Leading Contract Development and Manufacturing Organization (CDMO) MabPlex International, serving the global biopharmaceutical and biotechnology industries with sites in China and the United States, confirmed that an Investigational New Drug (IND) application has been approved by the U.S. Food and Drug Administration (FDA) for one of the company’s undisclosed customer’s proprietary antibody drugs.
The IND program is the means by which a pharmaceutical company obtains permission to start human clinical trials and to ship an experimental drug across state lines, within the United States, to clinical investigators before a marketing application for the drug has been approved.
MabPlex, which offers comprehensive (cell line) development, process characterization, conjugation optimization, cGMP manufacturing, and aseptic fill finish solutions for monoclonal antibody (mAb) and antibody-drug conjugate (ADC) therapeutics, was contracted to develop and manufacture the product from cell line development to IND filing in both the United States and China.
MabPlex’s high standards in development and cGMP quality management have been recognized by regulators in the United States, Australia and China, demonstrating the strength of the company’s global CDMO services…
For this project, MabPlex provided fully integrated services from cell line development to final drug product including a complete CMC package in support of the United States IND submission. Additionally, MabPlex will assist in completing the IND filing for the Chinese NMPA in the near future.
“This approval adds another milestone to our global repertoire of international IND submissions after the successful approval of an Australian IND last year,” noted Jianmin Fang, Ph.D, Chairman and Chief Executive Officer of MabPlex International.
“MabPlex’s high standards in development and cGMP quality management have been recognized by US, Australian and Chinese regulators demonstrating the strength of our global CDMO services,” Fang added.
Founded in 2013, MabPlex currently has two sites in China (Yantai and Shanghai) and one site in the United States (San Diego, CA).
Sutro Biopharma confirmed that the U.S. Food and Drug Administration (FDA) has concluded their 30-day review of the Investigational New Drug (IND) application for STRO-002 to be evaluated in a Phase I clinical study as a potential treatment for ovarian and endometrial cancer.
STRO-002 is an antibody-drug conjugate or ADC targeting folate receptor-α, a cell-surface protein expressed in 80% of gynecological cancers, including ovarian and endometrial cancers.
The investigational drug was developed using Sutro’s proprietary cell-free protein synthesis and site-specific conjugation platform, XpressCF+™, which enables precise design, rapid empirical optimization, and manufacture of site-specific ADCs.
In contrast to first-generation commercially available ADCs that comprise a mixture of imprecisely conjugated antibodies, Sutro’s proprietary and integrated cell-free protein synthesis and site-specific conjugation platform, XpressCF+™, led to the discovery of STRO-001 and STRO-002, and results in highly optimized ADCs comprising a single molecular species.
STRO-001 is a first-in-class ADC targeting CD74, a protein highly expressed in multiple myeloma and non-Hodgkin’s lymphoma, and is currently in a Phase I study.
STRO-002, , has been engineered to use Sutro’s novel, proprietary SC239 linker-payload, designed for increased stability and potency, which results in effective targeting of cancer cells and precise delivery of the payload.
“The ability to begin our Phase I clinical study marks an important milestone that expands Sutro’s clinical development pipeline and further validates our technology for the design of unique and potent antibody-drug conjugates,” said Bill Newell, Sutro’s Chief Executive Officer.
“The Phase I clinical trial of STRO-002 is expected to begin in early 2019 with the goal to investigate the safety, tolerability and preliminary anti-tumor activity of STRO-002 in patients with gynecologic malignancies,” Newell added.
Patients with ovarian cancer will be enrolled during the dose escalation-phase of the study, and two separate cohorts for ovarian and endometrial cancer will be evaluated during dose expansion.
“Ovarian and endometrial cancer patients need targeted treatment options with better tolerability and efficacy,” noted Wendel Naumann, MD, a Gynecologic Oncologist Professor, at the Levine Cancer Institute, Carolinas Medical Center.
In preclinical studies, STRO-002 effectively delivered its cytotoxin to targeted cancer cells without significant accumulation of a toxic metabolite in the blood. Testing of clinically relevant doses in non-human primates showed no evidence of ocular toxicity, a vexing problem associated with conventional ADCs containing standard tubulin-inhibiting agents.
“This is an important development for ADC-based cancer therapeutics and could provide new means to achieving greater anti-tumor activity in the clinic before the onset of dose-limiting side effects,” added Arturo Molina, MD, Sutro’s Chief Medical Officer.
Unlike first-generation ADCs, STRO-002 is a homogeneous, site-specific antibody-drug conjugate that incorporates a novel, proprietary linker-warhead, thereby enabling effective and precise payload delivery to targeted cancer cells. Preclinical studies demonstrated efficacy of STRO-002’s potent in vitro cytotoxicity in ovarian and endometrial cancer cell lines, and tumor growth inhibition in multiple in vivo ovarian and endometrial cancer models.
Safety studies conducted in non-human primates have shown tolerability at clinically relevant doses with no observed ocular toxicity.
Antibody drug conjugates (ADCs) are a relatively new type of drug that combines the targeting ability of a biologic with a highly potent cytotoxic agent.
This powerful combination promises to become a game-changer in the fight against cancer—potentially replacing broad spectrum chemotherapies with more specific, less damaging options. At the same time, because ADCs’ cell-killing drug payloads are thousands of times more toxic than conventional treatments, safety concerns are proportionally amplified. That makes gaining regulatory approval for first-in-man studies far more demanding than with a traditional biopharmaceutical.
While it’s natural for pharmaceutical developers to focus on toxicological and pharmacological findings from animal studies, far too often, early stage ADC developers underestimate the importance of their filing’s Chemistry Manufacturing and Controls (CMC) section. This may result in regulatory requests for additional information or unanticipated studies, which can delay or even permanently derail a promising program.
This white paper discusses a pragmatic approach to helping ADC developers ensure IND success. It highlights two main challenges:
Complexity of the ADC molecule
Insufficient CMC data
This publication outlines strategic and analytical approaches that can save time and effort, and help ensure that regulatory requirements for CMC data are satisfied. It suggests that the best way to accelerate the regulatory path to first-in-man studies is to focus the CMC development plan on three areas:
Critical Quality Attributes (CQA)
Frequently overlooked studies
1.0 Antibody-drug Conjugates and the IND Process
Before human clinical trials can commence in the United States, new drugs must go through a complicated and time-consuming Investigational New Drug (IND) application and approval process. An IND application must demonstrate complete pharmaceutical or biopharmaceutical analyses. In addition to extensive data from animal pharmacology, toxicology studies, clinical protocols and investigator information, it must include detailed Chemistry, Manufacturing and Controls (CMC) information on the manufacturing and stability of the clinical trial material (CTM).
When it comes to clinical studies with ADCs, additional scrutiny of CTM is to be expected. The inherent instability of biologics, together with the level of toxicity associated with an ADC’s small molecule payload have grave implications on patient safety. It is not surprising, then, that CMC data requirements and the level of analytical support needed to support an ADC program are substantially greater than with more traditional therapies.
According to the editors of ADC Review / Journal of Antibody Drug Conjugates, “One of the most critical aspects is to address all the unique issues involved in the submission of an IND completely, correctly, and in a timely fashion…” 
Incomplete or incorrect information can result in requests for additional studies, delaying the filing of a successful IND or worse—the financially motivated end to an otherwise promising program. But with a well-planned approach to testing and diverse technical/analytical expertise on your team, ADC developers can avoid these pitfalls and help ensure a seamless path to the clinic.
2.0 Why ADC development is so hard
According to the 2016 Nice Insight CDMO Outsourcing Survey, 57% of companies surveyed said they were developing ADCs, compared to 51% who said they have naked monoclonal antibodies (mAbs) in development. Another source states that 182 companies around the world have ADCs in their pipeline. Despite this surge, only four ADCs have been licensed to date. Plenty of examples exist of drugs that showed potential in early pre-clinical stages, but didn’t progress, and were terminated. Many of these failures were due to toxicity or incomplete characterization data.
This white paper deals with two of the most common challenges relating to IND approval for ADCs. These are:
The complexity of the ADC molecule itself, which is critical, as analysis of this complex structure informs decisions about its design and manufacture.
Lack of necessary CMC data on the clinical trial Material
2.1 Challenge #1 – The complexity of the ADC molecule
The analytical challenges unique to ADC development are numerous, but chief among them are the complexity and stability of the mAb, the very difficult synthesis and characterization of the small molecule payload (cytotoxic agent) and linker, the chemical linking chemistry, and different conjugations that may be involved. 
Understanding the structure and behavior of biologically derived molecules–and interpreting analytical findings to inform development decisions—requires a myriad of analytical techniques and experienced biopharmaceutical scientists.
Few Contract Manufacturing Organizations (CMOs) have the breadth of testing services required for full biopharmaceutical analysis. Not surprisingly, an estimated 70%-80% of ADC analysis is outsourced.
ADC analysis also requires expertise handling highly cytotoxic compounds. Because the potency of ADC payloads is much greater than biologic drugs, it is crucial to truly understand the role that each part of the ADC – mAb, linker and cytotoxic agent – plays in the toxicity, stability and safety of a new drug.
Linkers: improvements in linker design focus on serum stability and drug-to-antibody ratio (DAR). The overall concern with linkers is to produce more homogenous ADC populations by studying the conjugation between linker and mAb.
Payloads: choosing the right payload involves certain basic criteria, such as solubility, stability, and the likelihood of conjugation. But ascertaining the correct drug potency also has proven to be a critical factor. According to McCombs et al, “poor clinical efficacy of first-generation ADCs is attributed to sub-therapeutic levels of drug reaching the target.”
The IND analytical package must include not only assays and purity analyses, but also the drug-to-antibody ratio (DAR) and site(s) of conjugation. Only advanced biopharmaceutical analysis can supply this information.
Selecting the right analytical techniques is critical. Valliere- Douglass et al. suggest that conventional analytical methods used for standard biopharma characterization are not sufficient for ADCs. They outline the latest methods in mass spectrometry that have helped scientists fully characterize ADC drugs when conventional techniques fall short.
A list of analytical services and techniques necessary for ADC characterization is given in Part 4 of this white paper.
2.2 Challenge #2 – Failure to provide sufficient CMC data
One of the primary reasons IND submissions for new ADCs are delayed is because the biopharma company (or their contract service provider) fails to perform analyses in accordance with Chemistry, Manufacturing and Controls (CMC) guidelines.
This is because nine times out of ten, the drug developer lacks a clear plan for meeting CMC data requirements when mapping the development process.16 In fact, a key factor in streamlining your IND submission for a new ADC is finding a development partner who can help you articulate a well-planned CMC strategy early in the project.
Complete structural characterization, physico-chemical testing, and biophysical analysis of the antibody-drug conjugate are required. This includes the parent monoclonal antibody, as well as analysis of biological activity, toxicity, and stability of the drug product. Table 1 on the following page shows the structural analysis needed for the mAb intermediate.
As already mentioned, ADC analysis is more complex than traditional biopharmaceutical analysis. Multiple biopharma studies and analytical methods are required, as well as concurrent expertise in performing these techniques and interpreting the data.
Table :Necessary analysis of mAb to meet CMC guidelines, and corresponding analytical techniques
Bottom line: you may find traditional techniques used for biopharmaceutical analyses are quickly becoming obsolete. New, highly sensitive and specific technologies are becoming the standard, and are indispensable if you are to progress through the clinic ahead of your competition.
3.0 Why traditional approaches fall short
The complexity of the ADC molecule and lack of emphasis on CMC development strategy are the primary causes for delays in ADC IND approvals. But since most early stage developers lack internal analytical resources, they must partner with consultants or CROs who understand regulatory guidance and can help them navigate the IND process. They also need access to a full suite of cGLP and cGMP-compliant analytical testing services. But it can be difficult to find a partner with the experience and capabilities necessary to step into this role.
There are two primary reasons why the choice of outsourcing partners can be especially critical for ADC developers:
Older techniques are unable to provide the analyses necessary for ADC molecules – the stability of specific molecules cannot be determined, and a deep understanding of the molecule may not be possible.
Absence of a Plan
All too often, early stage developers lack a defined CMC strategy. When this is the case, archived samples often aren’t set aside, validation reports and studies are inconclusive, and compatibility studies are overlooked—all of which can lead to delays and/or insufficient data. In the absence of a clearly defined testing strategy, analytical methods are not in place to ensure the identity, strength, quality, purity and potency of the drug. These are required for every New Drug Application (NDA).
Finally, according to an article by Amer Alghabban in Pharmaceutical Outsourcing: “The way a pharmaceutical company contracts CROs/ CMOs has a critical and direct impact on a company’s realization of its goal”
Alghabban states that many manufacturers – 45.6% in one survey–have reported quality problems with their vendors, inexperience with regulatory requirements, and 49.1% of vendors were not able to keep their promises.
Ultimately, current practices fail to overcome the two challenges outlined in section 2 because ADC developers partner with the wrong CRO.
4.0 Three ways to streamline the IND Process for ADCs
There are proven ways to increase your chances of successfully filing an IND for a new ADC, and at the same time reduce the amount of effort and expense involved.
Complete characterization and protein analysis play the most important part in this process. This means characterizing attributes such as the drug-to-antibody ratio (DAR) and sites of conjugation. DAR is a critical factor for ADCs, because it represents the average number of drugs conjugated to the mAb. The DAR value influences the drug’s effectiveness, as low toxin loading lowers potency, and high toxin loading can negatively affect pharmacokinetics (PK) and toxicity. Sites of conjugation are important, because improving site-specific drug attachment can result in more homogeneous conjugates and allow control of the site of drug attachment.
There are several considerations that can accelerate time-toclinical trials for an ADC. These include:
Analyzing critical quality attributes, or CQA
Developing a defined testing plan to ensure no necessary studies are overlooked, such as compatibility and residual solvent analysis—and a schedule that ensures the most efficient and timely completion
Adopting platform approaches to ADC development
The following sub-sections will address each of these in turn.
4.1 Conduct Detailed Studies of Critical Quality Attributes
Critical quality attributes (CQA) are biological, chemical and physical attributes that are measured to ensure the final drug product maintains its quality, safety, and potency. The precursor to defining CQAs is complete characterization of the drug product and intermediates.
Currently, characterization of the mAb intermediate is already well defined, and includes studies such as:
Mass Analysis — Intact, reduced, deglycosylated
Peptide Map (UPLC–UHR QTof MS): sequencing, Post Translational Modifications (PTMs) and disulfide linkages
N-Glycan Profile Site, extent and structure of glycosylation
Differential scanning calorimetry
CQAs (relating to safety and efficacy of the drug) for an ADC product also include the following additional assays:
Appropriate analytical techniques
Drug-to-antibody ratio (DAR)
Drug load distribution
Peptide map-UPLC-UHR QToF
Peptide map-UPLC-UHR QToF
Linker payload structure
FTIR, UPLC/MS/MS, NMR
Table 2:CQAs for an ADC relating to safety and efficacy, and corresponding analytical techniques
Additional attributes considered CQA, due to their impact on health and efficacy include:
Free drug concentration
Free Drug Concentration
As mentioned earlier, the FDA is concerned primarily with human safety in regards to an IND submission. With ADCs, this means they are concerned with the concentration of free drug (toxin) in the final product — both on release and on stability. While the main advantage of ADCs is their targeted specificity, any free toxin introduced into the bloodstream is a serious threat to human health and safety. Therefore, any assay used to measure free drug concentration must be exceptionally sensitive (≤1 ng/ mL). This is typically performed by UPLC/MRM/MS.
Antigen binding is vital to the efficacy and specificity of an ADC. Non-specific binding results in the death of healthy cells and toxicity. Techniques to measure binding include:
Enzyme-linked immunosorbent assay (ELISA) – a biochemical technique for detecting and quantifying peptides, proteins and antibodies. Multiple formats can be utilized, but all incorporate binding of an antibody to the analyte resulting in a subsequent signal (UV, fluorescence, phosphorescence).
Electro-chemiluminescence (ECL) – a detection method based on luminescence from electrochemical reactions. ELISA and ECL can be used interchangeably, but ECL’s greater sensitivity allows it to be used in other studies, streamlining the IND process.
Surface Plasmon Resonance (SPR) – a label-free method used to monitor noncovalent molecular interactions in real time. Generally considered a poor candidate for antigen binding, due to poor inter-day precision.
While all of the physico-chemical analyses (CE, icIEF, SEC, etc.) provide an idea of the purity and stability of a single aspect of an ADC, they do not provide a measure of the functional stability of the entire molecule. Cell bioassays are the ultimate measure of an ADC’s activity, stability and 3-dimensional structure, as they measure the effect of all degradation pathways. Bioassays, by their very nature, are variable and are technique-dependent, making them difficult to utilize as part of your IND submission. While research quality bioassays are sufficient for drug development; a qualified, accurate cell bioassay is an absolute requirement for an IND application. Optimizing these assays to make them precise and robust requires expert and experienced scientists. They provide a method that can be confidently used for stability and post-IND formulation development. Upon IND approval, these studies should be initiated immediately, shortening formulation/ process optimization.
4.2 Perform Studies that are often overlooked
A successful IND depends on multiple studies – particularly relating to toxicology – that are often overlooked, or even neglected. This is due to a lack of planning early on in the process. And these oversights can result in delays of several months.
A number of overlooked studies should be performed prior to initiation of toxicology and other early clinical tests. These include:
Infusion set/syringe compatibility
Toxicology studies are performed at low doses and require greater sensitivity than release/stability assays. As required by the FDA, dose formulations must be assayed for toxicology studies, to ensure the correct dose is being delivered. The typical approach is ECL or ELISA. If ECL is developed for release, it is easily adapted to these studies, streamlining the overall IND process.
Infusion Set/Syringe Compatibility
Concern has been raised about the occurrence of critical incidents related to infusion sets. Every drug developer and CRO needs to establish a set of procedures to evaluate infusion sets from their vendors, particularly in terms of drug loss to surfaces. This includes filters, pre- and post-IV bags, and tubing. Multiple concentrations and durations should be tested.
According to the FDA: “The purpose of in-use stability testing is to establish a period of time during which a multiple-dose drug product may be used while retaining acceptable quality specifications once the container is opened.” 
The FDA recently announced a draft GIF #242 entitled “In- Use Stability Studies and Associated Labeling Statements for Multiple-Dose Injectable Animal Drug Products”. The draft will outline how to design and carry out in-use stability studies to support the in-use statements, for multiple-dose injectable drug products.22 While this focuses on animal and multi-dose studies, the draft also reflects the importance the FDA places on in-use stability for human trials, and yet they are often neglected during the IND process.
Multiple stability-indicating assays are required, including:
Size Exclusion Chromatography (SEC)
Micro Flow Imaging (MFI)
The linkage of the payload to the monoclonal antibody is an organic chemical event involving many of the typical solvents and catalysts. Therefore, similar to traditional pharmaceuticals, both residual solvents and heavy metals must be monitored on release of the drug substance. Typical assays include:
4.3 Adopt a “Platform’ Approach
The basic idea behind a platform approach is to leverage “prior knowledge” to reduce the effort needed to start clinical trials. It begins with identifying a class of molecules that show comparable characteristics, such as physico-chemical properties and stability profiles.
New candidates with characteristics that match known molecules can be treated as a “next-in-class” candidates. Once comparable characteristics are validated, developers can focus additional testing on areas of difference between the new candidate and historical likenesses—reducing testing requirements and at the same time further adding to the body of shared knowledge related to the platform, and increasing the platform’s robustness. Adopting a platform approach can significantly streamline IND testing requirements, accelerating time to clinic and reducing costs. According to Bradl et al., the platform approach enabled biopharmaceutical development for toxicological studies within 14 months after receiving DNA sequences.  After another six months, material from GMP facilities was provided for clinical studies. This resulted in a time requirement of 20 months from DNA to Investigational Medicinal Product Dossier.
Of course, a key element is actually identifying those molecules that match the definition of a “next-in-class” candidate. Careful planning in regards to methods, data, and documentation will provide a universal approach applicable to other antibody drug conjugates.
Standardization of instrumental parameters, data collection and data manipulation can speed up characterization. The necessary studies include:
1. QToF – An ultra-high resolution Quadrupole Time of Flight MS, coupled to a UPLC can provide the vast majority of characterization data. Powerful QToF software, designed specifically for proteins, deconvolutes complicated mass spectra, simplifying data interpretation. The QToF can determine:
Post translational modifications
Payload linkage sites
2. Release and Stability:
The majority of assays are similar for all ADCs: SDS CE, icIEF, SEC, UV, and DAR. Generic assays can be qualified directly and only modified/optimized if qualification criteria are not met.
Design method qualifications appropriate to Phase I and template protocols
Binding assays should all utilize ECL. The sensitivity of this technique allows it to be used for toxicology and compatibility studies, as well as release and stability.
Other investigations typically include prophylactic studies in anticipation of agency questions. While they are not necessarily required for the IND filing, having data to support responses to agency questions will prevent delays. By preparing data in an IND-ready format, you’ll ensure “drag and drop” of the data, greatly facilitating the process in the typical last minute rush to complete the IND.
5.0 Buyer’s Guide: Choosing the right CRO for Fast IND Submission and Approval
According to a report by Global Industry Analysts, Inc., the global biopharma market is estimated to reach U.S. $ 306 billion by the year 2020.25
With this continued market expansion, including antibody drug conjugate development, there is a greater need for contract lab support. Not only this, but there is a critical need for high-quality contract laboratory partners who understand the regulatory guidelines, can perform required risk assessments, and can develop, validate and execute challenging analytical procedures.
If you’re looking for help from a CRO to reduce risk, and increase your chances of a successful IND submission, here’s what you need to look for:
True loyalty and partnership
You need a CRO that will take complete ownership of your product, and not just treat it like another sample. A CRO that partners with you closely – and isn’t simply a vendor – means they form a core part of your team, and have a personal stake in your success. They’re hands-on, and keep you updated every step of the way. Whatever CRO you choose, be sure they make their experts available to you at all times. They should take part in meetings, telecons, kickoff calls, and be involved in every stage of the process.
Significant scientific expertise in biopharmaceutical development and biopharma services is a must. A large proportion of the CRO staff should be made up of Ph.D. scientists and biopharma veterans. The CRO should assign scientific advisors that act as connections between your team and theirs. Their expertise and scientific background means they can accurately map out the entire process, from development to IND submission.
The right experience
Ideally, your CRO should have experience supporting successful IND submissions under tight deadlines. They should also have a solid track record of working on multiple biopharma products over several years. These drugs should span a wide range, from monoclonal antibodies and antibody-drug conjugates, to biosimilars and pegylated proteins. All projects need to be backed by an exceptional regulatory record.
Flexibility is important when the unexpected happens. Your CRO needs to work closely with you to determine the best analytical approaches. Their flexibility (and scientific expertise) means the CRO can think outside the box when things don’t go according to plan. They can quickly identify alternative ways of getting things done. In fact, finding novel ways to characterize and understand biopharmaceutical behavior is often necessary to file a successful IND.
Full range of analytical biopharma services. The complexity and heterogeneity of ADCs mean they are exceptionally challenging to characterize. A full suite of analytical services is necessary to do this. Be sure to ask your CRO about their capabilities, and what biopharma services they offer. As mentioned in this white paper, you need to be sure your CRO won’t overlook anything, and can help you meet CMC regulations. Their scientists should be experts in these techniques and interpretation of their data. At a minimum, these techniques should include cell-based bioassay development and analysis by ultra high resolution QToF, as well as routine release and stability testing.
6.0 Case Study: CMC Suport for ADC Development Situation
Virtual client had very aggressive timelines for submitting INDs for two antibody drug conjugates within 12 months. The Client requested complete chemistry support for the CMC section of the IND
In collaboration with the client’s scientists, EAG proposed a fast-tracked method development and validation program to meet their timelines. EAG scientists performed complete characterization of the mAb and drug product, including complete sequencing, PTMs, and glycan analysis. Developed and validated multiple methods for release and stability including: icIEF, ELISA, cell bioassay, DAR, free drug, N-linked Glycan, SEC, CE-SDS, and HCP
All data was delivered to the client within the deadline, and both INDs were submitted on schedule
Both INDs were successful, and the FDA had no observations/ remarks regarding the EAG’s portion of the IND. Our client’s priorities changed during the study, requiring additional studies beyond the scope of the original project. We were able to accommodate these changes and still meet their deadlines. EAG scientists were fully involved in project kick-offs.
Finding a CRO who can partner with you to accelerate your antibody drug conjugate IND submission is challenging. It’s not easy to determine which CROs can truly partner with you to help you achieve your objectives.
This white paper has outlined two critical challenges with ADC development. Specifically, these challenges relate to successfully filing an IND. They are:
The complexity of the ADC molecule
Failing to meet CMC regulations
Given these challenges, there are 3 ways to streamline the IND process:
Characterize all critical quality attributes
Perform studies that are often overlooked
Adopt a platform approach
ADC, antibody drug conjugate; DAR, drug-to-antibody ratio; CMC, Chemistry Manufacturing and Controls; IND, Investigational New Drug; ELISA, Enzyme-linked immunosorbent assay; ECL, Electro-chemiluminescence; SPR, Surface Plasmon Resonance.
ADCs, Antibody-drug Conjugates, Characterization, Chemistry Manufacturing and Controls (CMC)
This work is published by InPress Media Group, LLC (Antibody Drug Conjugate Development: Keys to Rapid IND Submission and Approval) and is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Non-commercial uses of the work are permitted without any further permission from InPress Media Group, LLC, provided the work is properly attributed. Permissions beyond the scope of this license may be available at adcreview.com/about-us/permission.
EAG Laboratories is a global scientific services company providing testing, analytical and characterization services to clients across a vast array of technology- and life-science-related industries. The company has recently made significant investments to support its growing life science offering and customer base, including the recent expansion of its cGMP pharmaceutical laboratory in Columbia, Missouri.
This expansion is directly related to the biotech and pharmaceutical industry’s critical need for high-quality contract laboratory partners who understand the regulatory guidelines, can perform required risk assessments and have the capacity to execute validated analytical procedures. But it also gives the company’s experienced and dedicated team of highly skilled scientists a state-or-the-art environment to assist customers in successfully execute drug development projects.
To see how these developments impact the development of novel drugs, including ADCs, we sat down with Glenn Petrie, Ph.D., Senior Scientific Advisor at EAG Laboratories.
With over 20 years of experience in support of biotechnology projects, Petrie helps EAG Laboratories’ clients find answer to challenging development questions unique to large molecule development. He serves as the primary business development contact for the company’s biopharmaceutical development projects.
PH: In your experience, what are some of the unique challenges presented by the complex nature of antibody-drug conjugates?
GP: Understanding biopharmaceutical stability and demonstrating biological activity are some of the most critical and challenging aspects of the development of novel therapeutic agents. This is, however, becoming increasingly difficult as we move towards molecules with multiple mechanisms, such antibody-drug conjugates or ADCs. Antibody-drug conjugates combine the target specificity of a monoclonal antibody with the therapeutic activity of a highly potent cytotoxic anticancer agent utilizing a variety of (linker-) chemistries. This results in an extremely complex structure.
Our team of highly dedicated and experienced scientists collaborate with our clients, offering them their experience and expertise in developing and optimizing a variety of bioassay methods for product potency and stability.
“One of the most critical aspects is to address all the unique issues involved in the submission of an IND completely, correctly, and in a timely fashion…”
PH: What are some of the special considerations when developing analytical methods for ADCs?
GP: When developing methods for the analysis of ADCs our scientists take into account the unique properties of a specific ADC, including the drug-antibody ratio or DAR and drug load distribution.
DAR and drug load distribution are critical product quality attributes. A hydrophobic interaction chromatography or HIC method has been developed to determine the drug load distribution and DAR for a variety of ADCs. This is important, because, in general terms, DAR and drug load distribution determine the drug quantity to which a patient is exposed. In addition, each of these various drug loaded forms may differ in their pharmacokinetic/pharmacodynamic (PK/PD) characteristics.
Another important aspect to consider is that the linker chemistry applied and the cytoxin included in a specific ADC will modify the chemistry of the monoclonal antibody. As a result of changes to the surface of the antibody protein, this modification may result in increased antibody aggregation and/or decreased binding and cytotoxicity decreasing the overall stability and efficacy of the antibody-drug conjugate. SEC, antigen binding and cytotoxicity reflect overall 3-dimensional structure and are therefore Critical Quality Attributes.
PH: In March you presented a talk during BioPharma Asia 2017 in Singapore* discussing “ADC Development: Keys to Rapid IND Submission and Approval.” During the presentation you discussed the unique analytical challenges presented by the complex structure of antibody drug conjugates (ADCs) and the myriad of analyses required in support of a successful Investigational New Drug application (IND). How do you support your clients with an IND submission?
GP: One of the most critical aspects of an IND submission is to address all the the unique issues involved; to be complete, correct, and submit the IND in a timely fashion. Incomplete or incorrect information can cause delays in submission and may even result in a rejection of the IND. We use our extensive experience and expertise to support in our clients in submitting successful INDs. And we do that in an accelerated time frame.
During my presentation in Singapore I also highlighted proven ways to maximize the probability of a successful IND, while minimizing the time and effort required. This included critical biopharmaceutical characterization parameters and critical quality attributes or CQAs, but I also discussed studies that may be frequently overlooked, such as compatibility and residual solvents/metals/
PH: What makes an IND-submission for an ADC complex?
GP: It’s good to keep in mind there is no specific regulatory guidance to industry for ADC development. In their process to regulate ADCs, the FDA follows existing guidelines for both small drugs and monoclonal antibodies. As such, this process is a collaborative effort across product quality FDA-offices, including the Office of Biotechnology Products (OBP) and the Office of Pharmaceutical Quality (OPQ). OBP and OPQ primarily focus on the manufacturing of the antibody component of the ADC and the control strategy for the antibody intermediate, as well as for the drug substance and drug product. In addition, the small molecule review group at the OPQ is primarily responsibility for review of the adequacy of the payload and linker, conjugation reaction and aspects of the control strategy.
PH: What is the foremost important aspect the FDA – and other regulators are looking at?
GP: The FDA’s primary concern is safety. This is especially important for ADCs. These novel drugs contain one of many different cytotoxic agents that are far more potent than standard anticancer agents. And as a result of their cytotoxicity, with IC50 in the picomolar range. Consequently, to ensure the drug product, it is critically important to determine the concentration of unconjugated drug. The level of free, unconjugated, drug is usually in the 1-10 ng/mL range, making it very difficult to detect. Typically Liquid Chromatography-Multiple Reaction Monitoring/Mass Spectroscopy or LC-MRM-MS has the necessary sensitivity and specificity and will suffice. But ocassionally you’ll have to push sensitivity even further to determine free or conjugated drug down to 1 ng/mL or less.
PH: As part of your work, what should companies note about working with regulatory agencies – whether this involves the FDA or EMEA?
GP: One of the key aspects is to ask questions. Don’t be afraid to ask for clarification if the advice given is not clear. It will help you submit the correct information and avoid unnecessary and expensive mistakes.
But it is also important to understand the difference in the requirements of the different agencies. The EMEA, for example, places an emphasis on cell bioassays as part of their equivalent of an IND; the FDA does not. Generally the European requirements are more rigorous early in the process, but that difference quickly disappears in the following phases of the process.
PH: In your experience, why are pharma and biotechnology companies eager to work with companies like EAG?
GP:We’re scientists, not only by training, but as part of our natural curiosity. When we talk with a client, we speak scientist to scientist – and that is a real benefit because the biotech and pharmaceutical industries have a critical need for high-quality scientific partners who truly understand analytical methods and regulatory guidelines and can execute complex study designs. Our team of highly skilled scientists has a proven track record for efficiently advancing complex programs through the regulatory process. They bring deep insight to the analysis and characterization of ADCs, monoclonal antibodies, bispecifics, biosimilars, fusion proteins, pegylated proteins and other biotherapeutics.
When we start working with a client early in the development process we truly become an extension of their team. We are not just performing our analysis. It’s not just “throwing a report over the fence… ” as one might say. We become true collaborators. As a result we sometimes know more about our client’s molecules than the client.
In the end, I think that the reason why our industry partners want to work with us is our unique experience and dedicated – collaborating approach. That is what our clients are looking for and what makes EAG unique in the industry.
PH: What does the future of ADCs hold?
GP: Today there are only two ADCs on the market. But I expect that this will change quickly. When speaking with our clients my understanding is that most of them have very aggressive timelines. One of our clients mentioned that they expect submitting 2 INDs per year – each year – for years to come.
PH:…. that is a really big push…
GP: Yes it is. But according to some analysts this is still a conservative estimate. Several months ago, during World ADC Summit in San Diego, California, expert opinion seemed to suggest that there will be 15 ADCs on the market by 2020.
PH: How does that impact your work?
GP: Some of our clients have voiced legitimate concern when discussing the available capacity. But in reality, I don’t think that they need to worry. Earlier this year we started to increase our staff of scientists. We expect to increase the number of scientists by 35 – 40% this year and triple our dedicated cell bioassay capability in Columbia, Missouri.
With the strong commitment of our senior management team we expect to be ahead of the curve.
PH: Looking back over the last decade – what are some of the major accomplishments that excite you about the development of ADCs?
GP: The diversity in the development and the differences in the various platforms is exciting. For example, there are now two well established platforms developed by Seattle Genetics and the other by Immunogen. There is also considerable invested in the development of new cytotoxic payloads, additional linker technologies, and site-specific attachment.
I’m also excited about the improvement in controlling DAR. All these developments may help us to improve the efficacy and decreases the side effects of novel ADCs.
Looking towards the future, I can see the potential for applications beyond oncology and hematology.
That really excites me.
Last Editorial Review: March 27, 2017
* BioPharma Asia 2017, March 21 – 23, 2017, Suntec Convention, Singapore, Singapore
The U.S. Food and Drug Administration (FDA) cleared Mersana Therapeutics’ Investigational New Drug (IND) application to begin Phase I clinical trials for its lead investigational antibody-drug conjugate drug candidate XMT-1522. The investigational compound is Mersana’s first pipeline product, and defines a new class of HER2-targeted therapies. The investigational drug is based on Mersana’s Fleximer® technology.
The drug is being co-developed with Takeda Pharmaceutical. Under the terms of the agreement Mersana will, based on the FDA clearance of this IND, receive a $20 million milestone payment.
Earlier this year, in February, Takeda, through its wholly owned subsidiary Millennium Pharmaceuticals,, entered into a strategic partnership with Mersana to co-develop XMT-1522. The execution of the Phase I trial will be managed by Mersana. The company will also retain full commercial rights in the United States and Canada while Takeda will have rights in rest of world.
Current ADC Development
Industry-wide there are 60 antibody-drug conjugates in clinical development. Today 40 of these investigational compounds are in phase I trials, 16 in phase II and 4 phase III. Currently, there are two licensed ADCs on the market. In 2015 17 novel ADCs entered the clinic while an additional 7 have entered clinical trials in 2016. Many of the ADCs in phase I trials are not identifying a target disease, but are broadly recruiting for solid tumors. With more than 10 ADCs, breast and lung cancer are common diseases for ADCs. Of the 30 different targets for solid tumors 11 target breast cancer and 9 lung cancer.
A promising approach
“XMT-1522 represents a promising therapeutic approach for cancer patients with significant unmet medical needs and we are pleased to be in a position to move this therapy into clinical development.” said Donald A. Bergstrom, M.D. Ph.D., Chief Medical Officer, Mersana Therapeutics.
“We have designed a robust phase I program that will allow us to better understand the potential of XMT-1522 to address the needs of several patient groups who currently have limited options,” Bergstrom added.
Breast and Gastric cancers
XMT-1522 is an anti-HER2 antibody-drug conjugate. The investigational drug incorporates HT-19, a novel, proprietary anti-HER2 antibody optimized for cytotoxic payload delivery. The antibody is conjugated with Mersana’s Dolaflexin platform which includes its Fleximer biodegradable hydrophilic polymer technology and proprietary auristatin payload.
XMT-1522 provides a drug load or drug-to antibody ratio (DAR) of approximately 12 molecules per antibody, specifically designed to improve potency while simultaneously increasing tolerability. XMT-1522 has the potential to extend HER2-targeted therapy beyond the current HER2-positive populations into patients with lower levels of HER2 expression.
Early and pre-clinical results
Early and pre-clinical data presented during the 2015 annual meeting of the American Society for Cancer Research (AACR) and the 2015 San Antonio Breast Cancer Symposium (SABCS) has shown nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell. This data also shows that XMT-1522 is typically 1-3 logs more potent than ado-trastuzumab emtansine, also known as T-DM1 (Kadcyla®; Genentech/Roche), an antibody-drug conjugate consisting of the antibody trastuzumab linked to the cytotoxic agent emtansine, also called DM1, a thiol-containing maytansinoid.
Researchers at Mersana have further shown that XMT-1522 is active across a panel of 25 tumor cell lines  and in a range of models representing HER2+ disease where current HER2-targeted therapies, including ado-trastuzumab emtansine, are not active. XMT-1522 is also active in models representing HR+ and HR- HER2 IHC 1+ and 2+ disease. [Note]
In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well- tolerated doses. In one high HER2-expressing model of gastric cancer (800,000 HER2 receptors/cell), complete regressions was achieved with a single 1 mg/kg dose of XMT-1522. To achieve comparable activity with ado-trastuzumab emtansine, a dose of 10 mg/kg is required. 
One of the unique features of XMT-1522 is that it is non-competitive with existing therapies – trastuzumab or pertuzumab (Perjeta®; Genentech/Roche) – for HER2 binding. A triple combination of XMT-1522 + trastuzumab + pertuzumab in the same mouse xenograft model mentioned above, resulted in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis.
In a low HER2-expressing breast cancer model (79,000 HER2/cell) and gastric cancer model (22,000 HER2/cell) models, complete regressions was achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively while ado-trastuzumab emtansine is inactive at doses ≥10 mg/kg.
In non-human primates XMT-1522 demonstrated good stability of the drug-conjugate in plasma with t1/2 ~5 days (comparable to antibody t1/2) and minimal exposure to free payload.
One of the interesting findings in early and pre-clinical studies is that despite the high potency of XMT-1522 in low HER2 tumor models, researchers did not observe XMT-1522-related toxicity in critical HER2-expressing tissues including heart and lung. The preclinical data further support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2- directed therapies are not indicated. Finally, the combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2-driven tumors.
Phase I protocol
The Phase I protocol will evaluate XMT-1522 in patients with advanced HER2-positive breast and gastric cancer, as well as advanced breast cancer with low HER2 expression and HER2-expressing non-small cell lung cancer.
“Our partnership with Mersana exemplifies our approach of uniting Takeda’s experience in bringing novel oncology therapies to market with promising drug discovery technology like Mersana’s Fleximer to help drive science forward for patients with unmet medical needs,” noted Phil Rowlands, Interim Head, Oncology Therapeutic Area Unit, Takeda.
In addition to developing novel antibody-drug conjugates with select pharmaceutical partners, including Takeda, Mersana is also developing its own portfolio of next-generation Fleximer-ADCs with superior properties not found with current ADC technologies to address unmet needs and improve patient outcomes in multiple oncology indications.
[Note]: IHC or or Immunohistochemistry is a staining process performed on fresh or frozen breast cancer tissue removed during biopsy. It is used to show whether or not the cancer cells have HER2 receptors and/or hormone receptors on their surface. This information plays a critical role in treatment planning.
Goodwin Biotechnology, Inc., a biological contract development and manufacturing organization or CDMO specialized in bioprocess development and GMP manufacturing of biopharmaceuticals utilizing mammalian cell culture expression systems and bioconjugation technologies, has partnered with Aspyrian Therapeutics to complete process development, scale-up, and cGMP manufacturing of the Aspyrian’s novel Antibody Drug Conjugate (ADC).
San Diego-based start-up biotechnology company Aspyrian Therapeutics recently submitted – and received – U.S Food and Drug Administration’s (FDA) acceptance of an Investigational New Drug (IND) Application for RM-1929, their first-in-class, precision targeted therapy for cancer. The company is now recruiting patients to participate in a clinical study.
The current trials is a two-part, multicenter, open-label, dose-escalation, combination Phase I study of patients with recurrent Head and Neck Cancer (HNC), who, in the opinion of their physician, cannot be satisfactorily treated with surgery, radiation or platinum chemotherapy. The estimated study completion date is September 2016.
Part I of this study is a drug dose-escalation, fixed low fluency light application study to determine the drug dose that can be safely given to saturate the epidermal growth factor receptor (EGFR) at the tumor.
Part II of this trial is a light dose-escalation, fixed drug dosing infusion study to determine the optimal light application. In combination with the Part I drug dose, an optimal light application is needed to achieve clinical response with an acceptable safety profile. Interim analysis will be performed after completion of Part I and prior to initiation of Part II. 
Dual specificity Aspyrian’s first-in-class therapy uses an antibody conjugate is designed to precisely target cancer cells after which it is locally activated to elicit rapid anticancer responses. The dual specificity resulting from in-tumor activation of a tumor-selective conjugate promises to deliver oncologists the ability to achieve locoregional tumor control with minimal damage to surrounding healthy tissues and structures.
“This clinical trial is the first of its kind to evaluate a novel therapy with a two-step targeting approach designed to deliver unparalleled tumor specificity,” explained Merrill Biel, M.D. Ph.D., Chief Medical Officer of Aspyrian. “Recurrent head and neck cancer patients who have failed standard treatments of surgery, radiation, and chemotherapy currently have no further treatment options available. RM-1929 offers hope to these difficult-to-treat patients and has the added advantage that it targets their cancer while sparing damage to adjacent normal and healthy tissues that are critical to their health related quality of life, such as those required for normal speech and swallowing.”
RM-1929 was developed in collaboration with Hisataka Kobayashi, MD, Ph.D, and Peter L. Choyke at the National Cancer Institute (NCI), the inventors of the photoimmunotherapy platform.  “Activation of RM-1929 at the tumor triggers very rapid tumor destruction with exquisite specificity,” explained Miguel Garcia-Guzman, Ph.D., President and Chief Scientific Officer at Aspyrian. “RM-1929 provides oncologists a new, precision-targeted approach for the effective locoregional management of cancer either alone or in combination with other anticancer therapies.”
RM-1929 targets a specific cancer antigen that is highly expressed in carcinomas allowing treatment of a number of different cancer types, including cancers of the head and neck, esophagus, lung, colon, and pancreas. “The initiation of this clinical study marks a key milestone for Aspyrian, as we continue to develop a pipeline of precision-targeted therapies for the treatment of solid tumors. The broad therapeutic potential of our lead product candidate gives us the opportunity to expand the clinical program for RM-1929 into a range of cancer indications during 2016,” noted Scott Salka, Chief Executive Officer of Aspyrian.
Aspyrian Therapeutics is developing a novel class of precision oncologic drugs for the treatment of solid tumors based on the photoimmunotherapy platform licensed exclusively from the National Cancer Institute. The Near-Infrared Photoimmunotherapy (PIT) technology uses a target-specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IR700 (IRDye® 700DX), conjugated to monoclonal antibodies targeting epidermal growth factor receptors. The molecule is activated with a laser at the tumor site to induce rapid cancer cell destruction.
The new ADC technology platform permits highly efficient and selective ablation of tumor cells while sparing damage to critical structures adjacent to the tumor. Because the payload in this unique ADC is inert until it is both in the tumor and activated by a laser using visible light, it overcomes the dose-limiting adverse effects that have hindered the development of conventional, toxin-loaded ADC systems.
Benefiting patients is the fact that laser activation can be accomplished on an outpatient basis by either external illumination or by needle placement of an optical fiber to illuminate a deep-seated tumor from within. As part of their development program, Aspyrian is currently working with a number of monoclonal antibodies with the potential to treat various types of cancers, including head and neck, esophageal, lung, brain, pancreatic, colorectal, breast and ovarian.