Methods for formulating antibody-drug conjugate compositions

Abstract

To provide methods for formulating antibody drug conjugate compositions.SOLUTION: The present invention provides improved methods for formulating therapeutic compositions comprising an antibody drug conjugate ("ADC"), which reduce potency variability between batches of the ADC and provide administration of such therapeutic compositions within a narrow intended range. The present invention provides a novel method for formulating a therapeutic composition comprising an antibody drug conjugate ("ADC") based on the drug concentration, thereby narrowing the variability in potency between batches of the ADC, and minimizing toxicity and increasing the efficacy of a drug formulated according to the method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 044,592, filed September 2, 2014, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Antibodies that specifically bind to tumor surface antigens are used to deliver cytotoxic drugs in the form of antibody-drug conjugates (ADCs). Cytotoxic drugs are typically conjugated to antibodies at cysteine ​​or lysine residues. The number of drug molecules conjugated per antibody, also known as the drug-to-antibody ratio ("DAR"), is typically a distribution of species ranging from 0 to 8. The DAR of a manufactured batch of ADC is empirically determined using spectrophotometric measurements, and ADC therapeutic compositions typically contain a mixture of ADC species with different drug loads. Therefore, the DAR of an ADC batch represents the average DAR of the ADC species within the batch.

[0003] Both ADC and antibody cancer therapeutics are formulated based on trace antibody protein concentrations and must adhere to specifications. While formulation labels provide "trace" or target protein concentration information, the drug concentration in the vial can vary relative to the target antibody concentration due to acceptable variations in DAR while still adhering to acceptable standards. ADC potency is generally linear with respect to concentration. Unlike antibodies, ADCs have the added potential for variable potency due to DAR. Typical antibody concentration specifications and DARs allow for some batch-to-batch variation in the concentration of cytotoxic drugs in ADC product vials when patient dosing is based on trace antibody concentrations.

[0004] It is important that patients receive safe and effective ADC doses. Improved methods of formulating ADC compositions advantageously reduce batch-to-batch variability in potency, efficacy, and / or toxicity, ensuring that patients receive the ADC within the intended therapeutic range. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides a novel method for formulating therapeutic compositions comprising antibody-drug conjugates ("ADCs") based on drug concentration, thereby narrowing potency variability between batches of ADCs, minimizing toxicity, and increasing efficacy of drugs formulated according to this method.

[0006] The present invention is based, at least in part, on the discovery that the efficacy and toxicity of some ADCs are driven entirely or predominantly by the drug dose, rather than the antibody dose. Traditional methods for formulating antibody-based therapeutics, such as pharmaceutical compositions containing ADCs, rely on patient dosing based on antibody concentration. While this can be advantageous for compositions containing only antibodies, formulating an ADC using antibody concentration can cause the drug concentration to fluctuate and fall outside the desired range. ADC efficacy is generally linear with respect to the concentration of the bound drug, which is affected by both the antibody concentration and the DAR, as shown by the formula [drug] = DAR × [antibody]. The DAR, antibody, and drug each vary within a given acceptable range, as defined by a given ADC specification. However, because the drug and antibody are conjugated, variability in one component will affect the other components. For example, a ±10-20% variability in antibody concentration (within acceptable limits by industry standards) can lead to a ±10-20% variation in drug concentration, resulting in a ±20-40% variability in the potency of the formulation in the vial. A ±15% variation in DAR can result in a 15% higher or lower drug concentration, further varying potency. This effect may be particularly relevant for certain subsets of ADCs, where drug concentration has been demonstrated to be a major driver of toxicity and efficacy.

[0007] ADCs are linked to cytotoxic agents, also known as "drug" molecules, and the number of drug molecules conjugated per antibody molecule is represented by the term "drug-to-antibody" ratio ("DAR"). The DAR of a manufactured batch of ADC is empirically determined using spectrophotometric measurements by obtaining the ratio of the drug concentration to the antibody concentration. The DAR of a particular batch of ADC represents the average number of drugs conjugated to each antibody molecule within that batch. Typical DAR specifications for clinical development are in the range of ±10-15%. Traditionally, the first step in ADC formulation is to determine the molar concentrations of both the drug and antibody and calculate the DAR. The ADC is then formulated to a target antibody concentration, allowing the drug concentration to vary according to the manufactured DAR value as follows: [drug] = DAR × [antibody].

[0008] In contrast, the present invention is based on the discovery that variability in efficacy and toxicity can be minimized by formulating ADC compositions based on a target drug concentration defined by a fixed antibody concentration and a fixed DAR. Therefore, in cases where the potency, efficacy, and / or toxicity of an ADC have been demonstrated to be primarily driven by the amount of drug administered, an improved method for reducing variability in the concentration of a cytotoxic drug would be beneficial. Accordingly, the formulation method described below involves determining a target drug concentration at a fixed antibody concentration and a fixed DAR, and formulating the antibody-drug conjugate composition to achieve the target drug concentration. Such an improved formulation method ensures that the drug is administered to patients within the intended narrow range without the added risk of batch failure.

[0009] In one aspect, the present invention generally provides a method for reducing potency variability in an antibody-drug conjugate composition (e.g., by at least about 5%, 10%, 20%, or more). The method includes determining a target drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio; and formulating the antibody-drug conjugate composition to achieve the target drug concentration, thereby reducing potency variability in the composition. In one embodiment, the variability in drug concentration is about ±10%. In various embodiments, the variability is less than about ±5, 6, 7, 8, or 9%. In one embodiment, the method reduces batch-to-batch potency variation (e.g., by at least about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more). It should be noted that when variability is expressed as ±, it is intended to describe variation that is a particular percentage above or below a particular value. When variability is expressed as a single total value (e.g., at least 10%), it is intended to represent the difference between the maximum and minimum possible values. In another embodiment, the composition is a final formulation. In yet another embodiment, the drug concentration varies within the antibody specification concentration. In one embodiment, the antibody concentration is equal to the target antibody concentration ± less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. In another embodiment, the antibody concentration is equal to the target antibody concentration ± less than about 10%, 12%, 15%, or 20%.

[0010] In another different aspect, the present invention provides a method for reducing efficacy variability in a composition comprising an antibody-drug conjugate. The method comprises reducing efficacy variability in the composition by formulating the antibody-drug conjugate by targeting a variable drug concentration that falls in the midpoint of the overlap range between the specification range of antibody concentration and the specification range of drug concentration. In this case, the present invention provides a method for reducing efficacy variability in a composition containing an antibody-drug conjugate. The method comprises: (a) measuring the DAR of an antibody-drug conjugate composition; (b) determining the upper antibody specification limit and the lower antibody specification limit, where the upper antibody specification limit is the target antibody concentration plus the maximum variation allowed by the specification, and the lower antibody specification limit is the target antibody concentration minus the maximum variation allowed by the specification; (c) determining an upper limit of a defined drug specification and a lower limit of a defined drug specification, where the upper limit of the defined drug specification is the maximum variation allowed by the target drug concentration plus the specification, and the lower limit of the defined drug specification is the maximum variation allowed by the target drug concentration minus the specification; (d) Determine the drug specification upper limit (USL(drug)), calculated as follows: USL (drug) μg / mL = upper limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (e) Determine the lower specification limit (LSL(drug)) of the drug, calculated as follows: LSL (drug) μg / mL = Lower limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (f) comparing the USL(drug) calculated in step (d) with the upper limit of the drug specification defined in step (c) and selecting the lower of the two values ​​as the effective upper limit of the drug specification; (g) comparing the LSL(drug) calculated in step (e) with the lower limit of the drug specification defined in step (c) and selecting the higher of the two values ​​as the effective lower limit of the drug specification; and (h) Reducing potency variability in the antibody-drug conjugate composition by formulating the composition to a target drug concentration that is midway between the effective upper limit of the drug specification and the effective lower limit of the drug specification. In one embodiment, the method narrows the range between the upper and lower drug specification limits to about ±3-9%. In another embodiment, the method narrows the range between the upper and lower drug specification limits to about ±4%. In one embodiment, the maximum variation allowed by the specifications in step (b) is about ±15%. In another embodiment, the maximum variation allowed by the specifications in step (b) is less than about ±10, 11, 12, 13, or 14%. In one embodiment, the maximum variation allowed by the specifications in step (c) is about ±15%. In another embodiment, the maximum variation allowed by the specifications in step (c) is less than about ±10, 11, 12, 13, or 14%. In various embodiments, the antibody is a non-functional antibody. In various embodiments, the DAR is the lower DAR specification limit or the upper DAR specification limit. In various embodiments, the lower limit of the DAR specification is 2.3, 2.4, or 2.5. In various embodiments, the upper limit of the DAR specification is 2.9, 3.0, or 3.1.

[0011] In a related embodiment, for example, when the antibody is a functional antibody, the invention also provides a method of formulating an antibody drug conjugate that targets an antibody concentration that minimizes fluctuations in drug concentration when formulating an ADC by targeting a variable drug concentration that falls midway between the overlapping ranges of the antibody concentration specification range and the drug concentration specification range, thereby allowing for tighter control of the antibody concentration in the ADC formulation.

[0012] In another aspect, the present invention provides a method for formulating an antibody-drug conjugate composition, the method comprising: determining a target drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio; and formulating the antibody-drug conjugate composition to achieve the target drug concentration.

[0013] In yet another aspect, the present invention provides a method for reducing potency variability in a composition comprising an antibody-maytansinoid conjugate, the method comprising determining a target maytansinoid concentration at a fixed antibody concentration and a fixed maytansinoid-to-antibody ratio, and formulating the antibody-maytansinoid conjugate composition to achieve the target maytansinoid concentration, thereby reducing potency variability in the composition.

[0014] In yet another aspect, the invention provides a method for formulating an antibody-maytansinoid conjugate composition, the method comprising determining a target maytansinoid concentration at a fixed antibody concentration and a fixed maytansinoid-to-antibody ratio; and formulating the antibody-maytansinoid conjugate composition to achieve the target maytansinoid concentration.

[0015] In yet another aspect, the present invention provides a method for formulating an antibody-benzodiazepine (e.g., pyrrolobenzodiazepine or indolinobenzodiazepine) conjugate composition, the method comprising determining a target benzodiazepine concentration at a fixed antibody concentration and a fixed benzodiazepine-to-antibody ratio; and formulating the antibody-benzodiazepine conjugate composition to achieve the target benzodiazepine concentration.

[0016] In another aspect, the present invention provides a method for reducing efficacy variability in a composition containing an antibody-benzodiazepine (e.g., a pyrrolobenzodiazepine or indolinobenzodiazepine) conjugate. The method comprises: (a) measuring the DAR of an antibody-benzodiazepine (e.g., pyrrolobenzodiazepine or indolinobenzodiazepine) conjugate composition; (b) determining the upper antibody specification limit and the lower antibody specification limit, where the upper antibody specification limit is the target antibody concentration plus the maximum variation allowed by the specification, and the lower antibody specification limit is the target antibody concentration minus the maximum variation allowed by the specification; (c) determining an upper limit of a defined benzodiazepine specification and a lower limit of a defined benzodiazepine specification, wherein the upper limit of the defined benzodiazepine specification is the maximum variation allowed by the target benzodiazepine concentration plus the specification, and the lower limit of the defined benzodiazepine specification is the maximum variation allowed by the target benzodiazepine concentration minus the specification; (d) Determine the benzodiazepine specification upper limit (USL(drug)) calculated as follows: USL (drug) μg / mL = upper limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (e) Determine the benzodiazepine lower specification limit (LSL(drug)), calculated as follows: LSL (drug) μg / mL = Lower limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (f) comparing the USL(drug) calculated in step (d) with the upper limit of the benzodiazepine specification defined in step (c) and selecting the lower of the two values ​​as the effective upper limit of the benzodiazepine specification; (g) comparing the LSL(drug) calculated in step (e) with the lower limit of the benzodiazepine specification defined in step (c) and selecting the higher of the two values ​​as the effective lower limit of the benzodiazepine specification; and (h) Reducing potency variability in the antibody-benzodiazepine conjugate composition by formulating the composition to a target benzodiazepine concentration that is midpoint between the effective upper limit of benzodiazepine specification and the effective lower limit of benzodiazepine specification.

[0017] In yet another aspect, the present invention provides a method of administering to a subject within a narrow intended range, the method comprising providing an antibody-drug conjugate composition formulated according to the method of any of the above aspects, and administering said composition to a subject.

[0018] In yet another aspect, the invention provides a pharmaceutical composition containing an antibody-drug conjugate formulated according to the method of the above aspect, wherein the trace drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration is provided on the label.

[0019] In various embodiments of any of the above aspects or any other aspects of the invention described herein, the drug is a cytotoxic agent. Cytotoxic agents include, but are not limited to, tubulin inhibitors, DNA damaging agents, DNA cross-linking agents, DNA alkylating agents, and cell cycle or mitosis disruptors. Non-limiting examples of cytotoxic agents include maytansinoids; benzodiazepine compounds, such as pyrrolobenzodiazepines and indolinobenzodiazepines; and auristatins. In certain embodiments of the above aspects, the method reduces batch-to-batch potency variability (e.g., by at least about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more). In other embodiments, the composition is a final formulation. In yet other embodiments, the antibody concentration varies within the antibody specification concentration. In yet other embodiments of the above aspects, the method reduces batch-to-batch potency variability in manufacturing an antibody-maytansinoid conjugate. In various embodiments of the above aspects, the composition is allowed to vary in potency by about 10-40% (e.g., 10, 15, 20, 25, 30, 35, 40%). In other embodiments of the above aspects, the composition is allowed to vary in potency by about 10-20% (e.g., 10, 12, 15, 18, 20%). In still other embodiments of the above aspects, the antibody concentration specification is equal to the target ± less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. In other embodiments, the antibody concentration specification is equal to the target ± less than about 10%, 12%, 15%, or 20%. In various embodiments of the above aspects, the potency variability of the composition is reduced compared to when the antibody-drug conjugate composition is formulated based on antibody concentration. In various embodiments of the above aspects, the antibody concentration and conjugated drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration are determined by spectrophotometry. In various embodiments of the above aspects, the drug-to-antibody ratio is determined by size exclusion chromatography (SEC) or SEC-mass spectrometry (SEC-MS). In other embodiments of the above aspects, the efficacy or toxicity of the composition is independent of the drug-to-antibody ratio or antibody concentration, but depends on the total administered dose of the conjugated drug (e.g., maytansinoid, benzodiazepine compound, auristatin).In still other embodiments of the above aspects, the efficacy of the composition is independent or largely independent of the drug-to-antibody ratio. In various embodiments of the above aspects, the toxicity of the composition is independent or largely independent of the drug-to-antibody ratio. In various embodiments of the above aspects, the efficacy or toxicity depends or largely depends on the conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration. In various embodiments of the above aspects, the efficacy depends, substantially depends, or at least partially depends on the conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration. In various embodiments of the above aspects, the toxicity depends, substantially depends, or at least partially depends on the conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration. In various embodiments of the above aspects, the efficacy is independent, substantially independent, or at least partially independent of the antibody concentration. In various embodiments of the above aspects, toxicity is independent, substantially independent, or at least partially independent of antibody concentration. In various embodiments of the above aspects, efficacy and toxicity depend, substantially depend, or at least partially depend on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration and antibody concentration. In various embodiments of the above aspects, efficacy and toxicity depend less on antibody concentration than on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration. In various embodiments of the above aspects, efficacy depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration and antibody concentration. In various embodiments of the above aspects, toxicity depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration and antibody concentration.In various embodiments of the above aspects, efficacy depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration and antibody concentration, and toxicity depends, substantially depends, or at least partially depends on conjugate drug concentration. In various embodiments of the above aspects, efficacy depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration and antibody concentration, and toxicity depends, substantially depends, or at least partially depends on antibody concentration. In various embodiments of the above aspects, toxicity depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration and antibody concentration, and efficacy depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration. In various embodiments of the above aspects, toxicity depends, substantially depends, or at least partially depends on antibody concentration, and efficacy depends, substantially depends, or at least partially depends on conjugate drug (e.g., maytansinoid, benzodiazepine compound, auristatin) concentration. In various embodiments of the above aspects, the antibody-drug conjugate composition is formulated for injection. In various embodiments of the above aspects, the antibody-drug conjugate is formulated with a pharmaceutically acceptable parenteral vehicle. In various embodiments of the above aspects, the antibody-drug conjugate is formulated in a unit-dose injectable form.

[0020] In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method includes determining an upper specification limit (USL) and a lower specification limit (LSL). In certain embodiments, the calculated USL and LSL are determined using the following formulas: USL (drug) μg / mL = upper limit of antibody concentration specification × PAR × drug molecular weight × 1000 / antibody molecular weight LSL (drug) μg / mL = lower limit of antibody concentration specification × PAR × molecular weight of drug × 1000 / molecular weight of antibody.

[0021] In other embodiments of any of the above aspects, the cytotoxic compound or drug is a tubulin inhibitor, a DNA damaging agent, a DNA cross-linking agent, a DNA alkylating agent, or a cell cycle or mitosis disruptor.

[0022] In still other embodiments of any of the above aspects, the drug includes, but is not limited to, maytansinoids and maytansinoid analogs, benzodiazepine compounds (e.g., pyrrolobenzodiazepines and indolinobenzodiazepines; see also Table 1: compounds D1-D10 and DGN462), taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes such as calicheamicin, dolastatins and dolastatin analogs such as auristatins, tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino-doxorubicin.

[0023] In various embodiments of the above aspects, the maytansinoid is DM1, DM3, or DM4. In various embodiments of the above aspects, the benzodiazepine compound is selected from representative cytotoxic agents D1-D10 and DGN462, listed in Table 1 below.

[0024] [Table 1-1] [Table 1-2] It should be noted that other variations of the compounds listed in Table 1 (eg, sulfonated versions) are contemplated and would be readily apparent to one of ordinary skill in the art.

[0025] In various embodiments of the above aspects, the antibody may be a functional antibody or a non-functional antibody. Non-functional antibodies include, for example, huDS6 and antibodies that have only effector-mediated cell killing, such as huMovl9(M9346A), huAnti-CD123, huMy9-6(Z4681A), and huB4. Functional antibodies include, for example, huEGFR-7R and huCD37-3. In certain embodiments, the drug is a benzodiazepine compound and the antibody is a non-functional antibody. In certain embodiments, the drug is a maytansinoid and the antibody is a non-functional antibody.

[0026] In various embodiments of the above aspects, the linker is a cleavable linker such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), or N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP). In various embodiments of the above aspects, the linker is a non-cleavable linker such as 2-iminothiolane, acetylsuccinic anhydride, or succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC). The common linkers 2-iminothiolane and acetylsuccinic anhydride can be used as cleavable or non-cleavable linkers.

[0027] In various embodiments of the above aspects, the linker-antibody-drug conjugate is selected from the group consisting of huMovl9-sulfo-SPDB-DM4, huMovl9-sulfo-SPDB-Dl, huMovl9-D2, huMovl9-sulfo-SPDB-D10, huMovl9-sulfo-SPDB-DGN462, huMy9-6-sulfo-SPDB-Dl, huMy9-6-D2, huMy9-6-sulfo-SPDB-D10, huMy9-6-sulfo-SPDB B-DGN462, huAnti-CD123-sulfo-SPDB-Dl, huAnti-CD123-D2, huAnti-CD123-sulfo-SPDB-D10, huAnti-CD123-sulfo-SPDB-DGN462, huB4-SPDB-DM4, huDS6-SPDB-DM4, huCD37-3-SMCC-DMl, huCD37-50-SMCC-DMl, or huEGFR-7R-SMCC-DMl.

[0028] Other features and advantages of the invention will become apparent from the detailed description and claims.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology, vol. 1, pp. 111-114, 1997; ... Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings defined in these sources unless otherwise specified.

[0030] The term "adjusted ideal body weight (AIBW)" refers to a size descriptor that accounts for gender, total body weight, and height. AIBW can be calculated, for example, using the formula AIBW=IBW+0.4(kg body weight-IBW), where: Ideal weight (IBW) 1.IBW 1 (male)=0.9H 1 -88 2.IBW 1 (female)=0.9H 1 -92. ( 1 H=cm height; W=kg weight) IBW, LBW, and ADJ are Green and Duffull, British This is described in more detail in Journal of Clinical Pharmacology 58:119-133 (2004), the entire contents of which are incorporated herein by reference.

[0031] "Cytotoxic agent" refers to a small chemical compound, peptide, or nucleic acid molecule that is toxic to cells. In some embodiments described herein, for ease of reference, the term "drug" is used to refer to a cytotoxic agent. For example, in antibody-drug conjugates (ADCs), the term "drug" is used interchangeably with the term "cytotoxic agent." In certain embodiments, the cytotoxic agent (or "drug") is conjugated to an antibody. In one particular embodiment, the cytotoxic agent is a maytansinoid, such as DM1, DM3, or DM4. In other embodiments, cytotoxic agents include, but are not limited to, benzodiazepine compounds (e.g., pyrrolobenzodiazepines and indolinobenzodiazepines; see also Table 1: compounds D1-D10 and DGN462), taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes such as calicheamicin, dolastatins and dolastatin analogs such as auristatins, tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino-doxorubicin.

[0032] "Drug-to-antibody ratio (DAR)" means the average number of "drug" (i.e., cytotoxic agent) molecules conjugated per antibody. The DAR may be characterized using any method known in the art, including, but not limited to, spectroscopy, dynamic light scattering, size exclusion chromatography (SEC), size exclusion chromatography coupled with mass spectrometry (SEC-MS), and mass spectrometry.

[0033] "Maytansinoid-to-antibody ratio (MAR)" means the average number of maytansinoid molecules conjugated per antibody.

[0034] "Target antibody concentration" means the desired antibody concentration.

[0035] "Target drug concentration" or "target cytotoxic agent concentration" means the desired concentration of a drug or cytotoxic agent. It should be noted that the concentration of the drug or cytotoxic agent is largely calculated based on the complexed form of the drug, but may include small amounts of free or uncomplexed drug found in the sample.

[0036] "Target maytansinoid concentration" means the desired concentration of maytansinoid.

[0037] "Potency variability" refers to the differing potencies present in different batches of formulation. Potency variability is preferably reduced by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50% or more. Desirable.

[0038] "Formulation" refers to the final dosage form containing the active pharmaceutical ingredient. In one embodiment, the final formulation is a container (e.g., a vial) containing the antibody-drug conjugate of the present invention, alone or in combination with excipients.

[0039] "Specifications" means a set of standards that a drug or drug product must adhere to to be suitable for its intended use. Specifications are typically proposed by the manufacturer and approved by a regulatory agency (e.g., the FDA).

[0040] As used herein, a "functional antibody" refers to an antibody that affects cell death through a direct cell-killing mechanism, such as apoptosis or necrosis. A functional antibody has direct cell-killing activity in vivo without being conjugated to a drug (an "unmodified antibody"). Non-limiting examples of functional antibodies include the huEGFR-7R antibody and the huCD37-3 antibody. As used herein, a "non-functional antibody" refers to an antibody that (i) has unknown cell-killing activity in vivo (e.g., indirect or indirect cell-killing as an unmodified antibody, e.g., huDS6), or (ii) has indirect cell-killing activity as a result of effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), or (iii) has increased complex activity in vivo when effector functions are increased, or any combination of (i), (ii), and (iii). Non-functional antibodies may have anti-proliferative activity, for example, by blocking the binding of growth agents (e.g., growth factors). Non-limiting examples of non-functional antibodies with indirect cell killing activity include huMovl9, huMy9-6, and huB4.

[0041] By "huB4" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to CD19, such as human CD19. Exemplary huB4 antibodies of the invention can comprise the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0042] "huB4-SPDB-DM4" specifically binds to CD19 and is linked to the cytotoxic maytansinoid N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) linker. 2’ -Deacetyl-N 2’"huB4-SPDB-DM4" refers to an antibody-drug conjugate comprising a huB4 antibody conjugated to -(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4). huB4-SPDB-DM4 is described, for example, in U.S. Pat. No. 8,435,528 and International Patent Application Publication No. WO2004 / 103272, the entire contents of which are incorporated herein by reference.

[0043] "huMovl9" (also referred to as "M9346A") refers to a humanized antibody or epitope-binding fragment thereof that specifically binds to folate receptor alpha (also known as folate receptor 1 or "FOLR1" herein). The detailed sequence of huMovl9 is described in U.S. Pat. Nos. 8,557,966 and 8,709,432 and International Patent Application Publication No. WO2011 / 106528, the entire contents of which are incorporated herein by reference. Exemplary huMOV19 antibodies of the invention may comprise the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0044] "huMovl9-sulfo-SPDB-DM4" (also referred to as "IMGN853") specifically binds to FOLR1 and binds the cytotoxic maytansinoid N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sSPDB) via the disulfide-containing linker. 2’ -Deacetyl-N 2’"ADC huMovl9-sulfo-SPDB-DM4" refers to an antibody-drug conjugate comprising the huMovl9 antibody conjugated to -(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4). The ADC huMovl9-sulfo-SPDB-DM4 is described, for example, in Ab et al., AACR; Cancer Res 2011;71(8 Suppl):Abstract number 4576, and in U.S. Patent Nos. 8,557,966 and 8,709,432, and International Patent Application Publication No. WO2011 / 106528, the contents of each of which are incorporated herein by reference in their entirety.

[0045] "huDS6" refers to a humanized antibody or epitope-binding fragment thereof that specifically binds to the CA6 sialoglycotope on a Mucl mucin receptor (e.g., human Mucl) expressed by cancerous cells. Exemplary sequences of huDS6 are described in U.S. Pat. No. 7,834,155 and International Patent Application Publication Nos. WO 2005 / 009369 and WO 2007 / 024222, the entire contents of which are incorporated herein by reference. Exemplary huDS6 antibodies of the invention may comprise or consist of the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0046] "huMy9-6" (also referred to as "Z4681A") refers to a humanized antibody or epitope-binding fragment thereof that specifically binds to the leukocyte differentiation antigen CD33, such as human CD33. Exemplary sequences of huMy9-6 heavy chain variable region portions are described in U.S. Patent Publication No. 20060177455, the entire contents of which are incorporated herein by reference. Exemplary sequences of huMy9-6 light chain variable region portions are known in the art and are described in U.S. Patent Nos. 7,557,189, 7,342,110, 8,119,787, and 8,337,855, the entire contents of which are incorporated herein by reference. An exemplary huMy9-6 antibody of the invention may comprise or consist of the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0047] "huMy9-6-sulfo-SPDB-DGN462" (also referred to as "IMGN779") refers to an anti-huCD33 antibody conjugated via a cleavable disulfide linker to an indolinobenzodiazepine dimer containing a monoimine moiety called DGN462. [ka]

[0048] "huEGFR-7R" (also referred to as "J2898A") refers to a humanized antibody or epitope-binding fragment thereof that specifically binds to EGFR, such as human EGFR. Exemplary huEGFR-7R antibodies of the invention may comprise or consist of the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0049] "huEGFR-7R-SMCC-DM1" (also referred to as "IMGN289") refers to an antibody-drug conjugate comprising a huEGFR-7R antibody that specifically binds EGFR and is conjugated to the maytansinoid N(2')-deacetyl-N(2')-(3-mercapto-l-oxopropyl)-maytansine (DM1) via an N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) linker. The ADC huEGFR-7R-SMCC-DM1 is described, for example, in U.S. Pat. No. 8,790,649 and International Patent Application Publication No. WO 2012 / 058588, the entire contents of which are incorporated herein by reference.

[0050] By "huCD37-3" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to CD37, such as human CD37. Exemplary sequences for huCD37-3 are described in U.S. Pat. No. 8,765,917 and International Patent Application Publication No. WO 2011 / 112978, the entire contents of which are incorporated herein by reference. Exemplary huCD37-3 antibodies of the invention may comprise or consist of the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0051] "huCD37-3-SMCC-DM1" (also referred to as "IMGN529") refers to an antibody-drug conjugate comprising the humanized IgG1 antibody K7153A that specifically binds to CD37 covalently linked to the maytansinoid N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1) via the non-cleavable maleimide-derived thioether-based linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC).

[0052] By "huCD37-50" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to CD37, such as human CD37. Exemplary sequences for huCD37-50 are described in U.S. Pat. No. 8,765,917 and International Patent Application Publication No. WO 2011 / 112978, the entire contents of which are incorporated herein by reference. Exemplary huCD37-50 antibodies of the invention may comprise or consist of the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences: [ka]

[0053] "huAnti-CD123" means a humanized antibody or epitope-binding fragment thereof that specifically binds to CD123, such as human CD123. Exemplary huAnti-CD123 antibodies are described in U.S. Provisional Patent Application No. 62 / 186,161, the entire contents of which are incorporated herein by reference.

[0054] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, epitope-binding antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, single variable novel antigen receptor domain antibody fragments (VFv), and monoclonal antibodies. NAR , or V NARdomain), unibodies with deleted hinge regions, nanobodies, antibody fragments consisting of a single small variable antibody domain (Ablynx), CH domains (Hu et al., Cancer Res. 56:3055-3061, 1996); DuoBody®, a bispecific engineered IgG1 antibody containing (i) a stable hinge region that is non-permissive for in vivo Fab arm exchange and (ii) an IgG4-like CH3 domain engineered to be permissive for in vivo Fab arm exchange (see, e.g., WO2008 / 119353 and WO2011 / 131746); multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies; and Probodies, engineered masked monoclonal antibodies that remain inactive in healthy tissues but are specifically activated in disease microenvironments (e.g., cleaved by protease enrichment or specificity in the disease microenvironment) (Desnoyers et al., Sci Transl Med 5:207ral44, 2013), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies may belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of the heavy chain constant domains, designated α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have distinct and well-known subunit structures and three-dimensional configurations. The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variation (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). In addition, a combination of these two approaches may be used in the art of CDR determination.

[0055] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0056] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals characterized by unregulated cell proliferation of a cell population. Cancer includes blood cancers or solid tumors. More specifically, cancer is leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphocytic leukemia (ALL), e.g., B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)) or lymphoma (e.g., non-Hodgkin's lymphoma), myelodysplastic syndrome (MDS), melanoma, lung cancer (e.g., non-small cell lung cancer; NSCLC), ovarian cancer, endometrial cancer, peritoneal cancer, pancreatic cancer, breast cancer, prostate cancer, squamous cell carcinoma of the head and neck, and cervical cancer.

[0057] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide while possessing certain biochemical modifications that improve the analog's function compared to the native polypeptide. Such biochemical modifications may, for example, increase the analog's protease resistance, membrane permeability, or half-life without altering ligand binding. Analogs may also contain artificial amino acids.

[0058] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0059] In this disclosure, "comprises," "comprising," "containing," "having," and the like, have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meaning ascribed to them in U.S. Patent law, and the terms are open-ended, permitting the presence of things other than what is recited, provided that the basic or novel characteristics of what is recited are not altered by the presence of things other than what is recited, but excluding prior art embodiments.

[0060] "Detecting" refers to determining the presence, absence, or amount of the analyte being detected.

[0061] "Disease" means any condition or disease that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia and cancer, which may be treated with the compositions of the present invention.

[0062] By "effective amount" is meant the amount of agent required to ameliorate the symptoms of a disease compared to an untreated patient. The effective amount of the active agent(s) (e.g., antibody drug conjugate (ADC) or drug) used to practice the present invention in the therapeutic treatment of a disease will vary depending on the mode of administration, age, weight, and general health of the subject. Ultimately, the treating physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount.

[0063] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, while epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least three, more commonly at least five or 8-10 amino acids, in a unique spatial conformation.

[0064] "Formulating" refers to the process used to manufacture a formulation.

[0065] The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) of non-human species (e.g., mouse, rat, rabbit, hamster) are replaced with residues from the CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capacity (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some cases, Fv framework region (FR) residues of human immunoglobulins are replaced with corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capacity. Humanized antibodies can be further modified by substitution of additional residues either within the Fv framework regions and / or within the substituted non-human residues to improve and optimize the antibody's specificity, affinity, and / or potency. Generally, a humanized antibody comprises substantially all of at least one, typically two or three, variable domains containing all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. Humanized antibodies can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539.

[0066] The goal of humanization is to reduce the immunogenicity of xenogeneic antibodies, such as murine antibodies, for introduction into humans while maintaining full antigen-binding affinity and specificity of the antibody.

[0067] Humanized antibodies may be produced using a variety of techniques, such as resurfacing or CDR grafting. As used herein, resurfacing techniques use a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surfaces of antibody variable regions so that they resemble surfaces of known antibodies from the target host.

[0068] Schemes and methods for resurfacing antibodies, as well as other methods for reducing the immunogenicity of antibodies in different hosts, are disclosed in U.S. Pat. No. 5,639,641 (Pedersen et al.), which is incorporated herein by reference in its entirety. Briefly, in a preferred method, (1) an alignment of sites from a pool of antibody heavy and light chain variable regions is generated to provide a set of heavy and light chain variable region framework surface exposed positions, wherein the aligned sites for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) the set of heavy and light chain variable region framework surface exposed amino acid residues is largely closely identical to the set of rodent surfaces exposing the identified amino acid residues; (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is replaced with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5 Å of any atom of any residue in the complementarity-determining region of the rodent antibody; and (5) producing a humanized rodent antibody with binding specificity.

[0069] Antibodies can be humanized using a variety of other techniques, including CDR-grafting (EP 0239400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan EA, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka GM et al., 1994, Protein Engineering, 7(6):805-814; Roguska MA et al., 1994, PNAS, 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be produced by a variety of methods known in the art, including phage display. See also U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Patent Application Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, the foregoing references being incorporated herein by reference in their entireties.

[0070] The term "human antibody" means an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any technique known in the art. This definition of human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, such as, for example, an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0071] The term "antibody drug conjugate" or "ADC," as used herein, refers to a compound that is linked to a cell-binding agent (i.e., an antibody or fragment thereof). Typically, the cell-binding agent (e.g., an antibody) is covalently attached to the drug by a linker.

[0072] The terms "isolated," "purified," or "biologically pure" refer to a material that is free to varying degrees from components that normally accompany it as found in its natural state. "Isolated" indicates some degree of separation from the original source or environment. "Purified" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances so as not to substantially affect the biological properties of the protein or cause other adverse consequences, including any impurities. That is, a nucleic acid or peptide of the invention is purified if it is substantially free from cellular material, viral material, or culture medium, if produced by recombinant DNA techniques, or from chemical precursors or other chemicals, if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high-performance liquid chromatography). The term "purified" can indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. With respect to proteins that may be subject to modification (eg, phosphorylation or glycosylation), different modifications may result in different isolated proteins that can be separately purified.

[0073] A "linker" is a chemical moiety capable of linking a compound to a protein. In one embodiment, the linker connects a drug, such as a maytansinoid, to a cell-binding agent, such as an antibody or fragment thereof, via a stable covalent bond. The linker can be susceptible to or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and hydrophilic forms thereof, as described herein and known in the art.

[0074] Exemplary cleavable linkers include, but are not limited to, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), and disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP). Exemplary non-cleavable linkers include, but are not limited to, 2-iminothiolane, acetylsuccinic anhydride, and succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The common linkers 2-iminothiolane and acetylsuccinic anhydride can be used as cleavable or non-cleavable linkers.

[0075] A "monoclonal antibody" refers to a homogeneous antibody population involved in highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as both antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule containing an antigen-recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced by any technique, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animal techniques.

[0076] By "specifically binds" is meant a compound or antibody that recognizes and binds to the polypeptide of interest but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample that naturally contains a polypeptide of the invention.

[0077] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

[0078] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0079] Sequence identity is typically measured using sequence analysis software (e.g., the Genetics Computer Group's Sequence Analysis Software Package, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program uses a BLAST score to indicate closely related sequences. -3 ~e -100 can be used with probability scores between .

[0080] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, sheep, or cat.

[0081] The term "therapeutically effective amount" refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow or stop to some extent) cancer cell invasion into peripheral organs, inhibit (i.e., slow or stop to some extent) tumor metastasis, inhibit tumor growth to some extent, and / or ameliorate to some extent one or more symptoms associated with cancer. See the definition of "treating" herein. To the extent a drug inhibits the growth of and / or kills existing cancer cells, the drug may be cytostatic and / or cytotoxic. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Although not necessarily, a prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is typically used in subjects prior to or at an earlier stage of disease.

[0082] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to encompass any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0083] As used herein, the terms "treat," "treating," "treatment," and the like refer to the reduction or amelioration of a disease and / or its associated symptoms. It should be understood that treating a disease or condition does not require the complete elimination of the disease, condition, or its associated symptoms, although this does not exclude it.

[0084] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or clear from the context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0085] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean value. "About" is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0086] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as a single group or combination of listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0087] Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for reducing potency variability in an antibody-drug conjugate composition, comprising: (a) determining the target drug concentration at a fixed antibody concentration and a fixed drug-to-antibody ratio (DAR); and (b) reducing efficacy variability in the antibody-drug conjugate composition by formulating the composition to achieve the target drug concentration; The method comprising: (Item 2) 1. A method for reducing efficacy variability in a composition comprising an antibody-drug conjugate, comprising: (a) Determining the target antibody concentration and drug concentration at a fixed drug-antibody ratio (DAR); (b) targeting a variable drug concentration that identifies the midpoint of the range where the antibody concentration specification range and the drug concentration specification range overlap; (c) reducing efficacy variability in the antibody-drug conjugate composition by formulating the composition to the target variable drug concentration. The method comprising: (Item 3) 3. The method of claim 1 or 2, wherein the variability of the drug concentration is about ±10%. (Item 4) 3. The method of claim 2, wherein the variability of the antibody concentration is about ±10%. (Item 5) 5. The method of claim 3 or 4, wherein the variability is less than about ±5, 6, 7, 8, or 9%. (Item 6) 1. A method for reducing efficacy variability in a composition comprising an antibody-drug conjugate, comprising: (a) measuring the DAR of the antibody-drug conjugate composition; (b) determining an upper antibody specification limit and a lower antibody specification limit, the upper antibody specification limit being the target antibody concentration plus the maximum variation that the specification allows, and the lower antibody specification limit being the target antibody concentration minus the maximum variation that the specification allows; (c) determining an upper limit of a defined drug specification and a lower limit of a defined drug specification, the upper limit of the defined drug specification being the target drug concentration plus the maximum variation that the specification allows, and the lower limit of the defined drug specification being the target drug concentration minus the maximum variation that the specification allows; (d) Determine the drug specification upper limit (USL(drug)), calculated as follows: USL (drug) μg / mL = upper limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (e) Determine the lower specification limit (LSL(drug)) of the drug, calculated as follows: LSL (drug) μg / mL = Lower limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (f) comparing the USL(drug) calculated in step (d) with the upper drug specification limit defined in step (c) and selecting the lower of the two values ​​as the effective upper drug specification limit; (g) comparing the LSL(drug) calculated in step (e) with the lower limit of the drug specification defined in step (c) and selecting the higher of the two values ​​as the effective lower limit of the drug specification; and (h) reducing efficacy variability in the antibody-drug conjugate composition by formulating the composition to a target drug concentration that is midpoint between the effective upper limit of the drug specification and the effective lower limit of the drug specification. The method comprising: (Item 7) 7. The method of claim 6, wherein the method narrows the range between upper and lower specification limits for the drug and the antibody to about ±3-9%. (Item 8) 7. The method of claim 6, wherein the method narrows the range between upper and lower specification limits for the drug to about ±4%. (Item 9) 7. The method of claim 6, wherein the maximum variation allowed by the specifications in step (b) is about ±15%. (Item 10) 7. The method of claim 6, wherein the maximum variation allowed by the specifications in step (b) is less than about ±10, 11, 12, 13, or 14%. (Item 11) 7. The method of claim 6, wherein the maximum variation allowed by the specifications in step (c) is about ±15%. (Item 12) 7. The method of claim 6, wherein the maximum variation allowed by the specifications in step (c) is less than about ±10, 11, 12, 13, or 14%. (Item 13) 13. The method according to any one of items 1 to 12, wherein the antibody is a non-functional antibody. (Item 14) 14. The method according to any one of items 1 to 13, wherein the drug concentration varies within the antibody specification concentration. (Item 15) 15. The method according to any one of items 2 to 14, wherein the antibody concentration and the drug concentration are allowed to vary. (Item 16) 1. A method for formulating an antibody-drug conjugate composition, comprising: (a) determining the target drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio; and (b) formulating the antibody-drug conjugate composition to achieve the target drug concentration of step (a). The method comprising: (Item 17) 17. The method of any one of items 1 to 16, wherein the drug is a tubulin inhibitor, a DNA damaging agent, a DNA cross-linking agent, a DNA alkylating agent, or a cell cycle disruptor. (Item 18) 18. The method according to any one of items 1 to 17, wherein the drug is a maytansinoid. (Item 19) 18. The method according to any one of items 1 to 17, wherein the drug is a benzodiazepine compound. (Item 20) 20. The method of claim 19, wherein the benzodiazepine compound is a pyrrolobenzodiazepine or an indolinobenzodiazepine. (Item 21) 18. The method according to any one of items 1 to 17, wherein the drug is an auristatin. (Item 22) 22. The method according to any one of items 13 to 21, wherein the antibody is a non-functional antibody. (Item 23) 23. The method according to any one of items 1 to 17, 19, 20, and 22, wherein the drug is a benzodiazepine compound and the antibody is a non-functional antibody. (Item 24) 23. The method of any one of items 1 to 18, 20, and 22, wherein the drug is a maytansinoid and the antibody is a non-functional antibody. (Item 25) 22. The method according to any one of items 1 to 12 and 14 to 21, wherein the antibody is a functional antibody. (Item 26) 1. A method for reducing potency variability in a composition comprising an antibody-maytansinoid conjugate, comprising: (a) determining the target maytansinoid concentration at a fixed antibody concentration and a fixed maytansinoid-to-antibody ratio; and (b) reducing efficacy variability in the antibody-maytansinoid conjugate composition by formulating the composition to achieve the target maytansinoid concentration. The method comprising: (Item 27) 27. The method of any one of items 1 to 26, wherein the method reduces batch-to-batch potency variability in manufacturing the antibody-drug conjugate or antibody-maytansinoid conjugate. (Item 28) 27. The method according to any one of items 1 to 26, wherein the composition is a final formulation. (Item 29) 1. A method for formulating an antibody-maytansinoid conjugate composition, comprising: (a) determining the target maytansinoid concentration at a fixed antibody concentration and a fixed maytansinoid-to-antibody ratio; and (b) formulating the antibody-maytansinoid conjugate composition to achieve the target maytansinoid concentration. The method comprising: (Item 30) 1. A method of reducing potency variability in a composition comprising an antibody-benzodiazepine conjugate, comprising: (a) measuring the DAR of the antibody-benzodiazepine conjugate composition; (b) determining an upper antibody specification limit and a lower antibody specification limit, where the upper antibody specification limit is the target antibody concentration plus the maximum variation that the specification allows, and the lower antibody specification limit is the target antibody concentration minus the maximum variation that the specification allows; (c) determining an upper limit of a defined benzodiazepine specification and a lower limit of a defined benzodiazepine specification, the upper limit of the defined benzodiazepine specification being the target benzodiazepine concentration plus the maximum variation allowed by the specification, and the lower limit of the defined benzodiazepine specification being the target benzodiazepine concentration minus the maximum variation allowed by the specification; (d) Determine the benzodiazepine specification upper limit (USL(drug)) calculated as follows: USL (drug) μg / mL = upper limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (e) Determine the benzodiazepine lower specification limit (LSL(drug)), calculated as follows: LSL (drug) μg / mL = Lower limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (f) comparing the USL(drug) calculated in step (d) with the upper limit of the benzodiazepine specification defined in step (c) and selecting the lower of the two values ​​as the effective upper limit of the benzodiazepine specification; (g) comparing the LSL(drug) calculated in step (e) with the lower limit of benzodiazepine specification defined in step (c) and selecting the higher of the two values ​​as the effective lower limit of benzodiazepine specification; and (h) reducing efficacy variability in the antibody-benzodiazepine conjugate composition by formulating the composition to a target benzodiazepine concentration that is midpoint between the effective upper limit of the benzodiazepine specification and the effective lower limit of the benzodiazepine specification. The method comprising: (Item 31) 31. The method of claim 30, wherein the method narrows the range between upper and lower specification limits for the drug and the antibody to about ±3-5%. (Item 32) Item 33. The method of item 30, wherein the method narrows the range between the upper and lower specification limits to about ±4%. 31. The method of claim 30, wherein the maximum variation allowed by the specifications in step (b) is about ±15%. (Item 34) 31. The method of claim 30, wherein the maximum variation allowed by the specifications in step (b) is less than about ±10, 11, 12, 13, or 14%. (Item 35) 31. The method of claim 30, wherein the maximum variation allowed by the specifications in step (c) is about ±15%. (Item 36) 31. The method of claim 30, wherein the maximum variation allowed by the specifications in step (c) is less than about ±10, 11, 12, 13, or 14%. (Item 37) 37. The method according to any one of items 26 to 36, wherein the antibody is a non-functional antibody. (Item 38) 38. The method according to any one of items 6 to 15 and 30 to 37, wherein the DAR is the lower limit of the DAR specification or the upper limit of the DAR specification. (Item 39) 39. The method of claim 38, wherein the lower limit of the DAR specification is 2.3, 2.4, or 2.5. (Item 40) 39. The method of claim 38, wherein the upper limit of the DAR specification is 2.9, 3.0, or 3.1. (Item 41) 41. The method of any one of items 1 to 40, wherein the composition varies in potency by about 10 to 40%. (Item 42) 41. The method of any one of items 1 to 40, wherein the composition varies in potency by about 10-20%. (Item 43) 41. The method of any one of items 1 to 40, wherein the composition varies in potency by up to about 20%. (Item 44) 44. The method of any one of items 1 to 43, wherein the variability in composition efficacy is reduced compared to when the antibody-drug conjugate composition is formulated based solely on antibody concentration. (Item 45) 45. The method according to any one of items 1 to 44, wherein the antibody concentration and drug concentration are determined by spectrophotometry. (Item 46) 45. The method of any one of items 1 to 44, wherein the drug to antibody ratio is determined by size exclusion chromatography (SEC) or SEC-mass spectrometry (SEC-MS). (Item 47) 45. The method of any one of items 1 to 44, wherein the antibody-drug conjugate composition is formulated for injection. (Item 48) 45. The method of any one of items 1 to 44, wherein the antibody-drug conjugate is formulated with a pharmaceutically acceptable parenteral vehicle. (Item 49) 45. The method according to any one of items 1 to 44, wherein the antibody-drug conjugate is formulated in a unit-dose injectable form. (Item 50) 40. The method of any one of items 18 to 49, wherein the maytansinoid is DM1, DM3, or DM4. (Item 51) 51. The method according to any one of items 38 to 50, wherein the antibody is a non-functional antibody. (Item 52) 52. The method of claim 51, wherein the non-functional antibody is huMov19, huMy9-6, or huB4. (Item 53) 52. The method of claim 51, wherein the non-functional antibody is huDS6. (Item 54) 51. The method according to any one of items 26 to 50, wherein the antibody is a functional antibody. (Item 55) 55. The method of claim 54, wherein the functional antibody is huEGFR-7R or huCD37-3. (Item 56) 56. The method of any one of items 1 to 55, wherein the antibody-drug conjugate comprises a cleavable linker or a non-cleavable linker. (Item 57) 57. The method of claim 56, wherein the cleavable linker is N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), or disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP). (Item 58) 58. The method of claim 57, wherein the non-cleavable linker is 2-iminothiolane, acetylsuccinic anhydride, or succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). (Item 59) The antibody-drug conjugates are selected from the group consisting of huMovl9-sulfo-SPDB-DM4, huMovl9-sulfo-SPDB-Dl, huMovl9-D2, huMovl9-sulfo-SPDB-D10, huMovl9-sulfo-SPDB-DGN462, huMy9-6-sulfo-SPDB-Dl, huMy9-6-D2, huMy9-6-sulfo-SPDB-D10, huMy9-6-sulfo-SPDB-DGN462, and huAnti-CD123-sulfo-SPDB. 31. The method of any one of paragraphs 1, 2, 6, 16, 26, 29, or 30, wherein the antibody is huAnti-CD123-DM1, huAnti-CD123-D2, huAnti-CD123-sulfo-SPDB-D10, huAnti-CD123-sulfo-SPDB-DGN462, huB4-SPDB-DM4, huDS6-SPDB-DM4, huCD37-3-SMCC-DM1, huCD37-50-SMCC-DM1, or huEGFR-7R-SMCC-DM1. (Item 60) A method of administering to a subject within a narrow intended range, comprising: Administering to the subject an antibody-drug conjugate composition formulated according to the method of any one of items 1 to 59. The method comprising: (Item 61) 58. A pharmaceutical composition comprising a huMovl9-sulfo-SPDB-DM4 antibody-drug conjugate formulated according to the method of any one of items 1 to 18, 22, 24, 26 to 29, 31 to 52, 56, and 57, The pharmaceutical composition, wherein the trace amount of maytansinoid is provided on the label. (Item 62) 58. A pharmaceutical composition comprising a huMy9-6-sulfo-SPDB-DGN462 antibody-drug conjugate formulated according to the method of any one of items 2 to 17, 19, 20, 22, 23, 30 to 49, 51, 52, 56, and 57, The pharmaceutical composition, wherein the trace DGN462 concentration is provided on the label. [Brief explanation of the drawings]

[0088] [Figure 1] Graph showing the dependence of cytotoxic potency on the maytansinoid-to-antibody ratio (MAR) of huMovl9-sulfo-SPDB-DM4. huMovl9-sulfo-SPDB-DM4 is an immunoconjugate containing a humanized monoclonal antibody against FOLR1 (huMovl9) conjugated to the cytotoxic maytansinoid DM4 via a sulfo-SPDB linker. Cytotoxic potency is measured relative to the huMovl9-sulfo-SPDB-DM4 standard with a MAR of 3.4. % Potency = EC50 reference / EC50 test article × 100%. [Figure 2] 1 is a graph showing the dependence of cytotoxic potency on the concentration of huMovl9-sulfo-SPDB-DM4. [Figure 3] Two scatter plots and a table are provided simulating the estimated effect of drug-to-antibody ratio (DAR) on ocular toxicity ("ocular toxicity"). AIBW refers to adjusted ideal body weight. DAR was calculated based on drug administration levels. [Figure 4] We provide scatter plots and tables simulating the estimated effect of drug-to-antibody ratio (DAR) and concentration on ocular toxicity. DARs were calculated and the corresponding ADCs were not actually administered to patients. [Figure 5] Two graphs are included showing the lack of effect of DAR on median tumor volume in KB and IGROV-1 murine xenograft models when huMovl9-sulfo-SPDB-DM4 conjugates were administered at the same DM4 dose. Mice were administered huMovl9-sSPDB-DM4. All conjugates were administered at 25 μg / kg DM4 and varying antibody doses (higher for low-DAR conjugates, lower for high-DAR conjugates). Similar antitumor activity was observed regardless of the varying DAR and antibody doses. [Figure 6]1 is a graph showing the similar effect of DM4 dose on mouse body weight for conjugates with 9.0 DAR compared to 3.6 DAR when the administered DM4 dose was the same. All conjugates were administered at 1.4 mg / kg DM4 and varying antibody doses (higher for low DAR conjugates, lower for high DAR conjugates). [Figure 7] Figure 1 shows the effect of DM1 dose on mean body weight change for conjugates with various maytansinoid-to-antibody ratios when the administered DM1 dose was the same. All conjugates were administered at 3.0 mg / kg DM1 and varying antibody doses (higher for low DAR conjugates, lower for high DAR conjugates). Toxicity was similar for all conjugates regardless of DAR. [Figure 8] FIG. 1 is a graph showing in vivo toxicity studies in mice receiving antibody-SPDB-DM4 conjugates of various DARs, including ADC huDS6-SPDB-DM4 ("huDS6-DM4"); huB4-SPDB-DM4 ("huB4-DM4"); and huMy9-6-SPDB-DM4 ("huMy9-6-DM4"). [Figure 9A] Figures 9A and 9B are tables demonstrating the benefits of formulating ADC compositions based on DM4 concentration. Figure 9A shows the acceptable DM4 concentrations in μg / ml obtained when formulating antibody-drug conjugates at a target antibody concentration (5.0 ± 1.0 mg / ml) and a DAR of 3.4 ± 0.5 (circled). The DM4 concentration is 91.1 at a 5.0 mg / mL target antibody concentration and a 3.4 DAR target (boxed). Low potency variability (boxed region of DM4 concentration) was possible by formulating based on DM4 concentrations with a ± 10% specification. The target DAR, antibody, and DM4 concentration are boxed. In Figure 9B, the antibody concentration specification fails at the high-low DAR extremes (boxed region). The target DAR, antibody, and DM4 concentration are boxed. [Figure 9B]Figures 9A and 9B are tables demonstrating the benefits of formulating ADC compositions based on DM4 concentration. Figure 9A shows the acceptable DM4 concentrations in μg / ml obtained when formulating antibody-drug conjugates at a target antibody concentration (5.0 ± 1.0 mg / ml) and a DAR of 3.4 ± 0.5 (circled). The DM4 concentration is 91.1 at a 5.0 mg / mL target antibody concentration and a 3.4 DAR target (boxed). Low potency variability (boxed region of DM4 concentration) was possible by formulating based on DM4 concentrations with a ± 10% specification. The target DAR, antibody, and DM4 concentration are boxed. In Figure 9B, the antibody concentration specification fails at the high-low DAR extremes (boxed region). The target DAR, antibody, and DM4 concentration are boxed. [Figure 10A] 10A-10C are graphs showing the effect of formulating antibody-drug conjugate (ADC) batches by varying antibody concentration to achieve a target drug (DGN462) concentration. "USL" and "LSL" indicate the upper and lower specification limits for antibody concentration. DGN462 is an exemplary drug. Vertical lines (thin "I") indicate the upper and lower drug concentration limits. "DAR" indicates the drug-to-antibody ratio. [Figure 10B] 10A-10C are graphs showing the effect of formulating antibody-drug conjugate (ADC) batches by varying antibody concentration to achieve a target drug (DGN462) concentration. "USL" and "LSL" indicate the upper and lower specification limits for antibody concentration. DGN462 is an exemplary drug. Vertical lines (thin "I") indicate the upper and lower drug concentration limits. "DAR" indicates the drug-to-antibody ratio. [Figure 10C] 10A-10C are graphs showing the effect of formulating antibody-drug conjugate (ADC) batches by varying antibody concentration to achieve a target drug (DGN462) concentration. "USL" and "LSL" indicate the upper and lower specification limits for antibody concentration. DGN462 is an exemplary drug. Vertical lines (thin "I") indicate the upper and lower drug concentration limits. "DAR" indicates the drug-to-antibody ratio. [Figure 11A]Figures 11A-11C are graphs showing that formulating ADC compositions by varying antibody and drug concentrations (white ovals) narrows the acceptable specification range for both the antibody and drug compared to varying only the antibody specifications to achieve a target drug concentration (grey diamonds). [Figure 11B] Figures 11A-11C are graphs showing that formulating ADC compositions by varying antibody and drug concentrations (white ovals) narrows the acceptable specification range for both the antibody and drug compared to varying only the antibody specifications to achieve a target drug concentration (grey diamonds). [Figure 11C] Figures 11A-11C are graphs showing that formulating ADC compositions by varying antibody and drug concentrations (white ovals) narrows the acceptable specification range for both the antibody and drug compared to varying only the antibody specifications to achieve a target drug concentration (grey diamonds). DETAILED DESCRIPTION OF THE INVENTION

[0089] The present invention provides improved methods for formulating therapeutic compositions comprising antibody-drug conjugates ("ADCs") to narrow potency variability between batches of ADCs and / or to narrow drug and antibody specifications over a wider range of drug-to-antibody ratios (DARs).

[0090] In one aspect, the present invention is based, at least in part, on the discovery that the efficacy and toxicity of some ADCs are driven, in whole or in part, by the drug dose rather than the antibody dose. Formulating ADC compositions based on target drug concentrations advantageously minimizes potency variability in the final formulation and ensures that patients are dosed within the intended narrow range.

[0091] Traditionally, antibody-drug conjugate therapeutic compositions are formulated based on antibody concentration. Some variability is inherent in formulating antibody-drug conjugates based on antibody concentration, even if a given specification remains within an acceptable range. In particular, at the end of the ADC manufacturing process, the concentration of the antibody in the conjugate is measured and the conjugate is diluted to reach a target drug concentration based on a fixed antibody concentration. In practice, the antibody concentration in the final formulation is allowed to vary from the target concentration. In one example, formulation specifications allow for a ±20% variation in antibody base concentration (e.g., 4.0-6.0 mg / mL for a target antibody concentration of 5.0 mg / mL). Therefore, depending on the DAR, the ADC potency of the final formulation can vary by up to ±35%, potentially falling outside the desired range.

[0092] The formulation method reported herein below involves determining the drug concentration at a fixed antibody concentration and a fixed drug-to-antibody ratio and formulating the antibody-drug conjugate composition to achieve the desired drug concentration. Briefly, formulating the ADC composition based on drug concentration and adding, for example, a ±10% drug concentration specification significantly narrows the potency present in the final formulation to ±10% of the target drug concentration (±10% specification). Formulating the ADC based on drug concentration and ensuring that drug concentration does not vary by more than 10% ensures that potency varies only ±10%. Thus, the novel formulation method of the present invention eliminates the DAR potency dependency of formulating to a narrow range of drug concentrations. This formulation strategy only slightly increases the risk of antibody concentrations that are out of specification, significantly reducing the risk of a batch not meeting specifications. Such improved formulation methods ensure that patients are administered within the intended narrow range without substantially increasing the risk of ADC batches not meeting specifications.

[0093] In yet another aspect, the present invention provides a method for reducing efficacy variability in a composition comprising an antibody-drug conjugate. The method comprises formulating the antibody-drug conjugate by targeting variable concentrations of both the drug and antibody within overlapping ranges (i.e., having small variations (±4-9%) in both concentration values ​​rather than large variations (±10-15%) in one concentration), thereby reducing efficacy variability in the composition. In one embodiment, the small variations are about 4, 5, 6, 7, 8, or 9%. In other embodiments, the large variations are about 10, 11, 12, 13, 14, or 15%.

[0094] In another aspect, the present invention provides a method for reducing efficacy variability in a composition comprising an antibody-drug conjugate, the method comprising formulating the antibody-drug conjugate by targeting variable concentrations of either the drug or the antibody within a range where both specifications overlap, thereby reducing efficacy variability in the composition.

[0095] Antibody-drug conjugate formulations ADC cancer therapeutics are formulated similarly to antibody cancer therapeutics; i.e., based on antibody protein concentration. The formulation label will include information on the "microdose" or target concentration on which dosing is based (e.g., mg / kg or mg / m 2 Although the antibody concentration is typically given as a 10-20% target, typical specifications for antibody concentration are ±10%-20% of the target. Because ADC potency is generally linear with concentration, formulation potency can vary by up to ±20%. Unlike antibodies, ADCs have the additional potential for variable potency depending on the drug-to-antibody ratio (DAR). A typical DAR specification for early clinical development is ±15% of the target, allowing the amount of conjugated cytotoxicity to vary with a given antibody concentration. For most ADCs, a linear relationship can be demonstrated between DAR and potency, indicating that potency is partially or completely dictated by the concentration of the administered conjugated drug.

[0096] For many ADCs, toxicity can be demonstrated in rodents to be entirely dependent on the dose of the administered conjugated drug, regardless of the dose of the antibody. Therefore, toxicity is independent of the DAR as long as the administered dose of the conjugated drug is the same. For some ADCs in which the antibody does not have intrinsic anti-tumor activity, efficacy depends entirely on the dose of the drug. In such cases, efficacy is the same regardless of the DAR as long as the administered dose of the conjugated drug is the same. However, due to typical specifications for antibody concentration and DAR, the concentration of the conjugated drug can be somewhat varied. For some ADCs, even if the antibody has intrinsic anti-tumor activity or is considered, for example, a functional antibody, ADC efficacy may be driven more by the dose of the drug rather than the antibody.

[0097] In cases where it is possible to demonstrate that the efficacy and toxicity of an ADC are primarily driven by the amount of conjugated drug administered, it may prove beneficial to narrow the specification for the concentration of the conjugated drug rather than the antibody. Thus, the present invention provides a method for formulating a therapeutic composition based on the concentration of the drug rather than the concentration of the antibody. The target concentration of the drug is the drug concentration calculated at a fixed antibody concentration and a fixed DAR. A specification set near the target conjugated drug concentration dictates the acceptable potency variability in the formulation vial. Thus, a ±10% drug concentration specification narrows the acceptable potency variability to ±10%.

[0098] In other embodiments, therapeutic compositions can be formulated by targeting variable drug concentrations based on DAR and antibody specifications to achieve an ADC therapeutic composition that falls within the center of the effective specification range of antibody and drug concentrations, where both drug and antibody concentrations overlap. The center of the effective range achieved by targeting variable drug concentrations within ±5% of the target DAR can be substantially similar to the final formulated product when static drug concentrations are used. Improvements are realized when the DAR varies ±5-15% of the target DAR. Formulating therapeutic compositions by targeting variable drug concentrations at these upper and lower DAR limits can result in less variability for both drug (e.g., about ±4%) and antibody concentrations (e.g., about ±10%) compared to targeting static drug concentrations that vary antibody concentrations by about ±15%. Such ranges are useful for formulating ADC compositions, an example of which is huMy9-6-sulfo-SPDB-DGN462. In one embodiment, the invention features the use of the methods described herein to formulate huMy9-6-sulfo-SPDB-DGN462, an antibody-drug conjugate comprising DGN462 conjugated to the anti-CD33 antibody huMy9-6 via the cleavable disulfide linker s-SPDB. Other drugs useful in the invention include benzodiazepines, such as those depicted in Table 1 or variations thereof, as well as those according to the following structural formula: [ka] [ka]

[0099] antibody-drug conjugates The present invention relates to improved methods for formulating ADCs comprising an antibody (e.g., an antibody that binds to a tumor antigen) or antibody fragment disclosed herein linked or conjugated to a cytotoxic agent (e.g., a drug or prodrug), and functional equivalents thereof. A variety of antibodies can be used in the methods of the invention. In certain embodiments, the antibody specifically binds to an antigen or ligand such as FOLR1 (also known as FRα), CD33, CD123, CD19, MUC1, CA6, CD37, EGFR, and fragments of any of the above-listed polypeptides. In certain embodiments, the invention includes, but is not limited to, ADCs comprising any of the following antibodies: huMovl9, huMy9-6, huAnti-CD123, huB4, huDS6, huCD37-50, huCD37-3, and huEGFR-7R.

[0100] Suitable drugs or prodrugs are known in the art. The drug or prodrug may be cytotoxic. Cytotoxic drugs used in the ADCs of the present invention are any compounds that cause the death of cells (e.g., cancer cells), induce cell death, or reduce cell viability to some extent, including, for example, tubulin inhibitors, DNA damaging agents, DNA cross-linking agents, DNA alkylating agents, and cell cycle disruptors. In certain embodiments, suitable cytotoxic drugs include maytansinoids and maytansinoid analogs. Other suitable cytotoxic agents include, for example, benzodiazepines (e.g., pyrrolobenzodiazepines and indolinobenzodiazepines; see also Table 1: compounds D1-D10 and DGN462), taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes, such as calicheamicin, dolastatins and dolastatin analogs, such as auristatins, tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino-doxorubicin.

[0101] ADCs can be prepared using linking groups to link drugs or prodrugs to antibodies or functional equivalents. Suitable linking groups are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups.

[0102] The drug or prodrug may be linked to the antibody or fragment thereof, for example, via a disulfide bond. The linker molecule or cross-linker may contain a reactive chemical group capable of reacting with the antibody or fragment thereof. The reactive chemical group for reacting with the cell-binding agent may be, for example, N-succinimidyl ester and N-sulfosuccinimidyl ester. Furthermore, the linker molecule may contain a reactive chemical group such as a dithiopyridyl group that reacts with the drug to form a disulfide bond. Examples of linker molecules include N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see, e.g., Carlsson et al., Biochem. J., 173:723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see U.S. Publication No. 20090274713), and N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see, e.g., CAS Registry number Examples of suitable cross-linking reagents include acetylsuccinic anhydride (see 341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride, succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). For example, antibodies or cell-binding agents can be modified with cross-linking reagents, such that derivatized antibodies or cell-binding agents containing free or protected thiol groups are reacted with disulfide- or thiol-containing maytansinoids to produce conjugates. The conjugates can be purified by chromatography, including, but not limited to, HPLC, size exclusion, adsorption, ion exchange, and affinity capture, as well as dialysis or tangential flow filtration.

[0103] In one embodiment of the invention, the antibody is linked to the cytotoxic drug via a disulfide bond and a polyethylene glycol spacer to enhance the potency, solubility, or efficacy of the ADC. Such cleavable hydrophilic linkers are described, for example, in WO 2009 / 0134976. An additional benefit of this linker design is the desired high monomer ratio and minimal aggregation of the antibody-drug conjugate. Specifically contemplated in this embodiment are linkers containing a polyethylene glycol spacer ((CH2CHO) n=1~14 These conjugates are cell-binding agent and drug conjugates linked by disulfide groups (-SS-) with a narrow drug loading range of 2 to 8. These conjugates exhibit relatively high potency of biological activity against cancer cells and have desirable biochemical properties, such as high conjugation yield and high monomer ratio with minimal protein aggregation.

[0104] Many of the linkers disclosed herein are described in detail in U.S. Pat. Nos. 7,989,598; 8,163,888; 8,198,417; 8,236,319; 8,563,509; U.S. Patent Application Publication No. US20130029900 and International Patent Application Publication Nos. WO2009 / 0134976; WO2009 / 134977; and WO2012 / 177837, the entire contents of each of the foregoing patents and applications being incorporated herein by reference.

[0105] The invention includes embodiments in which about two to about eight drug molecules, such as maytansinoids, benzodiazepine compounds, auristatins, DNA alkylating agents, or other compounds of interest, are linked to an antibody or fragment thereof, resulting in more effective anti-tumor activity of the conjugate compared to drug loading of fewer or greater numbers of drugs linked to the same cell-binding agent.

[0106] In one embodiment, the drug to antibody ratio is on average about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.1, 11.2, 11.3, 11 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). Virtually any cytotoxic drug can be used in the ADC. In certain embodiments, cytotoxic agents useful in the present invention are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include esters of maytansinol and maytansinol analogs. Maytansinol and maytansinol analogs, as well as any drug that inhibits microtubule formation and is highly toxic to mammalian cells, are included.

[0107] In certain embodiments, the ADCs of the invention comprise a maytansinoid. Maytansinoids useful in the invention include N 2’ -Deacetyl-N 2’ -(3-mercapto-1-oxopropyl)-maytansine (DM1), N 2’ -Deacetyl-N 2’ (4-mercapto-1-oxopentyl)-maytansine (referred to as DM3), and N 2’ -Deacetyl-N 2’ -(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4). DM1 is represented by the following structural formula: [ka] See also U.S. Patent Publication No. 20130156796. DM4 is represented by the following structural formula: [ka] See also U.S. Patent Publication No. 20130156796.

[0108] Examples of suitable maytansinol esters include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids are described in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; Nos. 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497; and 7,473,796.

[0109] Another maytansinoid containing a side chain containing a sterically hindered thiol bond is N 2’ -Deacetyl-N 2’ (4-mercapto-1-oxopentyl)-maytansine (referred to as DM3), which is represented by the following structural formula (V): [ka]

[0110] Each of the maytansinoids taught in U.S. Patent Nos. 5,208,020 and 7,276,497 can also be used in the conjugates of the present invention, the entire disclosures of which are incorporated herein by reference. The carbon positions of exemplary maytansinoid structures are provided below: [ka]

[0111] Many positions in maytansinoids can serve as positions for chemically attaching a linking moiety. For example, the C-3 position bearing a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy, and the C-20 position bearing a hydroxy group are all expected to be useful. In some embodiments, the C-3 position serves as a position for chemically attaching a linking moiety, and in some particular embodiments, the C-3 position of maytansinol serves as a position for chemically attaching a linking moiety.

[0112] Some descriptions of how to generate such antibody-maytansinoid conjugates are provided in U.S. Patent Nos. 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein in its entirety.

[0113] Generally, a solution of an antibody in an aqueous buffer can be incubated with a molar excess of a maytansinoid bearing a disulfide moiety with a reactive group. The reaction mixture can be quenched by the addition of an excess of an amine (e.g., ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration.

[0114] The average number of maytansinoid molecules bound per antibody molecule can be determined by spectrophotometrically measuring absorbance at 252 nm and 280 nm and determining the molar antibody and drug concentrations. An exemplary calculation for huMovl9-sulfo-SPDB-DM4 is provided herein below. The average number of maytansinoid molecules per antibody molecule is then calculated by dividing the molar drug concentration by the molar antibody concentration. The average number of maytansinoid molecules / antibody can be, for example, 1 to 10 or 2 to 5. In some embodiments, the average number of maytansinoid molecules / antibody is 3.4.

[0115] In certain embodiments, the ADCs of the invention comprise a benzodiazepine. Benzodiazepines useful in the invention include, for example, pyrrolobenzodiazepines and indolinobenzodiazepines (see also Table 1: compounds D1-D10 and DGN462). In various embodiments of the above aspect, the benzodiazepine compound is selected from the representative cytotoxic agents D1-D10 and DGN462 listed in Table 1. DGN462 is described, for example, in U.S. Patent No. 8,765,740, the entire contents of which are incorporated herein by reference. Compound D2 is described, for example, in U.S. Provisional Patent Application No. 62 / 045,236 and in "Antibody-Drug Conjugates (ADCs) of Indolino-Benzodiazepine DNA-Alkylating Agents," 2015 AACR, Abstract no. Compound D2 is described, for example, in U.S. Provisional Patent Application No. 62 / 045,248 and in "Antibody-Drug Conjugates (ADCs) of Indolino-Benzodiazepine DNA-Alkylating Agents," 2015 AACR, Abstract number 652.

[0116] Pharmaceutical Composition The present invention further provides pharmaceutical compositions comprising one or more ADCs described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients.

[0117] Examples of antibody-drug conjugates for use in the pharmaceutical compositions of the present invention include, but are not limited to, huMovl9-sulfo-SPDB-DM4, huMovl9-sulfo-SPDB-Dl, huMovl9-D2, huMovl9-sulfo-SPDB-D10, huMovl9-sulfo-SPDB-DGN462, huMy9-6-sulfo-SPDB-Dl, huMy9-6-D2, huMy9-6-sulfo-SPDB-D10, huMy9-6-sulfo-SPDB-DGN462, DB-DGN462, huAnti-CD123-sulfo-SPDB-D1, huAnti-CD123-D2, huAnti-CD123-sulfo-SPDB-D10, huAnti-CD123-sulfo-SPDB-DGN462, huB4-SPDB-DM4, huDS6-SPDB-DM4, huCD37-3-SMCC-DM1, huCD37-50-SMCC-DM1, or huEGFR-7R-SMCC-DM1.

[0118] In certain embodiments, formulations are prepared by combining a purified ADC of the invention with a pharmaceutically acceptable vehicle (e.g., carrier, excipient) for storage and use (Remington, The Science and Practice of Pharmacy 20th Edition, Mack Publishing, 2000). The present invention provides formulations of such compositions based on drug concentration. In some embodiments, the ADCs of the present invention are provided in a suitable carrier, diluent, and / or excipient, e.g., 0.9% saline (0.9% w / v NaCl), 5% (w / v) dextrose; and may also contain a stabilizer such as Tween 20. In certain embodiments, the ADCs are provided in an IV bag or drug vial.

[0119] Other suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers such as phosphate, citric acid, acetic acid, succinic acid, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; and sugars such as sucrose, mannitol, trehalose, or sorbitol.

[0120] The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration may be intravenous, intraarterial, parenteral, including infusion; oral; transdermal; or intracranial (e.g., subthecal or intracerebroventricular).

[0121] Kits containing antibody-drug conjugates The present invention provides kits containing antibody-drug conjugates (ADCs) that can be used to practice the methods described herein. In certain embodiments, the kits contain the ADC in one or more containers, where the amount of ADC is based on drug concentration, and the amount of ADC varies from specification by as much as ±10%. Those skilled in the art will readily recognize that the disclosed ADCs can be easily incorporated into one of the established kit formats well known in the art. If desired, the kit can include instructions for using the ADC to treat a patient. The instructions can be printed directly on the container (if present), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container.

[0122] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully explained in such references as "Molecular Cloning: A Laboratory Manual," Second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987; "Current Protocols in Molecular Biology" (Ausbel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and thus may be considered in making and practicing the invention. Techniques particularly useful for particular embodiments are discussed in the following sections.

[0123] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. [Example]

[0124] Example 1: ADC in vitro efficacy depends on the amount of drug delivered to a cell or subject The anti-FOLR1 monoclonal antibody portion of huMovl9-sulfo-SPDB-DM4 targets and binds to the cell surface antigen FOLR1 (also known as FRα). After antibody-antigen interaction and internalization, the immune complex releases DM4, which binds to tubulin and disrupts microtubule assembly / disassembly dynamics, thereby inhibiting cell division and proliferation of FOLR1-expressing tumor cells. FOLR1, a member of the folate receptor family, is overexpressed on a variety of epithelial-derived cancer cells.

[0125] The in vitro potency of antibody-drug conjugates (ADCs) is linearly related to the drug-antibody ratio (DAR), also known as the maytansinoid-to-antibody ratio (MAR) (Figure 1). The data shown in Figures 1 and 2 were generated using huMovl9-sulfo-SPDB-DM4 as an exemplary ADC. In fact, there was a 31% potency shift (i.e., from 2.9 to 3.9) over one DAR, resulting in a calculated 29% difference in DM4 dose. Figure 2 shows the general dependence of cytotoxic potency on huMovl9-sulfo-SPDB-DM4 concentration. When the conjugate concentration is diluted to half the concentration of the standard, the cytotoxic potency is half that of the standard. Similarly, when the starting concentration of the conjugate is twice that of the standard, the cytotoxic potency is twice that of the standard. Details of specific cytotoxicity assays are provided in Example 6 below.

[0126] With a target DAR of 3.4, the acceptable variability range of the actual DAR present in the final formulation can vary from 2.9 to 3.9 (Figure 3).

[0127] Example 2: DM4 doses cause in vivo toxicity and efficacy With huMovl9-sulfo-SPDB-DM4, it is desirable to achieve the highest possible ADC level and achieve efficacy without approaching the ocular toxicity threshold. As shown in Figure 3, when the huMovl9-sulfo-SPDB-DM4 DAR is approximately 3.4 and the dose range is 3.3-7 mg / kg, 32% of patients were found to exceed the ocular toxicity threshold. When the huMovl9-sulfo-SPDB-DM4 DAR is 2.9 and the dose range is 3.3-7 mg / kg, 13% of patients are expected to exceed the ocular toxicity threshold. When the huMovl9-sulfo-SPDB-DM4 DAR is 3.4 and the dose range is 3.3-7 mg / kg, 32% of patients are expected to exceed the ocular toxicity threshold based on the reduction in DM4 dose they receive. If huMov19-sulfo-SPDB-DM4DAR is 3.9 and the dose ranges from 3.3 to 7 mg / kg, 48% of patients would be expected to exceed ocular toxicity levels based on the increasing DM4 dose they receive.

[0128] At 5 mg / kg huMovl9-sulfo-SPDB-DM4 (adjusted ideal body weight), where the DAR was 2.9, no patients exceeded the ocular toxicity threshold. However, when the DAR was 3.4 or 3.9 with 5 mg / kg huMovl9-sulfo-SPDB-DM4, 14% and 57% of patients exceeded the ocular toxicity threshold, respectively. Actual clinical data for the 3.4 DAR cohort (filled circles on the graph) are shown in Figure 4. The remaining data reflect simulated dose analysis.

[0129] Figure 4 illustrates the importance of ensuring patients receive a dose within the intended narrow range. Ideally, to ensure maximum efficacy and safety, patients receive a dose of huMov19-sulfo-SPDB-DM4 that approaches but does not exceed the ocular toxicity threshold.

[0130] As described in Examples 3 and 4, toxicity for huMovl9-sulfo-SPDB-DM4 depends on the amount of DM4 administered. Preclinical efficacy studies with huMovl9-sulfo-SPDB-DM4 showed that toxicity is independent of the DAR when the DM4 dose is the same. Furthermore, preclinical toxicity studies with huMovl9-sulfo-SPDB-DM4 and many other conjugates showed that toxicity is caused by the dose of ligated DM4, regardless of the DAR.

[0131] Example 3: Antitumor activity of huMovl9-sulfo-SPDB-DM4 was independent of DAR In vivo studies were performed to analyze huMovl9-sulfo-SPDB-DM4 activity in KB and IGROV-1 murine xenograft models (Figure 5). The KB cell line was established from HeLa cell contaminants. The KB cell line is used as a tumor model because it reproducibly forms tumors in nude mice and overexpresses the folate receptor. The IGROV-1 tumor model is derived from a human ovarian carcinoma.

[0132] HuMovl9-Sulfo-SPDB-DM4 with different DARs ranging from 2.5 to 4.1 was administered to mice bearing KB or IGROV-1 tumor xenografts at a DM4 dose of 25 μg / kg and variable antibody doses. As shown in Figure 5, the DAR did not affect efficacy as long as the same DM4 dose was administered. The results of this analysis showed that the DM4 dose determined efficacy in the FOLR1-positive KB and IGROV-1 tumor models regardless of the DAR.

[0133] Example 4: Toxicity was independent of drug-antibody ratio An in vivo study was conducted to evaluate the effect of the drug-antibody ratio on the maximum tolerated dose (MTD) of huMovl9-sulfo-SPDB-DM4 (Figure 6). Mice received huMovl9-sulfo-SPDB-DM4 at a fixed DM4 dose of 1400 μg / kg. The antibody dose was varied. Mouse body weight was monitored as a measure of toxicity. The administered ADC varied significantly in drug-to-antibody ratio (e.g., DAR 9.0 vs. DAR 3.6). Interestingly, as long as the same DM4 dose was administered, the DAR did not affect toxicity within the range of 3.6–9.0. Thus, toxicity was independent of the DAR.

[0134] In another in vivo toxicity analysis, the ADC huEGFR-7R-SMCC-DM1 was administered at a fixed DM1 dose of 3.0 mg / kg. The DAR was varied (e.g., 2.3, 3.5, 6.3, 10.1), but the DM1 dose was kept constant. Mean body weight (BW) change was monitored as an indicator of toxicity. Body weight loss was similar for the different DAR conjugates, indicating that toxicity was independent of DAR as long as the DM1 dose was kept constant (Figure 7).

[0135] This in vivo analysis was extended to antibody-SPDB-DM4 conjugates, including huDS6-SPDB-DM4, huB4-SPDB-DM4, and huMy9-6-SPDB-DM4 (Figure 8). The ADC huDS6-SPDB-DM4 (also known as "huDS6-DM4") is the humanized monoclonal antibody huDS6 linked to the potent cytotoxic maytansinoid DM4 via the cleavable disulfide crosslinker N-succinimidyl-4-2-pyridyldithiobutanoate (SPDB). The ADC huDS6-SPDB-DM4 targets solid tumors such as ovarian, breast, cervical, lung, and pancreatic cancers. The ADC huB4-SPDB-DM4 (also known as "huB4-DM4") is a novel antibody-drug conjugate consisting of a humanized monoclonal IgG1 anti-CD19 antibody (huB4) conjugated to DM4 via the cleavable disulfide crosslinker N-succinimidyl-4-2-pyridyldithiobutanoate (SPDB). The ADC huMy9-6-SPDB-DM4 (also known as "huMy9-6-DM4") is an ADC that specifically binds to CD33, a Siglec family antigen primarily expressed on myeloid cells. The ADC huMy9-6-SPDB-DM4 has undergone clinical evaluation for the treatment of acute myeloid leukemia.

[0136] As shown in Figure 8, the identified conjugates were administered at three to four different doses, and mouse survival rates were measured as an indicator of toxicity. The DAR ranges of the four conjugates were narrow (ranging from 3.49 to 4.0), so the LD 50 The range (the dose at which 50% of animals were lethal) was also narrow (1.6 to 2 mg / kg DM4 dose). Conjugates with different DARs were administered at an antibody dose of 80 mg / kg, and mouse survival rates were measured as an indicator of toxicity (Figure 8). The similar results for all conjugates indicate that toxicity is caused by the total DM4 dose administered, regardless of the DAR. That is, within the DAR range of 2.1 to 5.1, toxicity is not affected by different DARs.

[0137] Example 5: Formulating ADC therapeutic compositions based on drug concentration minimizes efficacy variations resulting from DAR variations. Traditionally, antibody-drug conjugate therapeutic compositions are formulated based on antibody concentration. Figure 9A illustrates the inherent variability in formulating antibody-drug conjugates based on antibody concentration, even while remaining within the acceptable range of specifications. In particular, at the end of the ADC manufacturing process, the antibody concentration is measured and diluted to reach the target drug concentration, which for huMov19-sulfo-SPDB DM4 is 5.0 mg / ml. In Figure 9A, the target antibody concentration (5.0 mg / ml) for huMov19-sulfo-SPDB-DM4 is boxed and the target DAR (3.4) is circled. At this target antibody concentration, the DM4 concentration is 91.1 μg / ml. In practice, the antibody concentration in the final formulation is allowed to vary from the target concentration. The antibody concentration in the final product could be as low as 4.0 mg / ml or as high as 6.0 mg / ml. Thus, depending on the DAR, the DM4 concentration in the final formulation could be reduced to 62.1 μg / ml or increased to 125.4 μg / ml, as indicated by the boxed areas of DM4 concentration.

[0138] Formulating the ADC composition based on DM4 concentration and adding a + / - 10% DM4 concentration specification significantly narrows the potency present in the final formulation to + / - 10% of the target DM4 concentration (highlighted; + / - 10% specification).

[0139] The trend for out-of-specification batches is shown in Figure 9B, highlighting the target DM4 concentration and DAR. DM4 concentration is shown at the top, DAR is shown on the left, and the resulting antibody concentration is shown in the highlighted box (5.0 mg / ml). If the antibody concentration varies from the target by more than ±20%, the batch is out-of-specification. The concentration of antibody present in the out-of-specification batches is shown in bold (Figure 9B). The risk of out-of-specification batches is fairly low.

[0140] In summary, current formulation specifications allow for ±20% variation in antibody base concentration (4.0-6.0 mg / mL). Therefore, ADC potency could vary by as much as ±35% depending on the DAR. By formulating the ADC based on DM4 concentration and varying the DM4 concentration by as much as ±10%, potency can vary by only ±10%. Therefore, the new formulation method eliminates DAR-potency dependence by formulating to a narrow range of DM4 concentrations. Such a formulation strategy only slightly increases the risk of a batch not meeting specifications due to an antibody concentration outside its specifications.

[0141] Example 6: DAR complex formulation The ADC huMovl9-sulfo-SPDB-DM4, which contains the huMovl9 antibody, an SPDB linker, and the cytotoxic drug DM4, is an example of an ADC whose in vitro potency, in vivo efficacy, and in vivo toxicity are independent of the DAR and are driven entirely by the concentration of administered DM4. Therefore, huMovl9-sulfo-SPDB-DM4 is a good candidate for formulation based on DM4 concentration rather than huMovl9 concentration. To test the hypothesis that this narrows formulation efficacy, a series of huMovl9-sulfo-SPDB-DM4 conjugates with a range of DARs were prepared. The conjugates were purified into a basic formulation buffer (10 mM sodium acetate, 9% (w / v) sucrose, pH 5.0), and the DM4 and huMovl9 antibody concentrations in each sample were measured spectrophotometrically at wavelengths of 252 nm and 280 nm, respectively. The molar concentrations of the DM4 and huMovl9 antibody containing conjugates were calculated as follows:

number

[0142] Each of the various DAR conjugates was formulated in two different ways: first, diluted with the base formulation buffer to achieve a target huMovl9 antibody concentration within the specification range of 5.0 mg / mL ± 20%; additionally, the various DAR conjugates were formulated to achieve a target DM4 concentration within the proposed specification of 91.1 μg / mL ± 10%. All samples were subjected to specific cytotoxicity assays.

[0143] The specific cytotoxicity assay involves incubating folate receptor 1 (FOLR1)-positive cells (KB) in the presence of medium containing serial dilutions of the huMovl9-sulfo-SPDB-DM4 drug conjugate in duplicate wells of a sterile, 96-well, flat-bottom, black tissue culture plate with a clear bottom. Each assay plate contains a series of standards, controls, and test articles at identical dilutions in wells with KB cells and medium-blank wells. After a 4-day incubation period at 37°C ± 2°C, plates are removed from the incubator and allowed to equilibrate to room temperature for 1 hour before being assayed using CellTiter-Glo™ Luminescent Cell Viability Reagent was added. After incubating the plates for an additional 2 hours, the luminescent signal on a Victor III plate reader was analyzed and recorded. CellTiter-Glo™ uses a unique, stable form of luciferase to measure ATP as an indicator of viable cells. The luminescent signal generated is directly proportional to the number of viable cells present in a well, which in turn is inversely proportional to the cytotoxicity of the drug in that well. Because the luciferase reaction requires ATP, conditions were created such that the amount of light generated is proportional to the amount of ATP present, which reflects the number of viable cells. The three plate data files were imported into PLA2.0 software, and the EC values ​​for the standards and test articles were calculated. 50 Values ​​were determined from a constrained four-parameter logistic curve fit using all six replicates for each sample. For samples meeting the acceptance criteria for slope difference and parallelism, the % relative potency of the test article was calculated using the IC4PL curve fit derived from the constrained four-parameter logistic curve fit. 50 The % efficacy is calculated as follows:

number

[0144] The results of these calculations are shown in Tables 2 and 3 (below). The standard used for the potency assays in Table 2 was Sample A, while the standard used for the potency assays in Table 3 was Sample F. A dilution series for each sample was generated assuming a trace concentration of 5 mg / mL huMovl9 to mimic how ADCs are administered in a clinical setting. When formulating huMovl9-sulfo-SPDB-DM4 ADCs to target a 5 mg / mL huMovl9 concentration (Table 2), there was a wide range of potency, as expected, with a total difference of 59.8–124.6%, or ~2×, between the highest and lowest potencies of the ADCs. This is in good agreement with the expected range of ±35%. In contrast to formulating huMovl9-sulfo-SPDB-DM4 ADCs with various DARs to target a DM4 concentration of 91.0 mg / mL, the resulting range of relative potencies was much narrower, ranging from 80.9–106.5%, or ~1×. This is in good agreement with the expected range of ±10%. Most of the measured potencies are within 15% of the expected value based on DM4 concentration. This is within the combined experimental error of both potency and concentration measurement assays. Together, these results demonstrate the advantage of formulating to a DM4 concentration target rather than a huMovl9 concentration target, as is typical for ADCs. [Table 2] [Table 3]

[0145] Example 7: Targeting variable antibody and drug concentrations narrows the specification window over a wider range of DAR. In some cases, it may be desirable to achieve small variations in both drug and antibody concentrations through fluctuations in the target drug concentration rather than large variations in non-target concentrations (e.g., antibody concentrations). Formulating ADC compositions using such methods maximizes the specification range by targeting the middle of the range where both antibody and drug concentrations overlap at a particular DAR value. In practice, drug specifications are narrower than antibody specifications (e.g., ±10% for drug vs. ±15% for antibody). Thus, allowing smaller variations in antibody and drug concentrations provides an additional control strategy for achieving tighter drug concentration specifications (rather than absolute targets) while minimizing the risk of a batch that is out of specification but is used perfectly safely.

[0146] The desirability of using the above-described methods to formulate an ADC of huMy9-6-sulfo-SPDB-DGN462 is demonstrated. The huMy9-6-sulfo-SPDB-DGN462 ADC is a CD33-targeted antibody-drug conjugate comprising the antibody huMy9-6 conjugated to the novel DNA alkylating agent, DGN462, via the cleavable disulfide linker, sulfo-SPDB.

[0147] In each of Figures 10A, 10B, and 10C, the upper and lower limits of the antibody specification range are indicated by dotted lines, the X-axis indicates the DAR of the drug batch, with a target DAR of 2.7; the vertical lines indicate the upper and lower limits of the DGN462 specification at a given DAR, and the dark gray diamonds on each vertical line indicate the overlap between the DGN462 specification range and the antibody specification range at a particular DAR. In Figure 11A, when the batch DAR is 2.7, the center of the DGN462 specification range falls neatly within the center of the antibody range. In Figure 10B, when the batch DAR is close to the target DAR, the fixed DGN462 target concentration required to achieve the antibody concentration falls well within the upper and lower antibody specification limits. In Figure 10C, when the DAR of a batch approaches the upper and lower DAR specification limits, 3.0-3.1 and 2.3-2.4, respectively, the amount of antibody required to achieve the DGN462 target concentration approaches the upper and lower antibody concentration specification limits, resulting in a fixed DGN462 concentration that approaches or exceeds the defined antibody concentration specification. Improved formulation methods are therefore desirable.

[0148] Figures 11A-11C show the improvement obtained by varying both DGN462 and antibody concentrations, particularly when the DAR approaches the upper and lower DAR specification limits (e.g., 3.0-3.1 and 2.3-2.4). Targeting a variable drug concentration identifies the middle of the range where the antibody concentration specification range and the drug concentration specification range overlap (Figure 11A). The center of the overall drug efficacy range is well below the upper antibody specification limit. Thus, targeting a variable DGN462 at the upper and lower DAR specification limits produces a more consistent product by limiting the target antibody concentration to + / - 10% variation instead of a full ±15% variation. In contrast, for batches where the DAR approaches the upper and lower limits, varying the antibody concentration to achieve a fixed DGN462 target concentration results in larger deviations from the target antibody concentration, increasing the risk of efficacy and toxicity variability (Figures 11A and 11B). Figure 11C shows the improvement provided by the method of targeting variable drug concentrations to the upper and lower limits of the DAR specification, with DGN462 and antibody concentrations maintained between narrower limits of 4% (white ovals) compared to using antibody concentration (grey diamonds) to achieve the target drug concentration (fixed target total drug exceeds the antibody upper specification limit at DAR 2.3).

[0149] The following formulas are useful for calculating upper and lower specification limits for a drug:

number

[0150] Table 4 shows the method used to calculate the upper and lower specification limits for DGN462 (USL DGN462, LSL DGN462). The upper antibody concentration specification limit is constant. The DAR is empirically determined for each batch of antibody drug conjugate. Calculation of the desired upper and lower specification limits was performed as follows:

number

[0151] The values ​​"2.30" and "1.70" define the upper and lower limits of the antibody specification. The denominator is the molecular weight of the antibody. [Table 4] - When LSL and USL are calculated at specific points, these limits may deviate from the proposed specifications. These outliers can be set to either LSL (34.0 mg / mL) or USL (41.5 mg / mL) using the > or < rule. -It is suggested that for formulation purposes, DAR be reported to two decimal places.

[0152] In the following example, an ADC is formulated by targeting a variable drug concentration. Here, the ADC contains the non-functional antibody huMovl9 (molecular weight 145676 g / mol) conjugated to D2 (molecular weight 961.05 g / mol), which has a target DAR of 2.7, an antibody concentration of 2.0 mg / mL, and a cytotoxic agent concentration of 39.2 μg / mL. The ADC is formulated to target a variable drug concentration and minimize the offset of the antibody concentration from the target. As shown in Figure 11A, targeting a variable drug concentration allows the resulting antibody concentration to vary by ±10% (1.8-2.2), compared to ±15% when using a static drug concentration (Figure 11A; see, e.g., Tables 4-7). Targeting a variable drug identifies the middle of the range where the antibody and drug specification ranges overlap (Figure 11A; see, e.g., Tables 4-7).

[0153] Table 5 shows the method used to calculate the upper and lower specification limits for D2 (USL D2, LSL D2). The upper and lower antibody concentration specification limits are fixed and the DAR is determined empirically. [Table 5]

[0154] In another example, an ADC is formulated to target variable cytotoxic agent concentrations. Here, the ADC comprises the non-functional antibody huEGFR-7R (molecular weight 144975 g / mol) conjugated to D1 (molecular weight 838 g / mol), with a target DAR of 2.7, an antibody concentration of 2.0 mg / mL, and a cytotoxic agent concentration of 34.3 μg / mL. The ADC is formulated to target variable cytotoxic agent concentrations and minimize the offset of the antibody concentration from the target.

[0155] Table 6 shows the method used to calculate the upper and lower specification limits for D1 (USL D1, LSL D1). The upper and lower antibody concentration specification limits are fixed and the DAR is determined empirically. [Table 6]

[0156] In yet another example, an ADC is formulated to target variable cytotoxic agent concentrations. Here, the ADC comprises the functional antibody huMy9-6 (molecular weight 146192 g / mol) conjugated to D10 (molecular weight 1062.22 g / mol), which has a target DAR of 2.7, an antibody concentration of 2.0 mg / mL, and a cytotoxic agent concentration of 44.0 μg / mL. The ADC is formulated to target variable cytotoxic agent concentrations and minimize the offset of the antibody concentration from the target.

[0157] Table 7 shows the method used to calculate the upper and lower specification limits for D1 (USL D1, LSL D1). The upper and lower antibody concentration specification limits are fixed and the DAR is determined empirically. [Table 7]

[0158] (Other embodiments) From the foregoing description, it will be apparent that the invention described herein can be modified and adapted to suit various uses and conditions, and such embodiments also fall within the scope of the following claims.

[0159] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as either a single element or as any combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0160] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for reducing potency variability in an antibody-drug conjugate composition, comprising: (a) measuring the drug-antibody ratio DAR of the antibody-drug conjugate composition; (b) determining an upper antibody specification limit and a lower antibody specification limit, the upper antibody specification limit being the target antibody concentration plus the maximum variation allowed by the specification, and the lower antibody specification limit being the target antibody concentration minus the maximum variation allowed by the specification; (c) determining an upper defined drug specification limit and a lower defined drug specification limit, the upper defined drug specification limit being the target drug concentration plus the maximum variation allowed in the specification, and the lower defined drug specification limit being the target drug concentration minus the maximum variation allowed in the specification; (d) Determining the upper limit of drug specification (USL drug) calculated as follows: USL (drug) μg / mL = upper limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (e) Determining the lower limit of drug specification (LSL drug), calculated as follows: LSL (drug) μg / mL = lower limit of antibody concentration specification × DAR × molecular weight of drug × 1000 / molecular weight of antibody (f) comparing the USL drug calculated in step (d) with the upper limit of drug specification defined in step (c) and selecting the lower of the two values ​​as the effective upper limit of drug specification; (g) comparing the LSL(drug) calculated in step (e) with the lower limit of the drug specification defined in step (c) and selecting the higher of the two values ​​as the effective lower limit of the drug specification; and (h) reducing efficacy variability in the antibody-drug conjugate composition by formulating the composition to a target drug concentration that is midpoint between the effective upper limit of the drug specification and the effective lower limit of the drug specification. The method comprising:

2. 10. The method of claim 1, wherein the method narrows the range between upper and lower specification limits for the drug and the antibody to about ±3-9%.

3. 10. The method of claim 1, wherein the method narrows the range between upper and lower specification limits for the drug to about ±4%.

4. 10. The method of claim 1, wherein the maximum variation allowed in the specifications in step (b) is less than 15%.

5. 10. The method of claim 1, wherein the maximum variation allowed in the specifications in step (b) is less than 10%.

6. 10. The method of claim 1, wherein the maximum variation allowed in the specifications in step (c) is 15%.

7. 10. The method of claim 1, wherein the maximum variation allowed in the specifications in step (c) is less than 10%.

8. The method of any one of claims 1 to 7, wherein the antibody is a non-functional antibody.

9. The method of any one of claims 1 to 8, wherein the antibody concentration and the drug concentration are allowed to vary.

10. The method of any one of claims 1 to 9, wherein the drug is a maytansinoid.

11. The drug is a maytansinoid, and the antibody is a non-functional antibody.

11. The method according to any one of claims 10 to 10.

12. 12. The method of any one of claims 1 to 11, wherein said method reduces batch-to-batch potency variability in manufacturing said antibody-drug conjugate.

13. 13. The method of any one of claims 1 to 12, wherein the antibody-drug conjugate composition exhibits reduced variability in efficacy compared to when the composition is formulated based solely on antibody concentration.

14. The method of any one of claims 1 to 13, wherein the antibody-drug conjugate comprises a cleavable linker.

15. 15. The method of claim 14, wherein the cleavable linker is N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), or the disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP).

16. the antibody-drug conjugate comprises an antibody that binds to folate receptor alpha and comprises a light chain and a heavy chain comprising the amino acid sequences of SEQ ID NOs: 4 and 5, respectively; 16. The method of any one of claims 1 to 15, wherein the antibody is conjugated to N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4) via the linker N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sSPDB).

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