New Antibody Technologies: An Overview of Recent Developments

Transcription

1 New Antibody Technologies: An Overview of Recent Developments Kouhei Tsumoto, Ph.D. Institute of Medical Science School of Engineering The University of Tokyo 1

2 New Antibody Technologies: An Overview of Recent Developments Recent advances in antibody technologies have made it possible to develop approved biopharmaceutical products in many therapeutic areas, including cancer and immune disorders; more than thirty monoclonal antibodies have been approved. In recent years, antibody drug conjugates (ADCs) are a class of therapeutics that combines the target specificity of an antibody with the potency of a chemotherapeutics. 2

3 New Antibody Technologies: An Overview of Recent Developments ADCs are monoclonal antibodies (mab) bearing cytotoxic drugs covalently bound via a chemical linker. Key ADC parameters to be addressed; mab to be used linker/drug selection conjugation sites, e.g. drug-to-antibody ratio (DAR). pharmacokinetic considerations and manufacturing issues 3

4 Key Characteristics of ADCs RAVI V. J. CHARI ACCOUNTS OF CHEMICAL RESEARCH (2008) 41: Kedan Lin & Jay Tibbitts Pharm Res (2012) 29:

5 New Antibody Technologies: An Overview of Recent Developments hurdles to be overcome selection of true target, e.g. cancer cell specific targets enhanced recruitment of bystander cell killing mechanism development of more economic production technologies with reliable quality. Kedan Lin & Jay Tibbitts Pharm Res (2012) 29: DOI /s y 5

6 ADCs in Clinical Development for Cancer Currently 34 ADCs are in clinical development for cancer. From Dr. Yamasaki (Tosoh), and Dr. Jackson (Igenica) 6

7 Table Approved ADCs and their product-related information ADCs Linker Drugantibody ratio (DAR) Mylotarg a (withdrew in 2010) Adcetris b (approved in 2011) Kadcyla c (approved in 2013) N-4-(4- acetylphenoxy) butanoic acid and N-acetyl-γcalicheamicin dimethl hydrazide Maleimidocaproyl -valine-citrulline 4-[Nmaleimidomethyl ] cyclohexane-1- carboxylate (MCC) Dosage form Excipients 2-3 Lyo Dextran 40, sucrose, NaCl and phosphate buffer ~4 Lyo Trehalose, polysorbate 80, and citrate buffer ~3.5 Lyo sucrose, polysorbate 20, sodium succinate ph upon reconstitution Package system? Single-use amber glass 6.6 Single-use glass vials 5.0 Single-use glass vial Product storage 2-8C 2-8C 2-8C (4h limit after recon at 2-8C) a b European public assessment report c 7

8 The limited number of ADC approvals is partly due to.. The limited number of ADC approvals is partly due to a few significant challenges still existing today such as (1) inadequate cellular uptake (<1%?) and (2) residual offtarget effects. To address the first challenge, the antibody needs to be designed to target one or more surface antigens with high binding affinity and high rate of internalization. The antibody target should be highly or overly expressed in the target cells. Significant differences in relative distribution of a particular antigen were found in different types of cancer cells and a minimum of number of antigens may be needed to initiate an effective action. 8

9 The limited number of ADC approvals is partly due to.. One can also use much smaller mab fragments such as scfv to covalently attach anti-cancer drugs. Limited uptake of antibodies would require use of cytotoxic agents, which are highly effective at low concentrations with clear mechanisms of action. For anti-tumor agents, these include disruption of DNA (such as calicheamicins and duocarmycins) and inhibition of microtubules (auristatins and maytansines). It is noted that different types of cancers may have different level of sensitivity to these tubulin-acting agents. 9

10 ADC Challenges 1. Formulation Challenges 2. Linker Chemistry Challenges 3. Challenges during product manufacturing and stability monitoring 10

11 ADC Challenges 1. Formulation Challenges 2. Linker Chemistry Challenges 3. Challenges during product manufacturing and stability monitoring 11

12 (1) Formulation challenges-1 ADCs contains three components mab, linker and drug. Among the three components, mab and the linkers are the first to be considered for formulation stabilization, as they are generally less stable than the small molecule drugs. 12

13 (1) Formulation challenges-2 mabs generally have reasonable stability and about half of all naked mab drug products are in a liquid form. Their stability can be negatively impacted during and/or after chemical modification for ADCs. Many of the small drug candidates are relatively hydrophobic such as calicheamicin and paclitaxel, and addition of hydrophobic group would enhance the hydrophobicity-driven aggregation tendency. In addition, conjugation via lysine residues would reduce the number of protein surface charges and reduce the hydrophilicity of the protein. Therefore, addition of such molecules to antibodies may impact negatively the stability of antibodies. 13

14 (1) Formulation challenges-3 Formation of protein aggregates was a major problem during preparation of antibody- calicheamicin conjugates, and the reaction conditions/additives had to be selected to reduce the tendency of conjugate aggregation during the attachment process. The paclitaxel (PTX)-monoclonal antibody (mab) conjugates (PTXMAbs) showed solubility limitations when the DAR >3, and addition of a hydrophilic component such as poly(ethylene glycol) (dpeg) would not result in any solubility issue as demonstrated in coupling PTX-L- Lys[(dPEG12)(3)-dPEG4]-dPEG6-NHS to the antibody. 14

15 (1) Formulation challenges-4 Conjugation of DM1 to Trastuzumab via lysine residues decreased the melting temperature for the CH2 transition of the mab and incubation of the conjugate led to an increase in aggregate level by approximately 5% after 14 days at 40 C while Trastuzumab alone did not show a significant change under the same condition. Covalent aggregation could occur in this case through reaction of the active maleimide moiety via Michael addition. 15

16 ADC Challenges 1. Formulation Challenges 2. Linker Chemistry Challenges 3. Challenges during product manufacturing and stability monitoring 16

17 Table Summary of linker chemistry Classifications based on different properties Linking sites Cleavability Cleaving mechanisms Lysine-linked Cleavable Acid-catalyzed Thio-linked Non-cleavable Thio-catalyzed - Natural thio Enzymecatalyzed - Engineered thio 17

18 18 Wakankar et al. mabs (2011)

19 Kedan Lin & Jay Tibbitts Pharm Res (2012) 29: DOI /s y 19

20 (2) Linker Chemistry Challenges-1 Some peptide linkers are relatively hydrophobic and can negatively influence the stability of ADCs. In the preparation of a mab and doxorubicin conjugate with Phe-Lys or Val-Cit as a linker, formation of a noncovalent dimer was observed immediately upon conjugation. Likely due to the hydrophobic nature of these peptides, addition of a hydrophilic methoxytriethylene glycol chain onto doxorubicin via a hydrazone bond greatly inhibited aggregation of the final conjugates. 20

21 (2) Linker Chemistry Challenges-2 Disulfide bonds in mabs are critical for maintaining the structural stability and function. Use of natural thios in mabs for conjugation with maleimides requires reduction of the intrachain disulfide bonds. Removing some interchain disulfide bonds would theoretically destabilize the mab, as only noncovalent association is available between the antibody heavy chains (HC) and light chains (LC). All the above examples demonstrate that ADCs may be destabilized during and/or after preparation. Significant amount of efforts may be needed in developing a stable formulation and/or robust process for successful commercialization. 21

22 Stabilization of linkers The linkers are stable enough to prevent generation of any amount of free cytotoxic drugs during formulation, manufacturing process, shipping, and storage. The fact is that most cleavable linkers discussed above possess limited in-vitro stability. For example, the conjugate vaccine amab-klh linked via succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) was stable in PBS (ph 7.2) only at -80C and significant release of free mab was seen at 4C in 6 months during storage. The linker stability can be significantly influenced by the neighboring groups. For example, hydrazone bond is generally stable at ph 7 but hydrolyzes rapidly at ph 5 to release the free drug. This is the case for hydrazone linking a polymer and a dye via a linker N-4-acetylphenyl maleimide, which showed a significantly faster rate of release of the dye at ph 5.0 than at ph 7.4 at 37C, but showed no difference between ph 7.4 and 5.0 via a heterobifunctional linker - 1-(N-3-propanal)- 4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA). Thioether oxidation was implicated in the linker instability and a different rate of oxidation was observed at different ph s. These examples demonstrate that the solution ph of ADCs is a critical parameter for evaluation in the development of a stable formulation. Lyophilization may have to be used if the linker instability is difficult to control. All three commercial ADC products are in lyophilized form. 22

23 ADC Challenges 1. Formulation Challenges 2. Linker Chemistry Challenges 3. Challenges during product manufacturing and stability monitoring 23

24 (3) Challenges during product manufacturing and stability monitoring ADCs are complex drug products. Purified drug substance intermediates include mabs, linkers, and small molecule drugs. Several reaction steps may have to be carefully controlled to make drug products consistently. Because of the nature of high toxicity of small molecule drugs, dedicated suites for handling these agents and ADCs may be required in a biopharmaceutical environment. Isolators may be ideal but operation may not be efficient. Prevention of cross contamination and good cleaning procedures have to be implemented. 24

25 (3) Challenges during product manufacturing and stability monitoring Due to the stringent requirement for aseptic processing and specific handling requirement for the cytotoxic agents, manufacturing processes may have to take place in different phases at different manufacturing sites. This will certainly make the manufacturing process complex, and time-consuming. Stability monitoring for ADCs would be different from a naked mab. In addition to quality attributes for mabs, additional ones for ADCs include at least DAR, drug distribution, free drug level, and related degradation products. Significant efforts on analytical characterization of ADCs are required. Assays used for the naked antibodies may not work for the conjugates. Direct analysis of protein-containing ADC samples on RP-HPLC can lead to column deterioration due to protein binding to the column. Removal of the interchain disulfides make it necessary to determine the intact mass of IgG1 conjugate under non-denaturing conditions. 25

26 Drug to Antibody Ratio (DAR) Drug Distribution Size Variant Analysis of Conjugates Charge-Based Separations Analysis of Unconjugated Drugs Peptide Mapping Analysis Biophysical Analysis 26

27 Drug to Antibody Ratio (DAR) UV/Vis spectroscopic analysis Hydrophobic interaction chromatography (HIC) Comparison of UV/Vis with UV-MALDI-TOF MS Drug Distribution UV-MALDI-TOF MS SEC/ESI-MS Ion-exchange chromatography (IEC), iso-electroscopic focusing electrophoresis (IEF) and capillary IEF (cief) RP-HPLC Size Variant Analysis of Conjugates SEC with organic solvent Capillary electrophoresis with SDS (CE-SDS) 27 Wakankar et al. mabs (2011)

28 Charge-Based Separations IEC and IEF Analysis of Unconjugated Drugs ELISA, CE, HPLC detected with laser-induced fluorescence (LIF), RP-HPLC-MS, LC/MS/MS, Peptide Mapping Analysis CE, capillary liquid chromatography (CLE) with LIF, RP- HPLC-MS, LC/MS/MS Biophysical Analysis Effects on higher order structure and stability; UV-Vis, near-uv-cd, Far-UV-CD, Thermal assay (DSC and DSF) Effects on solubility; aggregation propensity, e.g. DLS, MFI, AUC, etc. 28 Wakankar et al. mabs (2011)

29 Drug to Antibody Ratio (DAR) UV/Vis spectroscopic analysis Hydrophobic interaction chromatography (HIC) 29 Wakankar et al. mabs (2011)

30 Drug Distribution SEC/ESI-MS 30 Wakankar et al. mabs (2011)

31 RP-HPLC (A-D) and CE-SDS (E-H) RP-HPLC; reduced and alkylated CE-SDS; non-reduced Wakankar et al. mabs (2011) 31

32 Size Variant Analysis of Conjugates SEC with or w/o organic solvent Wakankar et al. mabs (2011) 32

33 Significant reduction of concentration of conjugated antibody PK of total antibody from ADC results from mixed effects of conjugated antibody with naked antibody Kedan Lin & Jay Tibbitts Pharm Res (2012) 29: DOI /s y 33

34 SEC and DSC They have used.. HIC SEC with MALS DAR by UV RP-HPLC DSC Far-UV CD CE-SDS Trp Fluorescence 34

35 35

36 DSC measurement Onset temperature is critical for description of thermal stability. BBRC 355, 751 (2007) 36

37 DSC 37

38 Analytical Methods for ADC: Perspectives Developments in mass spectroscopy; TOF analyzers, MS/MS, MS/MS/MS CE, imaged capillary isoelectric focusing (icif) system HPLC column technologies (SEC, HIC, IEC) Combination of ELISA, cell-based potency assays 38

39 Analytical Methods for ADC: Critical Quality Attributes (CQAs) There is an increasing body of literature devoted to understanding what attributes are critical. In the case of ADC Size variants (aggregates and fragments) Drug distribution and average DAR Free drug level Other CQAs; case-by-case, including charged variants, process impurities such as those related to the drug and the conjugation, and product isoforms Wakankar et al. mabs (2011) 39

40 Concluding Remarks ADCs have been proved to be an effective weapon on top of mab s in combating cancer. At the same time, development of a commercial ADC drug product presents specific challenges. These include, but not limited to, identification and selection of the right surface antigen, design and selection of a suitable linker, development of shelf-stable product formulation, establishment of suitable analytical methods for monitoring critical quality attributes, and optimization of the manufacturing process. Through combination of analytical and process knowledge, ADCs with consistent quality, safety and efficacy can be produced for clinical and commercial use. 40

41 Acknowledgement Laboratory of Physical Biochemistry Dr. Nagatoishi Dr. Akiba Dr. Jose MM Caaveiro Tsutomu Arakawa (APL, CA) Daisuke Ejima (Ajinomoto Inc.) Satoshi Ohtake (Pfizer) Izumi Kumagai (Tohoku Univ.) 41

42 Thank you for your kind attention! 42