CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/556,153 filed September 8, 2017, the contents of which are incorporated herein by reference.

BACKGROUND

[0002] Antibody-drug-conjugates (ADCs) are an emerging class of drug molecules.

Their ability to locate to a specific target and deliver a potent drug makes them an attractive option for developing a target based therapeutic product. ADCs are produced by chemically linking potent drug molecules to a monoclonal antibody via a selected chemical linker. The average number of drug molecules that are conjugated to the monoclonal antibody is called drug-to-antibody ratio, ("DAR"). DAR is an important quality attribute of ADC products, because it can impact product efficacy, safety and/or stability. Accordingly, methods of assessing DAR of ADC products in a reliable and high throughput manner are desirable.

DETAILED DESCRIPTION

[0003] The present disclosure provides methods of assessing DAR of ADC products that provide advantages over known methods. Specifically, methods of the disclosure can be used in high-throughput applications and/or without having to dilute ADC samples during the assessment.

UV-Vis and Beer-Lambert 's Law

[0004] DAR has traditionally been measured using UV-Vis spectroscopy (see, e.g.,

Chen, Methods Mol. Biol. 1045:267-73 (2013)). The basis for this analysis is the Beer- Lambert law, a direct proportional relationship between the absorbance and concentration of a substance:

A = eel, where A is the absorbance, ε is the extinction coefficient (a physical constant of the substance), I is the path length through the cell containing the analyte, and c is the concentration. [0005] DAR measurement for an ADC product using UV-Vis spectroscopy relies on the difference in absorption maxima for the antibody (e.g., 280 nm) and the absorption maxima for the drug (e.g., at 252 nm). For example, the average DAR can be calculated using the difference in the measured absorption at 280 nm and 252 nm for the conjugated material. Although the UV-Vis method is widely used in industry, it lacks the throughput needed for formulation screening studies. It also cannot be used without sample dilution, leading to errors associated with sample dilution.

[0006] The present disclosure is thus based, at least in part, on alternative methods for measuring DAR using size exclusion chromatography (e.g., UPLC) and slope spectroscopy. These methods were characterized and compared to UV-Vis spectroscopy with respect to reproducibility, precision and sensitivity. The data generated support use of UPLC-based DAR methods to overcome the throughput limitations of traditional UV-Vis methods.

Further, the slope spectroscopy based method can be used to analyze ADC samples without sample dilution.

UPLC-based methods

[0007] In one embodiment, size exclusion is used to determine DAR. In some embodiments, the methods disclosed herein comprise applying a sample comprising an antibody-drug conjugate to a size-exclusion chromatography matrix. In some embodiments, the methods disclosed herein comprise applying to and running a sample comprising an antibody-drug conjugate through a size-exclusion chromatography matrix. In some embodiments, a total amount of ADC sample is applied to a size exclusion matrix for analysis. For example, the following UPLC-based methodology was used to assess DAR.

Gradient Info: Isocratic

Flow Rate: 0.4 mL/min

Method Run Time: 12 minutes

Column Temperature: Not controlled

Wavelength of

280 nm and 252 nm (or Amax for conjugated drug)

Detection

Target Injection

6-150 μg ADC (no dilution)

Amount

[0008] Data collected at 280 nm were integrated using Empower' s Apex Track integration method with peak shoulder detection. The retention time integration range is molecule dependent, but is usually within 3-9 minutes. The peak with largest height and area was classified as "native", "main" or "monomer" peak. Any peaks eluting earlier than the "native" peak were classified as "HMW" peaks. Any peaks eluting later than the "native peak" were classified as "LMW" peaks.

[0009] The relative percentage of each species was calculated from the ratio of the area of individual peaks to the total area of all peaks. The relative percent area of the following was reported as an indicator of purity: % Total HMW, % Native (or Main or Monomer), and % Total LMW. The total area of all peaks was summed and used in subsequent DAR calculations. However, in some embodiments, only the area of the native peak is used.

[0010] Data collected at 252 nm were integrated using Empower' s Apex Track integration method with peak shoulder detection. The retention time integration range is molecule dependent, but is usually within 3-9 minutes. The total area of all peaks was summed and used in subsequent DAR calculations. However, in some embodiments, only the area of the native peak is used.

[0011] DAR was determined from the total peak area at 280 nm (Amax for the ADC) and the total peak area at 252 nm (Amax for the drug). Although 252 nm is a common Amax for drug conjugates used for ADCs, an appropriate wavelength can be selected for a specific conjugate, e.g., using known methods. The amount of drug bound to the antibody can be determined by the difference in total peak areas at these two wavelengths, using the naked antibody as a reference standard, if applicable. [0012] The following two equations (which were derived from the Beer-Lambert law) were verified and demonstrated consistency.

Equation 1 :

[0013] Equation 1 does not require the use of a naked antibody reference standard.

However, systematic determination of extinction coefficients (ε) for both the antibody and the drug at 252 nm is required. The extinction coefficient at a given wavelength can readily be calculated from the Beer-Lambert law by using a solution of either the antibody or the drug having a known concentration and measuring the absorbance at the given wavelength.

Equation 2:

.mAb

•280nm

DAR=

Total AreajSK

Total Area^^n * Total Area^o^m - Total Area^^ * Total Area^^

drug * r , ADC drug * _χ , _{A rp} „ADC

8252nm ^{1 0tal Area} 280nm ^{" 8} ₂₈ 0nm ^{1 0tal Area} 252nm

[0014] Equation 2 does not require extinction coefficient determination for the antibody at 252 nm, but it does require collection of UPLC data for a naked antibody reference standard.

[0015] Although UPLC has been exemplified, other size exclusion chromatography techniques can be used in methods described herein. Size exclusion chromatography generally refers to separation of molecules by size, where the chromatographic elution time is characteristic for a particular molecule. Additional methods include, e.g., SEC-HPLC, reversed phase (RP) HPLC, RP-UPLC.

[0016] In some embodiments, an ADC sample is not diluted prior to analysis by size exclusion chromatography (e.g., HPLC or UPLC). In some embodiments, an ADC sample does not require dilution prior to analysis by size exclusion chromatography as a total amount of the ADC sample is applied to the size exclusion chromatography matrix. In some embodiments, a sample containing about 1 μg/μL to about 500 μg/μL ADC is analyzed.

Slope spectroscopy-based methods [0017] In some embodiments, DAR is determined by calculating the concentrations of antibody and drug in an ADC sample. For example, slope spectroscopy is a known method for determining the absorbance of a solution at various path lengths. The values of the absorbance at various path lengths can then be used, based on the Beer-Lambert law, to calculate the concentration of a compound in the solution. Methods and systems employing slope spectroscopy are known (see, e.g., US Publ. No. 20120130649) and commercially available (see, e.g., SoloVPE (C Technologies, Inc., Bridgewater, NJ)). Such methods and systems were adapted to measure concentrations of antibody and drug in ADC preparations, from which a DAR was determined.

[0018] For example, an ADC sample can be placed in a vessel; a probe can be moved relative to the vessel such that the probe makes contact with the bottom of the vessel; the probe can be moved relative to the vessel such that the probe moves from the bottom of the vessel through the sample by a predetermined increment such that a preselected path length through the solution is obtained; an absorbance reading can be taken at an absorption maxima for the antibody; the probe can be moved repeatedly relative to the sample and a

measurement can be taken; a regression line can be generated from the absorbance and path length such that a slope of the regression line is obtained; and the concentration of the antibody can be determined by dividing the slope of the regression line by the extinction coefficient of the antibody. The steps can then be repeated using the absorption maxima for the drug to determine the concentration of the drug. DAR can be calculated from the determined drug concentration and antibody concentration.

[0019] In some embodiments, an ADC sample is not diluted prior to analysis by slope spectroscopy. In some embodiments, a sample containing about 0.1 μg/μL to about 500 μg/μL ADC is analyzed.

Antibody-Drug Conjugates

[0020] The term "antibody-drug conjugate" as used herein, refers to a protein that is created by linking an antibody to a biologically active cytotoxic payload or drug. Antibody- drug conjugates (ADC) are generally produced through chemical modification/coupling reactions known to those skilled in the art. Any antibody-drug conjugate can be analyzed using the methods described herein.

[0021] In some embodiments, an antibody-drug conjugate includes an anti-tumor antibody (see, e.g., Adler et al., Hematol. Oncol. Clin. North Am. 26:447-81 (2012); Li et al., Drug Discov. Ther. 7: 178-84 (2013); Scott et al., Cancer Immun. 12: 14 (2012); and

Sliwkowski et al., Science 341 : 1192-1198 (2013)). Table 1 presents a non-comprehensive list of certain human polypeptide antigens targeted by known, available antibody agents, and notes certain cancer indications for which the antibody agents have been proposed to be useful. Any of the antibodies in Table 1 can be included in an antibody-drug conjugate assessed using methods of the disclosure.

Table 1 :

CD138 (SYND1) BT062 Multiple Myeloma

CD 152 (CTLA-4) Ipilimumab Metastatic melanoma

CD221 (IGF1R) AVE 1642, IMC-A12, MK-0646, Glioma, lung, breast, head and neck,

R150, CP 751871 prostate and thyroid cancer

CD254 (RANKL) Denosumab Breast and prostate carcinoma

CD261 (TRAILR1) Mapatumumab Colon, lung and pancreas tumors and

haematological malignancies

CD262 (TRAILR2) HGS-ETR2, CS-1008

CD326 (Epcam) Edrecolomab, 17-1A, IGN101, Colon and rectal cancer, malignant ascites,

Catumaxomab, Adecatumumab epithelial tumors (breast, colon, lung)

CD309 (VEGFR2) IM-2C6, CDP791 Epithelium-derived solid tumors

CD319 (SLAMF7) HuLuc63 Multiple myeloma

CD340 (HER2) Trastuzumab, Pertuzumab, Ado- Breast cancer

trastuzumab emtansine

CAIX (CA9) cG250 Renal cell carcinoma

EGFR (c-erbB) Cetuximab, Panitumumab, Solid tumors including glioma, lung, breast, colon, and head and neck tumors nimotuzumab and 806

EPHA3 (HEK) KB004, IIIA4 Lung, kidney and colon tumors, melanoma, glioma and haematological malignancies

Episialin Epitumomab Epithelial ovarian tumors

FAP Sibrotuzumab and F19 Colon, breast, lung, pancreas, and head and neck tumors

HLA-DR beta Apolizumab Chronic lymphocytic leukemia, non- Hodkin's lymphoma

FOLR-1 Farletuzumab Ovarian tumors

5T4 Anatumomab Non-small cell lung cancer

GD3/GD2 3F8, chl4.18, KW-2871 Neuroectodermal and epithelial tumors gpA33 huA33 Colorectal carcinoma

GPNMB Glembatumumab Breast cancer

HER3 (ERBB3) MM- 121 Breast, colon, lung, ovarian, and prostate tumors

Integrin αΥβ3 Etaracizumab Tumor vasculature Integrin α5β1 Volociximab Tumor vasculature

Lewis-Y antigen hu3S193, IgN311 Breast, colon, lung and prostate tumors

MET (HGFR) AMG 102, METMAB, SCH90010i Breast, ovary and lung tumors

Mucin- 1/CanAg Pemtumomab, oregovomab, Breast, colon, lung and ovarian tumors

Cantuzumab

PSMA ADC, J591 Prostate Cancer

Phosphatidylserine Bavituximab Solid tumors

TAG-72 Minretumomab Breast, colon and lung tumors

Tenascin 81C6 Glioma, breast and prostate tumours

VEGF Bevacizumab Tumour vasculature

[0022] In some embodiments, an antibody-drug conjugate includes a drug that is one or more of pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, a drug is a chemotherapeutic agent useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents such as taxanes, maytansine and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil,

Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or analogs thereof (e.g., DM1),

Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin,

Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof.

[0023] In some embodiments, an antibody-drug conjugate assessed using a method of the disclosure is hLLl -doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN- 38, hPAM4-SN-38, hLLl-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P- Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLLl-Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, CMC-544, SAR3419, CDX-011, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG- 5 ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG- 7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, FMGN-853, IMGN-529, IMGN-901, vorsetuzumab mafodotin, or lorvotuzumab mertansine (see, e.g., Sassoon et al., Methods Mol. Biol. 1045: 1-27 (2013); Bouchard et al., Bioorganic Med.

Chem. Lett. 24: 5357-5363 (2014)).

Applications

[0024] Methods of the disclosure have a variety of applications and include, e.g., quality control at different stages of manufacture of a drug substance or drug product, analysis of an ADC preparation prior to and/or after completion of a drug substance or drug product manufacture (e.g., prior to or after distribution to a fill/finish environment or facility), prior to or after release of a drug substance or drug product into commerce (e.g., before distribution to a pharmacy, a caregiver, a patient, or other end-user). In some instances, an ADC preparation is a drug substance (an active pharmaceutical ingredient or "API") or a drug product (an API formulated for use in a subject such as a human patient). In some instances, an ADC preparation is from a stage of manufacture or use that is prior to release to care givers or other end-users; prior to packaging into individual dosage forms, such as syringes, pens, vials, or multi-dose vials; prior to determination that the batch can be commercially released, prior to production of a Certificate of Testing, Material Safety Data Sheet (MSDS) or Certificate of Analysis (CofA) of the preparation.

[0025] Assessments from methods described herein are useful for guiding, controlling or implementing a number of activities or steps in the process of making, distributing, and monitoring and providing for the safe and efficacious use of an ADC preparation. Thus, in an embodiment, e.g., responsive to the evaluation, e.g., depending on whether a criterion is met (e.g., a particular DAR, average DAR, and/or DAR range), a decision or step is taken.

Methods described herein may include making a decision: (a) as to whether an ADC preparation may be formulated into drug substance or drug product; (b) as to whether an ADC preparation may be reprocessed (e.g., the preparation may undergo a repetition of a previous process step); and/or (c) that the ADC preparation is not suitable for formulation into drug substance or drug product. In some instances, methods comprise: formulating as referred to in step (a), reprocessing as referred to in step (b), or rendering the preparation unusable for commercial release, e.g., by labeling it or destroying it, as referred to in step (c).