, WO 2013/123152) or a humanized Liv-1 antibody (see, for example, US 2013/0259860).

Drug load - r.

Referring again generally to ligand-linker-drug conjugates of formulas I, I' and II, the number of drug-linker units per ligand is represented as p. (In this context, the drug to drug-linker may be a cytotoxic agent.) In embodiments where the linkers are not branched, p is the number of drug-linker molecules per ligand (eg, antibody). When considering individual conjugates, p is an integer representing the number of drug-linker molecules per ligand. When considering a composition containing multiple conjugates, p is the average number of drug linkers per ligand (or in embodiments where the linkers are not branched, the average number of drug linker molecules per ligand (eg, antibodies)). The variable p is within the range of 4 to 20, typically 6 to 12, 8 to 12, or 8 to 16, or up to 20.

The average number of drug-linker units per ligand unit in the conjugation reaction preparation can be characterized by conventional means such as mass spectrometry, ELISA, HIC, and HPLC. The quantitative distribution of ligand-linker drug conjugates in terms of p can also be determined. In some cases, separation, purification, and characterization of homogeneous ligand-drug conjugates, where p is a specific index of the ligand-drug conjugate with other drug loads, can be achieved by means such as reverse phase HPLC or electrophoresis.

Activity analyzes.

There are a number of different assays that can be used to determine whether a ligand-drug conjugate has a cytotoxic effect on a cell line. In one example, a thymidine incorporation assay is used to determine whether a ligand-drug conjugate has a cytotoxic effect on a cell line. For example, cells at a density of 5000 cells/well of a 96-well plate are cultured for a 72-hour period and exposed to 0.5 µCi ³ H-thymidine for the final 8 hours of the 72-hour period, and incorporation ^{of 3} H-thymidine into the culture cells measured in the presence and absence of the ligand-drug conjugate. The ligand-drug conjugate has a cytotoxic effect on cells if the cultured cells have reduced ³ H-thymidine incorporation compared to similar cells cultured under the same conditions but not exposed to the ligand-drug conjugate.

(See also Klussman et al., Bioconjugate Chemistry 15:765-773 (2004); Doronina et al., Bioconjugate Chemistry 17:114-124 (2006)).

In another example, to determine whether a ligand-drug conjugate has a cytotoxic effect on a cell line, cell viability was determined by determining cell uptake of a dye, such as neutral red, trypan blue, or ALAMAR™ blue (see, for example, Page et al., 1993, Intl. J. of Oncology 3:473-476). In such an assay, cells are incubated in dye-containing media, cells are washed, and the remaining reflective dye

- 25 040940 cellular uptake of the dye, measured spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al., 1990, J. Nat'l Cancer Inst. 82:1107-12). Preferred ligand-drug conjugates include those with an IC ₅₀ value (defined as the concentration of mAb that results in 50% cell death) of less than 1000 ng/mL, preferably less than 500 ng/mL, more preferably less than 100 ng/mL, even more preferably less than 50 or even less than 10 ng/ml in a cell line.

Drug-linker compounds.

In another aspect, drug-linker compounds are provided having the formula

[d _e -----l ^h --l ^a JP' IV or a pharmaceutically acceptable salt or solvate thereof, wherein:

L ^a is a ligand attachment component; L ^H is an optionally branched hydrophilic linker, each L ^H branch has the formula

-AA1 - R ^L1 - R ^L2 - R ^L3 in which

AA1 is a hydrophilic amino acid that forms a cleavable peptide bond with the C-terminus of the DE unit to which it is attached;

R ^l1 is optional, and is a hydrophilic amino acid or an optionally substituted alkylene that can share an atom with LA in the presence of R ^L1 and the absence of RL ² and RL ³ ;

RL ² is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with LA in the presence of RL ² and the absence of RL ³ ; And

RL ³ is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with LA in the presence of RL ³ ;

index p' is an integer from 1 to 4; And

D _E is an effector moiety (as described herein);

wherein the ligand-linker-drug conjugate formed with the drug-linkers has a hydrophilicity index of less than or equal to 2; and the left and right L ^H lines show covalent attachments to the DE and LA units, respectively.

In a related aspect IV', there is provided drug-linker compounds having the following formula (D _e -----L ^H --L ^a P'IV' or a pharmaceutically acceptable salt or solvate thereof, wherein:

LA is a ligand attachment component;

L ^H is an optionally branched hydrophilic linker having the formula:

-ΛΑχ - R ^L1 - R ^L2 - R ^L3 in which

AA1 is a hydrophilic amino acid that forms a cleavable peptide bond with the C-terminus of the D _E unit to which it is attached;

R ^L1 is a hydrophilic amino acid or an optionally substituted alkylene that can share an atom with LA in the absence of RL ³ and RL ³ ;

RL ² is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with LA in the presence of RL ² and the absence of RL ³ ; And

R ^L3 is optional and is selected from a hydrophilic amino acid and an optionally substituted alkylene that can share an atom with LA in the presence of RL ³ ;

LA is a ligand attachment component;

index p' is an integer from 1 to 4; And

DE is an effector moiety (as described herein);

wherein the ligand-linker-drug conjugate formed with the drug-linkers has a hydrophilicity index of less than or equal to 2; and while the left and right lines L ^H show covalent attachments to the link DE and LA, respectively.

The various components of the ligand-linker-drug conjugates of formulas IV and IV are given in more detail below.

Drug link, DE.

Referring to formulas IV and IV', the drug unit, DE, is an effector moiety having the formula

- 26 040940

in which each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted -C ₁ -C ₈ -alkyl; with the proviso that both R ¹ and R ² are not H, except that both of R ³ and R ³ are not H;

R ³ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl;

R ³ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl and at least one of R ³ and R ³ is not H;

R ⁴ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl;

R ⁵ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl;

or R ⁴ and R ⁵ together form a carbocyclic ring and have the formula -(CR ^a R ^b )n- wherein R ^a and R ^b are independently selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl and n selected from the group consisting of 2, 3, 4, 5 and 6;

R ⁶ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl;

R ⁷ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl;

each R ⁸ is independently selected from the group consisting of H, -OH, optionally substituted -C ₁ -C ₈ alkyl, and optionally substituted -O-(C ₁ -C ₈ -alkyl);

R ¹² is selected from H, optionally substituted -C1-C8 alkyl, optionally substituted aryl, optionally substituted -X ¹ aryl, optionally substituted -C3- _C8 carbocycle, optionally substituted -X ¹ -(C3-C8 carbocycle), optionally substituted -C1-C8-alkylene-NH2, optionally substituted C3-C8 heterocycle and optionally substituted -X ¹ -(C3- _C8 heterocycle); and each X ¹ is independently -C ₁ -C ₁₀ alkylene.

In some related embodiments, R ¹² is not a phenylalanine and proline side chain. In some related embodiments, R ¹² is not a phenylalanine, methionine, tryptophan, or proline side chain.

In some embodiments, R ¹² is selected from naturally occurring L-amino acid side chains (other than proline) and glycine. In some additional embodiments, R ¹² is selected from naturally occurring L-amino acid side chains other than proline, glycine, or phenylalanine. In some additional embodiments, R ¹² is selected from naturally occurring L-amino acid side chains other than proline, glycine, tryptophan, methionine, and phenylalanine.

In some additional embodiments, R ¹² is selected from side chains of the hydrophilic amino acid group consisting of threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine, and triazolylalanine .

In some embodiments, R ¹² is a threonine side chain.

linker link.

Referring to the unit of the linker with formulas IV and IV, in some embodiments, the implementation of L ^A covalently linked to the sulfur atom of the ligand. In some aspects, the sulfur atom is that of a cysteine residue that forms an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is that of a cysteine residue that has been introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In additional aspects, the sulfur atoms to which ^LA is attached are selected from cysteine residues forming an interchain disulfide bond of the antibody and cysteine residues that have been introduced into the ligand unit (eg, by site-directed mutagenesis or chemical reaction).

AA ₁ forms a cleavable bond with an effector moiety, DE, such as a drug moiety. In embodiments where AA ₁ is attached to an amino acid from DE, AA ₁ forms a cleavable peptide bond with D _E . A cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches its target site. In other embodiments, AA ₁ forms an amide bond to the attachment site of an effector moiety that is susceptible to cleavage (eg, by proteases) when the conjugate reaches its target site. In some embodiments, AA1 is a hydrophilic amino acid, typically a naturally occurring amino acid, that is selected from the group consisting of glycine and the L forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine. In some embodiments, AA ₁ is glutamic acid.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L or D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-,

- 27 040940

-CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CHS^CO)-, -CHSSCHSSCHS^)- and -CHSSCHSSCHCO)-. In some additional embodiments, R ^L1 is selected from the group consisting of D-amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L1 is present and is an optionally substituted alkylene, it may be a C ₁ -C ₆ alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N(C ₁ -C ₃ -alkyl)-, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, R ^L1 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)CH(CH2NH2)-.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In some additional embodiments, when R ^L2 is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)R ^b -; where R ^a is selected from -CH2CH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CHCHSCHCHCHCO)-.

In embodiments where R ^L2 is present and is an optionally substituted alkylene, it may be C1-C6-alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl)-, -NH2 or -NH(C1-C3-alkyl). In some embodiments, R ^l2 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH ₂ NH ₂ )-.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)-, and -CHSSCHSSCHCO)-. In some additional embodiments, where R ^L3 is present, it is selected from the group consisting of the D-amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH ₂ CH ₂ CH ₂ CO ₂ H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CHSSCHSSCHCO)- and -CHCHSSCHCHCO)-.

In embodiments where R ^L3 is present and is an optionally substituted alkylene, it may be a C1-C6-alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl)-, -NH2 or -NH(C1-C3-alkyl). In some embodiments, R ^L3 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH ₂ NH ₂ )-.

In some embodiments of the above, AA1 is present and R ^L1 , R ^l2 and R ^L3 are absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, and R ^l2 and R ^l3 are absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, R ^l2 is present, and R ^L3 is absent.

In some embodiments of the above, AA1 is present, R ^L1 is present, R ^l2 is present, and R ^L3 is present.

In some embodiments of the above, AA1 is a hydrophilic amino acid and at least one of R ^L1 , R ^l2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 is glutamic acid and at least one of R ¹² and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 is glutamic acid, R ¹¹ is a hydrophilic amino acid, and at least one of R ¹² and R ^L3 is present and is an optionally substituted alkylene as defined above.

- 28 040940

In some embodiments of the above, AA1 and R ^L1 are a hydrophilic amino acid and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 and are a hydrophilic amino acid and R ^L1 and optionally R ^L2 are an optionally substituted alkylene as defined above.

In some embodiments of the above L ^H does not contain a glycine dipeptide (Gly-Gly), tripeptide or tetrapeptide. In some embodiments, L ^H does not include an Asn-(D)Lys peptide.

In some embodiments, L ^H comprises a modified peptide having two to four amino acids. The modified peptide contains an amino acid at position 1 (AA1) which is chosen to optimize the yield of the DE unit (eg, protease cleavage via an amide peptide bond). At one or both of the positions R ^L1 and R ^L2 is an amino acid that reorients the typical N to C bonds of peptides and facilitates the attachment of the last amino acid (e.g. R ^L2 or R ^L3 ) which, prior to attachment of the ligand unit, includes a protected α-amino group. like maleimide. An amino acid having an N-to-C bond is attached to the next group through its side chain. In some embodiments, this amino acid is an alpha amino acid. In other embodiments, it may be a beta or gamma amino acid. In some of these embodiments, the side chain is selected from -CH2NH2-, -CH2CH2NH2-, -CH2CH2CH2NH2- and -CH2CH2CH2CH2NH2-.

In some embodiments, L ^H , an amino acid having an N-to-C reverse bond (R ^L1 ), is attached to R ^L2 or R ^L3 , where R ^L2 or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene, in accordance with any of the embodiments described above.

In some embodiments, L ^H , an amino acid having an N-to-C reverse bond (R ^L1 ) is attached to R ^L2 , where R ^L2 is an optionally substituted alkylene, in accordance with any of the embodiments described above.

In some additional embodiments, L ^H is a hydrophilic, cleavable linker, each branch has the formula

where R ²¹ is selected from CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H, and R ²² is selected from -CH2NH ₂ , -CH ₂ CH ₂ NH ₂ , -CH ₂ OH and -CH ₂ CH ₂ OH. The wavy lines to the left and right show link attachments DE and LA, respectively.

In further embodiments, L ^H , or a branch thereof, has the formula

where R ²² is selected from -CH2NH2, -CH2CH2NH2, -CH2OH and -CH2CH2OH. In some embodiments, R ²² is selected from -CH2NH ₂ , -CH ₂ CH ₂ NH ₂ . The wavy lines to the left and right show link attachments DE and LA, respectively.

In some embodiments, L ^H , or a branch thereof, has the formula

The wavy lines to the left and right show link attachments DE and LA, respectively. In some embodiments, L ^H , or a branch thereof, has the formula

- 29 040940

The wavy lines to the left and right show link attachments DE and L ^A , respectively.

In some embodiments, L ^H , or a branch thereof, has the formula

The wavy lines to the left and right show link attachments DE and L ^A , respectively. In some embodiments, L ^H , or a branch thereof, has the formula

The wavy lines to the left and right show link attachments DE and L ^A , respectively. In some embodiments, L ^H , or a branch thereof, has the formula

The wavy lines to the left and right show link attachments DE and L ^A , respectively. In some embodiments, L ^H , or a branch thereof, has the formula

The wavy lines to the left and right show link attachments DE and L ^A , respectively. In some embodiments, L ^H , or a branch thereof, has the formula

The wavy lines to the left and right show link attachments DE and L ^A , respectively.

In some additional embodiments of the above, L ^H is a branched hydrophilic linker having the formula

- 30 040940 wherein each R ³¹ is independently selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2OH,

-CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and each of the bars shows the site of attachment to the link DE.

In some additional embodiments of the above, the LH is a branched hydrophilic linker having the formula

HN

while each of the strips shows the site of attachment to the link DE.

The L ^A subunit is described in more detail above.

Referring again to Formula IV and IV' drug-linker conjugates, in some further embodiments, the drug-linkers have a formula selected from

Specific embodiments include the following:

(IVa), and

or a pharmaceutically acceptable salt or solvate thereof.

Linkers.

In another aspect, hydrophilic linkers are provided having the formula (L ^H ) _p , - L ^A (VII) where L is an optionally branched hydrophilic linker having the formula

- AA _T - R ^L1 - R ^L2 - R ^L3 - ;

L ^a is a ligand attachment component; and p' is an integer from 1 to 4; and the lines to the left and to the right of LH show the attachment sites of the DE and L ^A link, respectively;

or a pharmaceutically acceptable salt or solvate thereof. Referring to Formula VII, in some embodiments, L ^A is covalently bonded to the sulfur atom of the ligand. L ^A may be, for example, a maleimide or a succimimide suitable for attachment to a sulfur atom.

AA1 can form a cleavable bond with an effector moiety of DE, such as a drug moiety. In embodiments where AA1 is attached to an amino acid from DE, AA1

- 31 040940 forms a cleavable peptide bond with DE. A cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches its target site. In other embodiments, ΆΛι forms an amide bond to the attachment site of an effector moiety that is susceptible to cleavage (eg, by proteases) when the conjugate reaches its target site. In some embodiments, ΆΆ1 is a hydrophilic amino acid, typically a naturally occurring amino acid, that is selected from the group consisting of glycine and the L forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine. In some embodiments, ΆΛι is glutamic acid.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-. In some additional embodiments, R ^L1 is selected from the group consisting of the D amino acid aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L1 is present and is an optionally substituted alkylene, it may be a C ₁ -C ₆ -alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N(C ₁ -C ₃ -alkyl)-, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, R ^L1 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH2NH2)-.

In embodiments where R ^L2 exists and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NHCH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-. In some additional embodiments, if R ^L2 is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L2 is present and is an optionally substituted alkylene, it may be a C1-C6 alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl)-, -NH2 or -NH(C1-C3-alkyl). In some embodiments, RL ² is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)-CH(CH2NH2)-.

In embodiments where RL ³ is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In some additional embodiments, where RL ³ is present, it is selected from the group consisting of the D amino acid aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L3 is present and is an optionally substituted alkylene, it may be C1-C6-alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C ₁ -C ₃ -alkyl)-, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, R ^L3 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)CH(CH2NH2)-.

In some embodiments of the above, ΆΆ1 is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments of the above, ΆΆ ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

- 32 040940

In some embodiments of the above, AA ₁ is present, R ^L1 is present, RL ² is present, and RL ³ is absent.

In some embodiments of the above, AA ₁ is present, R ^L1 is present, RL ² is present, and RL ³ is present.

In some embodiments of the above, AA ₁ is a hydrophilic amino acid and at least one of R ^L1 , RL ² and RL ³ is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is glutamic acid and at least one of R ^L1 , RL ² is a substituted alkylene as defined above.

and RL ³ is present and is optionally beyond In some embodiments of the above, AA1 is glutamic acid, ^RL1 is a hydrophilic amino acid, and at least one of RL ² and RL ³ is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ and R ^L1 are hydrophilic amino acids and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are an optionally substituted alkylene as defined above.

In some embodiments of the above, L ^H does not contain a glycine dipeptide (Gly-Gly), tripeptide, or tetrapeptide. In some embodiments, L ^H does not include an Asn-(D)Lys peptide.

In some embodiments, L ^H comprises a modified peptide having two to four amino acids. The modified peptide contains an amino acid at position 1 (AA ₁ ) which is chosen to optimize the yield of the D _E unit (eg, protease cleavage via an amide peptide bond). At one or both positions, R ^L1 and R ^L2 is an amino acid that changes the orientation of the typical N bonds of the peptides to C and facilitates the attachment of the last amino acid (for example, RL ² or RL ³ ), which, prior to attachment of the ligand unit, includes an α-amino group, protected as maleimide. An amino acid having an N-to-C bond is attached to the next group through its side chain. In some embodiments, this amino acid is an alpha amino acid. In other embodiments, it may be a beta or gamma amino acid. In some of these embodiments, the side chain is selected from -CH2NH2-, -CH2CH2NH2-, -CH2CH2CH2NH2- and -CH2CH2CH2CH2NH2-.

In some embodiments, L ^H , an amino acid having an N-to-C bond (R ^l1 ), is attached to RL ² or RL ³ , where RL ² and RL ³ is a hydrophilic amino acid or an optionally substituted alkylene, according to any of the embodiments described higher.

In some embodiments, L ^H , an amino acid having an N-to-C reverse bond (R ^l1 ), is attached to RL ² , where RL ² is a hydrophilic amino acid or an optionally substituted alkylene, in any of the embodiments described above.

In some additional embodiments, the implementation of L ^H is a hydrophilic, cleavable linker, each branch has the formula

where R ²¹ is selected from CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ²² is selected from -CH2NH ₂ , -CH ₂ CH ₂ NH ₂ , -CH ₂ OH and -CH2CH2OH. The wavy lines to the left and right show the sites of attachment to the link DE and L ^A , respectively.

In subsequent embodiments, L ^H or a branch thereof has the formula

where R ²² is selected from CH2NH2, -CH2CH2NH2, -CH2OH and -CH2CH2OH. In some embodiments, R ²² is selected from -CH2NH ₂ and -CH ₂ CH ₂ NH ₂ . The wavy lines to the left and right show the sites of attachment to the link DE and L ^a , respectively.

- 33 040940

In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right show the sites of attachment to the link DE and L ^A , respectively.

In some embodiments, L ^H or a branch thereof has the formula no.

But.

But.

But.

The wavy lines to the left and right show the sites of attachment to the unit DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right show the sites of attachment to the link DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right show the sites of attachment to the unit DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines on the left and right show the sites of attachment to the link D _E and

In some embodiments, L ^H or a branch thereof has the formula

L ^A , respectively The wavy lines on the left and right show the sites of attachment to the unit DE and L ^A , respectively In some embodiments, the implementation of L ^H or a branch thereof has the formula

- 34 040940

The wavy lines to the left and right show the sites of attachment to the link DE and L ^A , respectively.

In some additional embodiments of the above, L ^H is a branched hydrophilic linker having the formula

each R ³¹ is independently selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H, and -CH2CH2CH2CH2CO2H; and each of the strips shows the site of attachment to the link D _E .

In some additional embodiments of the above, L ^H is a branched hydrophilic linker having the formula

with each of the strips showing the site of attachment to the link D _E and the vertical dotted line indicates the site of attachment for the link of the ligand.

The L ^A subunit is described in more detail in this document.

In each of the embodiments shown above, each of the amine, hydroxyl, and carboxyl groups is optionally in a protected form. Suitable protecting groups are found, for example, in Greene and Wuts, Protective Groups in Organic Synthesis, J. Wiley & Sonsand, and are generally chosen to be removed independently of each other.

Linker-ligand conjugates.

In yet another aspect, linker-ligand conjugates are provided having the formula selected from ( [L ^H ] _P - - L ^A ) p - L (X) or a pharmaceutically acceptable salt or solvate thereof;

wherein:

L is a ligand that specifically binds to a target;

L ^H is an optionally branched hydrophilic linker, each L ^H branch has the formula

- AAi - R ^L1 - R ^L2 - R ^L3 -;

L ^a is a ligand attachment component;

the subscript p is an integer from about 4 to 20; and the subscript p is an integer from 1 to 4;

while the left and right lines L ^H indicate sites of attachment to the link D _E and L ^A , respectively.

AA1 can form a cleavable bond with an effector moiety of DE, such as a drug moiety. In embodiments where AA1 is attached to an amino acid from DE, AA1 forms a cleavable peptide bond with DE. A cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches its target site. In other embodiments, AA ₁ forms an amide bond to the attachment site of an effector moiety that is susceptible to cleavage (eg, by proteases) when the conjugate reaches its target site. In some embodiments, AA ₁ is a hydrophilic amino acid, typically a naturally occurring amino acid, that is selected from the group consisting of glycine and the L forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine. In some embodiments, AA ₁ is glutamic acid.

- 35 040940

In some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. L ^A may be, for example, a maleimide or succimimide suitable for attachment to a sulfur atom.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-. In some additional embodiments, R ^L1 is selected from the group consisting of D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L1 is present and is an optionally substituted alkylene, it may be a C ₁ -C ₆ alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N(C ₁ -C ₃ -alkyl)-, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, R ^L1 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)CH(CH2NH2)-.

In embodiments where R ^L2 exists and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L and D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NHCH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In some additional embodiments, when R ^L2 is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where R ^L2 is present and is an optionally substituted alkylene, it may be a C ₁ -C ₆ alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N(C ₁ -C ₃ -alkyl)-, -NH2 or -NH(C ₁ -C ₃ -alkyl). In some embodiments, R ^L2 is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)CH(CH2NH2)-.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it may be selected from the group consisting of glycine; L or D forms of aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-. In some additional embodiments, where RL ³ is present, it is selected from the group consisting of the D amino acids aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine; glycine; -NH-CH(R ^a )-CO-; and -NH-CH(COOH)-R ^b -; where R ^a is selected from -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH2OH, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ^b is selected from -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2C(O)-, -CH2CH2CH2C(O)- and -CH2CH2CH2CH2C(O)-.

In embodiments where RL ³ is present and is an optionally substituted alkylene, it may be a C1-C6 alkylene optionally substituted with 1-4 substituents selected from -NH-, -C(O)-, -COOH, -N (C1-C3-alkyl)-, -NH2 or -NH(C1-C3-alkyl). In some embodiments, RL ³ is ethylenediamine, -NH-CH(COOH)-CH2-NH- or -C(O)CH(CH ₂ NH ₂ )-. In some embodiments of the above, AA ₁ is present and R ^L1 , RL ² and RL ³ are absent.

In some embodiments of the above, AA ₁ is present and R ^L1 , RL ² and RL ³ are absent.

In some embodiments of the above, AA ₁ is present, and R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments of the above, AA ₁ is present, R ^L1 is present, RL ² is present, and R ^L3 is absent.

In some embodiments of the above, AA ₁ is present, R ^L1 is present, RL ² is present, and R ^L3 is present.

- 36 040940

In some embodiments of the above, AA1 is a hydrophilic amino acid and at least one of R ^L1 , R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA ₁ is glutamic acid and at least one of R ^L1 , R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 is glutamic acid, R ^L1 is a hydrophilic amino acid, and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 and R ^L1 are hydrophilic amino acids and at least one of R ^L2 and R ^L3 is present and is an optionally substituted alkylene as defined above.

In some embodiments of the above, AA1 is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as defined above.

In some embodiments of the above, L ^H does not contain a glycine dipeptide (Gly-Gly), tripeptide, or tetrapeptide. In some embodiments, L ^H does not include an Asn-(D)Lys peptide.

In some embodiments, L ^H comprises a modified peptide having two to four amino acids. The modified peptide contains an amino acid at position 1 (AA1) chosen to optimize the release of the DE unit (eg, by protease cleavage through an amide peptide bond). At one or both of the positions, R ^L1 and R ^L2 is an amino acid that reverses the typical N to C orientation of peptide bonds and facilitates the attachment of the last amino acid (e.g., R ^L2 or R ^L3 ) which, prior to attachment of the ligand unit, includes a protected α-amino group. like maleimide. An amino acid having an N-to-C bond is attached to the next group through its side chain. In some embodiments, this amino acid is an alpha amino acid. In other embodiments, it may be a beta or gamma amino acid. In some of these embodiments, the side chain is selected from -CH2NH2-, -CH2CH2NH2-, CH2CH2CH2NH2- and -CH2CH2CH2CH2NH2-.

In some embodiments, the L ^H amino acid having an N-to-C bond (R ^L1 ) is attached to R ^L2 or R ^L3 , where R ^L2 or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene, in accordance with any of the embodiments described higher.

In some embodiments, an L ^H amino acid having an N-to-C reverse bond (R ^L1 ) is attached to R ^L2 , where R ^L2 is an optionally substituted alkylene, in accordance with any of the embodiments described above.

In some additional embodiments, L ^H is a hydrophilic, cleavable linker, each arm has the formula ₀ co ₂ n R ²² amino acid 123 wherein R ²¹ is selected from CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and R ²² is selected from -CH2NH ₂ , -CH ₂ CH ₂ NH ₂ , -CH ₂ OH and -CH ₂ CH ₂ OH. The left and right wavy lines indicate the attachment to the link DE and LA, or branch L ^H , respectively.

In additional embodiments, L ^H or a branch thereof has the formula

in which R ²² is selected from -CH ₂ NH ₂ , -CH ₂ CH ₂ NH ₂ , -CH ₂ OH and -CH ₂ CH ₂ OH. In some embodiments, R ²² is selected from -CH2NH2 and -CH2CH2NH2. The left and right wavy lines indicate the attachment to the link DE and LA, or branch L ^H , respectively.

In some embodiments, L ^H or a branch thereof has the formula

- 37 040940

The wavy lines to the left and right indicate link positions DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right indicate link positions DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right indicate link positions DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right indicate link positions DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right indicate link positions DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right indicate link positions DE and L ^A , respectively. In some embodiments, L ^H or a branch thereof has the formula

The wavy lines to the left and right indicate link positions DE and L ^A , respectively.

In some additional embodiments of the above, L ^H is a branched hydrophilic linker having the formula

- 38 040940 where each R ³¹ is independently selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2OH,

-CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; and each of the bars shows the attachment to the DE unit, and the vertical dotted line shows the site of attachment

to the ligand unit.

In some additional embodiments of the above, the LH is a branched hydrophilic linker having the formula

with each of the strips showing the attachment to the DE unit and the vertical dotted line designating the site of attachment for the ligand unit.

L ^a - subunit described in more detail above.

In each of the embodiments above, each of the amine, hydroxyl, and carboxyl groups is optionally in a protected form. Suitable protecting groups are provided in Greene and Wuts, Protective Groups in Organic Synthesis, J. Wiley & Sons, and are generally chosen to be removed independently of each other.

(Vila)

where P is H or a protecting group and each of the remaining functional groups present in the R ¹ , R ² carboxylic acids shown are optionally protected and S is the sulfur atom of the ligand.

Treatment of diseases.

Also provided are methods of treating diseases using ligand-linker drug conjugates with any of the formulas described herein. The disease may be, for example, a cancer or an autoimmune disease. The ligand-linker-drug conjugates are administered in a therapeutically effective amount and according to a therapeutically effective schedule. In some aspects, the dose of the conjugate is equal to or less than that of a comparable doubly loaded conjugate. In some aspects, the dose of the conjugate is equal to or less than that of a comparable four-fold loaded conjugate. In some aspects, the dose of the conjugate is equal to or less than that of a comparable doubly loaded conjugate while the dosing schedule is the same or less frequent. In some aspects, the dose of the conjugate is equal to or less than that of a comparable four-fold loaded conjugate while the dosing schedule is the same or less frequent. In some additional aspects, the dose of the conjugate is greater and the dosing schedule is the same or less frequent than that of a comparable doubly loaded conjugate. In some additional aspects, the dose of the conjugate is less and the dosing schedule is the same as, or less frequent than, the comparable four-fold loaded conjugate. The comparator conjugate can be, for example, the same ligand-drug linker conjugate having a drug load of 2 or 4.

Treatment of a malignant neoplasm.

Ligand-linker-drug conjugates are useful for inhibiting the proliferation of tumor cells or cancer cells by inducing apoptosis in the tumor or cancer cells, or for treating cancer in a patient. Ligand-linker drug conjugates can be used, respectively, in a variety of settings for the treatment of malignancies. Ligand-drug conjugates can be used to deliver a drug to tumor cells or other cancer cells. Without being bound by theory, in one embodiment, the ligand unit of the ligand-linker drug conjugate specifically binds to a target (e.g., a cancer cell antigen) and the ligand-drug conjugate can be taken up (internalized) into the tumor cell or cancer cell by receptor-mediated endocytosis and other mechanism of internalization. The antigen may be attached to the tumor cell or cancer cell, or may be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, the drug (cytostatic agent) is released into the cell. In an alternative embodiment, the drug or drug moiety is cleaved from the ligand-linker-drug conjugate outside the tumor cell or cancer cell, and the drug or drug moiety subsequently enters the cell.

Ligand-linker-drug conjugates can provide conjugation-specific drug targeting to a tumor or malignancy, thereby reducing overall drug toxicity (when administered alone).

In one embodiment, the ligand unit specifically binds to a tumor cell or cancer cell. In another embodiment, the ligand unit specifically binds to a tumor cell or cancer cell antigen that is present on the surface of the tumor cell or cancer cell.

In another embodiment, the ligand unit specifically binds to a tumor cell or cancer cell antigen, which is an extracellular matrix protein associated with the tumor cell or cancer cell.

The specificity of the ligand unit for a particular tumor cell or cancer cell may be important in determining which tumors or malignancies are most effectively amenable to treatment.

Specific cancers that can be treated with ligand-linker-drug conjugates include, but are not limited to, solid tumors (eg, renal cell carcinoma, liver cancer, and skin cancer) and blood cancers (eg, acute myeloblastic leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), and multiple myeloma). Acute and chronic leukemias and lymphomas (such as Hodgkin's lymphoma and non-Hodgkin's lymphomas) can be treated.

Malignancies, including, but not limited to, a tumor, metastases, or other disease or disorder characterized by uncontrolled cell growth, can be treated or suppressed by administration of ligand-linker-drug conjugates.

In other embodiments, methods of treating cancer are provided, comprising administering to a patient in need thereof an effective amount of a ligand-drug conjugate and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one in which treatment of the malignancy does not reveal that the malignancy is refractory. In another embodiment, the chemotherapeutic agent is one that upon treatment of the cancer, the cancer is found to be refractory. The ligand-drug conjugates can be administered to a patient who has also undergone surgery as a treatment for cancer.

In some embodiments, the patient is also receiving additional treatment, such as radiation therapy. In a particular embodiment, the ligand-drug conjugate is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, a chemotherapeutic agent or radiation therapy is administered before or after administration of the ligand-drug conjugate.

The chemotherapeutic agent can be administered over a series of sessions or as a single dose. You can enter any of or a combination of chemotherapeutic agents, for example, the standard of therapeutic chemotherapeutic agents(VA).

In addition, methods are provided for treating cancer with a ligand-drug conjugate as an alternative to chemotherapy or radiation therapy, where chemotherapy or radiation therapy has been or may be too toxic, such as resulting in unacceptable or intolerable side effects for the individual being treated. The patient to be treated may, if desired, be treated with other anti-cancer treatment such as surgery, radiation therapy, or chemotherapy, depending on which treatment is acceptable or tolerable.

Treatment of autoimmune diseases.

Ligand-drug conjugates are useful for killing or inhibiting the replication of a cell(s) that causes or is involved in an autoimmune disease, or for treating an autoimmune disease. The ligand-drug conjugates can be used in a variety of settings, respectively, to treat an autoimmune disease in a patient. Ligand drug conjugates can be used to deliver drugs to target cells. Without being bound by theory, in one embodiment, the ligand-drug conjugate specifically binds to an antigen on the surface of the target cell, and the ligand-linker drug conjugate then enters the target cell via receptor-mediated endocytosis or other internalization mechanism. Once inside the cell, the drug unit is released. The released Drug unit is then free to move in the cytosol and stimulate cytotoxic or cytostatic activities. In an alternative embodiment, the drug is cleaved from the drug ligand conjugate from outside the target cell, and the drug or drug moiety subsequently enters the cell.

In one embodiment, the ligand moiety binds to an autoimmune antigen. In one aspect, the antigen is on the surface of a cell involved in an autoimmune condition.

In another embodiment, the ligand unit binds to an autoimmune antigen that is present on the cell surface.

In one embodiment, the ligand moiety binds to activated lymphocytes that are associated with an autoimmune disease state.

In a further embodiment, the ligand-drug conjugate kills or inhibits the multiplication of cells that produce autoimmune antibodies associated with a particular autoimmune disease.

Specific autoimmune diseases that can be treated with ligand-drug conjugates include, but are not limited to, Th2 lymphocyte-associated disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic scleroderma, and graft-versus-graft reaction). owner); disorders associated with Th1-lymphocytes (eg, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjoergren's syndrome, Hashimoto's thyroiditis, diffuse toxic goiter, primary biliary cirrhosis, Wegener's granulomatosis and tuberculosis); diseases associated with activated B lymphocytes (eg, systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes).

Also disclosed are methods of treating an autoimmune disease, including administering to a patient in need thereof an effective amount of a ligand-linker-drug conjugate and another therapeutic agent for treating a known autoimmune disease.

Treatment of infectious diseases.

Ligand-linker-drug conjugates are useful for killing or inhibiting the multiplication of a cell that causes an infectious disease or for treating an infectious disease. Ligand-linker-drug conjugates can be used, respectively, in various conditions for the treatment of an infectious disease in a patient. Ligandlinker-drug conjugates can be used to deliver a drug (eg, an antibiotic) to a target cell. In one embodiment, the ligand moiety binds to an infectious disease cell.

In one embodiment, the conjugates destroy or inhibit the proliferation of cells that cause a particular infectious disease.

Disclosed are methods for treating infectious diseases, including administering to a patient in need thereof a ligand-linker-drug conjugate and another therapeutic agent that is an agent against the pathogen.

- 41 040940

Compositions and methods of administration.

The present invention also provides pharmaceutical compositions comprising the ligand-linker-drug conjugates described herein and a pharmaceutically acceptable carrier. The ligand-linker-drug conjugates may be in any form that allows the compound to be administered to a patient for the treatment of a disorder associated with the expression of an antigen to which the ligand moiety specifically binds. For example, the conjugates may be in the form of a liquid or a solid. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous, intravenous, intramuscular, epigastric injection or infusion techniques. In one aspect, the compositions are administered parenterally. In another aspect, the compounds are administered intravenously.

Pharmaceutical compositions of ligand-linker-drug conjugates can be formulated to allow the compound to be bioavailable upon administration of the conjugate to a patient. The compositions may take the form of one or more dosage units, where, for example, a vial may be a single dosage unit.

The materials used in the preparation of pharmaceutical compositions may be non-toxic in the amounts used. For a person skilled in the art it is obvious that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on many factors. Appropriate factors include, without limitation, the type of animal (eg, human), the particular form of the compound, the route of administration, and the composition used.

The pharmaceutical compositions may, for example, be in the form of a liquid. The liquid may be useful for delivery by injection. Compositions for administration by injection or intravenous administration may also include one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and/or an isotonic agent.

Liquid compositions, whether solutions, suspensions or otherwise, may also include one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride solution, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, salts or esters of acetic acid, salts or esters of citric acid or salts or esters of phosphoric acid; detergents such as non-ionic surfactants, polyols; and tonic agents such as sodium chloride or dextrose. The composition for parenteral administration may be enclosed in an ampoule, a disposable syringe, or a single or multi-dose vial made of glass, plastic or other material. Saline is a typical adjuvant.

The injectable composition is preferably sterile.

The amount of conjugate that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined using standard clinical methods. In addition, in vitro or in vivo assays can be used to determine optimal dosage ranges. The precise dosage to be used in a pharmaceutical composition will depend on the route of administration and the severity of the disease or disorder, and the decision will be made according to the judgment of the practitioner and the circumstances of each patient.

The compositions contain an amount of the compound such that a suitable dosage is obtained. Typically, this amount is at least about 0.01% of the compound by weight of the composition.

For intravenous administration, the composition may contain from about 0.01 to about 10 mg of the ligand-linker-drug conjugate per kg of body weight of the animal. In one aspect, the composition may contain from about 0.01 to about 10 mg of the ligand-drug conjugate per kg of body weight of the animal. In another aspect, the amount of drug administered will be in the range of about 0.1 to about 7.5 mg compound/kg body weight.

Typically, the dosage of the compound administered to a patient is typically from about 0.01 mg/kg to about 10 mg/kg of the individual's body weight. In some embodiments, the dose administered to a patient is from about 0.01 mg/kg to about 7.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is between about 0.1 mg/kg and 5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is from about 0.1 mg/kg to about 4 mg/kg of the individual's body weight. In some embodiments, the dose administered is from about 0.1 mg/kg to about 3 mg/kg, or about 0.1 mg/kg to about 2 mg/kg of the individual's body weight. In some embodiments, the implementation of the administered dose is from about 0.5 mg/kg to about 5 mg/kg body weight of the individual. In some embodiments, the implementation of the administered dose is from about 1 mg/kg to about 5 mg/kg body weight of the individual. IN

- 42 040940 in some embodiments, the administered dose is from about 0.1 to 4 mg/kg, even more preferably from 0.1 to 3.2 mg/kg, or even more preferably from 0.1 to 2.7 mg/kg body weight of the individual during the treatment cycle.

Ligand-linker-drug conjugates can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (eg, oral mucosa, rectal and intestinal mucosa). The introduction can be systemic or local. Various delivery systems are known, eg encapsulation in liposomes, microparticles, microcapsules, capsules, and can be used to administer the compound. In some embodiments, more than one compound or composition is administered to a patient.

The term carrier means the diluent, adjuvant or vehicle with which the compound is administered. Such pharmaceutical carriers may be liquid, such as water. Carriers may be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a patient, the compound or compositions and pharmaceutically acceptable carriers are sterile. Water is a typical carrier when the compounds are administered intravenously. Salt solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene glycol, water, ethanol. The present compositions, if desired, may also contain small amounts of wetting or emulsifying agents or pH buffering agents.

In an embodiment, the conjugates are formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, in particular humans. As a rule, carriers or means of delivery for intravenous administration are sterile isotonic buffered aqueous solutions. If necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration, if necessary, may contain a local anesthetic, such as lignocaine, to relieve pain at the injection site. Typically, the ingredients are supplied either separately or mixed together in a dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. Where the conjugate is administered by infusion, it may be dispensed, for example, by means of an infusion bottle containing sterile pharmaceutical grade water or saline. In the event that the conjugate is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

Pharmaceutical compositions are typically formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practices (GMP) regulations of the US Food and Drug Administration.

Pharmaceutical compositions of the present invention comprise conjugates of a ligand drug of the present invention and a pharmaceutically acceptable carrier. In some preferred embodiments, all or substantially all or more than 50% of the ligand-drug conjugates present in the pharmaceutical composition contains a hydrolyzed thio-substituted succinimide. In some preferred embodiments, more than 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the ligand-drug conjugates present in the pharmaceutical composition , contains hydrolyzed thio-substituted succinimide.

Methods for preparing ligand-drug conjugates.

In another aspect, the present invention provides methods for preparing ligand drug conjugates, linkers, drug-linker conjugates, and linker-ligand conjugates.

In some embodiments, the methods of the present invention include the steps of providing a linker drug or linker unit as described herein by conjugating said linker drug or linker unit to a sulfhydryl group of the ligand unit to form a conjugate. In some additional embodiments, the thio-substituted maleimide or succinimide group(s) of the conjugate may undergo a hydrolysis reaction.

The rate of hydrolysis of the thio-substituted succinimide can be controlled by adjusting the reaction conditions after drug-linker ligand conjugation, for example by adjusting pH or temperature. In some embodiments of the present invention, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90%, or even 95% of the thio-substituted succinimide is hydrolyzed without controlling the reaction conditions, i. the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction. In some embodiments, all, substantially all, or at least

- 43 040940 at least 50, 60, 70, 80, 85, 90 or even 95% of the thio-substituted succinimide is hydrolyzed from within 20 minutes to 4 hours after conjugation, preferably from 20 minutes to 2 hours after conjugation. In typical embodiments, conjugation conditions are about pH 7.4 and temperature about 22°C.

In some embodiments, methods for preparing a ligand-drug conjugate include the steps of providing a drug-linker or linker unit; conjugating said drug-linker or linker unit with a sulfhydryl group of a ligand to form a ligand-drug conjugate containing a non-hydrolyzed thio-substituted succinimide, allowing the non-hydrolyzed thio-substituted succinimide to undergo a hydrolysis passage reaction, wherein all, substantially all, or at least 50, 60, 70, 80 or even 85% of the succinimide is hydrolyzed within 10 minutes to 4 hours after conjugation. In some embodiments, all, substantially all, or at least 50, 60, 70, 80, 85, 90, or even 95% of the succinimide is hydrolyzed at 10 minutes, 20, 40, 60, 90, or 120 minutes after conjugation. In some embodiments, the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction. In typical embodiments, conjugation conditions are about pH 7.4 and temperature about 22°C.

Assembly of ligand-drug conjugates.

The ligand-drug conjugates of the present invention can be assembled following the general scheme shown in FIG. 1.

Examples

General.

Unless otherwise noted, materials were obtained from commercial suppliers in the highest purity available and used without further purification. Anhydrous DMF and CH ₂ Cl ₂ were purchased from Aldrich. Fmoc-dolaproin-OH was specially synthesized by Albany Molecular Research, Inc. (Albany, New York). Dolavalin-Val-Dil-OH was prepared as described elsewhere. Fmoc-Dpr(ivDde)-OH and 2-chlorotrityl chloride, polyvinyl chloride resin (200-300 mesh, 1% DVB, 1 mmol/g displacement) was purchased from Novabiochem. Solid phase synthesis was carried out in plastic syringes (National Scientific Company) equipped with a filter cut from commercial medium pore grade PE sheet (Scienceware). A Burrell wrist action® shaker (Burrell Scientific, Pittsburgh, PA) was used for shaking. All reported solids are based on initial resin displacement and represent the weight balance of isolated pure material unless otherwise noted.

Preparative HPLC purification was performed on a Varian instrument equipped with a Phenomenex Synergy MAX-RP 4μ, 250x10 mm C12 reverse phase column eluted with 0.05% TFA in a water-acetonitrile gradient.

Mass spectra were obtained on a XEVO TOF MS connected to a Waters 2795 HPLC equipped with a Phenomenex Synergi 2.0x150 mm, 4 μm, 80 A reverse phase C12 column. % aqueous formic acid for 10 min, then isocratic 95% acetonitrile for 5 min at a flow rate of 0.4 ml/min.

The humanized h1F6 antibody specifically binds to the human CD70 antigen (Cancer Res 2006, 66(4), p. 2328; US Pat. No. 8,067,546). The humanized hBU12 antibody specifically binds to the human CD19 antigen (Blood, 2009, 113(18), p. 4362; US Pat. No. 7,968,687). Human kidney carcinoma cell lines, 786-0 and Caki-1, expressing human CD70, and transformed human follicular lymphoma cells DOHH2, expressing human CD19, were purchased from the American Type Culture Collection (ATCC; Manassas, VA). All cell lines were grown according to the supplier's recommendations and regularly tested for mycoplasma contamination.

Abbreviations: DPR means diaminopropionic acid; ivDde is L-(4,4dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-.

Example 1 General Synthesis Procedures.

Synthesis of maleimido-Dpr(Boc)-OH.

Nβ-Boc-L-2,3-diaminopropionic acid (1 mmol) and maleic anhydride (98 mg, 1 mmol) were dissolved in acetic acid (1 ml) in a 50 ml round bottom flask and the solution was stirred at room temperature for 3 h The solution was then concentrated to an oil under reduced pressure. The maleic acid intermediate was precipitated by adding ~10 ml CH ₂ O ₂ /hexane, 1/1, v/v, and the precipitate was collected by vacuum filtration. This material was then suspended in toluene (9 ml), followed by the addition of DMA (0.3 ml) and triethylamine (0.42 ml, 3 mmol). The mixture was stirred at 40-60° C. under N2 until all material was in solution. The vessel was then fitted with a condenser and the solution was heated to 120° C. and boiled for 4 hours over molecular sieves. The reaction mixture was filtered through a sintered glass funnel and concentrated almost to dryness under reduced pressure. The residue was dissolved in ethyl acetate (10 ml), transferred to a separating funnel and washed with 10% citric acid in water (2x10 ml) and

- 44 040940 saline solution (2x10 ml). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure and dried under high vacuum overnight to give the product as a white powder in 72% yield. 1H NMR (DMSO): δ 1.29 (s, 9H), 3.41 (m, 1H), 3.52 (m, 1H) 4.57 (dd, 1H).

6.97 (t, 1H), 7.07 (s, 2H). LCMS (ESI) calculated for (M+Na)+ 307.09; set m/z 307.17.

General procedures for the synthesis of auristatin-AA ₁ -MDpr.

Fig. 1 illustrates a typical synthesis of auristatin-(AA ₂ )-AA ₁ -MDpr drug linkers.

Resin load. To a 20 ml solid phase reaction vessel (plastic syringe with PET frit) was added 1 g of 2-chlorotrityl chloride resin (1 mmol, based on its manufacturer's label), followed by a solution of Fmoc-Dpr(ivDde)-OH or Fmoc-Lys(ivDde)-OH (1.5 mmol, 1.5 eq) and DIEA (1 mmol, 1 eq) in 10 ml dry CH ₂ C1 ₂ /DMF, 1/1, v/v. The vial was shaken for 5 min and then more DIEA (1.5 mmol, 1.5 eq) was added. The mixture was shaken for an additional 2 hours at room temperature. Methanol (2.5 ml) was added to quench unreacted areas. After 30 min, the resin was washed with DMF (5x10 ml), CH ₂ Cl ₂ (5x10 ml), ethyl ether (5x10 ml) and dried in vacuum.

Loading was determined by treating a small amount of resin (2-4 mg) with 20% piperidine/DMF (2 ml) for 2 hours in a volumetric flask (10 or 20 ml). The volume was adjusted with DMF; absorbance was measured at 301 nm. The load was calculated using the following equation:

Load (mmol / g) \u003d (vessel volume x Α ₃₀ ι) / ( 7800 x mg) x 1000

The average load was ~0.6 mmol/g

Fmoc removal stage. The resin containing the Fmoc-protected peptide was treated with 20% piperidine in DMF (10 ml/g resin) for 2 hours at room temperature. The resin was then washed with DMF (5x1 ml per gram of resin), CH ₂ Cl ₂ (5x1 ml per gram of resin), ethyl ether (5x1 ml per gram of resin) and dried in vacuo.

Accession stage. To the resin (1 eq) containing the deblocked N-terminal amino acid (AA) was added a solution of Fmoc-AA-OH (2 eq), HATU (2 eq) and DIEA (4 eq) in DMF (1 ml per gram of resin). The reaction vessel was shaken for 3-4 s. The resin was then washed in DMF (5x1 ml per gram of resin), CH ₂ Cl ₂ (5x1 ml per gram of resin), ethyl ether (5x1 ml per gram of resin) and dried in vacuo. Completion of the reaction was confirmed by a negative Kaiser test if necessary.

Attachment of the N-terminal dolovalin-Val-Dil-OH was performed in a similar manner.

Deprotection of ivDde and attachment of MDpr(Boe)-OH. After the addition of the dolovalin-Val-Dil-OH tripeptide, the resin was treated with 2% hydrazine/DMF (1 ml/g resin) for 2 hours at room temperature. The resin was then washed with DMF (5x1 ml per gram resin), CH ₂ Cl ₂ (5x1 ml per gram resin), ethyl ether (5x1 ml per gram resin) and dried in vacuo. A solution of Fmoc-MDpr(Boc)-OH (2 eq), HATU (2 eq) and DIEA (4 eq) in DMF (1 ml per gram of resin) was added to the resin, the mixture was shaken for 3 h at room temperature (RT) . Completion of the reaction was confirmed by a negative Kaiser test. The resin was washed with DMF (5x1 ml per gram of resin), CH ₂ Cl ₂ (5x1 ml per gram of resin), ethyl ether (5x1 ml per gram of resin) and dried in vacuo.

Resin cleavage and deprotection. The peptide-containing resin was treated with 20% TFA/CH ₂ C1 ₂ (2 ml/g resin) for 10 min at room temperature and the solution was collected in a round bottom flask. The resin was washed with 20% TFA/CH2Cl2 (2x0.5 ml per gram of resin). The combined solutions were left at room temperature for 3 hours. After deprotection, completion was confirmed by LC-MS. The volatiles were removed under reduced pressure on a Rotavap and the final product was purified by reverse phase preparative HPLC. All drug-linkers were obtained with >95% purity by reverse phase HPLC at 215 nm.

MA maleimide drug linkers were prepared in the same manner as described above using α-maleimidoacetic acid-NHS (Molecular Biosciences, Boulder, Colorado) instead of MDpr(Boc)-OH.

Drug linkers with an ethylenediamine (EDA) extender were prepared using a procedure similar to that previously reported (Bioconjugate Chem. 2008, 19, 1960-1963).

Example 2 Drug Linkers.

Drug linkers were synthesized as described above. The general formula looks like this:

Maleimides

MA: Y= H

MDpr: Y=CH ₂ NH ₂

Linker oud

EDA Statement 5: X-N, n-1

Dpr: X=COOH, n=1

Lys: X=COOH, n=4

- 45 040940 (In this formula, note that the designation AA2 and AA ₁ is reversed. R corresponds to R ¹² .)

In the next table. 1 summarizes the synthesis and characterization of various drug linkers. In the table, the first column (on the left) refers to the connection number. The second column (left) refers to the amino acid at the C-terminus of auristatin. The third, fourth and fifth columns refer to the components of the linker. Column 3 defines the amino acid constituents of the linker. In column 4, additional amino acid and/or non-amino acid components of the linker are defined. In column 5, the composition of the maleimide component of the linker is determined. The sixth column refers to the drug-linker yield. The seventh and eighth columns refer to the calculated and observed drug-linker masses as determined by mass spectroscopy. The last column (on the right) refers to the retention time HIC of ADCs containing drug-linkers as 8-fold loads (generally determined as described in Example 3).

Table 1

# Position 5 auristatin Linker Exit, % MS: Calculated (MH)+ MS: Defined, m/z HIC retention time (min) Amino acid(s) Amino acid or other extender Maleimides (MA or MDpr) 1 Phe No Dp g MA 14 969.6 969.7 NT 2 Phe Glu Dp g MA eleven 1098.6 1098.8 NT 3 Thr none Dp g MA 7.3 923.5 923.7 NT 4 Thr Glu Dp g MA θ,Ι 1052.6 1052.8 5 5 Phe Glu Dp g MDpr 20 1127.6 1127.4 NT 6 Thr Glu Dp g MDpr 20 1081.6 1081.7 4.5 7 Thr Glu Lys MDpr 36 1123.7 562.5 double charge 4.6 8 Triazole Glu Lys MDpr 28 1176.6 588.9 double charge NT 9 Phe Glu EDA MDpr thirty 1083.6 1083.9 5.8 10 Thr Glu EDA MDPr 23 1037.6 1037.8 NT eleven Phe lie EDA MDPr 25 1067.7 1067.9 6, 9 12 Thr lie EDA MDPr 20 1021.7 511.5 double charge 5.2 13 Triazole Glu EDA MDPr 43 1090.6 1091.6 4.9 14 asp Ala Lys MDPr 70 1079.6 1079.9 4.98 15 Glu Ala Lys MDPr 81 1093.6 1093.8 5.02 16 PhosphoTig Ala Lys MDPr 4.5 1145.6 1145.9 4.81 17 Asn AlaGlu Dpr MDPr 70 1165.6 1165.5 5 18 gin AlaGlu Dpr MDPr 40 1179.7 1180.09 5.1 19 asp AlaGlu Dpr MDPr 67 1166.6 1167.3 4.95 20 Glu Alaglu Dpr MDPr 72 1180.6 1180.96 4.98 21 hSer AlaGlu Dpr MDPr 71 1152.6 1152.7 5.1 22 Wai Oh AlaGlu Dpr MDPr 21 1166.7 1167.1 5.2 23 PhosphoTig AlaGlu Dpr MDPr 13 1232.6 1232.78 4.8 24 Pyrazole Glu Dpr MDPr 73 1117.6 1117.5 5.2 25 Triazole Glu Dpr MDPr 85 1118.6 1118.7 5 26 Asn Glu Dpr MDPr 91 1108.6 1108.5 4.9 27 asp Glu Dpr MDPr 90 1095.6 1095.4 4.8 28 Fur Glu Dpr MDPr eleven 1117.6 1117.6 5.6 29 Wai Oh Glu Dpr MDPr 22 1095.6 1095.7 5.1 thirty Ser Glu Dpr MDPr 86 1067.6 1068.3 4.9 31 hSer Glu Dpr MDPr 68 1081.6 1081.5 4.96 32 Thr IleLeu Dpr MDPr 30-50 1178.7 1178.8 6, 1 33 Phe IleLeu Dpr MDPr 30-50 1224.7 1224.9 7.4 34 Glu PheLeu Dpr MDPr 30-50 1240.7 1241.1 5.9 35 Thr LeuPhe Dpr MDPr 30-50 1212.7 1212.8 6.7 36 Phe LeuPhe Dpr MDPr 30-50 1258.7 1258.9 7.8 37 Glu PhePhe Dpr MDPr 30-50 1274.7 1274.8 6.1 38 Thr LysAla Dpr MDPr 30-50 1151.7 1151.9 4.9 39 Thr Lys Dpr MDPr 30-50 1080.6 1181.1 4.8 mc-MMAF 7.0 mc-vc-PABC-MMAF 8.2 mc-vc-PABC-MMAE 9.8

Abbreviations:

Ala refers to L-alanine; Asn refers to asparagine; Asp refers to L-aspartic acid; Gln refers to L-glutamine; Glu refers to L-glutamic acid; Ile refers to L-isoleucine; Leu refers to L-leucine; Lys refers to L-lysine; Phe refers to L-phenylalanine; PhosphoThr refers to L-phosphothreonine; Thr refers to L-threonine; hSer refers to L-hydroxyserine

- 46 040940

ValOH refers to L-hydrovaline

Pyrazole belongs to

Triazole belongs to

Fur refers to

MA refers to maleimidoacetyl; Dpr refers to diaminopropionic acid; MDpr refers to maleimidodiaminopropionic acid; EDA refers to diamine; mc-MMAF refers to the linker-maleimidocaproil MMAF; mc-vc-PABC-MMAF refers to maleimidocaproyl-valine-citrulline-paminobenzyl-carbamoyl MMAF; mc-vc-PABC-MMAE refers to maleimidocaproyl-valinecitrulline-p-aminobenzyl-carbamoylMMAE.

Example 3 Antibody Drug Conjugates Preparation of Antibody Drug Conjugates.

Typical h1F6 ADC.

Eight drug per antibody h1F6 antibody drug conjugates (ADCs) were prepared by fully reconstituting the antibody followed by reaction with the linker drug of interest. The antibody (10 mg/ml) was completely reconstituted by adding 10 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline (PBS) pH 7.4 (Invitrogen, Carlsbad, CA) with 1 mM diethylenetriaminepentaacetic acid ( DTPA), followed by incubation at 37°C for ~1 s. Excess TCEP was removed by a 10-fold dilution with PBS and antibody concentration, repeated 2 times using a centrifuge filter with a nominal molecular weight cutoff of 30 Kd (EMD Millipore, Billerica, Massachusetts). Complete reduction of the antibody was confirmed by reverse phase HPLC analysis, where the light and heavy chains were completely separated from the unreduced antibody. The linker drug (10 equivalents) was then added from a stock solution prepared in DMSO (10 mM). The reaction was left at room temperature for approximately 2 hours to allow conjugation and subsequent hydrolysis of the thiosuccinimide ring (MDpr). The reaction mixture was purified and buffer exchanged in PBS using PD-10 desalting columns (GE Healthcare, Piscataway, NJ). The drug/Ab ratio was evaluated by PLRP-MC analysis and is in the range of 7.8-8.0 drug/Ab. In addition, each ADC was analyzed by size exclusion chromatography, where HMW species are in the range of 0.5-2.0%.

Hydrophobic interaction chromatography.

ADC analysis was performed using hydrophobic interaction chromatography (HIC). HIC was performed by running a linear gradient from 0 to 100% of mobile phase B (MPA), where mobile phase A (MPA) consists of 1.5 M ammonium sulfate, 25 mM potassium phosphate, pH 7.0, and MPB consists of

- 47 040940 from 75% 25 mM potassium phosphate, pH 7.0, and 25% isopropanol. Separation was achieved using a Tosoh t-Butyl column (TSK-Gel Butyl-NPR 4, bx35mm, PN: 14947) heated to 25°C. Test samples were prepared by diluting 70 μg of ADC in MPA such that the total salt concentration is greater than or equal to 1.0 M ammonium sulfate in a total volume of 100 μl. Samples were injected into 90 μl and eluted using a 12 minute gradient. Monitoring at λ=280 nm. ADCs with greater hydrophobicity, or more drugs per molecule, elute at later retention times.

Fig. 4 and 5 show the results of the HIC analysis of various 8-fold loaded ADCs compared to the parental, unconjugated antibody (h1F6). ADCs were prepared as described above. The results generally show that increasing the hydrophilicity of auristatin, in combination with a hydrophilic linker, reduces the observed hydrophobicity of the conjugate.

In the next table. 2 shows the composition of various drug linkers of the table. 1 and the resulting 8-fold loaded antibody drug conjugate with the h1F6 antibody is analyzed. The HIC retention time (HIC RT) was determined as described above. h1F6 ADCs containing drug linkers MC-vc-PABC-MMAE, MC-vc-PABC-MMAF and MCMMAF were used as controls.

- 48 040940

1006 MC-vc-PABC-MMAE 9, 8 1251 MC-vc-PABC-MMAE 8.2 1269 MC-MMAF 7.0

Example 4 In vitro activity assays.

In vitro cytotoxicity assays were generally performed as previously described (see activity assays above). Briefly, cell cultures in logarithmic phase were harvested and cells were seeded at seeding densities of 500 to 10,000 cells/well according to predetermined conditions. After incubation for 24 hours, serial dilutions of test conjugates were added to allow for the reconstruction of surface proteins, and cultures were incubated for an additional 4 days. Cell growth and stain reduction to generate IC ₅₀ values were assessed using the Alamar Blue stain reduction assay (Biosource International, Camarillo, CA). Briefly, a 40% (w/v) solution of Alamar Blue was re-prepared in complete medium just prior to the addition of the cultures. Ninety-two hours after drug exposure, alamar blue solution was added to the cells to make up 10% of the culture volume. Cells were incubated for 4 hours and staining reduction was measured on a Fusion HT fluorescent plate reader (Packard Instruments, Meriden, Conn.).

Purified renal cancer cell lines 786-0 and Caki-1 were used. These cell lines expressed approximately 190,000 and 135,000 human CD70 molecules per cell, respectively. Drug linkers attached to the h1F6 antibody are described in Table 1. 1 and 2. Referring to the following table. 3A-c, h1F6 ADCs have an average drug load of 8 drugs/antibody unless otherwise noted. The hydrophilic h1F6 ADCs tested in these studies showed activity (IC ₅₀ values) comparable to or even better than the control h1F6-mcMMAF (1269).

Table 3A. Pooled IC ₅₀ results for auristatin-based ADC

ADC Dr/Ab Caki-1 786-0 hlF6-l 4 21 21 8 8 7 hlF6-2 4 17 21 8 7 7 hlF6-3 4 27 28 8 13 13 hlF6-4 4 20 21 8 eleven 9 hlF6-5 4 39 32 8 22 19 hlF6-6 4 23 26 8 7 9 hlF6-7 4 80 10 8 24 4 hlF6-8 8 19 4 hlF6-9 8 21 3 hlF6-10 8 57 5 hlF6-ll 8 9 1 hlF6-12 8 12 4 hlF6-13 8 60 5

DR/Ab refers to the average drug load;

IC ₅₀ are given in ng/ml.

Table 3B. Pooled IC ₅₀ results for auristatin-based ADC

ADC based auristatin Dr/Ab 786-0 Caki-1 hlF6-14 8.0 8 19 hlF6-15 8.0 8 16

- 49 040940

hlF6-16 8.0 9 12 hlF6-17 8.0 8 14 MF6-18 8.0 8 16 MF6-19 8.0 7 20 hlF6-20 8.0 7 9 MF6-21 hlF6-22 hlF6-23 hlF6-24 hlF6-25 hlF6-26 hlF6-27 hlF6-28 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 12 24 eleven eleven 14 9 7 7 22 21 18 33 34 19 15 21 hlF6-29 8.0 31 33 hlF6-30 8.0 12 18 hlF6-31 8.0 13 20 hlF6-6 8.0 eleven 12 hlF6-mcMMAF 4.0 44 42

Table 3C. IC ₅₀ for h1F6-based ADC, ng/ml

ADC based auristatin Dr/Ab 786-0 Pure RCC Caki-1 Pure RCC hlF6-32 8.0 5 3 hlF6-33 8.0 0.8 0.8 hlF6-34 8.0 3 6 hlF6-35 8.0 3 3 hlF6-36 8.0 0.7 0.5 hlF6-37 8.0 4 4 hlF6-38 8.0 3 3 hlF6-39 8.0 3 3 hlF6-6 8.0 7 5 hlF6-mcMMAF 4.0 34 12

Example 5 Pharmacokinetic Studies Radiolabeling of Antibodies and ADCs.

Pharmacokinetic (PK) experiments were performed using a radiolabeled antibody or ADC. Samples for the PK test were radioactively labeled using the following procedure. To a solution of the antibody or ADC in 500 mM potassium phosphate (pH 8.0) and 500 mM sodium chloride was added 55 pCi of N-succinimidyl propionate, [propionate-2,3-3H]- (Moravek Biochemicals, Cat. No.: MT 919, 80 Ci/mmol, 1 mCi/ml, 9:1 solution of hexane:ethyl acetate) per mg of antibody or ADC. The resulting mixture was vortexed and left at room temperature for 2 hours. The mixture was centrifuged at 4000xg for 5 minutes and the lower aqueous layer was removed and distributed into Amicon Ultra-15 centrifuge filter cells (Millipore, Cat. No.: UFC903024, nominal cut-off molecular weight 30 kDa). Unconjugated radioactivity was removed in 4 rounds of dilution and centrifugation at 4000xg. The resulting products were filtered through sterile 0.22 μm Ultrafree-MC centrifuge filter cells (Millipore, Cat. No.: UFC30GV0S) and final antibody or ADC concentrations were measured spectrophotometrically. The specific activity (µCi/mg) of each product was determined by liquid scintillation counting.

Pharmacokinetic studies.

The pharmacokinetic properties of the unconjugated antibody and various ADCs of this antibody (drug loading 8) have been investigated in several rodent models. In each experiment, 3 mg of radiolabeled antibody or ADC per kg of animal weight was administered via the tail vein. Each test sample was dosed once to animals in 3 replications. Blood was collected into K2EDTA tubes via saphenous vein or cardiac puncture for terminal sampling at various time points. Plasma was isolated by centrifugation for 10 min at 10000xg. 10 μl of plasma sample from each time point was added to 4 ml Ecoscint-A liquid scintillation cocktail (National Diagnostics) and total radioactivity was measured in a liquid scintillation counter.

The resulting disintegrations per minute were converted to μCi and the specific activity of the radiolabeled test samples was used to calculate the concentration of antibody or ADC remaining in plasma at each time point.

Referring to FIG. 2, the pharmacokinetic properties of h1F6 and its two hydrophilic ADCs were compared to those of three control ADCs. The hydrophilic ADCs were h1F6-4 (8-fold

- 50 040940 loaded with (aypistatin-T)-Glu-Dpr-MA) ₈ -h1F6) and h1F6-11 ((aurystatin F)-Ile-EDA-MDpr) ₈ -h1F6). The results indicated that the hydrophilic ADCs exhibited increased pharmacokinetic stability during the course of this study in mice. Hydrophilic auristatin with auristatin-T showed stability close to that of the unconjugated antibody. The h1F611 hydrophilic ADC construct showed improved PK stability compared to control, two of which included the monomethyl form of the same auristatin (auristatin F vs monomethyl auristatin F).

Referring to FIG. 3, the pharmacokinetic properties of hydrophilic conjugates of another monoclonal antibody were compared with those of the control conjugate, mAb-mcMMAF. All ADCs had an average drug loading of 8. Each of the hydrophilic ADCs showed improved pharmacokinetic stability compared to the control ADC.

The two hydrophilic ADCs of this were compared to the properties of three control ADCs. The hydrophilic ADCs were h1F6-8 (8-fold loaded (aypistatin-thiazole)-Glu-Lys-MDpr) ₈ h1F6) and h1F6-11 ((aurystatin F)-Ile-EDA-MDpr) ₈ - h1F6). The results show that hydrophilic ADCs exhibited improved pharmacokinetic stability during the course of this study in mice. In particular, the h1F6-11 hydrophilic construct showed improved stability compared to controls, two of which included the monomethyl form of the same auristatin (auristatin F vs monomethyl auristatin F).

Example 6 In Vivo Therapeutic Experiments.

786-0 cells were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia) and propagated under the culture conditions recommended by the ATCC. To form tumors, 786-0.5x106 cells were implanted into the right flank of female athymic nu/nu donor mice (Harlan, Indianapolis, IN). When the donor tumors were about 500 mm ³ , the mice were euthanized and the tumors excised under aseptic conditions, and the ~0.5x0.5 mm fragments were placed in a size 13 sterilized trocar for implantation in nu/nu mice. When tumors reached ~100 mm ³ , mice were randomly assigned to treatment groups.

To form DOHH2 tumors, 5x106 cells were implanted in the right flank of C.B.-17 SCID mice (Harlan, Indianapolis, Ind.). When tumors reached ~100 mm ³ , mice were randomly assigned to treatment groups.

Experimental groups were treated with intraperitoneal administration of the compounds at the indicated dose and schedule, or alternatively were not treated. Tumors were measured periodically and volumes were calculated using the formula V=((LxW ² )/2). Animals were euthanized when the tumors reached a volume of 1000 mm ³ or at the end of the study, whichever came first.

Tumor quadrupling times were chosen as end point time (TTE) which was determined by non-linear regression analysis of the exponential growth of each individual tumor growth dataset in each experimental animal. Mean tumor quadrupling time was calculated based on tumor volume at the start of treatment. Animals that did not reach the endpoint were assigned a TTE value equal to the last day of the study.

Statistical analysis was performed using Prism (GraphPad) software for Windows. The TTE Logrank test was used to analyze the significance of differences between the two groups, with differences considered significant at the 0.01<P<0.05 level and highly significant at P<0.01.

Referring to FIG. 6, the activity of 4-fold loaded and 8-fold loaded ADCs (4d/Ab and 8d/Ab, respectively) was tested in a single dose xenograft study in mice. First, with reference to the control, h1F6-mc-vc-PABC-MMAF, 4-fold loaded ADC gave better activity than 8-fold loaded ADC. In contrast, 8-fold loaded hydrophilic h1F6-6 (auristatin T-Glu-Dpr-MDPr) and h1F6-13 (auristatin thiazole-Glu-EDA-MDPr) ADCs showed greater activity than 4-fold loaded analogs.

Referring to FIG. 7, the activity of various 4-fold loaded and 8-fold loaded ADCs was tested in a single dose xenograft study in mice. Again in this model, the 8-fold loaded hydrophilic ADC hBU12-6 (Auristatin T-Glu-Dpr-MDPr) showed greater activity than its 4-fold loaded counterpart.

Referring to FIG. 8, the activity of various 4-fold loaded and 8-fold loaded ADCs was tested in a single dose xenograft study in mice. In this model, each of the 8-fold loaded h1F6-12 (auristatin T-Ile-EDA-MDPr) and h1F6-5 (auristatin F-Glu-DprMDPr) ADCs showed greater activity than the 4-fold loaded counterparts. 8-fold loaded ADC h1F6-11 (auristatin F-Ile-EDA-MDPr) showed a reverse trend.

Referring to FIG. 9, the activity of various 4-fold loaded and 8-fold loaded ADCs was tested in a single dose xenograft study in mice. In this model, 8-fold loaded ADCs h1F6-17, h1F6-20, h1F6-24, and h1F6-29 showed greater activity than 4-fold loaded analogs.

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Claims (9)

CLAIM 1. A hydrophilic drug-ligand conjugate compound having the formula D—---L ^H --L ^a V ^J p' or a pharmaceutically acceptable salt thereof, where L is a ligand, which is an antibody or antigen-binding fragment of an antibody that specifically binds to a target; where the target is the cell surface antigen of the target cell, each L ^a is a ligand attachment component covalently attached to the sulfur atom of the cysteine thiol group of the antibody, and L ^a has the structure where the wavy line indicates the point of attachment to L ^H and \\ indicates covalent attachment to the sulfur atom of the cysteine thiol group of the antibody or antigen-binding fragment of the antibody; each L ^H is a hydrophilic linker, with the structure where R ²¹ is selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2OH, -CH2CH2OH, -CH2CO2H, -CH2CH2CO2H, -CH2CH2CH2CO2H and -CH2CH2CH2CH2CO2H; And R ²² is selected from the group consisting of CH2NH2, CH2CH2NH2, CH2OH and CH2CH2OH; or with a structure selected from the group consisting of where in each L ^H structure, the left and right wavy lines indicate covalent attachment to DE and L ^a , respectively, where covalent attachment to DE occurs via a peptide bond cleaved by an intracellular protease when the hydrophilic compound of the drug-ligand conjugate reaches its target antigen and is internalized into the target cell; index p is an integer from 6 to 12, from 8 to 12, or from 8 to 16; index p' is the integer 1; and each DE is an auristatin with the formula: - 52 040940 where each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted -C ₁ -C8-alkyl; with the proviso that both R ¹ and R ² are not H, except that both of R ³ and R ³ are not H; R ³ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl; R ³ ' is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl and at least one of R ³ and R ³ is not H; R ⁴ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl; R ⁵ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl; or R ⁴ and R ⁵ together form a carbocyclic ring and have the formula -(CR ^a R ^b )n- wherein R ^a and R ^b are independently selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl, and n selected from the group consisting of 2, 3, 4, 5 and 6; R ⁶ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl; R ⁷ is selected from the group consisting of H and optionally substituted -C ₁ -C ₈ -alkyl; each R ⁸ is independently selected from the group consisting of H, -OH, optionally substituted -C1-C8 alkyl, and optionally substituted -O-(C ₁ -C ₈ -alkylα); R ¹² is selected from amino acid side chains selected from the group consisting of threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine, and thiazolylalanine; and wherein the hydrophilic drug-ligand compound has a hydrophilicity index of less than two, wherein the hydrophilicity index is measured as the retention time of the hydrophilic drug-ligand conjugate compound relative to the retention time of the unconjugated ligand under high performance liquid chromatography conditions, wherein, if the group is substituted, then it is substituted with one, two, three or four substituents selected from the group consisting of -halogen, -OR', -NR'R'', -SR', -SiR'R''R', -OC(O) R', -C(O)R', -CO ₂ R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'- C(O)NR''R''', -NR''C(O) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C (NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -NR'S(O) ₂ R'', -CN and -no ₂ where each R', R'' and R''' independently refers to hydrogen, unsubstituted C _1-8 alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C _1-8 αalkyl, C _{1- 8} alkoxy or C _1-8 thioalkoxy or unsubstituted aryl-C _1-4 alk strong groups; or if R' and R'' are attached to the same nitrogen atom, then they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. 2. A hydrophilic drug-ligand conjugate compound according to claim 1, wherein R ¹² is the side chain of L-threonine. 3. A hydrophilic drug-ligand conjugate compound according to claim 1 or 2, wherein the p index is 8. 4. The hydrophilic compound of the drug-ligand conjugate according to claim 1, wherein the p index is 10. 5. A hydrophilic drug-ligand conjugate compound according to claim 1, which has the structure where each S is a sulfur atom of the cysteine thiol group of an antibody or an antigen-binding fragment of an antibody. 6. The hydrophilic compound of the drug-ligand conjugate according to claim 1, which has the structure of any of the formulas Ia-Id: - 53 040940 where each S represents a sulfur atom of the cysteine thiol group of an antibody or an antigen-binding fragment of an antibody. 7. The hydrophilic compound of the drug-ligand conjugate according to claim 1 or 2, where -L ^A -L ^H has the formula where R ²¹ is selected from the group consisting of -CH2OH, -CH2CH2OH, -CH2CO2H, -CH ₂ CH ₂ CO ₂ H, -CH ₂ CH ₂ CH ₂ CO ₂ H and -CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H; And R ²² is -CH2NH2; and wavy lines on the left and right show covalent attachment to DE and antibody or antigen-binding antibody fragment, respectively; And S represents the sulfur atom of the cysteine thiol group of an antibody or an antigen-binding fragment of an antibody. 8. A method of treating a patient, comprising administering to the patient an effective amount of a hydrophilic compound of the drug-ligand conjugate according to any one of claims 1 to 7, wherein the patient has a malignancy, and wherein the cell surface antigen to which the antibody specifically binds or an antigen-binding fragment of an antibody of the hydrophilic compound drug-ligand conjugate is a cancer cell antigen. 9. The method of claim 8, wherein the effective amount of the hydrophilic compound of the drug-ligand conjugate is a dose of 0.1 to 10 mg/kg. -