Patent Description:
While a number of different drug classes have been evaluated for delivery via antibodies, only a few drug classes have proved sufficiently active as antibody-drug conjugates, while having a suitable toxicity profile, to warrant clinical development. One such class is the auristatins, related to the natural product dolastatin <NUM>. Representative auristatins include MMAE (N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine) and MMAF (N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine).

MMAE is an example of a cytotoxic agent that is active as a free drug, and is highly potent after conjugation to a monoclonal antibody (mAb) and release after internalization into cells. MMAE has been successfully conjugated to a mAb at the N-terminal amino acid of MMAE via a cathepsin B-cleavable peptide-based linker containing maleimidocaproyl-valine-citrulline (mc-vc-) and a self-immolative group, p-aminobenzyl-carbamoyl (PABC), to produce antibody-drug conjugates of the following structure, mAb-(mc-vc-PABC-MMAE)p. (In the preceding formula, p refers to the number of (mc-vc-PABC-MMAE) units per mAb or antibody. ) Upon cleavage of the bond between the vc peptide and the self-immolative PABC group, the PABC group releases itself from MMAE, liberating free MMAE.

Another auristatin, MMAF, is less active as a free drug (compared to MMAE), yet is highly potent after conjugation to an antibody, internalization and release into cells. MMAF has been successfully conjugated to a monoclonal antibody (mAb) at its N-terminal amino acid via a cathepsin B-cleavable peptide-based linker containing maleimidocaproyl-valine-citrulline (mc-vc-) and a self-immolative group p-aminobenzyl-carbamoyl (PABC) to produce antibody-drug conjugates of the structure, mAb-(mc-vc-PABC-MMAF)p. (p refers to the number of (mc-vc-PABC-MMAF) units per mAb or antibody). Upon cleavage of the bond between the peptide and the PABC subunit, the self-immolative PABC group releases itself from MMAF, liberating free MMAF.

MMAF is also active as a non-cleavable conjugate, containing the drug-linker maleimidocaproyl MMAF (mcMMAF). When this conjugate, mAb-(mcMMAF)p, is internalized into cells, the active species released is cys-mcMMAF. Because the linker is non-cleavable, the maleimidocaproyl and a cysteine residue of the antibody remain attached to the N-terminus of MMAF. MMAF was also 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 species, MMAF, is released following 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.

Therefore, another important factor in the design of antibody-drug conjugates is the amount of drug that can be delivered per targeting agent (i.e., the number of cytotoxic agents attached to each targeting agent (e.g., an antibody), referred to as the drug load or drug loading). Historically, assumptions were that higher drugs loads were superior to lower drug loads (e.g., <NUM>-loads vs <NUM>- loads). The rationale was that higher loaded conjugates would deliver more drug (cytotoxic agents) to the targeted cells. This rationale was supported by the observations that conjugates with higher drug loadings were more active against cell lines in vitro. Certain later studies revealed, however, that this assumption was not confirmed in animal models. Conjugates having drug loads of <NUM> or <NUM> of certain auristatins were observed to have similar activities in mouse models. See, e.g., <NPL>). Hamblett et al. further reported that the higher loaded ADCs were cleared more quickly from circulation in animal models. This faster clearance suggested a PK liability for higher loaded species as compared to lower loaded species. See Hamblett et al. In addition, higher loaded conjugates had lower MTDs in mice, and as a result had narrower reported therapeutic indices. In contrast, ADCs with a drug loading of <NUM> at engineered sites in a monoclonal antibody were reported to have the same or better PK properties and therapeutic indices as compared to certain <NUM>-loaded ADCs. For example, see <NPL>). Thus, recent trends are to develop ADCs with low drug loadings.

Alternative approaches to overcome the PK liability of higher loaded ADCs have been to append solubilizing groups to the ADCs. For example, polyethylene glycol polymers or other water soluble polymers have been included in linkers (e.g., between the drug and attachment site of an antibody) in an attempt to overcome PK liabilities. Another approach has been to append drug-polymers to an antibody, where each polymer contains a large number of drugs. These alternatives have not, however, necessarily achieved the desired result. In addition, appending solubilizing groups may increase manufacturing complexity of such conjugates.

There remains a need, therefore, for antibody drug conjugate formats (and more generally for formats for other conjugates), that allow for higher drug loading while maintaining other desirable characteristics of lower loaded conjugates, such as favorable PK properties. Surprisingly, the present invention addresses these needs.

The present invention relates to a Drug-Linker Compound as defined in appended claims <NUM> to <NUM>.

Disclosed herein are, inter alia, hydrophilic Ligand-Linker-Drug Conjugates. By designing the conjugates to have a hydrophilicity similar to the unconjugated targeting agent (e.g., a ligand such as an antibody), the conjugates retain the ability to provide pharmacokinetic (PK) properties similar to that of the unconjugated targeting agent in vivo. The conjugates can also have higher drug loadings (i.e., higher numbers of hydrophilic drug-linkers per targeting agent), as compared to lower loaded conjugates, while retaining such desirable PK properties and having the same or better activity in vivo. (For example, <NUM>-loaded or <NUM>-loaded conjugates can have the same or better PK properties than their <NUM> or <NUM> loaded counterparts, respectively; such <NUM>-loaded or <NUM>-loaded conjugates can have the same or better activity than their <NUM> or <NUM> loaded counterparts, respectively. ) Thus, targeting agents selected on the basis of certain desirable properties can be conjugated with drug linkers without substantially impairing such desirable properties as PK properties of the targeting agent alone. Also disclosed herein are methods of making and using such conjugates.

Also disclosed herein are methods of preparing and using the Drug-Linker compounds of the appended claims.

Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings. When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.

The term "hydrophilicity index" refers to a measure of the hydrophilicity of a conjugate relative to the hydrophilicity of the targeting agent alone (i.e., a Ligand, typically an antibody). The hydrophilicity index is measured as the retention time of a conjugate to that of the corresponding unconjugated targeting agent (alone) under high performance liquid chromatography (HPLC) conditions, as further described herein. For example, the retention time of a Ligand-Linker-Drug Conjugate, relative to the retention time of the unconjugated Ligand (typically an antibody), can be determined. In selected embodiments, the retention time of the conjugate is not greater than two minutes slower than the retention time of the unconjugated ligand, as determined as described in the examples (referred to as a hydrophilicity index of <NUM>). In certain embodiments, the retention time of the conjugate is not greater than one minute slower than the retention time of the unconjugated ligand, as determined as described in the examples (referred to as a hydrophilicity index of <NUM>). In certain embodiments, the retention time of the conjugate is not greater than one half minute slower than the retention time of the unconjugated ligand, as determined as described in the examples (referred to as a hydrophilicity index of <NUM>). If a different hydrophobic interaction column and/or method is used, it can be calibrated using conjugates from Tables <NUM> as references to determine reference conjugate mobilities (elution times) on the selected column and/or method. The determined reference mobilities on the selected hydrophobic interaction column and/or method can then be used to calculate a hydrophilicity index of a test article (as would be determined following Example <NUM>). For example, an auristatin T - Glu- Dpr - MA, an mc-MMAF and an mc-vc-PABC-MMAE drug linkers can be used to form conjugates to use as references. In another example, an auristatin T-Glu-Dpr-MA-h1F6 ADC, an h1F6-mc-MMAF and an h1F6-mc-vc-PABC-MMAE ADC can be used as references.

The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e. C<NUM>-<NUM> means one to eight carbons). 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, <NUM>-propenyl, crotyl, <NUM>-isopentenyl, <NUM>-(butadienyl), <NUM>,<NUM>-pentadienyl, <NUM>-(<NUM>,<NUM>-pentadienyl), ethynyl, <NUM>- and <NUM>-propynyl, <NUM>-butynyl, and the higher homologs and isomers. The term "cycloalkyl" refers to hydrocarbon rings having the indicated number of ring atoms (e.g., C<NUM>-<NUM>cycloalkyl) and being fully saturated or having no more than one double bond between ring vertices. "Cycloalkyl" is also meant to refer to bicyclic and polycyclic hydrocarbon rings such as, for example, bicyclo[<NUM>. <NUM>]heptane, bicyclo[<NUM>. <NUM>]octane, etc. The term "heterocycloalkane" or "heterocycloalkyl" refers to a cycloalkyl group that contain from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. The heterocycloalkane may be a monocyclic, a bicyclic or a polycylic ring system. Non limiting examples of heterocycloalkane groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, <NUM>,<NUM>-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, <NUM>-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrhydrothiophene, quinuclidine, and the like. A heterocycloalkane group can be attached to the remainder of the molecule through 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, as exemplified by -CH<NUM>CH<NUM>CH<NUM>CH<NUM>-. Typically, an alkyl (or alkylene) group will have from <NUM> to <NUM> carbon atoms, with those groups having <NUM> or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having four or fewer carbon atoms. Similarly, "alkenylene" and "alkynylene" refer to the unsaturated forms of "alkylene" having double or triple bonds, respectively.

As used herein, a wavy line, "<IMG>", that intersects a single, double or triple bond in any chemical structure depicted herein, represent the point attachment of the single, double, or triple bond to the remainder of the molecule.

The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl portions can be the same or different and can also be combined to form a <NUM>-<NUM> membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as dialkylamino or -NRaRb 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 stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C<NUM>-<NUM> haloalkyl" is mean to include trifluoromethyl, <NUM>,<NUM>,<NUM>-trifluoroethyl, <NUM>-chlorobutyl, <NUM>-bromopropyl, and the like.

The term "aryl" means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon group which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and the like. Substituents for each of the above noted aryl and heteroaryl ring systems, when described as 'substituted' are selected from the group of acceptable substituents described below.

The term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, and the like). Similarly, the term "heteroaryl-alkyl" is meant to include those radicals in which a heteroaryl group is attached to an alkyl group (e.g., pyridylmethyl, thiazolylethyl, and the like).

The above terms (e.g., "alkyl," "aryl" and "heteroaryl"), in some embodiments, will include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Unless otherwise indicated by context, substituents for the alkyl radicals (including those groups often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) can be a variety of groups selected from: -halogen, -OR', -NR'R", -SR', -SiR'R"R‴, -OC(O)R', -C(O)R', - CO<NUM>R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)<NUM>R', -NH-C(NH<NUM>)=NH, -NR'C(NH<NUM>)=NH, -NH-C(NH<NUM>)=NR', -S(O)R', -S(O)<NUM>R', - S(O)<NUM>NR'R", -NR'S(O)<NUM>R", -CN and -NO<NUM> in a number ranging from zero to (<NUM>'+<NUM>), where m' is the total number of carbon atoms in such radical. R', R" and R‴ each independently refer to hydrogen, unsubstituted C<NUM>-<NUM> alkyl, unsubstituted aryl, aryl substituted with <NUM>-<NUM> halogens, unsubstituted C<NUM>-<NUM> alkyl, C<NUM>-<NUM> alkoxy or C<NUM>-<NUM> thioalkoxy groups, or unsubstituted aryl-C<NUM>-<NUM> alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a <NUM>-, <NUM>-, <NUM>-, <NUM>-, or <NUM>-membered ring. For example, -NR'R" is meant to include <NUM>-pyrrolidinyl and <NUM>-morpholinyl.

Similarly, substituents for the aryl and heteroaryl groups are varied and are generally selected from: -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO<NUM>, - CO<NUM>R', -CONR'R", -C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O)<NUM>R', ,-NR'-C(O)NR"R‴, -NH-C(NH<NUM>)=NH, -NR'C(NH<NUM>)=NH, -NH-C(NH<NUM>)=NR', -S(O)R', - S(O)<NUM>R', -S(O)<NUM>NR'R", -NR'S(O)<NUM>R", -N<NUM>, perfluoro(C<NUM>-C<NUM>)alkoxy, and perfluoro(C<NUM>-C<NUM>)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<NUM>-<NUM> alkyl, C<NUM>-<NUM> haloalkyl, C<NUM>-<NUM> cycloalkyl, C<NUM>-<NUM> alkenyl, C<NUM>-<NUM> alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C<NUM>-<NUM> alkyl, and unsubstituted aryloxy-C<NUM>-<NUM> alkyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of from <NUM>-<NUM> carbon atoms.

The term "base" refers to a functional group that deprotonates water to produce a hydroxide ion. Exemplary bases are amines and nitrogen containing heterocycles. Representative bases include -N(R<NUM>)(R<NUM>) wherein R<NUM> and R<NUM> are independently selected from H or C<NUM>-<NUM> alkyl, preferably H or methyl,
<CHM>
<CHM>
<CHM>
<CHM>
wherein R<NUM>, R<NUM>, R<NUM> and R<NUM> are, at each occurrence, independently selected from hydrogen or C<NUM>-<NUM> alkyl, preferably H or methyl, and e is <NUM>-<NUM>. In some aspects, the base is a nitrogenous base.

The term "antibody" is used herein in the broadest sense and specifically covers 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 primarily two regions: a variable region and a constant region. The variable region specifically binds to and interacts with a target antigen. The variable region includes complementary determining regions (CDR) that recognize and bind to a specific binding site on a particular antigen. The constant region may be recognized by and interact with the immune system (see, e.g., <NPL>). An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. A monoclonal antibody can be, for example, human, humanized or chimeric.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

An "intact antibody" is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH<NUM>, CH<NUM>, CH<NUM> and CH<NUM>, as appropriate for the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.

An "antibody fragment" comprises a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')<NUM>, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), a fragment(s) produced by a Fab expression library, or an epitope-binding fragments of any of the above which specifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen or a microbial antigen).

An "antigen" is an entity to which an antibody specifically binds. An antigen can be, for example, proteinaceous (e.g., a protein, polypeptide or peptide), non-proteinaceous (e.g., a carbohydrate), or a combination of the two.

The terms "specific binding" and "specifically binds" mean that the targeting agent or Ligand, such as an antibody or antigen binding fragment, will bind in a highly selective manner with its corresponding target antigen and not with the multitude of other antigens. For an antibody, the antibody or antibody fragment typically binds with an affinity of at least about <NUM>×<NUM>-<NUM> M, and preferably <NUM>-<NUM> M to <NUM>-<NUM> M, <NUM>-<NUM> M, <NUM>-<NUM> M, or <NUM>-<NUM> M and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen having the same epitope.

The terms "inhibit" or "inhibition of" means to a reduce by a measurable amount, or to prevent entirely.

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

The term "substantial" or "substantially" refers to a majority, i.e. ><NUM>% of a population, of a mixture or a sample, preferably more than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM> %, <NUM>%, or <NUM>% of a population.

The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction inside a cell on a Ligand-Linker-Drug conjugate (e.g., an Antibody Drug Conjugate (ADC), or the like), whereby the covalent attachment (the Linker unit), between the DE moiety and the Ligand unit (e.g., an antibody (Ab)) is broken, resulting in the release of the DE unit. The cleaved moieties of the Ligand-Linker-Drug conjugate are thus intracellular metabolites.

The term "cytotoxic activity" refers to a cell-killing or a cytotoxic effect of a Ligand-Linker-Drug conjugate compound, typically via the released Drug unit, on the target cell. Cytotoxic activity may be expressed as the IC<NUM> value (also referred to as the half maximal inhibitory concentration), which is the concentration (molar or mass) per unit volume at which half the cells survive exposure 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 "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises cancerous cells.

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

Examples of a "patient" include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human.

The terms "treat" or "treatment," unless otherwise indicated by context, refer to therapeutic treatment and prophylactic measures to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, a stabilized (i. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder.

In the context of cancer, the term "treating" includes any or all of: inhibiting growth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term "treating" includes any or all of: inhibiting replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.

The phrase "pharmaceutically acceptable salt," as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound (e.g., a Drug, Drug-Linker, or a Ligand-Linker-Drug Conjugate). The compound can contain at least one amino group, and accordingly acid addition salts can be formed with the amino group. Exemplary 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, p-toluenesulfonate, and pamoate (i.e., <NUM>,<NUM>'-methylene-bis -(<NUM>-hydroxy-<NUM>- naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.

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 (ADCs), that have a hydrophilicity similar to that of the unconjugated Ligand (i.e., a targeting agent, such as an antibody or antigen binding fragment). By maintaining a conjugate hydrophilicity similar to that of the unconjugated Ligand, the resulting conjugates can have higher drug loadings (e.g., at least <NUM> or <NUM> drug linkers per Ligand), while maintaining certain desirable characteristics of the Ligand alone, such as reduced clearance in vivo, increased pharmacokinetic profile in vivo, increased exposure of the conjugates to the target cell(s), etc. Advantageously, such hydrophilic conjugates can be designed to have a 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 as Drug-Ligand conjugates, Ligand-Drug conjugates or Ligand Drug conjugates herein.

The hydrophilic linking groups (also referred to as Linkers or Linker units) of the Drug-Linker compounds of the invention are designed for increased hydrophilicity. The linking group allows efficient release of the cytotoxic agent (also referred to as a Drug unit or Drug) at the target cell, sufficient to induce cytotoxicity or a cytostatic effect in the case of a cytotoxic agent. The hydrophilic linkers are designed for efficient release of the Drug unit once a Ligand-Linker Drug conjugate comprising the Drug-Linker compound has been internalized into the target cell. Suitable recognition sites for release of the Drug unit by cleavage are those that allow efficient separation of the unit from the hydrophilic linking group. Herein, the recognition site is a peptide cleavage site. Examples of peptide cleavage sites include those recognized by intracellular proteases, such as those present in lysosomes. The cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches its targeted site.

The Drug units are auristatins that are designed to have increased hydrophilicity in combination with a hydrophilic Linker unit. The Drug units advantageously can be designed to have hydrophilic substituents, while retaining potent cytotoxic activity. Auristatin Drug units are attached at their C-terminal end to a Linker unit, as more fully described herein.

LH is a hydrophilic linker containing one, two, or three 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, LH is a hydrophilic linker comprising 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.

Conjugate hydrophilicity for Ligand-Drug Conjugates comprising a Drug-Linker compound according to the appended claims can be determined by comparing the hydrophilicity of the conjugate to that of the unconjugated targeting agent (i.e., Ligand or Ligand unit), referred to as the hydrophilicity index. Referring to the Examples, Example <NUM> discloses a preferred method for determining the hydrophilicity index of a conjugate. Alternatively, a different hydrophobic interaction column and/or method can be calibrated using conjugates from Tables <NUM> as references to determine reference conjugate mobilities (elution times) of the references on the selected column and/or method. The determined reference mobilities on the selected hydrophobic interaction column and/or method can then be used to calculate a hydrophilicity index of a test article (as would be determined following Example <NUM>). For example, an auristatin T - Glu- Dpr - MA, an mc-MMAF and an mc-vc-PABC-MMAE drug linkers can be used to form conjugates to use as references. In another example, an auristatin T-Glu-Dpr-MA-h1F6 ADC, an h1F6-mc-MMAF and an h1F6-mc-vc-PABC-MMAE ADC can be used as references.

Disclosed herein is a Ligand-Linker-Drug Conjugate comprising a Ligand unit and multiple Drug-Linker compounds attached to the Ligand unit. The Linker unit comprises a hydrophilic linker (LH) assembly, including a Ligand attachment component, such as via a thioether linkage. The Drug unit comprises a cytotoxic agent having an attachment component for connection to the Linker unit.

Drug-Linker compounds are provided wherein the Linker portion comprises a hydrophilic linker (LH) assembly having a Ligand attachment component (an attached maleimide moiety), suitable for reacting with a Ligand.

Ligand-Linker Drug Conjugates do not fall within the scope of the appended claims. However, Ligand-Linker Drug Conjugates may be formed comprising Drug-Linker Compounds of the appended claims.

Ligand-Linker-Drug conjugates having the following formula are disclosed herein:
<CHM>
or a pharmaceutically acceptable salt or solvate thereof,
wherein:.

Ligand-Linker-Drug conjugates having the following formula are also disclosed herein:
<CHM>
or a pharmaceutically acceptable salt or solvate thereof,
wherein:.

The Ligand-Linker-Drug conjugates of formulas I and I' are described in more detail below.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least four Linker-Drug units, wherein the Ligand unit and each of the Drug unit(s) are joined by a Linker unit(s) comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, each Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least six Linker-Drug units, wherein the Ligand unit and each of the Drug unit(s) are joined by a Linker unit(s) comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, each Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least eight Linker-Drug units, wherein the Ligand unit and each of the Drug unit(s) are joined by a Linker unit(s) comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, each Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The 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 joined by a Linker unit(s) comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, each Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The 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 joined by a Linker unit(s) comprising a hydrophilic linker (LH) assembly. The Linker units may attached to the Ligand unit via a thioether bond. For example, each Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

Referring to the Drug unit of formulas I and I', R<NUM> may be selected from H, optionally substituted -C<NUM>-C<NUM> alkyl, optionally substituted aryl, optionally substituted -X<NUM>aryl, optionally substituted -C<NUM>-C<NUM> carbocycle, optionally substituted -X<NUM>-(C<NUM>-C<NUM> carbocycle), optionally substituted -C<NUM>-C<NUM> alkylene-NH<NUM>, optionally substituted -C<NUM>-C<NUM> heterocycle and optionally substituted -X<NUM>-(C<NUM>-C<NUM> heterocycle).

In some aspects, R<NUM> is not the side chain of phenylalanine or proline. In some further aspects, R<NUM> is not the side chain of phenylalanine, methionine, tryptophan or proline.

In some aspects, R<NUM> is selected from the side chains of natural L-amino acids other than proline, and glycine. In some further aspects, R<NUM> is selected from the side chains of natural L-amino acids other than proline, glycine or phenylalanine. In some further aspects, R<NUM> is selected from the side chains of natural L-amino acids other than proline, glycine, tryptophan, methionine or phenylalanine.

In some further aspects, R<NUM> is selected from the side chains of the group of hydrophilic amino acids consisting of threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furyl alanine, threonine(PO<NUM>H<NUM>), pyrazolyl alanine, triazolyl alanine and thiazolyl alanine.

In some aspects, R<NUM> is the side chain of threonine.

Exemplary Drug units have the following formula, or a pharmaceutically acceptable salt thereof, wherein the wavy line indicates site of attachment to the Linker unit. In some exemplary units, the Drug unit is dimethyl- or monomethyl-auristatin F, as shown below:
<CHM>
<CHM>
or a pharmaceutically acceptable salt or solvate thereof.

Other exemplary Drug units are the dimethyl- or monomethyl forms of auristatin T. <CHM>
or
<CHM>
or a pharmaceutically acceptable salt or solvate thereof.

Referring to the Linker unit of formulas I and I', in some aspects LA is covalently linked to a sulfur atom of the Ligand. In some aspects, the sulfur atom is that of 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 (e.g., by site directed mutagenesis or chemical reaction). In further aspects, the sulfur atoms to which the LA's are attached are selected from cysteine residues that form an interchain disulfide bond of an antibody and cysteine residues that have been introduced into the Ligand unit (e.g., by site directed mutagenesis or chemical reaction).

AA<NUM> forms a cleavable bond with the Drug unit. In aspects where AA<NUM> is attached to an amino acid of the Drug unit, AA<NUM> forms a cleavable peptide bond with the Drug unit. The cleavable peptide bond is susceptible to cleavage by proteases when the conjugate reaches its target site. In some aspects, AA<NUM> is a hydrophilic amino acid, typically an amino acid that is selected from the group consisting of Glycine and L forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine. In some aspects, AA<NUM> is Glutamate.

In aspects where RL1 is present and is a hydrophilic amino acid, it can be selected from the group consisting of Glycine; L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; -NH - CH(Ra) - CO-; and -NH - CH(COOH) - Rb -; wherein Ra is selected.

In aspects where RL1 is present and is an optionally substituted alkylene, it can be a C<NUM> - C<NUM> alkylene, optionally substituted with <NUM>-<NUM> substituents selected from -NH-, -C(O)-, - COOH, -N(C<NUM> - C<NUM> alkyl), - NH<NUM> or -NH(C<NUM> - C<NUM> alkyl). In aspects, RL1 is ethylenediamine, -NH - CH(COOH)- CH<NUM> -NH - or -C(O) - CH(CH<NUM>NH<NUM>) -.

In aspects where RL2 is present and is a hydrophilic amino acid, it can be selected from the group consisting of Glycine; L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; -NH - CH(Ra)- C(O)-; and - NH- CH(COOH) - Rb -; wherein Ra is selected.

In aspects where RL2 is present and is an optionally substituted alkylene, it can be a C<NUM> - C<NUM> alkylene, optionally substituted with <NUM>-<NUM> substituents selected from -NH-, -C(O)-, - COOH, -N(C<NUM> - C<NUM> alkyl)-, -NH<NUM> or -NH(C<NUM> - C<NUM> alkyl). In some aspects, RL2is ethylenediamine, -NH - CH(COOH) - CH<NUM> -NH - or -C(O)- CH(CH<NUM>NH<NUM>) -.

In aspects where RL3 is present, and is a hydrophilic amino acid, it can be selected from the group consisting of Glycine; L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; -NH - CH(Ra)- C(O)-; and - NH- CH(COOH) - Rb -; wherein Ra is selected.

In some further aspects when RL3 is present, it is selected from the group consisting of the D amino acids of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; Glycine; -NH - CH(Ra)- C(O)-; and -NH - CH(COOH)- Rb -; wherein Ra is selected.

In aspects where RL3 is present and is an optionally substituted alkylene, it can be a C<NUM> - C<NUM> alkylene, optionally substituted with <NUM>-<NUM> substituents selected from -NH-, -C(O)-, - COOH, -N(C<NUM> - C<NUM> alkyl)-, -NH<NUM> or -NH(C<NUM> - C<NUM> alkyl). In some aspects, RL1 is ethylenediamine, -NH- CH(COOH)- CH<NUM> -NH - or -C(O)- CH(CH<NUM>NH<NUM>) -.

In some aspects of the above, AA<NUM> is present and RL1, RL2 and RL3 are absent.

In some aspects of the above, AA<NUM> is present, RL1 is present and RL2 and RL3 are absent.

In some aspects of the above, AA<NUM> is present, RL1 is present, RL2 is present and RL3 is absent.

In some aspects of the above, AA<NUM> is present, RL1 is present, RL2 is present and RL3 is present.

In some aspects of the above, AA<NUM> is a hydrophilic amino acid and at least one of RL1, RL2 and RL3 is present and is an optionally substituted alkylene, as set forth above.

In some aspects of the above, AA<NUM> is Glutamate and at least one of RL1, RL2 and RL3 is present and is an optionally substituted alkylene, as set forth above.

In some aspects of the above, AA<NUM> is Glutamate, RL1 is a hydrophilic amino acid and at least one of RL2 and RL3 is present and is an optionally substituted alkylene, as set forth above.

In some aspects of the above, AA<NUM> and RL1 are hydrophilic amino acids and at least one of RL2 and RL3 is present and is an optionally substituted alkylene, as set forth above.

In some aspects of the above, AA<NUM> is a hydrophilic amino acid and RL1 and optionally RL2 are an optionally substituted alkylene, as set forth above.

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

In some aspects, LH will include a modified peptide, having from two to four amino acids. The modified peptide has an amino acid in the <NUM>-position (AA<NUM>) that is selected to optimize release of the Drug unit (e.g., by protease cleavage via an amide peptide bond). In one or both of positions RL1 and RL2 is an amino acid that reverses the orientation of typical N to C linkages of peptides (forming amide bonds) and facilitates attachment of the last amino acid (e.g., RL2 or RL3) which, prior to attachment of the Ligand unit, includes an α-amino group protected as a maleimide. The amino acid having a reversed N to C linkage is attached to the next group via its side chain. In some aspects, this amino acid is an alpha amino acid. In other aspects, it can be a beta or gamma amino acid. In some of these aspects, the side chain is selected from -CH<NUM>NH<NUM> -, -CH<NUM>CH<NUM>NH<NUM> -, -CH<NUM>CH<NUM>CH<NUM>NH<NUM>-, and -CH<NUM>CH<NUM>CH<NUM>CH<NUM>NH<NUM>-.

In some aspects of LH, the amino acid having a reversed N to C linkage (RL1) is attached to RL2 or RL3, where RL2or RL3 is a hydrophilic amino acid or an optionally substituted alkylene, according to any of the aspects described above.

In some aspects of LH, the amino acid having a reversed N to C linkage (RL1) is attached to RL2, where RL2 is an optionally substituted alkylene, according to any of the aspects described above.

In some further aspects, LH is a hydrophilic, cleavable linker, each branch having the formula:
<CHM>
wherein R<NUM> is selected
from -CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>NH<NUM>, -CH<NUM>OH, -CH<NUM>CH<NUM>OH, -CH<NUM>CO<NUM>H, -CH<NUM>CH<NUM>CO<NUM>H, -CH<NUM>CH<NUM>CH<NUM>CO<NUM>H and -CH<NUM>CH<NUM>CH<NUM>CH<NUM>CO<NUM>H; and R<NUM> is selected from -CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>NH<NUM>, -CH<NUM>OH, and -CH<NUM>CH<NUM>OH. The left and right wavy lines indicate attachments to the Drug unit and LA, or the branch of LH, respectively.

In further aspects LH, or a branch thereof, has the formula:
<CHM>
wherein R<NUM> is selected from -CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>NH<NUM>, -CH<NUM>OH, and -CH<NUM>CH<NUM>OH. In some aspects, R<NUM> is selected from -CH<NUM>NH<NUM> and -CH<NUM>CH<NUM>NH<NUM>. The left and right wavy lines indicate attachments to the Drug unit and LA, or the branch of LH, respectively.

In certain aspects, LH, or a branch thereof, has the formula:
<CHM>.

The left and right wavy lines indicate attachments to the Drug unit and LA, respectively.

The left and right wavy lines indicate attachments to the Drug unit and LA, or the branch of LH, respectively.

In some further aspects of the above, LH is a branched hydrophilic linker having the formula:
<CHM>.

In some further aspects of the above, LH is a branched hydrophilic linker having the formula:
<CHM>
wherein each of the bars indicates attachment to a Drug unit, and the vertical dashed line indicates an attachment to a Ligand unit.

The LA subunit is described in more detail below.

The Ligand-Linker-Drug conjugates may have a formula selected from:
<CHM>
and
<CHM>
wherein S refers to a sulfur atom of the Ligand.

The Ligand-Linker-Conjugates may have the following structures:
<CHM>
<CHM>
<CHM>
<CHM>
wherein S refers to a sulfur atom of the Ligand, or a pharmaceutically acceptable salt or solvate thereof.

Ligand-Linker-Drug conjugates having the following formula are disclosed herein:
<CHM>
wherein:.

The Ligand-Linker-Drug conjugates of formula II are described in more detail below.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least four Linker-DE units, wherein the Ligand unit and each of the DE unit(s) are joined by a Linker unit comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, the Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least six Linker- DE units, wherein the Ligand unit and each of the DE unit(s) are joined by a Linker unit comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, the Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least eight Linker- DE units, wherein the Ligand unit and each of the DE unit(s) are joined by a Linker unit comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, the Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least ten Linker- DE units, wherein the Ligand unit and each of the DE unit(s) are joined by a Linker unit comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, the Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

The Ligand-Linker-Drug conjugates may comprise a Ligand unit and at least sixteen Linker- DE units, wherein the Ligand unit and each of the DE unit(s) are joined by a Linker unit comprising a hydrophilic linker (LH) assembly. The Linker units may be attached to the Ligand unit via a thioether bond. For example, the Linker unit may further comprise a hydrolyzed succinimide ring (or succinic acid) directly conjugated to the Ligand unit via a thioether linkage.

Referring to the Linker unit of formula II, in some aspects LA is covalently linked to a 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 (e.g., by site directed mutagenesis or chemical reaction). In further aspects, the sulfur atom(s) to which the LA's are attached are selected from cysteine residues that form an interchain disulfide bond of an antibody and cysteine residues that have been introduced into the Ligand unit (e.g., by site directed mutagenesis or chemical reaction).

AA<NUM> forms a cleavable bond with the DE unit. In aspects where AA<NUM> is attached to an amino acid of the DE unit, AA<NUM> 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 aspects, AA<NUM> forms an amide bond with an attachment site of the effector moiety (DE) that is susceptible to cleavage (e.g., by proteases) when the conjugate reaches its targeted site. In some aspects of formula II, AA<NUM> is a hydrophilic amino acid, typically a natural amino acid that is selected from the group consisting of Glycine and L forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine. In some aspects, AA<NUM> is Glutamate.

In aspects where RL1 is present and is a hydrophilic amino acid, it can be selected from the group consisting of Glycine; L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; -NH - CH(Ra)- C(O)-; and - NH - CH(COOH) - Rb -; wherein Ra is selected.

In aspects where RL1 is present and is an optionally substituted alkylene, it can be a C<NUM> - C<NUM> alkylene, optionally substituted with <NUM>-<NUM> substituents selected from -NH-, -C(O)-, - COOH, -N(C<NUM> - C<NUM> alkyl)-, -NH<NUM> or -NH(C<NUM> - C<NUM> alkyl). In some aspects, RL1 is ethylenediamine, -NH - CH(COOH) - CH<NUM> -NH - or -C(O)- CH(CH<NUM>NH<NUM>) -.

In aspects where RL2 is present and is a hydrophilic amino acid, it can be selected from the group consisting of Glycine; L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; -NH - CH(Ra)- C(O)-; and - NH - CH(COOH) - Rb -; wherein Ra is selected.

In some further aspects when RL2 is present, it is selected from the group consisting of the D amino acids of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; Glycine; -NH - CH(Ra) - C(O)-; and -NH - CH(COOH) - Rb -; wherein Ra is selected.

In aspects where RL2 is present and is an optionally substituted alkylene, it can be a C<NUM> - C<NUM> alkylene, optionally substituted with <NUM>-<NUM> substituents selected from -NH-, -C(O)-, - COOH, -N(C<NUM> - C<NUM> alkyl)-, -NH<NUM> or -NH(C<NUM> - C<NUM> alkyl). In some aspects, RL2 is ethylenediamine, -NH - CH(COOH) - CH<NUM> -NH - or -C(O)- CH(CH<NUM>NH<NUM>) -.

In aspects where RL3 is present, and is a hydrophilic amino acid, it can be selected from the group consisting of Glycine; L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; -NH - CH(Ra) - C(O)-; and - NH - CH(COOH) - Rb -; wherein Ra is selected.

In some further aspects when RL3 is present, it is selected from the group consisting of the D amino acids of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine; Glycine; -NH - CH(Ra) - C(O)-; and -NH - CH(COOH) - Rb -; wherein Ra is selected.

In aspects where RL3 is present, and is an optionally substituted alkylene, it can be a C<NUM> - C<NUM> alkylene, optionally substituted with <NUM>-<NUM> substituents selected from -NH-, -C(O)-, - COOH, -N(C<NUM> - C<NUM> alkyl)-, -NH<NUM> or -NH(C<NUM> - C<NUM> alkyl). In some aspects, RL3 is ethylenediamine, -NH - CH(COOH) - CH<NUM> -NH - or -C(O) - CH(CH<NUM>NH<NUM>) -.

In some further of the above, AA<NUM> is a hydrophilic amino acid and at least one of RL1, RL2 and RL3 is present and is an optionally substituted alkylene, as set forth above.

In some further aspects of the above, AA<NUM> and RL1 are hydrophilic amino acids and at least one of RL2 and RL3 is present and is an optionally substituted alkylene, as set forth above.

In some aspects, LH will include a modified peptide, having from two to four amino. The modified peptide has an amino acid in the <NUM>-position (AA<NUM>) that is selected to optimize release of the DE unit (e.g., by protease cleavage via an amide peptide bond). In one or both of positions RL1 and RL2 is an amino acid which reverses the orientation of typical N to C linkages of peptides and facilitates attachment of the last amino acid (e.g., RL2 or RL3) which, prior to attachment of the Ligand unit, includes an α-amino group protected as a maleimide. The amino acid having a reversed N to C linkage is attached to the next group via its side chain. In some aspects, this amino acid is an alpha amino acid. In other aspects, it can be a beta or gamma amino acid. In some aspects, the side chain is selected from -CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>CH<NUM>NH<NUM>, and -CH<NUM>CH<NUM>CH<NUM>CH<NUM>NH<NUM>.

In some aspects of LH, the amino acid having a reversed N to C linkage (RL1) is attached to RL2or RL3, where RL2or RL3 is a hydrophilic amino acid or an optionally substituted alkylene, according to any of the aspects described above.

In some further aspects, LH is a hydrophilic, cleavable linker, each branch having the formula:
<CHM>
wherein R<NUM> is selected.

In further aspects, LH, or a branch thereof, has the formula:
<CHM>
wherein R<NUM> is selected from -CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>NH<NUM>, -CH<NUM>OH, and -CH<NUM>CH<NUM>OH. In some aspects, R<NUM> is selected from -CH<NUM>NH<NUM> and -CH<NUM>CH<NUM>NH<NUM>. The left and right wavy lines indicate attachments to the DE unit and LA, respectively.

The left and right wavy lines indicate attachments to the DE unit and LA, or the branch of LH, respectively.

The left and right wavy lines indicate attachments to the DE unit and LA, respectively.

In some further aspects of the above, LH is a branched hydrophilic linker having the formula:
<CHM>
wherein each R<NUM> is independently selected from the group consisting of -CH<NUM>NH<NUM>, -CH<NUM>CH<NUM>NH<NUM>, -CH<NUM>OH, -CH<NUM>CH<NUM>OH, -CH<NUM>CO<NUM>H,
-CH<NUM>CH<NUM>CO<NUM>H, -CH<NUM>CH<NUM>CH<NUM>CO<NUM>H, and -CH<NUM>CH<NUM>CH<NUM>CH<NUM>CO<NUM>H; and each of the bars adjacent the R<NUM>indicates an attachment to a DE unit and the vertical dashed line indicates an attachment to a Ligand unit.

In some further aspects of the above, LH is a branched hydrophilic linker having the formula:
<CHM>
wherein each of the bars indicates attachment to a DE unit, and the vertical dashed line indicates an attachment to a Ligand unit.

In some further aspects of the above, a branched hydrophilic linker has the formula:
<CHM>.

Referring again to formulas I, I' and II (supra), LA is a Ligand attachment component. LA can be a maleimide or a hydrolyzed maleimide or succinimide group (illustrated below as a succinic acid moiety). When LA is attached to a Ligand unit, it may be a hydrolyzed maleimide or succinimide group (illustrated as a succinic acid moiety). Accordingly, LA may have the formula:
<CHM>
wherein the wavy line indicates the point of attachment to LH and the \\ indicates the point of attachment to L, the Ligand unit.

Alternatively, LA may have the formula:
<CHM>
wherein the wavy line indicates the point of attachment to LH and the \\ indicates the point of attachment to L, the Ligand unit.

LA may be the vestige of a maleimide group used for attachment of the Ligand portion. The design of LH and LA allows for facile addition of a Ligand unit, as well as providing an additional carboxylic acid group which increases the hydrophilicity of the Ligand-Drug Conjugate. Still further, the maleimide nitrogen becomes an α-amine of amino acid <NUM> (with reference to LH).

Referring again to formulas I, I' and II, the Ligand unit (L-) is a targeting agent that specifically binds to a target moiety. The Ligand can specifically bind to a cell component or to other target molecules of interest. The target moiety, or target, is typically on the cell surface. The Ligand unit may act to deliver the Drug unit to the particular target cell population with which the Ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides and peptides as well as non-proteinaceous agents such as carbohydrates. Suitable Ligand units include, for example, antibodies, e.g., full-length (intact) antibodies, as well as antigen binding fragments thereof.

Where the Ligand unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-proteinaceous 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.

LA may be covalently linked to a sulfur atom of the Ligand. The sulfur atom may be that of a cysteine residue that forms an interchain disulfide bond of an antibody. Alternatively, the sulfur atom may be that of a cysteine residue that has been introduced into the Ligand unit (e.g., by site directed mutagenesis or chemical reaction). The sulfur atoms to which the LA's are attached may be selected from cysteine residues that form an interchain disulfide bond of an antibody and cysteine residues that have been introduced into the Ligand unit (e.g., by site directed mutagenesis or chemical reaction). The cysteine residue may be introduced into the Fc region at position <NUM> according to the EU index numbering system as in Kabat (<NPL>).

The Ligand unit may form a bond with the maleimide present on LH via a sulfhydryl group of the Ligand to form a thio-substituted succinimide. The sulfhydryl group can be present on the Ligand in the Ligand's natural state, for example a naturally-occurring antibody, or can be introduced into the Ligand via chemical modification. Hydrolysis of the remaining succinimide produces the LA portion.

The Ligand unit may have one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and <NUM>-Iminothiolane hydrochloride (Traut's Reagent).

The Ligand unit can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups.

The sulfhydryl groups can be generated by reduction of the interchain disulfides. Accordingly, a Linker unit may be conjugated to a cysteine residue of the reduced interchain disulfides.

The sulfhydryl group may be chemically introduced into the antibody, for example by introduction of 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 factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-<NUM>, IL-<NUM>, 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 apoprotein from low density lipoprotein.

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 homogeneous populations of antibodies to a particular antigenic determinant (e.g., to a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. The antibodies include full-length antibodies and antigen binding fragments thereof. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., <NPL>; <NPL>; and <NPL>).

The antibody can be an antigen binding fragment, derivative or analog of an antibody that immunospecifically binds to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibody bound to tumor cells or matrix. In this regard, "antigen binding" means that the fragment, derivative or analog is able to specifically bind to the target moiety. Specifically, the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (see, e.g., <NPL>;<NPL>).

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

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. (See, e.g., <CIT>; and <CIT>. ) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region derived from a human immunoglobulin molecule. (See, e.g., <CIT>. ) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. <CIT>; <CIT>; <CIT>; <CIT>; International Publication No. <CIT>; <CIT>; <CIT>; <NPL>; <NPL>;<NPL>;<NPL>; <NPL>; <NPL>; and <NPL>; <NPL>;<NPL>; <CIT>; <NPL>; <NPL>; and <NPL>.

Completely human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains variable region genes, but which can express human heavy and light chain variable region genes.

Antibodies can have modifications (e.g., substitutions, deletions and/or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies can have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. <CIT>). Antibodies also can have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the Fc gamma receptor III.

Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.

Antibodies for the treatment of cancer can be used. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.

Antibodies for the treatment of an autoimmune disease can also be used. Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.

Useful antibodies can bind to a receptor or a receptor complex. The receptor or receptor complex can comprise, for example, an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.

The antibody may be a humanized CD70 antibody (see, e.g., <CIT>),a humanized CD19 antibody (see, e.g., <CIT>), a chimeric or humanized CD30 antibody (see, e.g., <CIT>), a humanized CD33 antibody (<CIT>), a humanized Beta6 antibody (see, e.g., <CIT>), or a humanized Liv-<NUM> antibody (see, e.g., <CIT>).

Referring again generally to the Ligand-Linker-Drug conjugates of formulas I, I' and II, the number of Drug-Linker units per Ligand is represented by p. (In this context, the drug of the Drug-Linker can be a cytotoxic agent. ) Where the linkers are not branched, p represents the number of Drug-Linker molecules per Ligand (e.g., antibody). When referring to individual conjugates, p is an integer representing the number of Drug-Linker molecules per Ligand. When referring to a composition containing multiple conjugates, p represents 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 (e.g., antibody)). The variable p ranges from <NUM> to <NUM>, typically <NUM> to <NUM>, <NUM> to <NUM> or <NUM> to <NUM>, or up to <NUM>.

The average number of Drug-Linker units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, HIC and HPLC. The quantitative distribution of Ligand-Linker-Drug conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Ligand-Drug Conjugates, where p is a certain value from Ligand-Drug Conjugate with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

There are a number of different assays that can be used for determining whether a Ligand-Drug Conjugate comprising a Drug-Linker Compound according to the appended claims exerts a cytotoxic effect on a cell line. In one example for determining whether a Ligand-Drug Conjugate exerts a cytotoxic effect on a cell line, a thymidine incorporation assay is used. For example, cells at a density of <NUM>,<NUM> cells/well of a <NUM>-well plate are cultured for a <NUM>-hour period and exposed to <NUM>µCi of <NUM>H-thymidine during the final <NUM> hours of the <NUM>-hour period, and the incorporation of <NUM>H-thymidine into cells of the culture is measured in the presence and absence of Ligand-Drug Conjugate. The Ligand-Drug Conjugate has a cytotoxic effect on the cells if the cells of the culture have reduced <NUM>H-thymidine incorporation compared to same cells cultured under the same conditions but not contacted with the Ligand-Drug Conjugate. (See also <NPL>); <NPL>).

In another example, for determining whether a Ligand-Drug Conjugate comprising a Drug-Linker compound according to the appended claims exerts a cytotoxic effect on a cell line, cell viability is measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g.,<NPL>). In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytoxicity (<NPL>). Preferred Ligand-Drug Conjugates include those with an IC<NUM> value (defined as the mAb concentration that gives <NUM>% cell kill) of less than <NUM> ng/ml, preferably less than <NUM> ng/ml, more preferably less than <NUM> ng/ml, even most preferably less than <NUM> or even less than <NUM> ng/ml on the cell line.

Drug-Linker compounds are disclosed herein, having the formula:
<CHM>
or a pharmaceutically acceptable salt or solvate thereof,
wherein:.

Drug-Linker compounds having the following formula are also disclosed herein:
<CHM>
or a pharmaceutically acceptable salt or solvate thereof,
wherein:.

The components of the Drug-Linker Compounds of formulas IV and IV', e.g., DE, and LH, can be as defined above in relation to Ligand-Linker-Drug conjugates.

Provided herein is a Drug-Linker Compound according to the appended claims having the formula:
<CHM>
or a pharmaceutically acceptable salt or solvate thereof,
wherein:.

In some embodiments, R is the side chain of threonine.

AA<NUM> forms a cleavable bond with the Drug unit. In particular, AA<NUM> forms a cleavable peptide bond with the Drug unit. The cleavable peptide bond is susceptible to cleavage by proteases. AA<NUM> is a hydrophilic amino acid, typically a natural amino acid that is selected from the group consisting of Glycine and L forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine. In some embodiments, AA<NUM> is Glutamate.

In embodiments where AA<NUM> is present, it is a hydrophilic amino acid selected from the group consisting of Glycine; and L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine. In some further embodiments, AA<NUM> is selected from the group consisting of D amino acids of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine.

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

In certain embodiments, LH has the formula:
<CHM>.

The left and right wavy lines indicate attachments to the Drug unit and maleimide, respectively.

In some further embodiments, the Drug-Linkers have a formula selected from:
<CHM>
wherein DE is the Drug Unit.

A specific embodiment includes the following:
<CHM>
(IVa) ; or a pharmaceutically acceptable salt or solvate thereof.

Ligand-Linker-Drug conjugates comprising Drug-Linker compounds as defined in the appended claims may be used to treat disease. The disease can be, for example, a cancer or an autoimmune disease. Such Ligand-Linker-Drug conjugates may be administered in a therapeutically effective amount and on a therapeutically effective schedule.

Disclosed herein are pharmaceutical compositions comprising: Ligand-Linker-Drug Conjugates comprising a Drug-Linker Compound as defined in the appended claims; and a pharmaceutically acceptable carrier. The Ligand-Linker-Drug Conjugates can be in any form that allows for the compound to be administered to a patient for treatment of a disorder associated with expression of the antigen to which the Ligand unit specifically binds. For example, the conjugates can be in the form of a liquid or solid.

Disclosed herein are methods of preparing Ligand-Drug Conjugates, Linkers, Drug-Linker and Linker-Ligand Conjugates.

The methods may comprise the steps of providing a Drug-Linker or Linker unit as described herein, conjugating said Drug-Linker or Linker unit to a sulfhydryl group of a Ligand unit to form a conjugate. The thio-substituted maleimide or succinimide group(s) of conjugate may undergo a hydrolysis reaction.

The methods for preparing a Ligand-Drug Conjugate may comprise the steps of providing a Drug-Linker or Linker unit; conjugating said Drug-Linker or Linker unit to a sulfhydryl group of a Ligand to form a Ligand-Drug Conjugate conjugate comprising a non-hydrolyzed thio-substituted succinimide; allowing the non-hydrolyzed thio-substituted succinimide to undergo a hydrolysis reaction, wherein all, substantially all, or at least <NUM>%, <NUM>%, <NUM>%, <NUM>% or even <NUM>% of the succinimide is hydrolyzed from <NUM> minutes to <NUM> hours following conjugation. The hydrolysis reaction may occur under the same reaction conditions as the conjugation reaction. The conjugation conditions may be a pH of about <NUM> and a temperature of about <NUM>° C.

The Ligand-Drug conjugates disclosed herein can be assembled following the general scheme outlined in <FIG>.

Any examples falling outside the scope of the appended claims are provided for better understanding the invention.

Unless otherwise noted, materials were obtained from commercial suppliers in the highest purity grade available and used without further purifications. Anhydrous DMF and CH<NUM>Cl<NUM> were purchased from Aldrich. Fmoc-Dolaproine-OH was custom synthesized by Albany Molecular Research, Inc. (Albany, NY). Dolavaline-Val-Dil-OH was prepared as described elsewhere. Fmoc-Dpr(ivDde)-OH and <NUM>-Chlorotrityl chloride resin (<NUM>-<NUM> mesh, <NUM>% DVB, substitution <NUM> mmol/gram) were purchased from Novabiochem. Solid phase synthesis was performed in plastic syringes (National Scientific Company) fitted with a filter cut out of fritware PE medium grade porous sheet (Scienceware). A Burrell wrist action® shaker (Burrell Scientific, Pittsburg, PA) was used for agitation. All solid-phase yields reported are based upon the initial substitution level of the resin and constitute a mass balance of isolated pure material, unless otherwise stated.

Preparative HPLC purifications were performed on Varian instrument equipped with C12 Phenomenex Synergy MAX-RP 4µ reversed phase column, <NUM> × <NUM>, eluting with <NUM>% TFA in a water-acetonitrile gradient.

Mass spectra data were obtained on a XEVO TOF MS interfaced to a Waters <NUM> HPLC equipped with a C12 Phenomenex Synergi <NUM> × <NUM>, <NUM>, <NUM>Å reverse-phase column. The eluent consisted of a linear gradient of acetonitrile from <NUM>% to <NUM>% in <NUM>% aqueous formic acid over <NUM>, followed by isocratic <NUM>% acetonitrile for <NUM> at flow rate <NUM>/min.

The humanized h1F6 antibody specifically binds to the human CD70 antigen (<NPL>; <CIT>). The humanized hBU12 antibody specifically binds to the human CD19 antigen (<NPL>; <CIT>). Human renal cell carcinoma cell lines <NUM>-O and Caki-<NUM> expressing human CD70, and human transformed follicular lymphoma DOHH2 cells expressing human CD19 were purchased from the American Type Culture Collection (ATCC; Manassas, Virginia). All cell lines were grown according to the suppliers' recommendations and routinely checked for mycoplasma contamination.

Abbreviations: DPR means diaminopropionic acid; ivDde is <NUM>-(<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dioxocyclohex-<NUM>-ylidene)-<NUM>-methylbutyl-.

Nβ -Boc-L-<NUM>,<NUM>-diaminopropionic acid (<NUM> mmol) and maleic anhydride (<NUM>, <NUM> mmol) were dissolved in acetic acid (<NUM>) in a <NUM> round bottom flask, and the solution was stirred at room temperature for <NUM> hours. The solution was then concentrated to an oil under reduced pressure. The maleic acid intermediate was precipitated by adding ~<NUM> CH<NUM>Cl<NUM>/hexane, <NUM>/<NUM>, v/v, and the precipitate was collected by vacuum filtration. This material was then suspended in toluene (<NUM>), followed by the addition of DMA (<NUM>), and triethylamine (<NUM>, <NUM> mmol). The mixture was stirred at <NUM>-<NUM> under N<NUM> until all material was in solution. The flask was then equipped with a condenser and the solution heated to <NUM> and refluxed for <NUM> hours over molecular sieves. The reaction mixture was filtered through a sintered glass funnel and concentrated to near dryness under reduced pressure. The residue was dissolved in ethyl acetate (<NUM>), transferred to a separatory funnel and washed with <NUM>% citric acid in water (<NUM> × <NUM>) and brine (<NUM> × <NUM>). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and dried under high vacuum overnight yielding product as a white powder with <NUM>% yield. <NUM>H NMR (DMSO): δ <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>) <NUM> (dd, <NUM>). <NUM> (t, <NUM>), <NUM> (s, <NUM>). LCMS (ESI) calcd. for (M+Na)+ <NUM>; found, m/z <NUM>.

<FIG> illustrates an exemplary synthesis of auristatin-(AA<NUM>)-AA<NUM>-MDpr drug-linkers.

Resin loading. In a <NUM> solid phase reaction vessel (plastic syringe with PET frit) was added <NUM> of <NUM>-chlorotrityl chloride resin (<NUM> mmol based on the manufacturer's label), followed by a solution of Fmoc-Dpr(ivDde)-OH or Fmoc-Lys(ivDde)-OH (<NUM> mmol, <NUM> equiv), and DIEA (<NUM> mmol, <NUM> equiv) in <NUM> of dry CH<NUM>Cl<NUM>/DMF, <NUM>/<NUM>, v/v. The vessel was shaken for <NUM>, and then more DIEA (<NUM> mmol, <NUM> equiv) was added. The mixture was shaken for additional <NUM> hours at RT. Methanol (<NUM>) was added to quench unreacted sites. After <NUM>, resin was washed with DMF (<NUM> × <NUM>), CH<NUM>Cl<NUM> (<NUM> × <NUM>), ethyl ether (<NUM> × <NUM>), and dried in vacuo.

Loading was determined by treating the small amount of resin (<NUM>-<NUM>) with <NUM>% piperidine/DMF (<NUM>) for <NUM> hours in volumetric flask (<NUM> or <NUM>). Volume was adjusted with DMF; absorption at <NUM> was measured. Loading was calculated by the following equation: <MAT>.

Fmoc removal step. Resin containing Fmoc-protected peptide was treated with <NUM>% piperidine in DMF (<NUM> per gram of resin) for <NUM> at room temperature. Then the resin was washed with DMF (<NUM> × <NUM> per gram of resin), CH<NUM>Cl<NUM> (<NUM> × <NUM> per gram of resin), ethyl ether (<NUM> × <NUM> per gram of resin), and dried in vacuo.

Coupling step. To the resin (<NUM> equiv) containing deprotected N-terminus amino acid (AA), a solution of Fmoc-AA-OH (<NUM> equiv), HATU (<NUM> equiv), and DIEA (<NUM> equiv) in DMF (<NUM> per gram of resin) was added. The reaction vessel was agitated for <NUM>-<NUM>. Then the resin was washed with DMF (<NUM> × <NUM> per gram of resin), CH<NUM>Cl<NUM> (<NUM> × <NUM> per gram of resin), ethyl ether (<NUM> × <NUM> per gram of resin), and dried in vacuo. Reaction completion was confirmed by negative Kaiser test where appropriate.

Coupling of N-terminal Dolavaline-Val-Dil-OH was performed in a similar way.

ivDde Deprotection and coupling of MDpr(Boc)-OH. After coupling of Dolavaline-Val-Dil-OH tripeptide, the resin was treated with <NUM>% hydrazine/DMF (<NUM> per gram of resin) for <NUM> hours at RT. Then the resin was washed with DMF (<NUM> × <NUM> per gram of resin), CH<NUM>Cl<NUM> (<NUM> × <NUM> per gram of resin), ethyl ether (<NUM> × <NUM> per gram of resin), and dried in vacuo. A solution of Fmoc-MDpr(Boc)-OH (<NUM> equiv), HATU (<NUM> equiv), and DIEA (<NUM> equiv) in DMF (<NUM> per gram of resin) was added to the resin and the mixture was shaken for <NUM> hours at room temperature (RT). Reaction completion was confirmed by negative Kaiser test. The resin was washed with DMF (<NUM> × <NUM> per gram of resin), CH<NUM>Cl<NUM> (<NUM> × <NUM> per gram of resin), ethyl ether (<NUM> × <NUM> per gram of resin), and dried in vacuo.

Cleavage off the resin and deprotection. Peptide-containing resin was treated with <NUM>% TFA/ CH<NUM>Cl<NUM> (<NUM> per gram of resin) for <NUM> at room temperature, and solution was collected in a round bottom flask. The resin was washed with <NUM>% TFA/CH<NUM>Cl<NUM> (<NUM> × <NUM> per gram of resin). Pooled solutions were left at RT for <NUM> hours. After deprotection, completion was confirmed by LC-MS. Volatiles were removed under reduced pressure on Rotavap, and the final product was purified by reverse phase preparative HPLC. All drug-linkers were obtained with ><NUM>% purity by reverse phase HPLC at <NUM>.

Drug-linkers with MA maleimide were prepared in a similar way as described above using α-maleimidoacetic acid-NHS (Molecular Biosciences, Boulder CO) instead of MDpr(Boc)-OH.

Drug-linkers with an ethylene diamine (EDA) stretcher were prepared by the procedure similar to the one reported earlier (<NPL>).

Drug linkers were synthesized as described above. The general formula was as follows:
<CHM>
(In this formula, note the designation of AA<NUM> and AA<NUM> is reversed. R corresponds to R<NUM>.

The following Table <NUM> summarizes the syntheses and characterizations of the various drug linkers. In the table, the first column (left) refers to the Compound Number. The second column (left) refers to amino acid at the C-terminus of the auristatin. The third, fourth and fifth columns refer to the components of the linker. In column <NUM>, the amino acid components of the linker are identified. In column <NUM>, additional amino acid and/or non-amino acid components of the linker are identified. In column <NUM>, the composition of the maleimide moiety of the linker is identified. The sixth column refers to the yield of the drug-linker. The seventh and eighth columns refer to the calculated and observed masses of the drug-linkers, as determined by mass spectroscopy. The last column (at right) refers to the HIC retention time of ADCs containing the drug linkers as <NUM> loads (generally determined as described in Example <NUM>).

Ala refers to L-alanine; Asn refers to asparagine; Asp refers to L-aspartate; Gln refers to L-glutamine; Glu refers to L-glutamate; 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;.

h1F6 antibody-drug conjugates (ADCs) with eight drugs per antibody were prepared by full reduction of the antibody followed by reaction with the desired drug-linker. The antibody (<NUM>/mL) was fully reduced by addition of <NUM> molar equivalents of tris(<NUM>-carboxyethyl)phosphine (TCEP) in phosphate buffered saline (PBS) pH <NUM> (Invitrogen, Carlsbad, CA) with <NUM> diethylenetriaminepentaacetic acid (DTPA), followed by incubation at <NUM> for ~<NUM>. Excess TCEP was removed by 10X dilution with PBS and concentration of the antibody, repeated <NUM> times using a <NUM> KD MWCO spin filter (EMD Millipore, Billerica, MA). Full reduction of the antibody was confirmed by reversed phase HPLC analysis where the light and heavy chains are completely resolved from unreduced antibody. The drug-linker (<NUM> equivalents) was then added from a stock solution prepared in DMSO (<NUM>). The reaction was allowed to stand at room temperature for approximately <NUM> hours to allow for conjugation and subsequent thiosuccinimide ring hydrolysis (MDpr). The reaction mixture was purified and buffer-exchanged into PBS using PD-<NUM> desalting columns (GE Healthcare, Piscataway, NJ). The drug/Ab ratio of the final product was estimated by PLRP-MS analysis and ranged from <NUM> - <NUM> drugs/Ab. In addition, each ADC was analyzed by size exclusion chromatography where HMW species ranged from <NUM> - <NUM>%.

Analysis of the ADCs was performed using Hydrophobic Interaction Chromatography (HIC). HIC is performed by running a linear gradient from <NUM>-<NUM>% Mobile Phase B (MPB) where Mobile Phase A (MPA) consists of <NUM> ammonium sulfate, <NUM> potassium phosphate, pH <NUM>, and MPB consists of <NUM>% <NUM> potassium phosphate, pH <NUM>, <NUM>% isopropanol. Separation was achieved using a Tosoh t-Butyl column (TSK-Gel Butyl-NPR <NUM> × <NUM>, PN: <NUM>) heated to <NUM>. Test articles were prepared by diluting <NUM>µg of ADC into MPA such that the total salt concentration is greater than or equal to <NUM> ammonium sulfate at a total volume of <NUM>µL. Samples were injected at <NUM>µL and eluted using a <NUM> minute gradient. Monitoring at λ=<NUM>. ADCs with greater hydrophobicity, or a greater number of drugs per molecule, elute at later retention times.

<FIG> and <FIG> show the results of HIC analyses of various <NUM> loaded ADCs, as compared to the parent, unconjugated antibody (h1F6). The ADCs were prepared as described above. The results generally show that increasing the hydrophilicity of the auristatin, in combination with a hydrophilic linker, decreases the apparent hydrophobicity of the conjugate.

The following Table <NUM> summarizes the compositions of various drug linkers of Table <NUM> and analyses of the resulting <NUM>-loaded antibody drug conjugates with antibody h1F6. The HIC retention time (HIC RT) was determined as described above. h1F6 ADCs containing the drug linkers MC-vc-PABC-MMAE, MC-vc-PABC-MMAF and MC-MMAF were used as controls.

In vitro cytotoxicity assays were performed generally as described previously (see supra, Activity Assays). Briefly, log phase cultures of cells were collected and cells plated at seeding densities ranging from <NUM> - <NUM>,<NUM> cells/well according to pre-determined conditions. After incubating <NUM> hours to allow surface protein reconstitution, serial dilutions of test conjugates were added and cultures incubated further for <NUM> days. Assessment of cellular growth and dye reduction to generate IC<NUM> values was done using Alamar Blue (Biosource International, Camarillo, CA) dye reduction assay. Briefly, a <NUM>% solution (wt/vol) of Alamar Blue was freshly prepared in complete media just before cultures were added. Ninety-two hours after drug exposure, Alamar Blue solution was added to cells to constitute <NUM>% culture volume. Cells were incubated for <NUM>, and dye reduction was measured on a Fusion HT fluorescent plate reader (Packard Instruments, Meriden, CT).

<NUM>-<NUM> renal and Caki-<NUM> clear cell renal cancer cell lines were used. These cell lines expressed approximately <NUM>,<NUM> and <NUM>,<NUM> human CD70 molecules per cell, respectively. The drug linkers attached to the h1F6 antibody are described in Tables <NUM> and <NUM>. Referring to the following Tables 3A-C, the h1F6 ADCs have an average drug loading of <NUM> drugs/antibody, unless otherwise indicated. The hydrophilic h1F6 ADCs tested showed activity (IC<NUM> values) comparable to, or better than, the control, h1F6-mcMMAF (<NUM>), in these studies.

Pharmocokinetic (PK) experiments were performed using radiolabeled antibody or ADC. PK test articles were radiolabeled using the following procedure. To a solution of antibody or ADC in <NUM> potassium phosphate (pH <NUM>) and <NUM> sodium chloride was added <NUM>µCi N-succinimidyl propionate, [propionate-<NUM>,<NUM>-<NUM>]- (Moravek Biochemicals, Cat. No.: MT <NUM>, <NUM> Ci/mmol, <NUM> mCi/mL, <NUM>:<NUM> hexane:ethyl acetate solution) per mg of antibody or ADC. The resulting mixture was vortexed and left at room temperature for <NUM> hours. The mixture was centrifuged at <NUM>,<NUM> x g for <NUM> minutes and the lower aqueous layer was removed and split into Amicon Ultra-<NUM> Centrifugal Filter Units (Millipore, Cat. No.: UFC903024, <NUM> kDa MWCO). Unconjugated radioactivity was removed by <NUM> rounds of dilution and centrifugation at <NUM>,<NUM> x g. The resulting products were filtered through sterile <NUM> Ultrafree-MC Centrifugal Filter Units (Millipore, Cat. No.: UFC30GV0S) and the final antibody or ADC concentration was measured spectrophotometrically. The specific activity (µCi/mg) of each product was determined by liquid scintillation counting.

The pharmacokinetic properties of an unconjugated antibody and various ADCs of the that antibody (drug loading of <NUM>) were examined in several rodent models. In each experiment, <NUM> of radiolabeled antibody or ADC per kg of animal weight were injected via the tail vein. Each test article was dosed once in <NUM> replicate animals. Blood was drawn into K2EDTA tubes via the saphenous vein or by cardiac puncture for terminal bleeds at various time points. Plasma was isolated by centrifugation for <NUM> minutes at <NUM>,<NUM> x g. A <NUM>µL of sample of plasma from each time point was added to <NUM> Ecoscint-A liquid scintillation cocktail (National Diagnostics) and the total radioactivity was measured by liquid scintillation counting. The resulting disintegrations per minute values were converted to µCi and the specific activity of the radiolabeled test articles was used to calculate the concentration of antibody or ADC remaining in the plasma at each time point.

Referring to <FIG>, pharmacokinetics properties of h1F6 and two hydrophilic ADCs thereof were compared to the properties three control ADCs. The hydrophilic ADCs were h1F6-<NUM> (<NUM>-loaded (auristatin-T)-Glu-Dpr-MA)<NUM> - h1F6) and h1F6-<NUM> ((auristatin F)-Ile-EDA-MDpr)<NUM> - h1F6). The results show that the hydrophilic ADCs exhibited improved pharmacokinetic stability over the course of this mouse study. The hydrophilic auristatin with an auristatin-T exhibited stability close to that of the unconjugated antibody. The hydrophilic design of ADC h1F6-<NUM> exhibited improved pK stability compared to the controls, two of which include a monomethyl form of the same auristatin (auristatin F vs monomethyl auristatin F).

Referring to <FIG>, pharmacokinetics properties of hydrophilic conjugates of another monoclonal antibody were compared to the properties of a control conjugate, mAb-mcMMAF. All of the ADCs had an average drug loading of <NUM>. Each of the hydrophilic ADCs exhibited improved pharmacokinetic stability, as compared to the control ADC.

Two hydrophilic ADCs thereof were compared to the properties three control ADCs. The hydrophilic ADCs were h1F6-<NUM> (<NUM>-loaded (auristatin-thiazole)-Glu-Lys-MDpr)<NUM> - h1F6) and h1F6-<NUM> ((auristatin F)-Ile-EDA-MDpr)<NUM> - h1F6). The results show that the hydrophilic ADCs exhibited improved pharmacokinetic stability over the course of this mouse study. In particular, the hydrophilic design of h1F6-<NUM> exhibited improved stability compared to the controls, two of which include a monomethyl form of the same auristatin (auristatin F vs monomethyl auristatin F).

<NUM>-O cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and propagated in culture conditions recommended by ATCC. To establish <NUM>-O tumors, <NUM> × <NUM><NUM> cells were implanted into the right flank of athymic nu/nu female donor mice (Harlan, Indianapolis, IN). When donor tumors were approximately <NUM><NUM>, mice were euthanized and tumors were aseptically excised and ~<NUM> × <NUM> fragments were loaded into a sterilized <NUM>-gauge trocar for implantation into nu/nu mice. When tumors reached ~<NUM><NUM>, mice were randomly allocated to treatment groups.

To establish DOHH2 tumors, <NUM> × <NUM><NUM> cells were implanted into the right flank of C. -<NUM> SCID mice (Harlan, Indianapolis, IN). When tumors were approximately ~<NUM><NUM>, mice were randomly allocated to treatment groups.

Experimental groups were treated via intraperitoneal injection with compounds at the dose and schedule indicated or alternatively left untreated. Tumors were measured periodically and volumes were calculated using the formula V= ((L × W<NUM>)/<NUM>). Animals were euthanized when tumors reached the volume of <NUM><NUM> or at the end of the study, whichever came first.

Tumor quadrupling times were chosen as time to endpoint (TTE), which was determined by using the non-liner regression analysis for exponential growth of each individual tumor growth data sets from each experimental animal. The median tumor quadrupling time was calculated based on the tumor volume at the beginning 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 conducted using Prism (GraphPad) software for Windows. The Logrank test of the TTE was used to analyze the significance of the differences between the two groups, with differences deemed significant at <NUM>≤ P ≤ <NUM>, and highly significant at P ≤ <NUM>.

Referring to <FIG>, the activity of <NUM>-loaded and <NUM>- loaded ADCs (4d/Ab and 8d/Ab, respectively) were tested in single dose, mouse xenograft studies. First, referring to the control, h1F6-mc-vc-PABC-MMAF, the <NUM>-loaded ADC gave better activity than the <NUM>-loaded ADC. In contrast, <NUM>-loaded ADCs of hydrophilic h1F6-<NUM> (auristatin T-Glu-Dpr-MDPr) and h1F6-<NUM> (auristatin thiazole-Glu-EDA-MDPr) both exhibited greater activity than the <NUM>-loaded counterparts.

Referring to <FIG>, the activity of different <NUM>-loaded and <NUM>- loaded ADCs were tested in single dose, mouse xenograft studies. Again in this model, the <NUM>-loaded hydrophilic ADC of hBU12-<NUM> (auristatin T-Glu-Dpr-MDPr) exhibited greater activity than its <NUM>-loaded counterpart.

Referring to <FIG>, the activity of various <NUM>-loaded and <NUM>- loaded ADCs were tested in single dose, mouse xenograft studies. In this model, <NUM>-loaded ADCs of h1F6-<NUM> (auristatin T-Ile-EDA-MDPr) and h1F6-<NUM> (auristatin F-Glu-Dpr-MDPr) both exhibited greater activity than the <NUM>-loaded counterparts. <NUM>-loaded ADC h1F6-<NUM> (auristatin F-Ile-EDA-MDPr) exhibited the opposite trend.

Claim 1:
A Drug-Linker Compound having the formula:
<CHM>
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R is the side chain of a hydrophilic amino acid that is selected from the group consisting of threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furyl alanine, threonine(PO<NUM>H<NUM>), pyrazolyl alanine, triazolyl alanine, and thiazolyl alanine;
AA<NUM> is a hydrophilic amino acid selected from the group consisting of Glycine and L forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine, and Alanine;
AA<NUM> is a hydrophilic amino acid selected from the group consisting of Glycine and L or D forms of Aspartate, Glutamate, Asparagine, Glutamine, Histidine, Lysine, Arginine, Serine and Alanine, or is absent;
X is H and subscript n is <NUM>, X is COOH and subscript n is <NUM>, or X is COOH and subscript n is <NUM>; and
Y is H or CH<NUM>NH<NUM>, provided that: when X is H, Y is CH<NUM>NH<NUM>.