Methods for formulating antibody drug conjugate compositions

Abstract

The present invention provides improved methods for formulating therapeutic compositions comprising antibody drug conjugates ("ADCs") that reduce variability in potency between ADC batches and provide for administration of the therapeutic compositions within a narrow, predetermined range. The present invention provides novel methods for formulating therapeutic compositions comprising antibody drug conjugates ("ADCs") based on drug concentration, thereby reducing variability in potency between ADC batches, minimizing toxicity, and increasing the efficacy of drugs formulated according to the methods.

Description

[0001] This application is a divisional application of the invention patent application with application number 201580047269.8, application date September 2, 2015, applicant is IMUNOGEN Co., Ltd., and invention title is “Method for preparing antibody drug conjugate composition”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62 / 044,592, filed September 2, 2014, which is incorporated herein by reference in its entirety. Background of the Invention

[0005] 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 usually conjugated to antibodies at cysteine ​​or lysine residues. The number of drug molecules conjugated to each antibody, also known as drug to antibody ratio ("DAR"), is typically a species distribution within the range of 0-8. The DAR of the ADC manufactured in a batch is empirically determined using spectrophotometric measurements, and ADC therapeutic compositions typically contain a mixture of different ADC species in terms of drug loading. Therefore, the DAR of an ADC batch represents the average DAR of the ADC species within the batch.

[0006] ADC cancer therapeutics and antibody cancer therapeutics are both formulated based on nominal antibody protein concentrations and must meet specifications. Although drug labels provide information about "nominal" or target protein concentrations, the drug concentration in the vial can vary relative to the target antibody concentration because, even when acceptance criteria are met, variations in DAR are permitted. The potency of ADCs is generally linear with respect to concentration. Unlike antibodies, ADCs have additional potency variability potential due to DAR. When patient dosing is based on nominal antibody concentrations, the typical specifications of antibody concentration and DAR cause the concentration of the cytotoxic drug in the ADC product vial to vary slightly from batch to batch.

[0007] It is important that patients receive a safe and effective dose of ADC. Improved methods of formulating ADC compositions would advantageously reduce batch-to-batch variability in potency, efficacy, and / or toxicity and ensure that patients receive an ADC within the intended therapeutic range. Summary of the Invention

[0008] The present invention provides a novel method for formulating therapeutic compositions comprising antibody drug conjugates ("ADCs") based on drug concentration, thereby reducing variability in potency between batches of ADCs, minimizing toxicity, and increasing the efficacy of drugs formulated according to this method.

[0009] The present invention is based, at least in part, on the discovery that the efficacy and toxicity of some ADCs are driven entirely or primarily by the concentration of the administered drug rather than by the antibody concentration. Conventional methods for formulating pharmaceutical compositions containing antibody-based therapeutics including ADCs have relied on dosing patients based on antibody concentration. While this may be advantageous for compositions containing only antibodies, formulating ADCs using antibody concentration can result in drug concentration variations and potentially fall outside the desired range. The efficacy of an ADC is generally linear with respect to the concentration of the attached drug, which can be affected by both the antibody concentration and the DAR, as illustrated by the formula [drug] = DAR * [antibody]. The DAR, antibody, and drug each vary within a given and acceptable range as defined by a given ADC specification. However, because the drug and antibody are linked, variability in one component can affect the other. For example, a variability of ±10-20% in antibody concentration within the acceptable range according to industry standards will result in a ±10-20% variation in drug concentration, which can result in a ±20-40% variability in the efficacy of the drug in the vial. A ±15% variation in DAR will add further variation in potency, as this will result in a 15% increase or decrease in drug concentration. This effect may be particularly relevant for a subset of specific ADCs where drug concentration has been shown to be the primary driver of toxicity and efficacy.

[0010] A cytotoxic agent, also known as a "drug" molecule, is attached to the ADC, and the number of drug molecules conjugated per antibody molecule is represented by the term 'drug to antibody' ratio ("DAR"). The DAR of a manufacturing batch of ADCs is determined empirically using spectrophotometric measurements by obtaining the ratio of drug concentration to antibody concentration. The DAR of a particular batch of ADCs represents the average number of drugs attached to each antibody molecule within that batch. Typical DAR specifications for clinical development are within the range of ±10-15%. Conventionally, the initial step in formulating an ADC is to determine the molar concentrations of both the drug and the antibody, and to calculate the DAR. The ADC is then formulated to obtain the target antibody concentration, thereby varying the drug concentration according to the manufacturing DAR value as follows:

[0011] [Drug] = DAR * [Antibody].

[0012] In contrast, the present invention is based on the discovery that by formulating ADC compositions based on target drug concentrations determined at a fixed antibody concentration and a fixed DAR, variability in efficacy and toxicity can be minimized. Therefore, in cases where the efficacy, efficacy, and / or toxicity of an ADC have been shown to be primarily driven by the amount of drug administered, improved methods for reducing the variability in cytotoxic drug concentrations would be beneficial. Thus, the following formulation method comprises determining a target drug concentration at a fixed antibody concentration and a fixed DAR, and formulating an antibody drug conjugate composition to achieve the target drug concentration. The improved formulation method ensures that patients are dosed within a narrow predetermined drug range without the risk of additional batch failures.

[0013] In one aspect, the present invention generally provides a method for reducing the potency variability of an antibody drug conjugate composition (e.g., at least about 5%, 10%, 20% or more), the method involving determining a target drug concentration at a fixed antibody concentration and drug-antibody ratio; and formulating the antibody drug conjugate composition to obtain the target drug concentration, thereby reducing the potency variability of 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., at least about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more). It should be noted that when the variation is expressed as ±, it is intended to describe a variation that is higher or lower than a specified value by a specific % . When the variation is expressed as a single total value (e.g., at least 10%), it is intended to represent the difference between the maximum potential value and the minimum potential value. In another embodiment, the composition is a finished drug product. In 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%.

[0014] In another different aspect, the present invention provides a method for reducing the potency variability of a composition comprising an antibody drug conjugate. The method involves formulating an antibody drug conjugate with a variable drug concentration as a target, wherein the variable drug concentration falls at the midpoint of a range where the antibody concentration specification range and the drug concentration specification range overlap, thereby reducing the potency variability of the composition. In this regard, the present invention provides a method for reducing the potency variability of a composition containing an antibody drug conjugate, comprising: (a) measuring the DAR of the antibody drug conjugate composition; (b) determining an antibody upper specification limit and an antibody lower specification limit, wherein the antibody upper specification limit is the target antibody concentration plus the maximum variation allowed by the specification, and the antibody lower specification limit is the target antibody concentration minus the maximum variation allowed by the specification; (c) determining a defined drug upper specification limit and a defined drug lower specification limit, wherein the defined drug upper specification limit is the target drug concentration plus the maximum variation allowed by the specification, and the defined drug lower specification limit is the target drug concentration minus the maximum variation allowed by the specification; (d) calculating a drug specification upper limit (USL(drug)) as follows:

[0015]

[0016] (e) Determine the calculated lower specification limit for drug (LSL(drug)) as follows:

[0017]

[0018] (f) comparing the calculated USL(drug) of step (d) with the defined upper drug specification limit of step (c), and selecting the lower of the two values ​​as the effective upper drug specification limit; (g) comparing the calculated LSL(drug) of step (e) with the defined lower drug specification limit of step (c), and selecting the higher of the two values ​​as the effective lower drug specification limit; and (h) formulating the antibody drug conjugate composition to a target drug concentration that is midway between the effective upper drug specification limit and the effective lower drug specification limit, thereby reducing the potency variability of the composition. In one embodiment, the method reduces the range of the upper and lower specification limits of the drug to about ±3-9%. In another embodiment, the method reduces the range of the upper and lower specification limits of the drug 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 specification 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 at the lower limit of the DAR specification or at the upper limit of the DAR specification. 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.

[0019] In a related aspect, for example, when the antibody is a functional antibody, the present invention also provides a method for formulating an antibody drug conjugate by targeting a variable antibody concentration that falls at the midpoint of a range where the antibody concentration specification range and the drug concentration specification range overlap, thereby targeting an antibody concentration that will minimize fluctuations in drug concentration when formulating the ADC. This allows for tighter control of the antibody concentration in the ADC formulation.

[0020] In another aspect, the present invention provides a method of formulating an antibody drug conjugate composition, the method involving determining a target drug concentration at a fixed antibody concentration and drug-to-antibody ratio; and formulating the antibody drug conjugate composition to achieve the target drug concentration.

[0021] In another aspect, the present invention provides a method of reducing the potency variability of a composition comprising an antibody maytansinoid conjugate, the method comprising determining a target maytansinoid concentration at a fixed antibody concentration and maytansinoid to antibody ratio; and formulating the antibody maytansinoid conjugate composition to achieve the target maytansinoid concentration, thereby reducing the potency variability of the composition.

[0022] In another aspect, the present invention provides a method of formulating an antibody maytansine conjugate composition, the method comprising determining a target maytansine concentration at a fixed antibody concentration and maytansine to antibody ratio; and formulating the antibody maytansine conjugate composition to achieve the target maytansine concentration.

[0023] In another aspect, the invention provides a method of formulating an antibody-benzodiazepine (e.g., pyrrolobenzodiazepine or indolinolobenzodiazepine) conjugate composition, the method comprising determining a target benzodiazepine concentration at a fixed antibody concentration and benzodiazepine to antibody ratio; and formulating the antibody-benzodiazepine conjugate composition to achieve the target benzodiazepine concentration.

[0024] In another different aspect, the present invention provides a method for reducing potency variability of a composition containing an antibody-benzodiazepine (e.g., pyrrolobenzodiazepine or indolinolobenzodiazepine) conjugate, comprising: (a) measuring the DAR of the antibody-benzodiazepine (e.g., pyrrolobenzodiazepine or indolinolobenzodiazepine) conjugate composition; (b) determining an antibody upper specification limit and an antibody lower specification limit, wherein the antibody upper specification limit is the target antibody concentration plus the maximum variation allowed by the specification, and and the antibody lower specification limit is the target antibody concentration minus the maximum variation allowed by the specifications; (c) determining a defined benzodiazepine upper specification limit and a defined benzodiazepine lower specification limit, wherein the defined benzodiazepine upper specification limit is the target benzodiazepine concentration plus the maximum variation allowed by the specifications, and the defined benzodiazepine lower specification limit is the target benzodiazepine concentration minus the maximum variation allowed by the specifications; (d) determining a calculated benzodiazepine upper specification limit (USL(drug)) as follows:

[0025]

[0026] (e) Determine and calculate the lower specification limit (LSL(drug)) for benzodiazepines as follows:

[0027]

[0028] (f) comparing the calculated USL(drug) of step (d) with the defined benzodiazepine upper specification limit of step (c), and selecting the lower of the two values ​​as the effective benzodiazepine upper specification limit; (g) comparing the calculated LSL(drug) of step (e) with the defined benzodiazepine lower specification limit of step (c), and selecting the higher of the two values ​​as the effective benzodiazepine lower specification limit; and (h) formulating the antibody-benzodiazepine conjugate composition to a target benzodiazepine concentration that is midway between the effective benzodiazepine upper specification limit and the effective benzodiazepine lower specification limit, thereby reducing variability in the potency of the composition.

[0029] In another aspect, the invention provides a method for administering a drug to a subject within a narrow predetermined range, the method involving providing an antibody drug conjugate composition formulated according to the method of any previous aspect, and administering the composition to the subject.

[0030] In another aspect, the invention provides a pharmaceutical composition containing an antibody drug conjugate formulated according to the method of the previous aspect, wherein the nominal drug (eg, maytansinoid, benzodiazepine, auristatin) concentration is provided on the label.

[0031] In various embodiments of any previous aspect or any other aspect of the present 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 maytansine-like compounds; benzodiazepine compounds, such as pyrrolobenzodiazepines and indolinebenzodiazepines; and auristatins. In specific embodiments of the previous aspects, the method reduces batch-to-batch potency variations (e.g., at least about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more). In other embodiments, the composition is a finished drug. In other embodiments, the antibody concentration varies within the antibody specification concentration. In other embodiments of the previous aspects, when producing an antibody maytansine conjugate, the method reduces batch-to-batch potency variability. In various embodiments of the previous 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 previous aspects, the composition is allowed to vary in potency by about 10-20% (e.g., 10, 12, 15, 18, 20%). In 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 previous aspects, the variability in composition potency is reduced relative to when the antibody drug conjugate composition is formulated based on antibody concentration. In various embodiments of the previous aspects, the antibody concentration and conjugated drug (e.g., maytansine, benzodiazepine, auristatin) concentration are determined by spectrophotometric measurements. In various embodiments of the previous aspects, the drug to antibody ratio is determined by size exclusion chromatography (SEC) or by SEC-mass spectrometry (SEC-MS). In other embodiments of the previous aspects, the efficacy or toxicity of the composition does not depend on the drug to antibody ratio or antibody concentration, but rather depends on the total administered dose of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin). In other embodiments of the previous aspects, the efficacy of the composition does not depend or does not substantially depend on the drug to antibody ratio. In various embodiments of the previous aspects, the toxicity of the composition does not depend or does not substantially depend on the drug to antibody ratio. In various embodiments of the previous aspects, the efficacy or toxicity depends or substantially depends on the conjugated drug (e.g., maytansine, benzodiazepine, auristatin) concentration. In various embodiments of the previous aspects, the efficacy depends on, substantially depends on, or at least partially depends on the conjugated drug (e.g., maytansine, benzodiazepine, auristatin) concentration.In various embodiments of the previous aspects, toxicity depends on, substantially depends on, or at least partially depends on the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin). In various embodiments of the previous aspects, efficacy does not depend on, substantially does not depend on, or at least partially does not depend on the antibody concentration. In various embodiments of the previous aspects, toxicity does not depend on, substantially does not depend on, or at least partially does not depend on the antibody concentration. In various embodiments of the previous aspects, efficacy and toxicity depend on, substantially depends on, or at least partially depends on the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin) and the antibody concentration. In various embodiments of the previous aspects, efficacy and toxicity depend on, substantially depends on, or at least partially depends on the antibody concentration compared to the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin). In various embodiments of the previous aspects, efficacy depends on, substantially depends on, or at least partially depends on the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin) and the antibody concentration. In various embodiments of the previous aspects, toxicity depends on, depends approximately on, or depends at least in part on the conjugated drug (e.g., maytansinoid, benzodiazepine, auristatin) concentration and the antibody concentration. In various embodiments of the previous aspects, efficacy depends on, depends approximately on, or depends at least in part on the conjugated drug (e.g., maytansinoid, benzodiazepine, auristatin) concentration and the antibody concentration, and toxicity depends on, depends approximately on, or depends at least in part on the conjugated drug concentration. In various embodiments of the previous aspects, efficacy depends on, depends approximately on, or depends at least in part on the conjugated drug (e.g., maytansinoid, benzodiazepine, auristatin) concentration and the antibody concentration, and toxicity depends on, depends approximately on, or depends at least in part on the antibody concentration. In various embodiments of the previous aspects, toxicity depends on, depends approximately on, or depends at least in part on the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin) and the antibody concentration, and efficacy depends on, depends approximately on, or depends at least in part on the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin). In various embodiments of the previous aspects, toxicity depends on, depends approximately on, or depends at least in part on the antibody concentration, and efficacy depends on, depends approximately on, or depends at least in part on the concentration of the conjugated drug (e.g., maytansine, benzodiazepine, auristatin). In various embodiments of the previous aspects, the antibody drug conjugate composition is formulated for infusion. In various embodiments of the previous aspects, the antibody drug conjugate is formulated with a pharmaceutically acceptable parenteral vehicle. In various embodiments of the previous aspects, the antibody drug conjugate is formulated in a unit dose injectable form.

[0032] In various embodiments of any previous aspect or any other aspect 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 formula:

[0033]

[0034]

[0035] 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 disrupting agent.

[0036] In other embodiments of any of the above aspects, the drug includes, but is not limited to, maytansine and maytansine analogs, benzodiazepine compounds (e.g., pyrrolobenzodiazepines and indolinolobenzodiazepines; see also Table 1: compounds D1-D10 and DGN462), taxoids, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes (such as calicheamicin), dolastatin and dolastatin analogs. analogs (including auristatins), tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino-doxorubicin.

[0037] In various embodiments of the previous aspects, the maytansinoid is DM1, DM3, or DM4. In various embodiments of the previous aspects, the benzodiazepine compound is selected from the representative cytotoxic agents D1-D10 and DGN462 listed in Table 1 below.

[0038] Table 1. Benzodiazepine compounds

[0039]

[0040]

[0041] It should be noted that other variations of the compounds listed in Table 1 (eg, sulfonated forms) are also contemplated and will be readily apparent to those skilled in the art.

[0042] In various embodiments of the previous aspects, the antibody may be a functional antibody or a non-functional antibody. Non-functional antibodies include, for example, huDS6 and antibodies with only effector-mediated cell killing, such as huMov19 (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 maytansine, and the antibody is a non-functional antibody.

[0043] In various embodiments of the previous aspects, the linker is a cleavable linker, such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 4-(2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB), or N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP). In various embodiments of the previous aspects, the linker is a non-cleavable linker, such as 2-iminothiolane, acetyl succinic anhydride, 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC). The universal linkers 2-iminothiolane and acetyl succinic anhydride can be used as cleavable or non-cleavable linkers.

[0044] In various embodiments of the foregoing aspects, the linker antibody drug conjugate is huMov19-Sulfo-SPDB-DM4, huMov19-Sulfo-SPDB-D1, huMov19-D2, huMov19-Sulfo-SPDB-D10, huMov19-Sulfo-SPDB-DGN462, huMy9-6-Sulfo-SPDB-D1, huMy9-6-D2, huMy9-6-Sulfo-SPDB-D10, huMy9-6-Sulfo-SPDB-D GN462, 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.

[0045] Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

[0046] definition

[0047] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention pertains. The following references provide general definitions of many of the terms used herein by those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular 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 and Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, as used herein, the following terms have the meanings ascribed to them.

[0048] The term "adjusted ideal body weight (AIBW)" refers to a body descriptor that takes into account sex, total body weight, and height. AIBW can be calculated, for example, using the formula AIBW = IBW + 0.4 (weight in kilograms - IBW), where:

[0049] Ideal body weight (IBW)

[0050] 1.IBW 1 (Male)=0.9 1 -88

[0051] 2.IBW 1 (Female) = 0.9H 1 -92.

[0052] ( 1 H = height in cm; W = weight in kg)

[0053] IBW, LBW, and ADJ are discussed in more detail in Green and Duffull, British Journal of Clinical Pharmacology 58: 119-133 (2004), which is herein incorporated by reference in its entirety.

[0054] In the case of "cytotoxic agent", it means a small molecule 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 the case of an antibody drug conjugate (ADC), the term "drug" can be used interchangeably with the term "cytotoxic agent". In specific embodiments, a cytotoxic agent (or "drug") is conjugated to an antibody. In a specific embodiment, the cytotoxic agent is a maytansine, such as DM1, DM3 or DM4. In other embodiments, cytotoxic agents include, but are not limited to, benzodiazepine compounds (e.g., pyrrolobenzodiazepines and indolinolobenzodiazepines; see also Table 1: compounds D1-D10 and DGN462), taxanes, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes (such as calicheamicin), dolastatins and dolastatin analogs (including auristatins), tomamycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino-doxorubicin.

[0055] By "drug to antibody ratio (DAR)" is meant the average number of "drug" (i.e., cytotoxic agent) molecules conjugated per antibody. The DAR is 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.

[0056] By "maytansinoid to antibody ratio (MAR)" it is meant the average number of maytansinoid molecules conjugated per antibody.

[0057] By "target antibody concentration" is meant the desired antibody concentration.

[0058] By "target drug concentration" or "target cytotoxic agent concentration," it is meant the desired drug or cytotoxic agent concentration. It should be noted that the concentration of the drug or cytotoxic agent is calculated primarily based on the conjugated form of the drug, but may include small amounts of free or unconjugated drug found in the sample.

[0059] By "target maytansinoid concentration" is meant the desired maytansinoid concentration.

[0060] By "potency variability," it is meant the different potencies that exist in different batches of a drug product. It is desirable to reduce potency variability by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50% or more.

[0061] By "drug product" is meant a finished dosage form containing an active pharmaceutical ingredient. In one embodiment, the finished drug product is a container (eg, a vial) containing an antibody drug conjugate of the invention, alone or in combination with an excipient.

[0062] By "specifications," it is meant a set of criteria that a drug or pharmaceutical product must meet in order to be acceptable for its intended use. Specifications are typically set forth by the manufacturer and agreed to by a regulatory agency, such as the FDA.

[0063] As used herein, "functional antibody" is intended to refer to an antibody that affects cell death by a direct cell killing mechanism (such as apoptosis or necrosis). A functional antibody has direct cell killing activity in vivo when not conjugated to a drug ("naked antibody"). Non-limiting examples of functional antibodies include huEGFR-7R antibody and huCD37-3 antibody. As used herein, "non-functional antibody" is intended to refer to an antibody that (i) has no known cell killing activity in vivo (e.g., as a naked antibody, has no direct or indirect cell killing, such as huDS6) or (ii) has indirect cell killing activity due to effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), or (iii) has increased conjugate activity in vivo when effector function increases, or any combination of (i), (ii), and (iii). A non-functional antibody may have anti-proliferative activity, such as by blocking the binding of a proliferator (e.g., a growth factor). Non-limiting examples of non-functional antibodies having indirect cell-killing activity include huMov19, huMy9-6, and huB4.

[0064] By "huB4" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to CD19, such as human CD19. An exemplary huB4 antibody of the invention may include the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences:

[0065] huB4 LC (SEQ ID NO: 1)

[0066]

[0067] huB4HC (SEQ ID NO: 2)

[0068]

[0069]

[0070] By "huB4-SPDB-DM4" is meant a huB4 antibody that specifically binds to CD19 conjugated to a cytotoxic maytansine N-succinimidyl ester via the linker 4-(2-pyridyldithio)butyrate (SPDB). 2 -Deacetyl-N 2’ -(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4) antibody drug conjugate. huB4-SPDB-DM4 is described, for example, in U.S. Patent No. 8,435,528 and International Patent Application Publication No. WO2004 / 103272, which are incorporated herein by reference in their entirety.

[0071] By "huMov19" (also referred to as "M9346A"), it is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to folate receptor alpha (also referred to herein as folate receptor 1 or "FOLR1"). The detailed sequence of huMov19 is described in U.S. Patent Nos. 8,557,966 and 8,709,432 and International Patent Application Publication No. WO2011 / 106528, which are incorporated herein by reference in their entireties. Exemplary huMOV19 antibodies of the present invention may include the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences:

[0072] huMov19 LC v1.00 (SEQ ID NO: 3)

[0073]

[0074]

[0075] huMov19 LC v1.60 (SEQ ID NO: 4)

[0076]

[0077] huMov19 HC (SEQ ID NO: 5)

[0078]

[0079] By "huMov19-sulfo-SPDB-DM4" (also referred to as "IMGN853") is meant a huMov19 antibody that specifically binds to FOLR1 conjugated to a cytotoxic maytansine N-succinimidyl ester via a disulfide linker containing 4-(2-pyridyldithio)-2-sulfobutyrate. 2 '-Deacetyl-N 2Antibody drug conjugates of '-(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4). The ADC huMov19-sulfo-SPDB-DM4 is described, for example, by Ab et al., AACR; Cancer Res 2011; 71(8 Suppl): Abstract No. 4576, as well as U.S. Patent Nos. 8,557,966 and 8,709,432, and International Patent Application Publication No.: WO2011 / 106528, each of which is incorporated herein by reference in its entirety.

[0080] By "huDS6" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to the CA6 sialoglycotope on the Muc1 mucin receptor (e.g., human Muc1) expressed by cancerous cells. Exemplary sequences of huDS6 are described in U.S. Patent No. 7,834,155 and International Patent Application Publication Nos.: WO2005 / 009369 and WO2007 / 024222, which are incorporated herein by reference in their entireties. The huDS6 antibodies of the present 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:

[0081] huDS6 LC (SEQ ID NO: 6)

[0082]

[0083] huDS6 HC (SEQ ID NO: 7)

[0084]

[0085] By "huMy9-6" (also referred to as "Z4681A") is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to the leukocyte differentiation antigen CD33, such as human CD33. Exemplary sequences of the huMy9-6 heavy chain variable region portion are described in U.S. Patent Publication No. 20060177455, which is incorporated herein by reference in its entirety. Exemplary sequences of the huMy9-6 light chain variable region portion 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, which are incorporated herein by reference in their entirety. 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:

[0086] huMy9-6LC (SEQ ID NO: 8)

[0087]

[0088] huMy9-6HC (SEQ ID NO: 9)

[0089]

[0090] By "huMy9-6-sulfo-SPDB-DGN462" (also known as "IMGN779") is meant an anti-huCD33 antibody conjugated via a cleavable disulfide linker to an indolinobenzodiazepine dimer containing a monoimine moiety known as DGN462.

[0091]

[0092] By "huEGFR-7R" (also referred to as "J2898A") is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to EGFR, such as human EGFR. Exemplary huEGFR-7R antibodies may comprise or consist of the following CDRs (shown in bold and underlined) or the following light chain (LC) and heavy chain (HC) sequences:

[0093] huEGFR-7R LC v1.0(SEQ ID NO:10)

[0094]

[0095] huEGFR-7R LC vl.01(SEQ ID NO:11)

[0096]

[0097] huEGFR-7R HC (SEQ ID NO: 12)

[0098]

[0099]

[0100] By "huEGFR-7R-SMCC-DM1" (also referred to as "IMGN289") is meant an antibody drug conjugate comprising a huEGFR-7R antibody that specifically binds to EGFR conjugated to the maytansine N(2')-desacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1) via the linker 4-(maleimidomethyl)cyclohexanecarboxylic acid N-succinimidyl ester (SMCC). The ADC huEGFR-7R-SMCC-DM1 is described, for example, in U.S. Patent No. 8,790,649 and International Patent Application Publication No. WO2012 / 058588, which are incorporated herein by reference in their entireties.

[0101] By "huCD37-3" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to CD37, such as human CD37. Exemplary sequences of huCD37-3 are described in U.S. Patent No. 8,765,917 and International Patent Application Publication No. WO2011 / 112978, which are incorporated herein by reference in their entireties. An exemplary huCD37-3 antibody of the present 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:

[0102] huCD37-3 LC (SEQ ID NO: 13)

[0103]

[0104] huCD37-3 HC v1.0(SEQ ID NO:14)

[0105]

[0106] huCD37-3 HC v1.1(SEQ ID NO:15)

[0107]

[0108] By "huCD37-3-SMCC-DM1" (also referred to as "IMGN529") is meant an antibody drug conjugate comprising a humanized IgG1 antibody K7153A that specifically binds to CD37 covalently linked to the maytansine-like N(2')-desacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1) via a maleimide-derived thioether-based non-cleavable linker, 4-[N-maleimidomethyl]cyclohexane-1-carboxylic acid succinimidyl ester (SMCC).

[0109] By "huCD37-50" is meant a humanized antibody or epitope-binding fragment thereof that specifically binds to CD37, such as human CD37. Exemplary sequences of huCD37-50 are described in U.S. Patent No. 8,765,917 and International Patent Application Publication No. WO2011 / 112978, which are incorporated herein by reference in their entireties. An exemplary huCD37-50 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:

[0110] huCD37-50 LC (SEQ ID NO: 16)

[0111]

[0112] huCD37-50 HC (SEQ ID NO: 17)

[0113]

[0114] By "huAnti-CD123," it is meant 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 Application Serial No. 62 / 186,161, which is incorporated herein by reference in its entirety.

[0115] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies; complete monoclonal antibodies; epitope-binding antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments); single-chain Fv (scFv) mutants; immunoglobulin novel antigen receptor antibodies (IgNARs), which include single variable novel antigen receptor domain antibody fragments (V NAR or V NAR domain); monoclonal antibodies in which the hinge region has been removed; nanobodies; antibody fragments consisting of a single monomeric variable antibody domain (Ablynx); minibodies, which are engineered antibody fragments comprising an scFv linked to a CH domain (Hu et al., Cancer Res. 56:3055-3061, 1996); These are bispecific modified IgG1 antibodies comprising (i) a stable hinge region that does not allow in vivo Fab arm exchange and (ii) an IgG4-like CH3 domain that is modified to allow in vivo Fab arm exchange (see, for example, WO2008 / 119353 and WO2011 / 131746); multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies; proteolytically activated antibodies, which are recombinant masked monoclonal antibodies that remain inert in healthy tissues but are specifically activated in disease microenvironments (e.g., cleaved by proteases that are enriched or unique in disease microenvironments) (see Desnoyers et al., Sci Transl Med 5:207ra144, 2013); chimeric antibodies; humanized antibodies; human antibodies; fusion proteins comprising an antigenic determinant portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, as long as the antibody exhibits the desired biological activity. Based on the identity of the heavy chain constant domains of the antibody, which are respectively referred to as α, δ, ε, γ and μ, an antibody can have any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM or any of their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Different classes of immunoglobulins have different 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, alone or in combination. The variable region of the heavy and light chains is each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.); and (2) methods 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 methods is sometimes used in the art to determine CDRs.

[0116] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an 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.

[0117] The terms "cancer" and "cancerous" refer to or describe a physiological condition in which a cell population in a mammal is characterized by unregulated cell growth. Cancer can include hematological cancers or solid tumors. More specifically, cancer is a leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia (ALL) (such as acute B lymphoblastic leukemia (B-ALL)), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)) or lymphoma (e.g., non-Hodgkin 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, head and neck squamous cell carcinoma, and cervical cancer.

[0118] By "analog," we mean a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while possessing certain biochemical modifications that enhance the analog's function relative to the naturally occurring polypeptide. Such biochemical modifications may increase the analog's protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs may include unnatural amino acids.

[0119] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody derived from another species (usually human) to avoid eliciting an immune response in that species.

[0120] In this disclosure, “comprises / comprising,” “containing,” and “having,” etc. may have the meanings ascribed to them in U.S. patent law and may mean “includes / including,” etc.; “consisting essentially of / consists essentially” likewise have the meanings ascribed to them in U.S. patent law, and the terms are open ended, allowing for the presence of more than what is recited as long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited, but excluding prior art embodiments.

[0121] "Detecting" refers to identifying the presence, absence, or amount of an analyte to be detected.

[0122] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia and cancer to be treated with the compositions of the present invention.

[0123] By "effective amount," it is meant the amount of a pharmaceutical agent required to improve the symptoms of a disease relative to an untreated patient. The effective amount of an active agent (e.g., an antibody drug conjugate (ADC) or drug) used in practicing the present invention to therapeutically treat a disease varies depending on the mode of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0124] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen that is capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope can be formed by both contiguous amino acids and non-contiguous amino acids that are adjacent by tertiary folding of the protein. Epitopes formed by contiguous amino acids are generally retained after protein denaturation, while epitopes formed by tertiary folding are generally lost after protein denaturation. An epitope typically includes at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation.

[0125] By "formulation" is meant the process used to produce a drug product.

[0126] The term "humanized antibody" refers to various forms of non-human (e.g., murine) antibodies, which are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof containing minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from complementary determining regions (CDRs) are replaced with residues from CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and ability (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, the Fv framework region (FR) residues of human immunoglobulins are replaced with corresponding residues from non-human species with the desired specificity, affinity, and ability. Humanized antibodies can be further modified to refine and optimize antibody specificity, affinity and / or ability by replacing the extra residues in the Fv framework region and / or the non-human residues replaced.In general, humanized antibodies will comprise substantially all at least one, and usually two or three variable domains, and the variable domains contain all or substantially all corresponding to the CDR district of non-human immunoglobulin, and all or substantially all FR districts are those with human immunoglobulin consensus sequence.Humanized antibodies also can comprise at least a portion of immunoglobulin constant region or domain (Fc), typically at least a portion of constant region or domain of human immunoglobulin.The example of the method for producing humanized antibodies is described in U.S. Patent No. 5,225,539.

[0127] The goal of humanization is to reduce the immunogenicity of xenogeneic antibodies, such as murine antibodies, for introduction into humans while maintaining the full antigen-binding affinity and specificity of the antibody.

[0128] Several techniques such as resurfacing and CDR grafting can be used to generate humanized antibodies. As used herein, resurfacing technology uses a combination of molecular modeling, statistical analysis, and mutagenesis to change the non-CDR surface of the antibody variable region to resemble the surface of known antibodies of the target host.

[0129] Strategies and methods for antibody resurfacing, as well as other methods for reducing the immunogenicity of antibodies in different hosts, are disclosed in US Pat. No. 5,639,641 (Pedersen et al.), which is hereby incorporated by reference in its entirety. Briefly, in a preferred method, (1) a pool of antibody heavy and light chain variable regions are aligned to provide a set of heavy and light chain variable region framework surface exposed positions, wherein the aligned positions are at least about 98% identical for all variable regions; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues of a rodent antibody (or fragment thereof) is determined; (3) a set of heavy and light chain variable region framework surface exposed amino acid residues that are most closely identical to the set of rodent surface exposed amino acid residues is identified; (4) the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3) are replaced with the set of heavy and light chain variable region framework surface exposed amino acid residues determined in step (2), except for those amino acid residues that are within 5 angstroms of any atom of any residue in the complementarity determining regions of the rodent antibody; and (5) a humanized rodent antibody having binding specificity is generated.

[0130] Antibodies can be humanized using a variety of other techniques, including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Patent 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. Patent No. 5,565,332). Human antibodies can be prepared by a variety of methods known in the art, including phage display methods. See also U.S. Patent 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 (each of which is herein incorporated by reference in its entirety).

[0131] The term "human antibody" means an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. This definition of a 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, antibodies comprising a murine light chain and a human heavy chain polypeptide.

[0132] As used herein, the term "antibody drug conjugate" or "ADC" refers to a compound linked to a cell-binding agent (i.e., an antibody or fragment thereof). Typically, the cell-binding agent (e.g., an antibody) is covalently bound to the drug via a linker.

[0133] The terms "isolated," "purified," or "biologically pure" refer to a substance that is free to varying degrees from components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation greater than isolated. A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. In other words, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" refers to a nucleic acid or protein that produces essentially one band in an electrophoretic gel. For proteins that can undergo modification, such as phosphorylation or glycosylation, different modifications can produce different isolated proteins that can be purified individually.

[0134] "Linker" is any chemical part that can make compound be connected to protein. In one embodiment, the linker makes a drug such as maytansine be connected to a cell binding agent such as an antibody or its fragment in a stable covalent manner. Under the conditions where the compound or antibody remain active, the linker can be susceptible to acid-induced cracking, light-induced cracking, peptidase-induced cracking, esterase-induced cracking and disulfide bond cleavage or generally have resistance to the cracking. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile 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.

[0135] Exemplary cleavable linkers include, but are not limited to, 3-(2-pyridyldithio) propionate N-succinimidyl ester (SPDP), 4-(2-pyridyldithio) butyrate N-succinimidyl ester (SPDB), 4-(2-pyridyldithio) 2-sulfobutyrate N-succinimidyl ester (sulfo-SPDB), and the disulfide 4-(2-pyridyldithio) pentanoate N-succinimidyl ester (SPP). Exemplary non-cleavable linkers include, but are not limited to, 2-iminothiolane, acetyl succinic anhydride, and 4-[N-maleimidomethyl] cyclohexane-1-carboxylic acid succinimidyl ester (SMCC). The universal linkers 2-iminothiolane and acetyl succinic anhydride can be used as cleavable or non-cleavable linkers.

[0136] "Monoclonal antibody" refers to a homogeneous antibody population that is directed to highly specific recognition and binding of a single antigenic determinant or epitope. This is different from polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising an antigen recognition site. In addition, "monoclonal antibody" refers to the antibody prepared in many ways, including but not limited to by hybridomas, phage selection, recombinant expression, and transgenic animals.

[0137] By "specifically binds," it is meant that the compound or antibody recognizes and binds to the target polypeptide but does not substantially recognize and bind to other molecules in a sample, such as a biological sample, that naturally includes the polypeptide of the present invention.

[0138] Nucleic acid molecules suitable for use in the methods of the present invention include any nucleic acid molecules that encode a polypeptide of interest or a fragment thereof. The nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide that is "substantially identical" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules suitable for use in the methods of the present invention include any nucleic acid molecules that encode a polypeptide of the present invention or a fragment thereof. The nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide that is "substantially identical" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule.

[0139] 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 of the amino acid sequences described herein) or nucleic acid sequence (e.g., any 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 level or, in the case of nucleic acids, to the sequence used for comparison.

[0140] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package from Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). The 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 method for determining the degree of identity, the BLAST program can be used, wherein in e -3 With e -100 Probability scores between indicate closely related sequences.

[0141] By "subject" is meant a mammal, including but not limited to a human or a non-human mammal, such as a bovine, equine, canine, ovine, or feline.

[0142] The term "therapeutically effective amount" refers to an amount of an antibody or other drug that is effective in "treating" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down or stop to some extent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down or stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. See the definition of "treat" herein. Insofar as a drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. A "prophylactic effective amount" refers to an amount that is effective to achieve the desired prophylactic result at the necessary dosage and for the necessary period of time. Typically, but not necessarily, because the prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactic effective amount will be less than the therapeutically effective amount.

[0143] 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0144] As used herein, the term "treat / treating / treatment" and the like refers to alleviating or improving a disorder and / or symptoms associated therewith. It should be understood that, although not excluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0145] Unless expressly stated or obvious from the context, as used herein, the term "or" should be understood as inclusive. Unless expressly stated or obvious from the context, as used herein, the terms "a", "an" and "the" should be understood as singular or plural.

[0146] Unless expressly stated or clear from the context, as used herein, the term "about" should be understood as within the normal range of tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood as 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 otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0147] Reciting a list of chemical groups in any definition of a variable herein includes defining that variable as any single group or combination of the listed groups. 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.

[0148] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1Graph illustrating the dependence of the cytotoxic potency of huMov19-Sulfo-SPDB-DM4, an immunoconjugate comprising a humanized monoclonal antibody (huMov19) directed against FOLR1 conjugated to the cytotoxic maytansine DM4 via a Sulfo-SPDB linker, on the maytansine to antibody ratio (MAR). Cytotoxic potency was measured relative to a huMov19-Sulfo-SPDB-DM4 reference standard having a MAR of 3.4. Percent potency = reference EC 50 / Test EC 50 *100%.

[0150] Figure 2 is a graph showing the dependence of cytotoxic potency on the concentration of huMov19-sulfo-SPDB-DM4.

[0151] Figure 3 Two scatter plots and a table are provided to simulate the possible effects of drug to antibody ratio (DAR) on eye toxicity ("oculotoxicity"). AIBW refers to adjusted ideal body weight. DAR was calculated based on drug dosing levels.

[0152] Figure 4 A scatter plot and table are provided to simulate the possible effects of drug-to-antibody ratio (DAR) and concentration on ocular toxicity. The DAR was calculated without actually administering the corresponding ADC to patients.

[0153] Figure 5 Included are two figures showing the lack of effect of DAR on median tumor volume in KB and IGROV-1 murine xenograft models when huMov19-sulfo-SPDB-DM4 conjugates were administered at the same DM4 dose. Mice were dosed with huMov19-sSPDB-DM4. All conjugates were dosed at 25 μg / kg DM4 and variable antibody doses (higher for low DAR conjugates and lower for high DAR conjugates). Similar anti-tumor activity was observed regardless of the variable DAR and antibody dose.

[0154] Figure 6 Figure 2 is a graph showing that DM4 dose has similar effects on murine body weight for a conjugate with a 9.0 DAR compared to a conjugate with a 3.6 DAR when the same DM4 dose is administered. All conjugates were dosed at 1.4 mg / kg DM4 and variable antibody doses (higher for low DAR conjugates and lower for high DAR conjugates).

[0155] Figure 7Figure 2 is a graph showing that for conjugates with various maytansine to antibody ratios, DM1 dose has a similar effect on mean body weight change when the same DM1 dose is administered. All conjugates were administered at 3.0 mg / kg DM1 and variable antibody doses (higher for low DAR conjugates and lower for high DAR conjugates). Regardless of DAR, the toxicity of all conjugates was similar.

[0156] Figure 8 is a graph showing in vivo toxicity studies conducted in mice receiving antibody-SPDB-DM4 conjugates with various DARs, including ADCs huDS6-SPDB-DM4 ("huDS6-DM4"); huB4-SPDB-DM4 ("huB4-DM4"); and huMy9-6-SPDB-DM4 ("huMy9-6-DM4").

[0157] Figure 9A and 9B is a table showing the advantages of preparing ADC compositions based on DM4 concentration. Figure 9A The allowable DM4 concentration (in μg / ml) when formulating the antibody drug conjugate at the target antibody concentration (5.0 ± 1.0 mg / ml) and DAR 3.4 ± 0.5 (circled) is shown. At the target antibody concentration of 5.0 mg / mL and the 3.4 DAR target (boxed), the DM4 concentration was 91.1. By formulating based on a DM4 concentration with a ± 10% specification, the potency variation was lower (boxed area of ​​DM4 concentration). The target DAR, antibody, and DM4 concentrations are boxed. Figure 9B In the figure, at the high-low DAR extremes, the antibody concentration specification fails (boxed area). The target DAR, antibody and DM4 concentrations are boxed.

[0158] Figures 10A-10C Figure 2 is a graph showing the effect of formulating an antibody drug conjugate (ADC) batch by varying the antibody concentration to achieve a target drug (DGN462) concentration. "USL" and "LSL" represent the upper and lower specification limits for antibody concentration. DGN462 is an exemplary drug. The vertical lines (the extended "I") represent the upper and lower limits for drug concentration. "DAR" represents the drug-to-antibody ratio.

[0159] Figures 11A-11C Graph showing that formulating ADC compositions by varying antibody and drug concentrations (open ovals) narrows the allowable specification range for both antibody and drug relative to varying only the antibody specification to achieve a target drug concentration (grey diamonds). Detailed Description of the Invention

[0160] The present invention provides improved methods for formulating therapeutic compositions comprising antibody drug conjugates ("ADCs"), thereby reducing variability in potency between batches of ADCs and / or over a wider range of drug-to-antibody ratios (DARs).

[0161] 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 dose of the drug administered rather than the dose of the antibody. Formulating ADC compositions based on target drug concentrations advantageously minimizes variability in the potency of the finished drug product and ensures that dosing to patients occurs within a narrow, predetermined range.

[0162] Conventionally, antibody drug conjugate therapeutic compositions have been formulated based on antibody concentration. A certain variability is inherent in the formulation of antibody drug conjugates based on antibody concentration, even when maintained within the permissible range of a given specification. 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 the target drug concentration based on a fixed antibody concentration. In practice, the antibody concentration in the finished drug product is allowed to vary from the target concentration. In one example, the formulation specification allows a variation of ±20% based on the concentration of the antibody (e.g., 4.0-6.0 mg / mL is allowed for a target antibody concentration of 5.0 mg / mL). Therefore, depending on the DAR, the ADC potency of the finished drug product may vary by as much as ±35% and may fall outside the desired range.

[0163] 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. In short, formulating the ADC composition based on drug concentration and adding, for example, a ±10% drug concentration specification significantly reduces the potency present in the finished drug product to ±10% (±10% specification) of the target drug concentration. By formulating the ADC based on drug concentration and allowing a variation of up to 10% in drug concentration, only a ±10% potency variation is allowed. Therefore, the novel formulation method of the present invention eliminates DAR potency dependence by formulating to obtain a narrow drug concentration range. This formulation strategy only slightly increases the risk that the antibody concentration will be outside the specification, and therefore the risk of a batch failing to meet the specification is quite low. The improved formulation method ensures that the patient is dosed within a narrow predetermined range without substantially increasing the risk that a batch of ADC will fail to meet the specification.

[0164] On the other hand, the present invention provides a method for reducing the potency variability of a composition comprising an antibody drug conjugate. The method involves formulating an antibody drug conjugate with a variable drug concentration and a variable antibody concentration within a range where two specifications overlap (i.e., by having a small change (± 4-9%) in both concentration values ​​rather than a large change (± 10-15%) in one concentration, thereby reducing the potency variability of the composition. In one embodiment, the small change is about 4, 5, 6, 7, 8, or 9%. In other embodiments, the large change is about 10, 11, 12, 13, 14, or 15%.

[0165] In another aspect, the present invention provides a method for reducing potency variability of a composition comprising an antibody drug conjugate, wherein the method involves formulating the antibody drug conjugate with a target of a variable drug concentration or a variable antibody concentration within a range where the two strengths overlap, thereby reducing potency variability of the composition.

[0166] Antibody Drug Conjugate Formulation

[0167] ADC cancer therapeutics are formulated similarly to antibody cancer therapeutics; that is, based on antibody protein concentration. Although drug labels give information about the "nominal" or target concentration on which to base dosing (e.g., based on mg / kg or mg / m2), ADCs are formulated similarly to antibody cancer therapeutics; that is, based on antibody protein concentration. 2 ), but a typical specification for antibody concentration is target ±10-20%. The potency of ADCs is generally linear with respect to concentration, so the potency of a drug product can vary by ±20%. Unlike antibodies, ADCs have additional potency variability potential due to the drug-to-antibody ratio (DAR). A typical DAR specification in early clinical development is target ±15%, which will vary the amount of attached cytotoxic agent for a given antibody concentration. For most ADCs, a linear relationship between DAR and potency can be demonstrated, indicating that potency is partially or completely governed by the concentration of the conjugated drug administered.

[0168] For many ADCs, it has been demonstrated in rodents that toxicity is entirely dependent on the dose of the administered conjugate drug, independent of the dose of the antibody. Thus, as long as the administered dose of the conjugate drug is the same, toxicity is independent of the DAR. For some ADCs in which the antibody does not possess intrinsic anti-tumor activity, efficacy is entirely dependent on the dose of the drug. In such cases, efficacy is the same as long as the administered dose of the conjugate drug is the same, regardless of the DAR. However, typical specifications for antibody concentration and DAR result in slight variations in the concentration of the conjugate drug. For some ADCs in which the antibody possesses intrinsic anti-tumor activity or is, for example, considered a functional antibody, ADC efficacy may still be driven more by the dose of the drug than by the antibody.

[0169] In cases where the efficacy and toxicity of an ADC can be shown to be primarily driven by the amount of conjugated drug administered, narrowing the specifications for the concentration of the conjugated drug rather than the antibody can prove beneficial. Therefore, the present invention provides methods for formulating therapeutic compositions based on the concentration of the drug rather than the antibody. The target concentration of the drug will be the drug concentration calculated at a fixed antibody concentration and a fixed DAR. Specifications set close to the target conjugated drug concentration will dictate the allowable potency variation in drug product vials. Thus, a drug concentration specification of ±10% will narrow the allowable potency variation to ±10%.

[0170] In other embodiments, therapeutic compositions can be formulated by targeting a variable drug concentration based on the DAR and antibody specifications to achieve an ADC therapeutic composition, wherein the variable drug concentration falls within the center of the effective specification range of the antibody concentration and the drug concentration, where both overlap. At a DAR within ±5% of the target, the center of the effective range achieved by targeting a variable drug concentration can be roughly similar to the final formulated product if a static drug concentration is used. Improvements are achieved when the DAR varies between ±5-15% of the target DAR. At these upper and lower DAR limits, formulating a therapeutic composition by targeting a variable drug concentration can provide less variability in both drug concentration (e.g., about ±4%) and antibody concentration (e.g., about ±10%) relative to targeting a static drug concentration that would vary the antibody concentration by approximately ±15%. Such ranges are suitable for formulating ADC compositions, examples of which include 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, which is an antibody drug conjugate comprising DGN462 conjugated to the anti-CD33 antibody huMy9-6 via a cleavable disulfide linker, s-SPDB. Other drugs suitable for use in the present invention include benzodiazepines, such as those shown in Table 1 or variations thereof, and represented by the following structural formula:

[0171]

[0172] Antibody-drug conjugates

[0173] The present invention relates to improved methods for formulating ADCs comprising antibodies (e.g., antibodies that bind to tumor antigens) or antibody fragments as disclosed herein and their functional equivalents linked or conjugated to a cytotoxic agent (e.g., a drug or prodrug). A variety of antibodies can be used in the methods of the present invention. In certain embodiments, the antibodies specifically bind to antigens or ligands such as FOLR1 (also known as FRα), CD33, CD123, CD19, MUC1, CA6, CD37, EGFR, and fragments of any of the polypeptides listed above. In certain embodiments, the present invention includes, but is not limited to, ADCs comprising any of the following antibodies: huMov19, huMy9-6, huAnti-CD123, huB4, huDS6, huCD37-50, huCD37-3, and huEGFR-7R.

[0174] Suitable drugs or prodrugs are known in the art. Drugs or prodrugs can be cytotoxic. The cytotoxic drugs used in the ADC of the present invention can be any compound that causes cell (such as cancer cell) death, or induces cell death, or reduces cell viability in a certain way, and include, for example, microtubule inhibitors, DNA damaging agents, DNA cross-linking agents, DNA alkylating agents and cell cycle disruptors. In specific embodiments, suitable cytotoxic drugs include maytansine and maytansine analogs. Other suitable cytotoxic drugs include, for example, benzodiazepines (e.g., pyrrolobenzodiazepines and indolinolobenzodiazepines; see also Table 1: compounds D1-D10 and DGN462), taxanes, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes (such as calicheamicin), dolastatins and dolastatin analogs (including auristatins), tomamycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino-doxorubicin.

[0175] ADCs can be prepared by 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.

[0176] The drug or prodrug can be linked to the antibody or its fragment, for example, via a disulfide bond. The linker molecule or cross-linking agent may include a reactive chemical group that can react with the antibody or its fragment. The reactive chemical group used to react with the cell binding agent may be, for example, N-succinimidyl ester and N-sulfosuccinimidyl ester. In addition, the linker molecule comprises a reactive chemical group that reacts with the drug to form a disulfide bond, such as a disulfide pyridyl linker molecule including, for example, 3-(2-pyridyldithio) propionic acid N-succinimidyl ester (SPDP) (see, for example, Carlsson et al., Biochem. J., 173:723-737 (1978)), 4-(2-pyridyldithio) butyric acid N-succinimidyl ester (SPDB) (see, for example, U.S. Patent No. 4,563,304). , 4-(2-pyridyldithio) 2-sulfobutyric acid N-succinimidyl ester (sulfo-SPDB) (see U.S. Publication No. 20090274713), 4-(2-pyridyldithio) pentanoic acid N-succinimidyl ester (SPP) (see, e.g., CAS Reg. No. 341498-08-6), 2-iminothiolane, or acetyl succinic anhydride, 4-[N-maleimidomethyl] cyclohexane-1-carboxylic acid succinimidyl ester (SMCC). For example, the antibody or cell binding agent can be modified with a cross-linking reagent, and the antibody or cell binding agent thus obtained containing free or protected thiol groups is then reacted with a disulfide or thiol-containing maytansinoid to produce a conjugate. The conjugate can be purified by chromatography (including but not limited to HPLC, size exclusion, adsorption, ion exchange, and affinity trapping), dialysis, or tangential flow filtration.

[0177] In one aspect of the invention, an antibody is linked to a cytotoxic drug via a disulfide bond and a polyethylene glycol spacer that enhances the potency, solubility, or efficacy of the ADC. Such a cleavable hydrophilic linker is described, for example, in WO2009 / 0134976. An additional benefit of this linker design is that the antibody-drug conjugate has a desired high monomer ratio and minimal aggregation. Specifically contemplated in this aspect are the use of a cleavable hydrophilic linker carrying a polyethylene glycol spacer ((CH2CH2O) n＝1-14 ) disulfide groups (-S-S-) linked cell-binding agents and drugs with a narrow drug loading range of 2-8, which are described to show relatively high and potent bioactivity against cancer cells and have the desirable biochemical properties of high conjugation yield and high monomer ratio as well as minimal protein aggregation.

[0178] Many of the connectors disclosed herein are described in detail in U.S. Patent Nos. 7,989,598; 8,163,888; 8,198,417; 8,236,319; 8,563,509; U.S. Patent Publication No.: US20130029900 and International Patent Application Publication Nos. WO2009 / 0134976; WO2009 / 134977; and WO2012 / 177837; the contents of each of the above-mentioned patents and applications are incorporated herein by reference in their entirety.

[0179] The present invention includes aspects in which from about 2 to about 8 drug molecules (e.g., maytansinoids, benzodiazepine compounds, auristatins, DNA alkylating agents, or other compounds of interest) are linked to an antibody or fragment thereof, and the anti-tumor effect of the conjugate is significantly more potent than a drug payload having fewer or higher numbers of drugs linked to the same cell-binding agent.

[0180] In one aspect, 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, 7.8, 7.9, 8.0, 8.1, 8. In some embodiments, the cytotoxic agent of the present invention is a cytotoxic drug. In some embodiments, the cytotoxic drug ...

[0181] In certain embodiments, the ADC of the present invention comprises a maytansine. Maytansine suitable for use in the present invention includes but is not limited to N 2’ -Deacetyl-N 2’ -(3-mercapto-1-oxopropyl)-maytansine (DM1), N 2’ -Deacetyl-N 2’ (4-Mercapto-1-oxopentyl)-maytansine (known as DM3) and N 2’ -Deacetyl-N 2’ -(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4).

[0182] DM1 is represented by the following structural formula:

[0183]

[0184] See also U.S. Patent Publication No. 20130156796.

[0185] DM4 is represented by the following structural formula:

[0186]

[0187] See also U.S. Patent Publication No. 20130156796.

[0188] Examples of suitable maytansinol esters include those having modified aromatic rings and those having modifications at other positions. Suitable maytansinoids are disclosed 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; 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.

[0189] Another maytansinoid comprising a side chain containing a sterically hindered thiol bond is N 2’ -Deacetyl-N 2’ (4-Mercapto-1-oxopentyl)-maytansine (referred to as DM3) is represented by the following structural formula (V):

[0190]

[0191] The various types of maytansine taught in U.S. Patent Nos. 5,208,020 and 7,276,497 can also be used in the conjugates of the present invention. In this regard, the entire disclosure of U.S. Patent Nos. 5,208,020 and 7,276,697 is incorporated herein by reference. The carbon positions of an exemplary maytansine structure are provided below:

[0192]

[0193] Many positions on maytansine can serve as the position in order to chemically connect linking moiety.For example, the C-3 position with hydroxyl, the C-14 position modified with hydroxyl, the C-15 position modified with hydroxyl and the C-20 position with hydroxyl are all expected to be applicable.In some embodiments, the C-3 position serves as the position in order to chemically connect linking moiety, and in some specific embodiments, the C-3 position of maytansinol serves as the position in order to chemically connect linking moiety.

[0194] Several descriptions for producing 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.

[0195] In general, a solution of the antibody in an aqueous buffer solution can be incubated with a molar excess of a maytansine-like compound having a disulfide moiety carrying a reactive group. The reaction mixture can be quenched by adding an excess of an amine (such as ethanolamine, taurine, etc.). The maytansine-antibody conjugate can then be purified by gel filtration.

[0196] The average number of maytansine molecules bound per antibody molecule can be determined by spectrophotometrically measuring absorbance at 252 nm and 280 nm and determining the molar concentration of the antibody and the molar concentration of the drug. An exemplary calculation for huMov19-sulfo-SPDB-DM4 is shown below. The average number of maytansine molecules per antibody is then calculated by dividing the molar concentration of the drug by the molar concentration of the antibody. The average number of maytansine molecules per antibody can be, for example, 1-10 or 2-5. In some embodiments, the average number of maytansine molecules per antibody is 3.4.

[0197] In certain embodiments, the ADC of the present invention comprises a benzodiazepine. Suitable benzodiazepines for use in the present invention include, for example, pyrrolobenzodiazepines and indolinobenzodiazepines (see also Table 1: compounds D1-D10 and DGN462). In various embodiments of the previous aspects, 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, which is incorporated herein by reference in its entirety. Compound D2 is described, for example, in U.S. Provisional Application Serial No. 62 / 045,236 and "Antibody-Drug Conjugates (ADCs) of Indolino-Benzodiazepine DNA-Alkylating Agents", 2015 AACR, Abstract No. 652. Compound D2 is described, for example, in U.S. Provisional Application Serial No. 62 / 045,248 and “Antibody-Drug Conjugates (ADCs) of Indolino-Benzodiazepine DNA-Alkylating Agents,” 2015 AACR, Abstract No. 652.

[0198] Pharmaceutical composition

[0199] 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 are suitable for inhibiting tumor growth and treating cancer in human patients.

[0200] Exemplary antibody drug conjugates for use in the pharmaceutical compositions of the present invention include, but are not limited to, huMov19-Sulfo-SPDB-DM4, huMov19-Sulfo-SPDB-D1, huMov19-D2, huMov19-Sulfo-SPDB-D10, huMov19-Sulfo-SPDB-DGN462, huMy9-6-Sulfo-SPDB-D1, huMy9-6-D2, huMy9-6-Sulfo-SPDB-D10, huMy9-6-Sulfo-SPDB-DGN462, B-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.

[0201] In certain embodiments, the preparation is prepared by combining the purified ADC of the present 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 for the formulation of the composition based on the drug concentration. In some embodiments, the ADC of the present invention is provided in a suitable carrier, diluent and / or excipient, such as 0.9% saline (0.9% w / v NaCl), 5% (w / v) dextrose; and may also contain a stabilizer such as Tween 20. In a specific embodiment, the ADC is provided in an IV bag or a pharmaceutical vial.

[0202] Other suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers such as phosphate, citrate, acetate, succinate, 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 dextrins; chelating agents such as EDTA; and sugars such as sucrose, mannitol, trehalose, or sorbitol.

[0203] The pharmaceutical compositions of the present invention can be administered in a number of ways for local or systemic treatment. Administration can be parenteral, including intravenous, intraarterial, or infusion; oral; transdermal; or intracranial (eg, intrathecal or intraventricular).

[0204] Kits comprising antibody drug conjugates

[0205] The present invention provides kits comprising antibody drug conjugates (ADCs) that can be used to perform the methods described herein. In certain embodiments, the kit includes an ADC in one or more containers, wherein the amount of ADC is based on the drug concentration, and wherein the amount of ADC varies by up to ±10% from the specification. Those skilled in the art will readily recognize that the disclosed ADCs can be readily incorporated into a well-known established kit format in the art. If necessary, the kit may include instructions for using the ADC to achieve patient treatment. The instructions may be printed directly on the container (when present), or in the form of a label affixed to the container, or in the form of a separate paper, brochure, card, or folded printed matter provided in or with the container.

[0206] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the knowledge of the skilled artisan. Such techniques are fully described in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques can be applied to produce the polynucleotides and polypeptides of the present invention and, therefore, can be considered in making and practicing the present invention. Particularly applicable techniques for specific embodiments will be discussed in subsequent sections.

[0207] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening and treatment methods of the invention and are not intended to limit the scope of what the inventors regard as their invention. Example

[0208] Example 1: ADC in vitro potency depends on the amount of drug delivered to cells or subjects

[0209] The anti-FOLR1 monoclonal antibody portion of huMov19-sulfo-SPDB-DM4 targets and binds to the cell surface antigen FOLR1 (also known as FRα). Following antibody-antigen interaction and internalization, the immunoconjugate releases DM4, which binds to tubulin and disrupts microtubule assembly / disassembly dynamics, thereby inhibiting cell division and cell growth of FOLR1-expressing tumor cells. FOLR1, a member of the folate receptor family, is overexpressed on a variety of epithelial cancer cells.

[0210] The in vitro potency of antibody drug conjugates (ADCs) is linearly correlated with the drug-antibody ratio (DAR), also known as the maytansinoid-to-antibody ratio (MAR). Figure 1 huMov19-sulfo-SPDB-DM4 was used as an exemplary ADC to generate Figure 1 and 2 Indeed, over one DAR (ie, 2.9-3.9) there was a 31% variation in potency resulting from a 29% calculated difference in the dose of DM4. Figure 2 The general dependence of cytotoxic potency on huMov19-sulfo-SPDB-DM4 concentration was demonstrated. When the conjugate concentration was diluted to half the concentration of the reference standard, the cytotoxic potency was half that of the reference standard. Similarly, when the starting concentration of the conjugate was twice that of the reference standard, the cytotoxic potency was doubled relative to the reference standard. Details of the specific cytotoxicity assay are provided herein below in Example 6.

[0211] At a target DAR of 3.4, the acceptable range of variability allows the actual DAR present in the finished drug product to vary between 2.9 and 3.9 ( Figure 3 ).

[0212] Example 2: DM4 dose drives toxicity and efficacy in vivo

[0213] For huMov19-sulfo-SPDB-DM4, it may be desirable to obtain as high an ADC level as possible without approaching the threshold of ocular toxicity to achieve efficacy. Figure 3 As shown in Figure 2, when the huMov19-Sulfo-SPDB-DM4 DAR was approximately 3.4 and the dose range was between 3.3 and 7 mg / kg, 32% of patients were found to be above the ocular toxicity level. When the huMov19-Sulfo-SPDB-DM4 DAR was 2.9 and the dose range was between 3.3 and 7 mg / kg, 13% of patients would be expected to be above the ocular toxicity level. When the huMov19-Sulfo-SPDB-DM4 DAR was 3.4 and the dose range was between 3.3 and 7 mg / kg, 32% of patients would be expected to be above the ocular toxicity level based on the fact that they would receive a reduced DM4 dose. When the huMov19-Sulfo-SPDB-DM4 DAR was 3.9 and the dose range was between 3.3 and 7 mg / kg, 48% of patients would be expected to be above the ocular toxicity level based on the fact that they would receive an increased DM4 dose.

[0214] At 5 mg / kg huMov19-sulfo-SPDB-DM4 (adjusted ideal body weight), none of the patients exceeded the ocular toxicity threshold when the DAR was 2.9. However, at 5 mg / kg huMov19-sulfo-SPDB-DM4, 14% and 57% of the patients exceeded the ocular toxicity threshold when the DAR was 3.4 or 3.9, respectively. Figure 4 Actual clinical data for the 3.4 DAR cohort are shown in FIG (closed circles on the graph). The remaining data reflect simulated dose analysis.

[0215] Figure 4 This illustrates the importance of ensuring that patients receive a dose within a narrow, predetermined range. Ideally, to ensure maximum efficacy and safety, patients would receive a dose of huMov19-sulfo-SPDB-DM4 that approaches, but does not exceed, the threshold for ocular toxicity.

[0216] As discussed in Examples 3 and 4, for huMov19-Sulfo-SPDB-DM4, toxicity depends on the amount of DM4 administered. Preclinical efficacy studies with huMov19-Sulfo-SPDB-DM4 showed no DAR dependence when the DM4 dose was the same. Furthermore, preclinical toxicity studies with huMov19-Sulfo-SPDB-DM4 and a number of other conjugates showed that toxicity was driven by the dose of attached DM4 and was independent of the DAR.

[0217] Example 3: The antitumor activity of huMov19-sulfo-SPDB-DM4 is not dependent on DAR

[0218] In vivo studies were performed to analyze the activity of huMov19-sulfo-SPDB-DM4 in KB and IGROV-1 murine xenograft models ( Figure 5 The KB cell line was derived from a HeLa cell line of tumor cells. It is used as a tumor model because it forms tumors with reproducible characteristics in nude mice and overexpresses the folate receptor. The IGROV-1 tumor model is derived from human ovarian cancer.

[0219] HuMov19-sulfo-SPDB-DM4 with different DARs ranging from 2.5 to 4.1 were administered to mice bearing KB or IGROV-1 tumor xenografts at a dose of 25 μg / kg of DM4 and variable antibody doses. Figure 5 As shown in , DAR did not affect efficacy as long as the same DM4 dose was administered. The results of this analysis indicate that DM4 dose determines efficacy in the FOLR1-positive KB and IGROV-1 tumor models, regardless of DAR.

[0220] Example 4: Toxicity is not dependent on the drug-antibody ratio

[0221] In vivo studies were performed to evaluate the effect of the drug-antibody ratio on the maximum tolerated dose (MTD) of huMov19-sulfo-SPDB-DM4. Figure 6 Mice received huMov19-sulfo-SPDB-DM4 at a fixed DM4 dose of 1400 μg / kg, with varying antibody doses. Mouse body weight was monitored as a measure of toxicity. The administered ADCs varied widely in drug-to-antibody ratio (e.g., DAR 9.0 vs. DAR 3.6). Interestingly, a DAR range of 3.6-9.0 did not affect toxicity, as long as the same DM4 dose was administered. Therefore, toxicity was not dependent on the DAR.

[0222] 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) changes were monitored as an indicator of toxicity. Weight loss was similar for the different DAR conjugates, indicating that toxicity was not dependent on the DAR as long as the DM1 dose was kept constant ( Figure 7 ).

[0223] 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 ). ADC huDS6-SPDB-DM4 (also referred to as "huDS6-DM4") is a humanized monoclonal antibody huDS6 linked to the potent cytotoxic maytansine DM4 via a cleavable disulfide crosslinker, 4-2-pyridyldithiobutyric acid N-succinimidyl ester (SPDB). ADC huDS6-SPDB-DM4 targets solid tumors such as ovarian, breast, cervical, lung, and pancreatic cancers. ADC huB4-SPDB-DM4 (also referred to as "huB4-DM4") is a novel antibody-drug conjugate consisting of a humanized monoclonal IgG1 anti-CD19 antibody (huB4) linked to DM4 via a cleavable disulfide crosslinker, 4-2-pyridyldithiobutyric acid N-succinimidyl ester (SPDB). ADC huMy9-6-SPDB-DM4 (also referred to as "huMy9-6-DM4") is an ADC that specifically binds to CD33, a sialic acid-binding immunoglobulin-like lectin (Siglec) family antigen expressed primarily on myeloid cells. ADC huMy9-6-SPDB-DM4 has been clinically evaluated for the treatment of acute myeloid leukemia.

[0224] like Figure 8As shown in , the indicated conjugates were administered at 3-4 different doses, and the mouse survival rate was measured as an indicator of toxicity. The DAR range of the four conjugates was narrow (ranging from 3.49 to 4.0); therefore, the LD 50 The range (dose lethal to 50% of the animals) was also narrow (DM4 doses between 1.6-2 mg / kg). Conjugates with different DARs were administered at a dose of 80 mg / kg antibody, and mouse survival was measured as an indicator of toxicity ( Figure 8 ). Regardless of DAR, similar results for all conjugates indicate that toxicity is driven by the total DM4 dose administered. That is, within the DAR range of 2.1 to 5.1, toxicity is not affected by different DARs.

[0225] Example 5: Formulating ADC therapeutic compositions based on drug concentration to minimize potency changes due to changes in DAR.

[0226] Conventionally, antibody drug conjugate therapeutic compositions have been formulated based on antibody concentration. Figure 9A This demonstrates the inherent variability in formulating antibody drug conjugates based on antibody concentration, even when kept within the permissible range of specifications. Specifically, at the end of the ADC manufacturing process, the antibody concentration was measured and diluted to achieve a target antibody concentration of 5.0 mg / ml for huMov19-sulfo-SPDB-DM4. Figure 9A In the figure, for huMov19-sulfo-SPDB-DM4, the target antibody concentration (5.0 mg / ml) 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 finished drug product is allowed to vary from the target concentration. The antibody concentration in the finished product can be as low as 4.0 mg / ml or as high as 6.0 mg / ml. Therefore, as shown by the boxed area of ​​DM4 concentration, depending on the DAR, the DM4 concentration in the finished drug product can be as low as 62.1 μg / ml or as high as 125.4 μg / ml.

[0227] Formulating the ADC composition based on DM4 concentration and adding a + / - 10% DM4 concentration specification significantly narrowed the potency present in the finished drug product to + / - 10% of the target DM4 concentration (highlighted; + / - 10% specification).

[0228] The tendency of batches to fail to meet specifications is shown in Figure 9B, where the target DM4 concentration and DAR are highlighted. The DM4 concentration is shown at the top, the DAR is shown on the left, and the resulting antibody concentration is shown in the highlighted box (5.0 mg / ml). When the antibody concentration varied by more than ±20% from the target, that batch was out of specification. The concentration of antibody present in batches that failed to meet specification is shown in bold ( Figure 9B ). The risk of a batch not meeting specifications is very low.

[0229] In summary, current formulation specifications allow for a ±20% variation in antibody concentration (4.0-6.0 mg / mL). Therefore, depending on the DAR, ADC potency can vary by as much as ±35%. By formulating the ADC based on DM4 concentration and allowing a maximum ±10% variation in DM4 concentration, only a ±10% variation in potency is tolerated. Therefore, the novel formulation approach eliminates DAR potency dependency by formulating to obtain a narrow DM4 concentration range. This formulation strategy only slightly increases the risk that a batch will fail to meet specifications due to an antibody concentration outside its specification.

[0230] Example 6: DAR conjugate formulation

[0231] huMov19-Sulfo-SPDB-DM4, an ADC comprising the huMov19 antibody, an SPDB linker, and the cytotoxic drug DM4, is an example of an ADC in which in vitro potency, in vivo efficacy, and in vivo toxicity are not dependent on the DAR but are instead driven entirely by the concentration of DM4 administered. Therefore, huMov19-Sulfo-SPDB-DM4 is a good candidate for formulation based on DM4 rather than huMov19 concentration. To test the hypothesis that this would narrow the potency of the drug, a series of huMov19-Sulfo-SPDB-DM4 conjugates with a range of DARs were prepared. The conjugates were purified into basic formulation buffer (10 mM sodium acetate, 9% (w / v) sucrose, pH 5.0), and the DM4 and huMov19 antibody concentrations of each sample were measured spectrophotometrically at wavelengths of 252 nm and 280 nm, respectively. The molar concentrations of DM4 and huMov19 antibodies constituting the conjugates were calculated as follows:

[0232]

[0233]

[0234] Each of the various DAR conjugates was formulated in two different ways: one was diluted with base formulation buffer to achieve a target huMov19 antibody concentration within the specification range of 5.0 mg / mL ± 20%. In addition, each DAR conjugate was 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.

[0235] The specific cytotoxicity assay involves incubating folate receptor 1 (FOLR1) positive cells (KB) in duplicate wells of a sterile 96-well flat-bottom black tissue culture plate with a clear bottom in the presence of medium containing a dilution series of huMov19-sulfo-SPDB-DM4 drug conjugate. Each assay plate contains a reference, control, and test article series at the same dilution in wells with KB cells and a medium blank. After a 4-day incubation period at 37°C ± 2°C, the plates are removed from the incubator and allowed to equilibrate to room temperature for 1 hour before adding CellTiter-Glo TM Luminescent cell viability reagent. The plate was incubated for an additional 2 hours before analysis and recording of the luminescent signal on a Victor III plate reader. CellTiter-Glo TM ATP is measured as an indicator of viable cells using a unique stable form of luciferase. The luminescent signal generated is directly proportional to the number of viable cells present in the well, and is also inversely proportional to the cytotoxicity of the drug in that well. Because the luciferase reaction requires ATP, various conditions have been created to make the amount of light generated proportional to the amount of ATP present, which reflects the number of viable cells. The 3 plate data files were entered into PLA 2.0 software, and all 6 replicates of each sample were used to determine the EC values ​​for the reference and test article using a constrained 4-parameter logistic curve fit. 50 For samples that passed the acceptance criteria of slope difference and parallelism, the relative % potency of the test article was reported based on the IC50 obtained from the constrained 4PL curve fit. The percent potency was calculated as follows.

[0236]

[0237] If the test article EC 50 Lower than the reference standard EC 50 , this indicates that the test article has greater potency than the reference standard, and the calculated % potency will be greater than 100%. Conversely, if the test article EC 50 Greater than the reference standard EC 50 , which indicates that the test article has less potency than the reference standard and the calculated % potency will be less than 100%.

[0238] The results of these calculations are shown in Tables 2 and 3 (below). The reference standard used for potency determinations in Table 2 is Sample A, while the reference standard used for potency determinations in Table 3 is Sample F. A dilution series for each sample was generated assuming a nominal huMov19 concentration of 5 mg / mL to simulate how the ADC would be dosed in a clinical setting. When huMov19-sulfo-SPDB-DM4 ADCs were formulated to target a huMov19 concentration of 5 mg / mL (Table 2), a wide range of potencies was observed, as expected: 59.8-124.6%, resulting in a total of approximately 2-fold difference between the most potent and least potent ADCs. This is in good agreement with the expected range of ±35%. In contrast, when huMov19-sulfo-SPDB-DM4 ADCs were formulated with various DARs to target a DM4 concentration of 91.0 mg / mL, a much narrower range of relative potencies was achieved: 80.9-106.5%, totaling approximately 1-fold. This is in good agreement with the expected range of ±10%. Most measured potencies were within 15% of the expected value based on DM4 concentration. This is within the combined experimental error of both the potency and concentration measurement assays. Overall, these results show the advantages of formulating to achieve a DM4 concentration target rather than a huMov19 concentration target, as is typical for ADCs.

[0239] Table 2. huMov19-sulfo-SPDB-DM4 conjugates made at various DARs (±15%) and formulated to target various huMov19 concentrations (±20%).

[0240]

[0241] Table 3. huMov19-sulfo-SPDB-DM4 conjugates manufactured at various DARs (±15%) and formulated to target various DM4 concentrations (±10%).

[0242]

[0243]

[0244] Example 7: Tightening the specification window over a wider DAR range by targeting variable antibody and drug concentrations.

[0245] In some cases, it may be desirable to vary the target drug concentration to achieve smaller variations in both drug and antibody concentrations, rather than significant variations in non-target concentrations (e.g., antibody concentrations). By targeting the midpoint of the range where both antibody and drug concentrations overlap at a specific DAR value, the ADC composition is formulated using the method to maximize the specification range. In practice, drug specifications are tighter than antibody specifications (e.g., ±10% (for drugs) versus ±15% (for antibodies)). Therefore, 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 batches that are completely safe for use despite not meeting specifications.

[0246] The desirability of formulating ADCs using the above methods is shown for huMy9-6-Sulfo-SPDB-DGN462, a CD33-targeting antibody drug conjugate comprising the antibody huMy9-6 conjugated to the novel DNA alkylating agent DGN462 via a cleavable disulfide linker Sulfo-SPDB.

[0247] exist Figure 10A 、 10B In each of Figures 1 and 10C, the upper and lower limits of the antibody specification range are indicated by dashed lines, the X-axis indicates the DAR of a batch of drug, where the target DAR is 2.7; the vertical lines show the upper and lower limits of the DGN462 specification at a given DAR, and the dark grey diamond on each vertical line indicates the overlap between the DGN462 specification range and the antibody specification range at a particular DAR. Figure 11A In the case of a batch with a DAR of 2.7, the center of the DGN462 specification range falls exactly in the center of the antibody range. Figure 10B In the case of a batch with a DAR close to the target DAR, the antibody concentration required to achieve a fixed DGN462 target concentration falls well within the upper and lower specification limits of the antibody. Figure 10C In the present invention, 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 fixed DGN462 concentration approaches or exceeds the defined antibody concentration specification because the amount of antibody required to obtain the target DGN462 concentration is close to the upper and lower specification limits of the antibody concentration. Therefore, an improved formulation method may be desirable.

[0248] Figures 11A-11C The improvements obtained by varying both DGN462 concentration and antibody concentration are shown—particularly when the DAR is close to the upper and lower DAR specification limits (e.g., 3.0-3.1 and 2.3-2.4). Targeting variable drug concentrations identifies the midpoint of the range where the antibody concentration specification range and the drug concentration specification range overlap ( Figure 11A The center of the effective range of total drug is well below the antibody upper specification limit. Therefore, at the upper and lower DAR specification limits, the approach targeting variable DGN462 limits the target antibody concentration to + / - 10% variation from the target, rather than the full ±15%, resulting in a more consistent product. In contrast, for batches where the DAR is close to 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 and increases the risk of variability in efficacy and toxicity ( Figure 11A and 11B ). Figure 11C Illustrate the improvement provided by the approach targeting variable drug concentration at the upper and lower DAR specification limits, compared to an approach where antibody concentration was used to obtain the target drug concentration (grey diamonds) (where the fixed target total drug exceeded the upper specification limit for the antibody at a DAR of 2.3), where DGN462 concentration and antibody concentration were maintained between narrower limits of 4% (open ovals).

[0249] The following equations can be used to calculate the upper and lower specification limits for a drug.

[0250]

[0251]

[0252] Table 4 illustrates the method used to calculate the upper and lower specification limits for DGN462 (USL DGN462, LSL DGN462). The upper specification limit for the antibody concentration is constant. The DAR for a batch of antibody drug conjugates is determined empirically. The calculation based on the desired upper and lower specification limits is performed as follows:

[0253]

[0254]

[0255] The values ​​"2.30" and "1.70" define the upper and lower specification limits for the antibody. The denominator is the molecular weight of the antibody.

[0256] Table 4. Calculated DGN462 concentrations based on antibody specification limits

[0257]

[0258] Formulated according to DGN462 (based on variable targets)

[0259]

[0260]

[0261] - When calculating LSL and USL at certain points, these limits will fall outside the proposed specifications

[0262] - These outliers can be set to LSL (34.0 mg / mL) or USL (41.5 mg / mL) using the > or < rule

[0263] - For formulation purposes, it is recommended to report the DAR to the second decimal place

[0264] In the following examples, ADCs were formulated by targeting variable drug concentrations. Here, the ADC included a non-functional antibody huMov19 (MW 145676 g / mol) conjugated to D2 (MW 961.05 g / mol), with 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 ADCs were formulated to target variable drug concentrations to minimize deviations from the target antibody concentration. Figure 11A As shown in , the antibody concentration obtained was varied by ±10% (1.8-2.2) targeting variable drug concentrations, compared to ±15% when using static drug concentrations ( Figure 11A ; See, e.g., Tables 4-7). Targeting the variable drug identified the midpoint of the range where the antibody specification range and drug specification range overlapped ( Figure 11A ; See, e.g., Tables 4-7)

[0265] Table 5 below illustrates the method used to calculate the upper and lower specification limits (USL D2, LSL D2) for D2. The antibody concentration upper and lower specification limits were constant, and the DAR was determined empirically.

[0266] Table 5.

[0267] Calculated D2 concentration based on antibody specification limits

[0268]

[0269] Formulated according to D2 (based on variable targets)

[0270]

[0271] In another embodiment, an ADC was formulated by targeting a variable cytotoxic agent concentration. Here, the ADC included a functional antibody huEGFR-7R (MW 144975 g / mol) conjugated to D1 (MW 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 was formulated to target a variable cytotoxic agent concentration to minimize deviation from the target antibody concentration.

[0272] Table 6 below illustrates the method used to calculate the upper and lower specification limits (USL D1, LSL D1) for D1. The antibody concentration upper and lower specification limits were constant, and the DAR was determined empirically.

[0273] Table 6.

[0274] Calculated D1 concentration based on antibody specification limits

[0275] Formulated according to D1 (based on variable targets)

[0276]

[0277]

[0278] In another embodiment, an ADC was formulated by targeting a variable cytotoxic agent concentration. Here, the ADC included a functional antibody huMy9-6 (MW 146192 g / mol) conjugated to D10 (MW 1062.22 g / mol), with 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 was formulated to target a variable cytotoxic agent concentration to minimize deviation from the target antibody concentration.

[0279] Table 7 below illustrates the method used to calculate the upper and lower specification limits (USL D1, LSL D1) for D1. The antibody concentration upper and lower specification limits were constant, and the DAR was determined empirically.

[0280] Table 7.

[0281] Calculated D10 concentration based on antibody specification limits

[0282] Formulated according to D10 (based on variable targets)

[0283]

[0284]

[0285] Other implementation plans

[0286] From the foregoing description, it will be apparent that the invention described herein can be varied and modified to adapt it to various uses and conditions. The embodiments are also within the scope of the following claims.

[0287] Reciting a list of elements in any definition of a variable herein includes defining that variable as any single element or combination (or subcombination) of the listed elements. Recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0288] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. Sequence Listing <110> Imuinojin Company <120> Methods for formulating antibody drug conjugate compositions <130> 365738.1009WO1 (00022) <140> PCT / US2015 / 048152 <141> 2015-09-02 <150> 62 / 044,592 <151> 2014-09-02 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 211 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 1 Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Arg Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Met Glu Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 100 105 110 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 115 120 125 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 130 135 140 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 145 150 155 160 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 165 170 175 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 180 185 190 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 195 200 205 Gly Glu Cys 210 <210> 2 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Asn Phe 50 55 60 Gln Gly Lys Ala Lys Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 3 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 3 Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30 Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser 65 70 75 80 Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95 Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 4 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 4 Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30 Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95 Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 5 <211> 447 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 6 <211> 213 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 6 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Ser Ala His Ser Ser Val Ser Phe Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Ser Leu Ala Ser Gly Val Pro Ala Arg Phe Gly Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Phe Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 7 <211> 447 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 Gln Ala Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Gly Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Asp Ser Val Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 8 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30 Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45 Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 9 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 10 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu His Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 11 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu His Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 12 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Cys Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Thr Tyr Thr Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Ala Pro Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 13 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Arg Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Asn Val Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Tyr Trp Gly Thr Thr Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 444 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 14 Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Pro Ser Leu Lys 50 55 60 Ser Arg Leu Ser Ile Lys Lys Asp His Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Gly Gly Tyr Ser Leu Ala His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 15 <211> 444 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 15 Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ser Leu Lys 50 55 60 Ser Arg Leu Ser Ile Lys Lys Asp His Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Gly Gly Tyr Ser Leu Ala His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 16 <211> 213 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Thr Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Pro Tyr Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asp Asn Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 17 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Phe Ala Trp His Trp Ile Arg Gln His Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Leu Tyr Ser Gly Ser Thr Val Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn His Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly

Claims

1. A method of producing a composition comprising an antibody drug conjugate, wherein the method reduces potency variability between production batches, wherein the antibody drug conjugate comprises a drug-antibody ratio between an upper specification limit (DAR) and a lower specification limit (DAR), wherein the upper specification limit (DAR) is the target DAR plus a 5-15% variation from the specification setting, and the lower specification limit (DAR) is the target DAR minus a 5-15% variation from the specification setting, wherein the composition has an antibody concentration between an upper specification limit for antibody concentration, wherein the upper specification limit for antibody concentration is the target antibody concentration set for the specification plus the maximum variation allowed for the specification, and the lower specification limit for antibody concentration is the target antibody concentration minus the maximum variation allowed for the specification, and wherein the composition has a drug concentration between an upper drug concentration limit and a lower drug concentration limit, wherein the upper drug concentration limit is a target drug concentration plus a 10% variation, and the lower drug concentration limit is a target drug concentration minus a 10% variation, wherein the target drug concentration is the drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio, The method includes: (a) determining a target drug concentration, wherein the target drug concentration is the drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio; (b) formulating the antibody drug conjugate to produce a composition comprising a drug concentration between said upper drug concentration limit and said lower drug concentration limit, thereby reducing the variability of the drug concentration of said composition between production batches to ±10%, The antibody drug conjugate comprises a maytansinoid linked to an antibody.

2. A method for reducing batch-to-batch potency variation of a composition comprising an antibody drug conjugate, wherein the antibody drug conjugate comprises a drug-antibody ratio between an upper specification limit (DAR) and a lower specification limit (DAR), wherein the upper specification limit (DAR) is the target DAR plus a 5-15% variation from the specification setting, and the lower specification limit (DAR) is the target DAR minus a 5-15% variation from the specification setting, wherein the composition has an antibody concentration between an upper specification limit for antibody concentration, wherein the upper specification limit for antibody concentration is the target antibody concentration set for the specification plus the maximum variation allowed for the specification, and the lower specification limit for antibody concentration is the target antibody concentration minus the maximum variation allowed for the specification, and wherein the composition has a drug concentration between an upper drug concentration limit and a lower drug concentration limit, wherein the upper drug concentration limit is a target drug concentration plus a 10% variation, and the lower drug concentration limit is a target drug concentration minus a 10% variation, wherein the target drug concentration is the drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio, The method includes: (a) determining a target drug concentration, wherein the target drug concentration is the drug concentration at a fixed antibody concentration and a fixed drug-antibody ratio; (b) formulating the antibody drug conjugate to produce a composition comprising a drug concentration between said upper drug concentration limit and said lower drug concentration limit, thereby producing a composition comprising an antibody drug conjugate having a drug concentration variability of ±10%, The antibody drug conjugate comprises a maytansinoid linked to an antibody.

3. The method of claim 1 or 2, wherein the antibody drug conjugate comprises a non-functional antibody.

4. The method of claim 1 or 2, wherein the antibody drug conjugate comprises an antibody comprising light chain CDR1-3, the amino acid sequences of which are shown at positions 24-38, 54-60, and 93-101 of SEQ ID NO: 4, respectively; and heavy chain CDR1-3, the amino acid sequences of which are shown at positions 31-35, 50-66, and 99-107 of SEQ ID NO: 5, respectively.

5. The method of claim 1 or 2, wherein the antibody drug conjugate comprises an antibody comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is as shown in SEQ ID NO: 4 and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:

5.

6. The method according to claim 4, wherein The maytansinoid is DM4.

7. The method according to claim 5, wherein The maytansinoid is DM4.

8. The method of claim 1 or 2, wherein the antibody drug conjugate is huMov19-sulfo-SPDB-DM4.

9. The method of claim 8, wherein the target drug concentration is 91.1 μg / ml.

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