Peer-Reviewed Articles on ADCs |

Abstract
Antibody Drug Conjugates (ADC) are a rapidly expanding area of pharma company pipelines. They combine the targeting of an antibody with the potency of a small molecule. Such a simple and elegant approach has far reaching consequences for the IT infrastructures that were established and implemented for antibody and small molecule drug discovery. The ability to track data associated with ADCs is critical for projects to conduct structure-activity relationships (SAR) and ultimately be successful. Herein we describe a simple approach to assigning a unique ID number to ADCs that involves only minimal modification to the established registration processes for the separate antibody and small molecules components.

1.0 Introduction
Antibody drug conjugates represent an increasingly important area for drug discovery.[1] They combine the best components of both antibodies and drugs.[2] The antibody provides the selective targeting of the therapeutic while the highly potent drug drives a high efficacious response.[3]

In addition to the many challenging discovery and development complexities presented by these hybrid biologic-small molecule entities, data management also needs to be addressed. While drug discovery has implemented effective software solutions for registration of the individual components of an ADC (i.e. antibody, drug), the ability to describe and register the combined (ADC) product presents interesting challenges for current IT infrastructures, particularly in instances where the existing component registration workflows do not accommodate each other and may additionally have evolved in completely distinct software environments.

The ability to track data associated with ADCs is critical for projects to conduct structure-activity relationships (SAR) and ultimately be successful. While a covalent bond elegantly joins the worlds of antibody and small molecule, the marriage of these two domains in the cheminformatics arena represents a significant undertaking.

**Figure 1.0:** (a) Linker-Drug MC-Val-Cit-PABC-MMD, a lysosomally cleavable dipeptide linker that releases monomethyl dolastatin 10 (MMD); (b) Linker-Drug MC-MMD, a non-cleavable linker that releases Cys-MC MMD in the lysozyme. (Click to enlarge Figure).

2.0 Registration of small molecules
Registration is the process of assigning corporate identifiers to unique entities for the purpose of tracking them through discovery pipelines. For small molecules, registration is routine. Card systems were originally used, but the process has since been computerized. Small molecules are registered after their chemical structures have been determined; this requirement essentially provides that for a corporate ID to be assigned to a structure the corresponding compound must have been made.

Each structurally unique molecular entity is assigned its own corporate ID. Additionally each batch, or lot, of compound material is assigned a unique lot number.

The relationship between the physical material and a lot number is always immutable. Almost always the registration system enforces a rule that the relationship between a structure and its corporate ID, once assigned, is also immutable. Since the structure must be determined prior to registration the need for changes are rare. When changes do occur, they result in the lot(s) being assigned a different corporate ID.

The registration system will normally allow for the registration of materials of unknown structure, usually by requiring that such materials be assigned a unique name, but also by allowing a special character (e.g. ‘X’) to represent an unknown component of an otherwise determined structure. The virtual registration of compounds without physical lots can also be permitted, but in these cases a different class of identifiers may be assigned.

Culturally, registration is ingrained into the thinking of chemists. In the past, productivity was sometimes assessed by the number of compounds registered. Since pharmacologically active compounds in discovery rarely have trivial names, the corporate ID serves as a substitute, being used in publications, patent applications, internal documents and presentations.

3.0 Registration of biologics
For biologics, the process of registration has been defined much more recently. For developers, the first instinct was to mirror the behavior of small molecule registration systems. This was challenging for a number of reasons.

Biological macromolecules are large and an absolute representation of their chemical structure is intractable. For proteins, the amino-acid sequence can be used as a surrogate for structure. However biological proteins, especially those that are secreted from the cell, are not simply polypeptides. Many proteins are post-translationally modified (e.g. by glycosylation). In most cases, the absolute structure of the glycans and their points of attachment will not be known, and a batch of protein may well be heterogeneous in respect of its post-translational modifications.

Usual practice in biologics registration is to use the amino acid sequence as the uniqueness-determining representation of the chemical structure. Variations in glycosylation may very well occur between lots of material. Exceptions can be made if scientists intend to make a purified form of post-translationally modified protein that differs substantially from the bulk form; such proteins can be assigned unique corporate IDs.

The registration of biologics is procedurally different in that the structure of the registered material is not always independently determined (the sequence of the protein is derived from the encoding plasmid and rarely verified by mass spectroscopy prior to registration). In many cases, the sequence of the protein will not be determined at all before a corporate ID is needed to track assay results (e.g. an antibody derived from a hybridoma). In these cases, the unique identifying information is essentially the process by which the biologic was produced (e.g. isolated from that hybridoma cell line) rather than an explicitly determined state. A consequence of this is that changes in the identifying information for a protein are much more common than for small molecules.

There are two approaches to addressing this challenge. One is to maintain the rule for small molecules that the relationship between identifying information and corporate ID is immutable once assigned. Such a system must endure the inconvenience of tube re-labelling and record modification should lots of material require a change of corporate ID.

The alternative approach is to conserve the relationship between a batch of material and its corporate ID whenever possible. In this approach the identifying information for a corporate ID can be changed provided no lots exist for which the old information remains correct. A consequence of this approach is that 2 corporate IDs can become synonymous if one is modified to have the same identifying information as another, and in this case the entities merge and retain a single preferred corporate ID.

Although the second approach may seem more reasonable for biologics, situations where a corporate ID can be assigned to a material by both state and process are very complex, and for this reason we at Abbvie have moved from the second approach to the first.

Registration is a more recent practice for biologics and the metadata that needs to be collected for each registered material is more complex than for small molecules. Consequently, processes must be designed to keep data entry as simple as possible and to ensure that it is carried out by the person most likely to know the required information. Biologists typically are less comfortable using numeric identifiers as substitutes for trivial names. They often rather prefer information-rich names (e.g. Mouse anti-Human KDR [IgG1/kappa]). We enforce uniqueness of these names, so that each corporate ID maps to a single name, but also allow a more free text lot name where variations between lots of the same material can be captured. However, lot consistency is important in any discovery endeavor and this should be an exception.

4.0 Registration of ADCs
Since ADCs comprise a small molecule component and a biologics component, information about them already resides in both the small molecule and biologics registration systems. The small molecule component itself comprises a payload (the active small molecule drug) and a linker (used to connect the drug to the protein). The payload, the linker and a reagent in which the payload and linker are attached all exist as chemical reagents and can therefore be registered. In practice, the linker, as a commercial off the shelf reagent that is not independently tested, is rarely registered. Uncertainties about the molecular nature of each of the components reside in their own systems.

For example, if we do not know the sequence of an antibody that is to be conjugated, then its corporate ID in the biologics registration system will be definite, but assigned by process. Similarly, if we do not know the structure of the combined payload/linker, perhaps because it is proprietary to a collaborator, then the small molecule corporate ID will be definite, but assigned on the basis of a unique name.

**Figure 2.0:** X-combo is a virtual compound with X representing the antibody to which the Linker-Drug is conjugated that enables GBRS to determine if an ADC is unique. (Click to enlarge Figure).

At Abbvie, two Accelrys products are used for registration. The Global Biologic Registration System (GBRS) is used to register antibodies. This uses the amino acid sequence to determine whether or not an antibody is unique and assigns both a PR# as its corporate ID (for PRotein), for example, PR-123456 and an individual lot#.

For small molecule registration, the software A-coder is used. This determines uniqueness based on chemical structure and assigns both an A# as the corporate ID (i.e. , A-1307119.0 where the .0 signifies it is the free base) and an individual lot#.

The same number sequence is used by both software packages removing the possibility of identical PR- and A-numbers.

When research into ADCs was initiated at Abbvie, it was recognized immediately that to ensure data integrity a registration process would need to be implemented. Unfortunately, neither GBRS nor A-coder had the required functionality to perform registration of ADCs alone. GBRS was not chemically intelligent and thus unable to determine uniqueness of the ADC. A-coder was only designed for small molecules and was not able to handle the large amino acid sequences of the antibody.

To minimize the impact on already established workflows for both antibodies and small molecules, a solution that leveraged both GBRS and A-coder was desired.

The first decision was that ADCs would be assigned a DC# (for Drug-Conjugate) as its corporate ID. This decision was taken so that as soon as a scientist saw data associated with the moniker A- (small molecule), PR- (protein) or DC- (ADC) the type of molecule would be immediately apparent.

Next, the decision of whether GBRS or A-coder would be used to register ADCs was addressed. Recognizing that the inventory management of ADCs was more similar to inventory management for biologics than to that for small molecules, GBRS was selected. GBRS was also selected as it enabled more sophisticated metadata capture for biologic entities and was the newer of the two registrations platforms at Abbvie.

As GBRS did not possess the chemical intelligence to determine the uniqueness of an ADC, a mechanism that enabled this was required. The solution was to use the combination of the PR# from the antibody and the A# from the linker-drug to define a unique ADC in the name field of GBRS.

For the example in Figure 2.0 “ADC-123456-1307119” would be entered in the name field of GBRS. As both the antibody and linker-drug identifiers would be generated by their respective registration systems designed to handle the appropriate entities, all of AbbVie’s registration rules would be applied appropriately.

While in principle this would provide a way to determine uniqueness of an ADC, there was a catch. Unfortunately, during conjugation the linker-drug structure is chemically modified which leaves the possibility for two unique linker-drugs to give rise to equivalent ADCs. For example, as shown in Figure 2, Linker-drug A contains a bromine, while Linker-drug B has an iodine resulting in a unique A# for each compound. During conjugation, the halogen is displaced by the antibody with both linker-drugs affording the same ADC. However, by this method of annotation GBRS would perceive that the two reactions produced different ADCs, as the two combinations of PR# from the antibody and A# from the linker-drug are unique.

This complication was resolved by introduction of a virtual compound called the “X-combo”. This virtual compound has an X representing the antibody and the chemical structure of the linker-drug after conjugation to the antibody (Figure 2.0). During registration, this enables A-coder to determine whether the X-combo is unique and to generate a corresponding A#. In GBRS, the combination of antibody PR# and X-combo A# in the name field can then be used to determine if this is a unique ADC or one that has already been registered and assign the correct DC#.

**Figure 3.0:** Step 1 of association process: structure of retrieval the linker-drug. (Click to enlarge Figure)

GBRS creates an ADC registration event when the scientist provides both an antibody and X-combo corporate ID. GBRS assigns a DC corporate ID based upon three pieces of information: 1) antibody corporate ID (PR-#), 2) small molecule X-Combo (A#), 3) drug-to-antibody ratio (DAR). A DAR2 and DAR4 molecule of the same antibody and X-combo will be assigned 2 different DC corporate ID’s. If an already existing antibody and X-combo have been registered this will become a new batch of material.

In order to facilitate SAR on the ADC and its individual components (antibody, linker, drug), the appropriate A#, PR# and DC# for an ADC had to be associated together. To aid in this association, the ADC Component Association Tool was developed to enable this in collaboration with Discngine. The ADC component is achieved in a simple 5 step procedure.

First, the structure of the linker-drug is retrieved using the A# (Figure 3.0). Next, the drug is identified either by modification of the retrieved linker-drug structure or using the A#.

The mechanism of action of the drug is also selected from a drop-down list at this stage. If the mechanism of action of the drug has not previously been registered, a new mechanism of action term can be entered manually and it is then captured in the drop-down list (Figure 4.0).

**Figure 4.0:** Step 2 of association process: identification of the payload.(Click to enlarge Figure)

As the structure of both the linker-drug and drug are known, the linker is then automatically identified by the software (Figure 5).

The ADC Association Tool identifies the linker structure from the Combo molecule based upon what chemical structure was identified as the drug during the previous step and removing this from the Combo chemical structure leaving the linker chemical structure.

The shorthand name for the linker is selected from a drop down list, for example, MC-Val-Cit-PABC. If the linker has not previously been registered, a new linker term can be entered manually and it is then captured in the drop-down list. Then the type of linker, for example, dipeptide or non-cleavable, is also captured. For linker-drugs with a non-cleavable linker, the free drug is not likely to exist. As a result, for these linker-drugs, the cysteinylated analogue is registered to represent the active species that is released from the lysosome (Figure 5.0).

**Figure 5.0:** Step 3 of association process: automatic identification of linker. (Click to enlarge Figure)

The final step is exemplification of the X-combo structure. The software retrieves the structure of the linker-drug, which can then be modified to represent the chemical structure of the linker-drug after conjugation to the antibody, with X representing the antibody (Figure 6.0).

Finally, the ADC Component Association Tool registers the X-combo in A-coder thereby conforming to AbbVie’s registration process rules on structure. The association between the ADC components along with the additional criteria on MOA and linker are stored in a custom ORACLE database. The element table in the A-coder registration system was modified to allow the X-combo molecules to contain the element X, which represents the antibody. The ADC Association Tool sends all of the metadata required for the X-combo molecule registration and assignment of its corporate ID.

**Figure 6.0:** Step 4 of association process: exemplification of X-combo structure.Having identified the 4 components of the ADC in the final step of the association process, a summary of the data from steps 1 to 4 is provided for the user to check (and edit if necessary) prior to registration. (Click to enlarge Figure)

Having created an association between all the components of an ADC, it is now possible to data mine on any aspect of an ADC. For example, one can easily search for all the ADCs with non-cleavable linkers that contain drug A-1581855. To enable substructure searching of ADCs, the structure of the X-combo was associated with the DC# of the ADC on the chemistry cartridge.

Figure 7.0 shows an example of ADCs with an MOA of auristatin. Due to the complexity and size of the structure of X-combos and linker-drugs, their visualization is not optimal. The use of metadata fields like linker, type and MOA can therefore be used to identify the structural variations within a set of ADCs being visualized.

Having associated all the components of an ADC facilitates comprehensive evaluation of SAR. All in vitro, in vivo and PK data can be uploaded to the corporate database and associated, at the lot level, with the relevant ADC component. Then, for example, it is possible to correlate the cell efficacy of the ADC with that of the free drug or the naked antibody.

5.0 Maleimide Hydrolysis
A known liability of ADCs using Cys-maleimide conjugation is the loss of the linker-drug through a reverse Michael reaction. Scientists at Genentech [4] published data showing 2 important facts:

hydrolysis of the maleimide ring affords a stable attachment;
the environment surrounding the cysteine influences hydrolysis of the maleimide ring.

They showed that sites with a positively charged environment promoted hydrolysis of the maleimide ring. Seattle Genetics [5] published data on maleimide hydrolysis showing that both a basic moiety proximal to the maleimide and also a short alkyl chain between the maleimide and amide can catalyze ring hydrolysis at basic pH. Pfizer [6] have nicely shown that a PEG spacer between the maleimide and amide enables base catalyzed ring hydrolysis.

**Figure 7.0:** Association of ADC components enables SAR visualization, for example, ADCs with auristatin as the MOA. (Click to enlarge Figure)

Maleimide ring hydrolysis is also achieved for linker-drugs with an ethyl spacer between the maleimide and valine by treatment at pH 9 for 3 days. The ring hydrolyzed maleimide structure is captured during registration of the X-combo (Figure 8.0).

Hydrolysis of the maleimide ring after conjugation can afford two possible hydrolyzed products. For clarity when visualizing the ADC structure only a single product with the X positioned alpha to the amide from the maleimide ring (as depicted in Figure 8.0) is captured in the database.

6.0 DAR Homogeneity
Having initially defined the criteria to determine a unique ADC as the combination of PR-# (antibody) + A-# (X-combo), it was decided that DAR should also be included. To enable data mining of this information, a minor modification to GBRS was made which added separate fields for aggregation, DAR and DAR separation.

**Figure 8.0:** X-combo is registered as the ring hydrolyzed maleimide structure (X represents the antibody). (Click to enlarge Figure)

ADCs produced by conjugation to inter-chain cysteines results in a heterogeneous DAR population. To improve both quality and consistency of ADCs synthesized at AbbVie, routine separation of the DAR species by hydrophobic interaction chromatography (HIC) was implemented. To enable immediate recognition of whether an ADC was a heterogeneous or DAR separated population, a simple terminology was adopted. For a heterogeneous DAR population the DAR was reported to one decimal place, for example, DAR 3.6. For a specific DAR peak following separation by HIC the DAR was reported as a whole number, for example, DAR 4.

7.0 Site of Conjugation
The final consideration was how to register ADCs when the site of conjugation is known, for example, with cysteine deletion and/or addition mutants. In these cases, the site of conjugation is captured in the antibody structure during the registration process for the antibody. As this is a novel antibody, it receives a different PR# to the native antibody so GBRS will recognize this and determine that the ADC is unique.

**Figure 9.0:** DAR Nomenclature: a) heterogeneous DAR designated by use of the decimal place, DAR 3.6; b) purified DAR designated by use of whole number, DAR 4. (Click to enlarge Figure)

To make this mutation more readily apparent, the mutated amino acid along with its location is captured in the name field during registration in GBRS. For example “ADC-123456-1307119-CYS237” would be entered in the name field to designate conjugation at CYS237. Using this format for entries in the name field not only ensures the correct identification of this ADC by the registration system, it also provides immediate clarity of the amino acid mutation(s).

8.0 Summary
A custom and novel ADC registration process has been implemented with minimal modification to AbbVie’s small or large molecule registration systems software or compound workflow. This new process enables in-depth SAR interrogation based on all components of the ADC, including the ability to perform searches based on the structure of the linker-drug. A simple terminology was implemented to discriminate between heterogeneous and separated DAR populations as well as other ADC property metadata.

Abbreviations:
ADC, antibody drug conjugate; Cit, citrulline; Cys, cysteine; DAR, drug to antibody ratio; GBRS, global biologics registration system; HIC, hydrophobic interaction chromatography; IT, information technology; MC, maleimide-caproyl; MOA, mechanism of action; MMD, monomethyl dolastatin 10; PABC, para-amino benzylic carbamate; SAR, structure-activity relationship; Val, valine.

August 14, 2017 | Authors: Adrian D. Hobson,* [a] Jeremy C. Packer, [b] Chris C. Butler [b] and Dirk A. Bornemeier.[b]
[a] AbbVie Bioresearch Center, 381 Plantation Street, Worcester, MA 01605
[b] AbbVie, Inc., 1 North Waukegan Road, North Chicago, IL 60064

Corresponding Author:
* adrian.hobson@abbvie.com

Author Contributions:
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Funding Sources
ADH, JCP, CCB and DAB are employees of AbbVie (or Abbott Laboratories prior to separation) and may own AbbVie/Abbott stocks or stock options and participated in the interpretation of data, review, and approval of the publication. The financial support for this work was provided by AbbVie.

Acknowledgements:
We acknowledge Doug Pulsifier, Robert Gregg, Michael Huang, Sreekumar Menon, Randy Metzger, Hetal Patel, Teresa Rosenberg, Jennifer Van Camp and Philip Hajduk for their input with this project.

Original manuscript received: July 24, 2017 | Manuscript accepted for Publication: August 3, 2017 | Published online August 14, 2017 | DOI: 10.14229/jadc.2017.14.08.002

Last Editorial Review: August 11, 2017

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Abstract
Armed with cytotoxic payloads, antibody-drug conjugate (ADC) becomes able to kill its naked-antibody-resistant tumor cell. When ADC circulates in the plasma, complete detachment of conjugated drug due to continuous deconjugation process results in accumulating naked antibody, the drug-detached carrier. In this study, we investigated naked-antibody-impaired cytotoxic effect of ADC.

The cytotoxic effect of HER2-targeted RC-48 ADC (Remegen Ltd, Yantai, China) co-existed with its naked antibody against the naked-antibody-resistant HER2-positive SKOV3 cell line was analyzed. The effective ADC EC₅₀ increased as naked antibody concentration increased, which confirmed the impairment in vitro. Assuming antitumor effect and cytotoxic effect were impaired to the same degree, we roughly assessed clinical efficacy impairment by analyzing pharmacokinetic profile of T-DM-1 (ado-trastuzumab emtansine, Kadcyla® | Genentech/Roche; one of the four antibody-drug conjugates approved by the U.S. Food and Drug Administration).

The inferred clinical efficacy impairment was significant during the whole time after intravenous administration, suggesting a promising room for improvement in ADC efficacy by eliminating the circulating naked antibody.

A novel experimental data driven bystander effect modified competitive antagonism model was trained to explain the cytotoxic data and predict the effective ADC EC₅₀ when its naked antibody existed. Because naked antibody co-existence is prevalent amongst all ADC pipelines, this research may deserve a clinical evaluation.

1.0 Introduction
Antibody therapeutics have been booming for decades due to their outstanding clinical performance in treating cancer. However, almost all antibody therapies lead to drug resistance over time due to various mechanisms. To win the battle against cancer was still challenging. The recent emergence of break-through therapies gave us real hope of conquering cancer. The antibody-guided, drug-loaded, missile-like next generation therapeutic, antibody-drug conjugate (ADC), belongs to one of those promising therapies [1].

**Figure 2.1a** *(Click here to enlarge)*

ADC is comprised of monoclonal antibody, the navigation element, targeting receptors over-expressed on cancer cell surface, and cytotoxic chemical drug, the warhead element, released after ADC, the missile, specifically entering tumor cell by internalization process. [2] A favorable property of ADC is that it can kill those tumor cells having evolved resistance to conventional naked antibody, which is almost the last hope of conventional-antibody-resistant patients with relapse. [3][4]

In most cases, on every ADC, the number of drugs conjugated, denoted as drug antibody ratio (DAR), is not uniformed, often ranging from 0 to 8, which can be measured in vivo. [5] When T-DM1 circulated in blood, an accumulation of naked antibody (DAR=0) was observed and explained by models[6].

**Figure 2.1b** *(Click here to enlarge)*

Co-existence of ADC and its naked antibody after intravenous administration was a common phenomenon amongst all ADC therapeutic agents [7]. The extent of co-existence was dependent on stability of linker between monoclonal antibody and chemical drug. [8]

So far, the effect of co-existing naked antibody on ADC cytotoxic efficacy was unknown. Naked antibody, generally ineffective due to cell resistance, competes with ADC for binding to the same antigen receptor, therefore hampers ADC from entering target cell, and leads to impaired efficacy[1]. From the present point of view such impairment could be neglected since naked antibody accounted for only a small percentage of overall total antibody.[1]

However, we identified such impairment through co-treatment assays, and we inferred pronounced clinical disadvantage based on certain assumptions. A novel data driven model, which considered the bystander effect, was raised to explain the experimental results. Our results suggested a possibility to improve the ADC efficacy by reducing circulating naked antibody, which might deserve further clinical investigation.

2.0 Results
To the best our knowledge, co-treatment of ADC and naked antibody on drug resistant cell has not been reported elsewhere. Competitive antagonism model tailored for ADC and its naked antibody has not been reported either.


*Naked antibody concentration* (ng·mL-1)	Effective ADC EC₅₀ (ng·mL-1)
0	1,560
11.5	1,695
115.0	2,229
1,150.0	4,253
11,500.0	7,844
115,000.0	11,241
Table 2.1

2.1 In vitro cytotoxicity of ADC co-existed with naked antibody

HER2-positive SKOV3 cell line, HER2-targeted RC-48 ADC and HER2-targeted RC-48 naked antibody were obtained from Remegen Ltd, Yantai, China [3]. SKOV3 cells were seeded at 1500 cells/well and allowed to grow 8 hours, then moved away initial culture medium before adding therapeutics. ADC and naked antibody were both added to wells in 42 paired combinations: final concentration RC-48 ADC at 0–2,000,000 ng/mL (0, 640 ng/mL, 3,200 ng/mL, 16,000 ng/mL, 80,000 ng/mL, 400,000 ng/mL, 2000000 ng/mL) and RC-48 naked antibody at 0-115000 ng/mL (0, 11.5 ng/mL, 115 ng/mL, 1,150 ng/mL, 11,500 ng/mL, 115,000 ng/mL). Cells treated with therapeutic-free culture medium (ADC and naked antibody both at 0) were used as negative control. ADC were incubated with cells for 72 hours before viability test. BIMAKE CCK-8 was used to determine the viability of cells (Fig. 2.1a and Fig. 2.1b).

As shown, SKOV3 cell was resistant to RC-48 naked antibody (Fig. 2.1a). The minimum cell viability was 20% and the effective ADC EC₅₀ was estimated by linear interpolation (Fig. 2.1b). In Fig. 2.1b, A line at 60% cell viability (half of effect), parallel to the x axis, intersected all broken lines to obtain effective ADC EC₅₀ values. When no naked antibody existed, the ADC EC₅₀ was 1,560 ng/mL. However, when naked antibody concentration was at 11.5 ng/mL, 115 ng/mL, 1,150 ng/mL, 11,500 ng/mL and 115,000 ng/mL, the effective ADC EC₅₀ was 1,695 ng/mL, 2,229 ng/mL, 4,253 ng/mL, 7,844 ng/mL and 11,241 ng/mL, respectively (Table 2.1).

2.2 In silico cytotoxicity of ADC co-existed with naked antibody

2.2.1 Schild equation
According to Schild equation, the drug-response logistic curve will be shifted by drug-ratio units when drug’s antagonist exists. [9] The equation is applicable if agonist A and antagonist B satisfy[10]: (1) The antagonist, B, is a true antagonist that, alone, does not change the conformation of the receptor; (2) Binding of agonist, A, and antagonist, B, is mutually exclusive at every binding site; (3) B has the same affinity for every binding site; (4) The observed response is the same if the occupancy of each site by A is the same, regardless of how many sites are occupied by B; (5) Measurements are made at equilibrium.
ADC is so similar to its naked antibody that we can suppose they share the same binding affinity (dissociation constant), molecular weight and internalization process. [11] The only difference is that ADC, the agonist, releases payload, while its naked antibody, the antagonist, does not, which means they satisfy the first four prerequisites.

The bias from Schild model caused by last unsatisfied prerequisite can be modified by bystander effect.

The unmodified Schild equation predicts the effective ADC EC₅₀ as follows:

In Eq. 2.1, refers to the effective ADC EC₅₀ when naked antibody co-exists at concentration [nkdAb], and KD the dissociation constant. Here the EC₅₀ of RC-48 ADC on SKOV3 was 1560 ng/mL as estimated by linear interpolation and the KD was 70 ng/mL as reported. [3]

**Figure 2.2** *(Click on image to enlarge)*

Co-existing naked antibody concentration [nkdAb] was set to 0-115000 ng/mL (0, 11.5 ng/mL, 115 ng/mL, 1150 ng/mL, 11500 ng/mL, 115000 ng/mL) and unmodified effective ADC EC₅₀ could be predicted.

2.2.2 Bystander effect modification
ADC incubation usually takes 3 to 7 days from binding to receptors to having targeted cell killed. [2]

Such long-lasting killing process resulted in remarkable bystander effect, where released payload entered bystanding cells and killed them, which led to failure to satisfy the last prerequisite in Schild equation and therefore the far-smaller-than-predicted experimental EC₅₀ results (Table 2.2 and Fig. 2.3).


Naked antibody concentration	Experimental Results	Schild equation results	Our model results
ng·mL-1	ng·mL-1	ng·mL-1	ng·mL-1
0	1,560	1,560	*
11.5	1,695	1,816	1,485
115.0	2,229	4,123	2,886
1,150.0	4,253	27,189	4,286
11,500.0	7,844	257,846	5,686
115,000.0	1,1241	2,564,417	7,087
Table 2.2

Since bystander effect increased as ADC concentration increased [12], we hypothesized that effective cell killing and original one had logarithmic correlation in our training model, rather than linear correlation in Schild equation.

**Figure 2.3** (Click on Image to enlarge)

In another word, the drug-response logistic curve was shifted by logarithm of naked antibody concentration when naked antibody existed (Fig. 2.2).

The modified equation predicts the effective ADC EC₅₀ as follows:

In Eq. 2.2, B was defined as bystander constant, which was trained to 13 by experimental data (assuming Max=80% and n=1) using MATLAB. The effective ADC EC₅₀ results of experiment, Schild equation and our model are shown in Table 2.2 and Fig. 2.3.

**Figure 2.4** (Click on image to enlarge)

The comparison of all data points amongst three results is shown in Fig. 2.4.

The training algorithm is presented later.

2.2.3 Bystander constant training algorithm
The bystander constant B was trained by experimental results Rij, the cell viability matrix after incubation by naked antibody at concentration [nkdAb]i and ADC at concentration [ADC]j (Fig. 2.5).

**Figure 2.5** (Click on image to enlarge)

P_ij(B) is the output of model (Eq. 2.3) with bystander constant B, iteratively increasing from initial value 2 to optimal value such that the sum of square of difference between every model output and experimental data point is minimum (Eq. 2.4).

3.0 Discussion
Previous study neglected the impact of naked antibody on ADC efficacy due to its small percentage of total antibody (5%)[1].

However, our study showed that the impact of naked antibody should not be overlooked. When naked antibody percentage (defined by P in Eq. 3.1) was 0.7%, 4.9%, 21.3%, 59.5%, 91.1%, the effective ADC EC50 increased (defined by I in Eq. 3.2) by 8.7%, 42.9%, 172.6%, 402.8%, 620.6%, respectively.

That was to say, for example, if naked antibody accounted for 4.9% of total antibody, a 42.9% extra dose more than pure ADC was used to kill the cell. However, the naked antibody percentage wasn’t immobile. As a T-DM1 PK profile showed, the naked antibody percentage continuously increased over time[6].

**Figure 3.1a, 3.1b and 3.1c** (Click here to enlarge)

When our results (I-P graph by linear interpolation, Fig. 3.1a) was applied to the T-DM1 profile (Fig. 3.1b), we roughly estimated how much more dose was wasted on antagonism over time. The result showed that the T-DM1 efficacy was impaired by 26.4% (Fig. 3.1c) immediately after intravenous administration, and became worse and worse.

Whether such impairment could be confirmed in vivo or in clinical trial was unknown. But when ADC and its naked antibody were co-administrated into animal, a better drug distribution was observed due to alleviation of binding-site barrier[13].

As for computational work, we gave a new competitive antagonism model which could explain and predict the effective ADC cytotoxicity when naked antibody existed, since Schild equation was no longer applicable.

Because bystander effect varied from cell to cell and ADC to ADC, we generated the model by experimental data, which was universal for all cytotoxicity modelling using the same type of cell and ADC. In our model, the bystander constant was the base of logarithm relation, which might be meaningful elsewhere in the field of ADC.

Abbreviations:
ADC, antibody drug conjugate; DAR, drug-to-antibody ratio; T-DM1, ado-trastuzumab emtansine; nkdAb, naked antibody.

Keywords:
drug-detached naked antibody, ADCs efficacy impairment, antagonism, Schild model, bystander effect.

August 14, 2017 | Authors: Nanfang Hong [1], Jianmin Fang * [1]
[1] School of Life Science and Technology, Tongji University, Shanghai, China

Corresponding Author:
* jfang@tongji.edu.cn

Author Contributions:
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Acknowledgements:
The author thanks to his mentor Jianmin Fang for his guidance; Remegen Ltd for materials support; Renhao Li, Fei Tao, Jie Li and Yan Feng for their technical assistance; Qi Liu, Hua Gu and Lei Huang for their helpful discussion.

How to cite:
Hong N, Fang J, Drug-detached Naked Antibody Impairs ADC Efficacy (2017),
DOI: 10.14229/jadc.2016.09.04.001.

Original manuscript received: May 12, 2017 | Manuscript accepted for Publication: August 3, 2017 | Published online September 4, 2017 | DOI: 10.14229/jadc.2016.09.04.001.

Last Editorial Review: September 1, 2017

Featured Image: Science and medical research, chemical laboratory. Courtesy: © Fotolia. Used with permission.

This work is published by InPress Media Group, LLC (Drug-detached naked antibody impairs ADC efficacy) 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.

The post Drug-detached Naked Antibody Impairs ADC Efficacy appeared first on ADC Review.

Abstract
With the increasing need for high-quality, state-of-the-art cancer care reaching more people than can be accommodated by National Cancer Institute (NCI)-designated and other academic cancer centers, a new breed of hybrid center has been emerging over the last several years. Although there does not appear to be an official consensus on what proportion of cancer patients are treated in community settings, some sources indicate that it may exceed 70% [1], making it an important goal to provide the same standards of care for all patients regardless of treatment setting. This paper describes four institutions that have been identified as “hybrid academic-community cancer centers,” and explains what differentiates them from traditional academic or community settings.

1.0 Hybrid Center Identified and Defined
The hybrid academic-community cancer center concept was first identified, named, and described in a series of articles [2][3][4][5] initially appearing in MedPage Today in August 2015 following an observation made during that year’s American Society of Clinical Oncology (ASCO) annual meeting in Chicago.These hybrid centers shared several common features:

Most recruited respected academic-clinician leaders from NCI-designated comprehensive cancer centers.
They were established at large, well-respected, financially secure not-for-profit regional community hospital systems interested in increasing quality of care and standards of practice.
They sought to infuse greater academic rigor into their programs, develop translational research programs, enroll more patients from their respective communities into clinical trials, and provide the best cancer care to populations not served by traditional academic centers.
They often adapted existing resources through collaborative efforts rather than reinventing the wheel.

Originally, four such centers were identified: Carolinas HealthCare System’s Levine Cancer Institute, Charlotte, NC; Christiana Care Health System’s Helen F. Graham Cancer Center and Research Institute, Newark, Del. [6]; Inova Schar Cancer Institute, Falls Church, Va.; Gibbs Cancer Center and Research Institute, Spartanburg Regional Healthcare System, Spartanburg, SC.

Baptist Health South Florida’s Miami Cancer Institute, Miami, Fla., and West Cancer Center, Memphis, Tenn., were identified in later articles.

During the course of the interviews for the articles and subsequent discussions between the author of the MedPage articles (ETR) and the hybrid center directors it was determined that the hybrid concept could benefit from a full-day interactive session among the hybrid leaders sharing best practices, as well as respective aspirations and challenges facing them.

2.0 Hybrid Symposium
In late February 2017, Miami Cancer Institute hosted a “Hybrid Symposium: the Nature and Nurturing of Hybrid Academic-Community Cancer Centers” featuring presentations and discussions by four hybrid directors: West’s Lee S. Schwartzberg, MD, ISCI’s Donald L. “Skip” Trump, MD, Gibbs’s Timothy Yeatman, MD, and MCI’s Michael Zinner, MD. Session moderators were Dr. Zinner, and symposium organizer Eric T. Rosenthal, the journalist who wrote the original articles.

Invitations to participate were extended to each of the hybrid center directors identified in the series, and four were able to attend. Other attendees included medical, executive and professional staff from the four centers; CME/CE credit was offered.

3.0 The Conference Objectives included

Recognizing and discussing the emerging presence of hybrid academic-community cancer centers.
Reviewing similarities and differences between existing models that vary according to individual centers’ strengths and needs.
Identifying and recommending new concepts inspired by models and practices of other centers.
Formulating a plan for future and ongoing collaboration in support of further development of hybrid academic-community cancer centers.

After the hybrid center directors provided respective overviews of their centers, a series of roundtable discussions commenced dealing with issues of common concern regarding: funding research at hybrid centers; relating to academic partners; dealing with culture clash; balancing the medical and scientific mission with fiscal realities; recruiting and retaining staff; branding and marketing centers within hospital systems and in communities; conclusions and next steps.

The participants agreed about the core common features defining their centers as hybrids, but one remarked that, “if you’ve seen one hybrid center, then you’ve seen one hybrid center,” noting that there is not and should not be only one model.

Hybrids must be adaptable, and build upon their individual strengths, community needs, and collaborations with academic partners. The one-size-fits-all model does not apply here. Their major goals should be enhancing access to quality cancer care and clinical trials, and providing oncologists and other health care practitioners with the opportunity to practice medicine without some of the restraints of academia while still participating in clinical trials and other research activities. The day’s discussion highlighted many similarities as well as differences.

4.0 Self-Descriptions
All centers involved in the conference met the basic criteria established for hybrid academic-community cancer centers, but this is how each center self-described its respective hybrid model:

Gibbs referred to itself as a “next-gen” cancer center. Its focuses on clinical research and trial activities, as well as a basic research wet-lab effort with DNA sequencing expertise related to colorectal cancer. With community partners Gibbs developed the Guardian Research Network (GRN), dedicated to precision medicine and democratizing clinical trials. GRN has 85 hospitals sharing millions of documents and EMRs to enhance drug development to search discrete data and non-structured text for specific inclusion and exclusion criteria for clinical trials to rapidly enroll patients using centralized architecture. Gibbs has relatively few therapeutic trials, but is involved in diagnostic, screening, and prevention trials, and has enrolled more than 700 patients on a single diagnostic trial.

ISCI is developing a patient-centered, clinical-translational research program to bring high-quality, interdisciplinary, ambulatory and inpatient cancer to the Inova Health System, a successful traditional, “hospital-centric” community model.

MCI has married the best community oncology practices in South Florida with Memorial Sloan Kettering Cancer Center’s standards of care, resources, capabilities, and clinical trials.

West Cancer Center is a hybrid center with its board of directors equally represented by former West Clinic clinicians and its CEO, Methodist hospital executives, and University of Tennessee officials. Some financial aspects are still monitored by the hospital system, but West anticipates evolving more fully into all aspects of its overall program.

5.0 Dedicated Facilities and Programs
All the centers have dedicated cancer center facilities on their main campuses built within the last several years.

West Clinic, a private 30-year-old multi-specialty oncology group, is currently affiliated with Methodist LeBonheur Health and University of Tennessee Health Science Center. Its 125,000-square-foot facility was completed in 2015 and houses comprehensive cancer services. Administrative offices are located off-site, and the hospital system is building another 75,000 square-foot cancer facility near the medical school. West has 12 locations in a 90-mile radius staffed by more than 100 providers.

Gibbs Cancer Center and Research Institute, with four locations, is a division of the 600-plus-bed Spartanburg (SC) Regional Health System. Gibbs has 250,000 square-feet dedicated to its cancer center and Beardon-Josey Breast Health Center, With Edward Via College of Osteopathic Medicine, Gibbs constructed a 7,500 square-foot basic research laboratory for personalized cancer treatment, colorectal cancer biology, cancer stem cells, and regenerative medicine.

Inova Health System has five hospitals, four with hospital-based radiation facilities and a fifth radiation facility in partnership with another community hospital partner and five ambulatory hematology-oncology practice sites in Northern Virginia. Inova is currently refurbishing Exxon Mobil’s former world headquarters to house a 240,000 square-foot ambulatory interdisciplinary cancer center. New construction has also begun for a 120,000 square-foot building for radiology, radiation oncology, and a two-vault proton beam facility, and another 240,000 square-foot building is being retrofitted to serve as research laboratories, with one-third dedicated to cancer. The Schar Family provided a $50 million gift to name the cancer institute and support program development and recruitment.

Miami Cancer Institute is part of the eight-hospital Baptist Health South Florida system, serving South Florida. MCI opened a $430-million four-story, 440,000-square-foot ambulatory cancer center in late-2016, connected by a pedestrian bridge to the Baptist Hospital of Miami, a 770-bed secondary/tertiary hospital. It has 113 examination rooms, 60 infusion rooms, eight infusion beds, a pediatric infusion suite, and a comprehensive radiation oncology program that includes proton therapy, MR-LinAcs, infusion, gamma knife, cyber knife, tomo- and true beam radiation therapy machines.

6.0 Recruitment of Leadership
Three of the four centers participating in the symposium had specifically recruited leaders from NCI-designated comprehensive cancer centers.

Dr. Yeatman was recruited in 2012 from Moffitt Cancer Center, where he was Professor of Surgery and EVP for Translational Research. He managed a $100 million Merck-Moffitt partnership, and cofounded M2Gen to manage the partnership. He is Center Director, President of GCC&RI, and President and CSO of GRN.

Prior to joining ISCI in 2015, Dr. Trump had been president and CEO of Roswell Park Cancer Institute.

MCI began recruiting Dr. Zinner a number of years before it began construction of its new facility. He joined MCI in 2016 after serving as clinical director of the Dana Farber-Brigham and Women’s Cancer Center, Surgeon-in-Chief at Brigham and Women’s Hospital, and Moseley Professor of Surgery at Harvard Medical School.

Dr. Schwartzberg has spent most of his professional career at the West Clinic.

(Derek Raghavan, MD, PhD, of Carolinas HealthCare System’s Levine Cancer Institute, was recruited from the Cleveland Clinic; and Christiana Care Health System’s Helen F. Graham Cancer Center recruited Nicholas Petrelli, MD, from Roswell Park Cancer Institute. (2))

7.0 Changes in Academic Partnerships
MCI formally joined the Memorial Sloan Kettering Cancer Alliance in January 2017 as its third member. It has a direct relationship with Florida International University Medical School with joint appointments including basic laboratory work done at FIU by MCI faculty and staff.

Inova has had a long-standing relationship with Virginia Commonwealth University for medical student education, and faculty have VCU appointments. In 2016, Inova and the University of Virginia announced development of an education and research affiliation to pursue novel programs in broad scientific research areas, education and eventually business and entrepreneurship. A critical component of the Inova-UVa relationship is the synergistic partnership between ISCI and the UVa NCI-designated cancer center with the potential to seek joint NCI comprehensive center designation. Inova also collaborates with George Mason University (GMU), Shenandoah University, and NCI, and is opening a joint proteomics facility with GMU.

Gibbs has had formal relationships with multiple academic institutions in both South Carolina and North Carolina, including the Medical University of South Carolina, Duke University, Wake Forest University, University of North Carolina, and Edward Via College of Osteopathic Medicine. Gibbs recently expanded its partnerships with five more health systems in the GRN, and is considering exposing surgical residents to a research year. There are no current plans for medical, surgical, or radiation oncology fellowships.

In 2012 West entered into a professional service agreement and co-management agreement with Methodist LeBonheur Health, which was serving as the major clinical affiliate of University of Tennessee Health Science Center and its College of Medicine. West assumed all responsibilities for the hematology-oncology division, formally operated by a different private practice group. The three partners evolved into the West Cancer Center, responsible for all clinical, research, and educational cancer services, including the subspecialty fellowship program. A proposed structure is being developed to integrate all clinical and academic staff as members of the cancer center, with the goal of creating a standalone center within the matrix organization.

8.0 Volume- vs. Value-Based Systems
All the centers noted that hematology-oncology is one of the most financially successful medical service areas. These centers also manage a substantial number of patients with benign hematologic problems, posing organizational and staffing challenges. Research funding has come through philanthropy, federal and foundation grants, and pharmaceutical company grants and clinical trials.

The centers are all extremely interested in changing from volume-based to value-based reimbursement. ISCI has value-driven aspirations and opportunities, and hopes to develop a health system based on value-based care. MCI plans to expand the existing Accountable Care Organization throughout its system. Gibbs is volume-based and dealing with the challenge of allowing physicians’ “protected time” for clinical research when clinical duties require most of their effort. West is transitioning to a value-based system through participation in the CMMI Oncology Care Model and other alternative payment models.

9.0 Fundraising
Historically Inova has not had a robust fundraising arm, and although ISCI is without debt, the development of centers of excellence in cancer, heart, neurosciences, and genomics has presented the need and opportunity to feature notable accomplishments to expand their respective fundraising profiles. In response, ISCI is developing a $100 million campaign. MCI does not currently have a well-developed fundraising program, but it is also without any debt from its new $430-million facility. West is close to raising $15 million in pledges in support of its research and goal of seeking NCI designation.

10.0 Marketing
Marketing has been a challenge for all centers. ISCI is engaged with the Inova Health System to market the cancer center’s unique capabilities. It considers Georgetown’s Lombardi Cancer Center, George Washington University Cancer Center, and Johns Hopkins its biggest competitors.

MCI is challenged by making itself a known entity in South Florida, Latin America, and the Caribbean, and has the most competition from University of Miami’s Sylvester Cancer Center.

Gibbs is involved in helping patients understand the high quality of care offered at its center with 10 disease-focused programs, bench laboratory research, and clinical trial activity. It competes primarily with a larger local hospital system that is less focused on cancer.

West’s greatest marketing challenge is explaining to patients three-entity partnership when each institution had its own reputation in the community. It cited a minority of patient “leakage” to academic medical centers.

11.0 Cultural Clashes
The various hybrid models have fostered their share of cultural clashes at all the institutions. At Gibbs, it was manifested when community medical oncologists became more involved in clinical trials. ISCI cited differences among administrative staff from community hospitals, its flagship tertiary-care facility, and faculty and staff with a patient-centered ambulatory and translational research mission. MCI has seen cultural clashes between the academic and community cultures, between both salaried professional and private practitioners, and between its center and regional system, which nonetheless provides excellent support. West has had a fairly smooth integration of its three-partnership consolidation, but still faces a conceptual cultural clash between private practitioners and fulltime salaried professionals, although it has not affected clinical care.

12.0 NCI-Designation Goals
The institutions have all begun aggressive research and educational efforts with different aspirations toward achieving future NCI designation.

Inova Health System began developing a “hybrid pathway” in 2010 former NCI director John Niederhuber, MD, was recruited to establish the Inova Translational Medicine Institute, a genomics program focusing on bioinformatics infrastructure. ISCI is developing clinical and translational research that emphasizes target and biomarker delineation, drug discovery, and development and rational therapeutics with a genomic base, and has recruited a drug development team to focus on new and more efficient approaches to developing and devaluating new therapies. Strengths currently include a clinical trials operation lead by. Joan Schiller, MD, recruited from UT Southwestern Simmons Cancer Center, accruing almost 200 patients per year, and a DoD-funded gynecologic oncology translational center led by Larry Maxwel, MD and Thomas Conrads, PhD. IHS has ACGME-certified postgraduate medical training programs in internal medicine, surgery, pediatrics, and ob-gyn. ISCI will be eligible to apply for a hematology-oncology fellowship program in 2018 and surgical oncology and radiation oncology programs shortly thereafter. NCI designation was not an original goal of the ISCI leadership, but the partnership of the Inova Health System and UVa offers the opportunity to develop a formal relationship in cancer that would entail pursuit of NCI comprehensive center designation.

MCI established its Center of Genomic Medicine after recruiting Jeffrey Boyd, PhD, from Fox Chase Cancer Center. The clinical molecular diagnostic facility offers next-generation sequencing, bioinformatics, curation, and clinical reports, and houses a bio-repository, providing high-quality, well-characterized cancer-related human biological materials for research. MCI is building a clinical trials program for translational research, including a phase 1 program. There are no plans for basic research on its campus, but such activities are available through affiliations with MSKCC and Florida International University. MCI plans to add population sciences and cancer control.

The center currently has 32 open trials with the number increasing, and has submitted a grant for investigator-initiated trials.

MCI currently has a small GME program with FIU and has hired a GME consultant to help expand its program to include both ACGME-approved and non-ACGME sub-specialty fellowships.

The institute does not intend to seek NCI-designation for at least five years.

Gibbs became one of the original NCI Community Cancer Center Program members in 2007. In 2005 Gibbs became an exclusive affiliate of MD Anderson Cancer Center, and established the Bearden-Josey Center for Breast Health in 2008. In 2014 the center received a five-year grant as part of the NCI Clinical Oncology Research Program. Rather than seeking NCI designation Gibbs intends to pursue disease-focused clinical and research programs) without the substantial associated costs and development time required for designation.

West has been involved in research activities for several decades through industry trials and as a previous National Cancer Institute – Community Clinical Oncology Program (NCI-CCOP), and is a full member of Southwest Oncology Group (SWOG). After affiliating with Methodist and UT, the center had nearly 40 open therapeutic research trials, mostly focused on phase 2 and 3 studies with a few phase 1 trials. It has since upgraded its research activities to include more than 50 staff and a full-time research director. Trials have expanded beyond medical oncology to include surgical and radiation oncology, translational work, and observational studies. Plans are under development for a more comprehensive bio-repository program. A scientific review committee evaluates all protocols and has launched the infrastructure to support investigator-initiated trials. The center is integrating university laboratories and scientists to focus on translational research and has recruited Neil Hayes, MD, from the University of North Carolina to head its Institute for Cancer Research.

West assumed management of the UT hematology/oncology fellowship program in 2012 and subsequently received full GME accreditation. Heme/onc fellowships have expanded to 15, with additional research fellowship positions planned for next year. West has also initiated a radiation oncology residency, and a gynecology oncology fellowship, and plans to begin surgical oncology and breast surgical oncology fellowships in 2018.

The center is highly focused on achieving NCI designation, anticipating between five to seven years to develop its assets to apply for designation, with comprehensive status planned within 10 years.

13.0 Future Goals
There was consensus that ongoing discussions among hybrid leaders had value, and that symposia should be continued on a rotating basis among the centers, with additional centers invited as they embrace the hybrid model. Interest was expressed for more cooperation among the centers, including clinical trials, population health, and sharing pathways.

Abbreviations:
ASCO, American Society of Clinical Oncology; GMU, George Mason University; GRN, Guardian Research Network; NCI, National Cancer Institute; NCI-CCOP, National Cancer Institute – Community Clinical Oncology Program; SWOG, Southwest Oncology Group.

Keywords:
Hybrid academic-community cancer centers; NCI-designated cancer center; Academic cancer center, Community Cancer Center.

November 27, 2017 | Authors: Eric T. Rosenthal * [1], Lee Schwartzberg, MD [2], Donald L. Trump, MD [3], Timothy J. Yeatman, MD [4], and Michael Zinner, MD [5]

[1] EvocaTalk® Research & Reports | [2] West Cancer Center | [3] Inova Schar Cancer Institute | [4] Gibbs Cancer Center and Research Institute | [5] Miami Cancer Institute

Corresponding Author:
* Eric T. Rosenthal, EvocaTalk® Research & Reports, 1404 Remington Road, Wynnewood, PA 19096, etr@evocatalk.com

Author Contributions:
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Acknowledgements:
The ideas and opinions expressed herein are those of the authors.
Authors’ Disclosures of Potential Conflicts of Interest: None
Author Contributions: All authors.
Conception and design: All authors
Collection and assembly of data: Eric T. Rosenthal
Manuscript writing: All authors
Final approval of manuscript: All authors

How to cite:
Rosenthal ET, Schwartzberg L, Trump DL, Yeatman TJ, Zinner M.
The Nature and Nurturing of Hybrid Academic-Community Cancer Centers (2017)
DOI: 10.14229/jadc.2017.11.27.001.

Original manuscript received: October 15, 2017 | Manuscript accepted for Publication: November 1, 2017 | Published online November 27, 2017 | DOI: 10.14229/jadc.2017.11.27.001.

Independent Review:
This article was submitted for peer reviewed and approved for publication by an independent editorial review board.

Last Editorial Review: November 24, 2017

Featured Image: Hybrid Academic-Community Cancer Center. Courtesy: © Fotolia. Used with permission.

This work is published by InPress Media Group, LLC (The Nature and Nurturing of Hybrid Academic-Community Cancer Centers) 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.

The post The Nature and Nurturing of Hybrid Academic-Community Cancer Centers appeared first on ADC Review.

The technology behind antibody-drug conjugates (ADCs) has been around for many years, but so far is without widespread commercial success. Penelope Drake and David Rabuka of Catalent Biologics assess the history and progress to date, and look at what might be preventing ADCs from reaching their full potential.

Abstract
Two decades ago, antibody-drug conjugates or ADCs were hailed as a major breakthrough, especially in the area of oncology therapeutics. The concept of delivering a potent drug payload directly to the site of the tumor for maximum effect with minimal damage caused to non-cancerous cells was viewed as, if not the Holy Grail of cancer treatment, at least a significant advance towards precision medicine. However, the concept has proved difficult to translate into clinical success.

1.0: Introduction
The first ADC reached the market in 2000, but to date, the U.S. Food and drug Administration (FDA) has approved only four ADC therapeutics. The two most recent were granted approval in 2017, and could mark the start of a new era in which ADCs begin to realize their full potential.

The two drugs approved most recently by the FDA are inotuzumab ozogamicin (Besponsa®) and gemtuzumab ozogamicin (Mylotarg®). Mylotarg, the very first marketed ADC, was originally approved in 2000 for treatment of CD33-positive acute myeloid leukemia (AML).

However, treatment-related toxicity concerns led to its withdrawal from the market in 2010, but it has now been re-approved with a lower recommended dose and altered dosing schedule.

Besponsa was approved for treatment of relapsed/refractory acute lymphoblastic leukemia (ALL).[1,2] They join brentuximab vedotin (Adcetris®), an anti-CD30 monomethyl auristatin E (MMAE) conjugate approved in 2011 to treat relapsed/refractory Hodgkin lymphoma and systemic anaplastic large cell lymphoma, and ado-trastuzumab emtansine (Kadcyla®), an anti-HER2 DM1 conjugate approved in 2013 to treat HER2+ metastatic breast cancer. Kadcyla is currently the only FDA-approved ADC for the treatment of solid tumors.

2.0: An Hybrid Entity
An ADC is very much a hybrid entity, combining both biologic and small molecule characteristics, and consisting of an antibody scaffold covalently modified with a variable number of small-molecule payloads, joined by a chemical linker. The antibody delivers the small molecule specifically to the intended cell type by targeting an antigen that is selectively expressed on tumor cells and internalizes upon antibody engagement. To be an effective therapy, all of these parts of the ADC must be optimized.

Changes to the linker can have a significant effect on the biophysical and functional performance of the ADC, and there are two main conjugation approaches for attaching linkers to antibodies, resulting in either heterogeneous or site-specific payload placement. Currently, the ADC clinical pipeline is still dominated by heterogeneous conjugates, although the functional and analytical advantages of site-specific conjugation [3] are now being recognized.

The average ratio of conjugated payload to antibody is referred to as the drug-to-antibody ratio (DAR) and this has a strong influence on both the efficacy and toxicity of an ADC. High-DAR ADCs can have poor biophysical characteristics that reduce efficacy and increase toxicity, but these effects can be mitigated using certain conjugation and linker technologies.[3]

3.0 Clinically-tested Payloads
To date, the majority of clinically-tested ADC payloads are either antimitotic/microtubule inhibiting, such as auristatins, maytansinoids and tubulysin, or DNA alkylating (e.g., pyrrolobenzodiazepines, indolinobenzodiazepines, calicheamicins, duocarmycins), although a few other interesting payloads with novel mechanisms of action have been introduced (irinotecan derivatives and α-amanitin).

The past five years however, have seen a dramatic change in the ADC clinical pipeline as preclinical technological advances have started to feed into clinical-stage projects. In early 2013, of the 20 ADCs in the clinic, nearly 80% were heterogeneous conjugates with payloads of antimitotic drugs, namely auristatins or maytansinoids. But between 2013 and 2017, the number of ADCs in clinical trials more than tripled [4], with site-specific ADCs accounting for nearly 15% of the total. There has also been a trend away from antimitotic payloads towards more potent cytotoxic drugs, particularly DNA alkylators.

The proportion of antimitotic payloads fell from 80% to 65% overall, and accounted for only one-third of site-specific ADCs. This decline can be attributed in part to the unimpressive clinical results of ADCs bearing antimitotic payloads.

According to a recent review [4], nearly 40% of ADCs bearing maytansine, monomethyl auristatin E (MMAE), or monomethyl auristatin F (MMAF) that entered clinical trials were later discontinued, presumably due to lack of efficacy or (rarely) excessive toxicity.

However, the highly potent DNA alkylating payloads carry an increased risk to patients and the fine line between potency and safety is one that scientists and regulators are still striving to achieve. The first site-specific ADC to reach the clinic, vadastuximab talirine, is an anti-CD33 antibody conjugated through engineered cysteine residues in the heavy chain to yield a DAR 2 molecule and is the first clinical ADC to bear a pyrrolobenzodiazepine (PBD) payload, a highly potent DNA alkylator.

It began clinical phase 1 trials in mid-2013, but the phase 3 trial was recently terminated due to toxicity concerns[5], even though the drug showed a 70% complete remission rate for AML patients.[6]

4.0: Mechanisms of toxicity
Meaningful improvements in ADC technology are expected to continue as preclinical studies focus on understanding the mechanisms of ADC toxicity, developing approaches for reducing off-target toxicities, and improving patient outcomes through changes in both ADC composition and clinical trial study design.

As yet, most clinical experience has been with ADCs carrying antimitotic payloads, which show prominent organ toxicities in the hematopoietic compartments and in the liver. Much less is known about the clinical effects of dosing DNA alkylators, although targeting of the hematopoietic compartments has been shown in clinical trials.

A deeper understanding is needed of the absorption, distribution, metabolism, and excretion (ADME) and drug metabolism and pharmacokinetics (DMPK) fates of both the intact conjugate and its small molecule component. Knowing where the drug goes and how it is processed will enable connections to be drawn with commonly observed clinical toxicities.

A 2015 review of toxicity studies [7] concluded that ADC toxicity was not driven by target antigen but rather by linker/payload: ADCs sharing the same linker/payload composition tended to reach the same maximum tolerated dose, even when their target antigens showed endogenous expression in completely different tissue/organ compartments.

This sobering observation revealed how much progress still needs to be made to achieve specific cytotoxic payload delivery to tumor cells without damaging healthy tissues. But it also offers a possible explanation for the high failure rate of 2013 era ADCs.

It is likely that the lack of clinical benefit observed for some ADCs was the result of an inability to dose to an efficacious level due to off-target toxicities driven by the linker/payload.

If ADC off-target toxicity can be controlled, then it is likely that the maximum tolerated dose can be increased, perhaps leading to better clinical response to treatment.

How to cite:
Drake P, Rabuka D, ADCs – The Dawn of a New Era? (2018),
DOI: 10.14229/jadc.2018.08.27.001.

Original manuscript received: July 25, 2018 | Manuscript accepted for Publication: August 21, 2018 | Published online August 27, 2018 | DOI: 10.14229/jadc.2018.08.27.001.

Last Editorial Review: August 25, 2018

Featured Image: Medical research | Microscope. Courtesy: © Fotolia. Used with permission.

This work is published by InPress Media Group, LLC (ADCs – The Dawn of a New Era?) 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.

Copyright © 2010 – 2018 InPress Media Group. All rights reserved. Republication or redistribution of InPress Media Group content, including by framing or similar means, is expressly prohibited without the prior written consent of InPress Media Group. InPress Media Group shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. ADC Review / Journal of Antibody-drug Conjugates is a registered trademarks and trademarks of InPress Media Group around the world.

The post ADCs – The Dawn of a New Era? appeared first on ADC Review.

Antibody-drug conjugation (ADC) technology has been around for several decades but has yet to reach its full potential in terms of clinical success. In this second article, Penelope Drake and David Rabuka, of Catalent Biologics, discuss how the learning curve of recent years is opening a promising way forward for ADCs. The first article of this series was published online in August 27, 2018.

One of the factors that has held back the wider use of ADCs as therapeutics is the difficulties encountered in striking a balance between payload efficacy and dose-limiting toxicities in off-target tissues. According to a survey of papers in the literature where ADCs with the same linker/payload but different drug-to-antibody ratios were dosed such that the amount of payload delivered was held constant but the amount of antibody varied, it appeared that dosing with more antibody resulted in improved efficacy.[1] This improvement may have been due to better ADC tumor penetration, which in turn may point the way towards improving efficacy outcomes without dosing more drug, thus widening the therapeutic window. If this is the case, then there are implications for preclinical, and perhaps clinical, study design.

Another area that is gaining increasing attention is the potential of the adaptive immune system to augment or complement in vivo efficacy of ADCs, particularly with respect to testing combination therapies of ADCs dosed along with checkpoint inhibitor drugs.[2] Given that many ADC payloads induce immunogenic cell death in their targets, there are distinct possibilities for synergy. There are also several examples of clinically-tested ADCs where clinical response was uncoupled from target antigen expression[3-5], suggesting that an innate immune-based mechanism may be at work.

Combination therapies
Combination therapies also merit further investigation, and in particular, combinations of drugs whose mechanisms of actions intersect with tumor biology have the potential to improve efficacy. For example, in recent work by Immunomedics, preclinical studies demonstrated a rationale for co-dosing an ADC along with small-molecule drugs that inhibit multidrug resistance (MDR) efflux activity in order to overcome ADC drug resistance due to tumor upregulation of MDR efflux transporters.[6]

The choice of target antigen will affect both the efficacy and toxicity of an ADC. A relatively new approach is to target the cancer stem cells or tumor-initiating cells (TICs) that propagate disease. Various biological markers exist for TIC identification, and two have been selected as ADC target antigens, with the furthest advanced of these being delta-like protein 3 (DLL3), recognized by the ADC rovalpituzumab tesirine, currently being tested in phase 3 clinical trials for the treatment of small-cell lung carcinoma.[7] Also being investigated as an ADC target is the protein tyrosine kinase 7 (PTK7), expressed on TICs isolated from patient-derived tumor xenografts (PDX) representing several solid tumor types. The ADC caused tumor growth inhibition in several PDX models and was also shown to reduce the frequency of TICs in tumor tissue over time.[8]

Another novel approach to controlling tumors is to limit their blood supply by targeting tumor-specific vasculature. For example, the antigen CD276 is expressed on both tumor cells and tumor endothelial cells in some cancers, but not on endothelium in healthy tissues. It has been hypothesized that an ADC that simultaneously eliminates both populations within the tumor environment would yield greater overall tumor control.[9]

Recent advances in linker technology could also improve the success rate of ADCs. The linker plays a vital role in joining the antibody to the small molecule payload, as it must be stable during ADC circulation within the bloodstream without compromising biological potency. The structure of the payload will dictate which reactive chemical groups may be used for ligation, with primary and secondary amines currently being most commonly accessed. Research continues to broaden functional group accessibility in this field.

Traceless linkers
Payloads that lose biological potency when the core chemical structure is modified require the use of traceless linkers. These systems consist of a cleavage event (the trigger) followed by the self-immolation event that releases the free payload. The kinetics of both cleavage and immolation can vary according to the structure of the linker and payload.

For payloads that tolerate chemical elaboration, non-cleavable linkers offer an opportunity to adjust payload functionality. For example, work has been carried out on a triglycyl peptide linker designed to overcome some of the biological limitations currently imposed on the efficacy of non-cleavable conjugates. [10] The work aimed to limit the extent of lysosomal proteolysis required for payload liberation, improve payload transit from the lysosome into the cytosol, and hinder payload transit from the extracellular space into neighboring cells. Use of the triglycyl design effectively turned the linker into a cleavable, but not traceless, system that was uncharged at low pH (in the lysosome) but negatively charged at neutral pH (in the cytosol). The study highlights some of the complex biology that underlies successful delivery of a cytotoxic payload to its site of action within a target cell.

Improving linker stability
A consensus is growing in the field that the conjugation site can affect the biophysical and functional outcomes of ADCs. It is a known effect of site-specific payload placement that conjugation at certain positions can improve linker stability, with the hypothesis being that particular conjugation environments can “shield” the linker from access to enzymatic activity such as proteases and esterases. Recent work carried out by Pfizer using site-specific conjugation of a new spliceostatin payload, thailanstatin A, at a range of locations revealed that the activity of this particular payload is unusually dependent on the conjugation site. [11] Studies are underway to explain this phenomenon.

ADCs have yet to live up to their full clinical potential, but many more tools are now available to optimize their development. These include fully human/humanized monoclonal antibodies, site-specific conjugation approaches, a range of potent cytotoxic payloads with various mechanisms of action, versatile linker technologies, and sophisticated analytics. Some ADCs currently in later stages of the clinical pipeline have shown encouraging results and may lead to additional approvals in the near-term.

Beyond oncology
It should also be noted that the therapeutic areas of opportunity for ADCs are not limited to oncology. For example, an antibody-antibiotic conjugate has been shown to be more effective than the free antibiotic payload for treating infections caused by drug-resistant bacteria. [12] ADCs and related conjugates could also help to improve treatment of chronic conditions, such as autoimmune and cardiovascular diseases, by using selective payload delivery to reduce side-effects.

Technologies are also on the horizon that aim to achieve targeted drug delivery in the absence of an internalizing antigen. One such approach involves the use of cytotoxic payloads that can induce cell death by mediating signals at the cell surface. [13] Another involves a two-step drug-delivery method whereby the targeting and delivery steps are functionally and temporally uncoupled; initially an antibody against a non-internalizing target antigen delivers the payload to the cell surface, then the payload release is induced by a systemically-delivered small molecule. [14]

Based on these innovations, it is only a matter of time until creative solutions find their way into the clinic, leading to a new and exciting phase of ADC therapeutics.

How to cite:
Drake P, Rabuka D, ADCs – Look Forward to a Potent Future (2018),
DOI: 10.14229/jadc.2018.09.27.001.

Original manuscript received: July 25, 2018 | Manuscript accepted for Publication: August 21, 2018 | Published online September 27, 2018 | DOI: 10.14229/jadc.2018.09.27.001.

Last Editorial Review: September 26, 2018

Featured Image: Medical research | Test tubes in medical research. Courtesy: © Fotolia. Used with permission.

This work is published by InPress Media Group, LLC (ADCs – Look Forward to a Potent Future) 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.

The post ADCs – Look Forward to a Potent Future appeared first on ADC Review.

When the first antibody-drug conjugate (ADC) was approved by the U.S. Food and Drug Administration (FDA) in 2000,[1] only a handful of patent applications claiming ADCs had been published.[2] As research continues to progress and the scientific community’s appreciation for the power of ADCs has grown, so have the numbers. FDA has now approved at least four ADCs,[3] and hundreds more are in development.[4] The number of patent applications has also grown, with the U.S. Patent and Trademark Office (USPTO) publishing over two hundred patent applications with claims to ADC inventions in the last two years alone.[5]

But filing an application with the USPTO does not guarantee that a patent will be obtained. Among other requirements, inventions worthy of U.S. patent protection must not have been obvious to a person of ordinary skill in the art at the time of invention (or, under current U.S. patent law, at the time the patent application was filed). In considering whether an invention would have been obvious, the USPTO will consider what was already known in the art, how the claimed invention differs from what was already known, and whether the differences would have been obvious. An invention may be deemed nonobvious if, for example, there was no motivation to modify what was known or no reasonable expectation of success in achieving the claimed invention, or if the invention enjoys commercial success or demonstrates results that would have been unexpected at the time of invention.

Four ways to demonstrate nonobviousness of an ADC invention are to show that (1) the claimed antibody, drug, or linker was not previously known; (2) a person having ordinary skill in the art would not have been motivated to modify known components to achieve the claimed ADC; (3) the skilled artisan would have had no reasonable expectation of success; or (4) the claimed ADC demonstrates unexpected results. These types of arguments have been presented to the USPTO in ADC-based patent applications, often in combination with each other and with amendments to the pending claims.

Provided below are three examples of patents that issued after such nonobviousness arguments were made to the USPTO: U.S. Patent Nos. 8,603,483 (the ’483 patent); 9,308,278 (the ’278 patent); and 9,850,312 (the ’312 patent). Companies seeking patent protection for their own ADC inventions should consider these and other examples when developing their own nonobviousness positions. The authors have not independently analyzed the obviousness of the claims discussed below, but provide these merely as examples of strategies used to secure allowance of claims directed to ADCs before the USPTO. Readers are encouraged to seek legal counsel in considering their own ADC inventions and these examples.

Example 1: Arguments of No Motivation, No Reasonable Expectation of Success, and Unexpected Results During the Prosecution of U.S. Patent No. 8,603,483 [6]

The USPTO issued the ’483 patent to Janssen Biotech, Inc. and ImmunoGen, Inc. on December 10, 2013, with claims to ADCs, pharmaceutical compositions comprising the ADCs, articles of manufacture comprising the ADCs, methods of producing the ADCs, methods of treating cancer using the ADCs, and methods of inhibiting the growth of cancer cells using the ADCs. For example, independent claim 1 is as follows:

1. An antibody-drug conjugate of the formula:

wherein the antibody is a human alphaV integrin specific antibody, and said antibody is capable of being internalized by a cell expressing said alphaV integrin, and wherein said antibody comprises (i) all of the heavy chain complementary determining region (CDR) amino acid sequences of CNTO 95 as shown in SEQ ID NOS: 1, 2, and 3, and (ii) all of the light chain CDR amino acid sequences of CNTO 95 as shown in SEQ ID NOS: 4, 5, and 6; and wherein the maytansinol is esterified at C-3; R1 and R2 are Me; X1 and X2 are H[;] n is 2; p is 2; and m is 3-4, and the pharmaceutically acceptable salts and esters thereof.

On June 3, 2011, during prosecution of the application that issued as the ’483 patent, the USPTO examiner rejected the then-pending claims for obviousness over combinations of four references. According to the examiner, the first reference taught an immunoconjugate comprising the antibody of CNTO 95 linked to a cytotoxin, the second reference taught that blockade of integrin receptors by CNTO 95 inhibited the growth of new blood vessels in vitro and growth of human melanoma tumors in nude mice, and the third reference taught that CNTO 95 has antitumor and antiangiogenic activity in vivo.

The examiner wrote that the invention of the then-pending claims differed from these teachings only by the recitation that the conjugate has the formula of [C‑L]m‑A, wherein C is DM4 (R1 and R2 are Me and n=2). According to the examiner, the fourth reference taught a conjugate comprising the huMy9-6 monoclonal antibody chemically coupled to maytansinoid DM4 via an N-succinimidyl 4-(2-yridyldithio)butanoate, and it would have been obvious to one of ordinary skill in the art to substitute hyMy9-6 antibody with the CNTO-95 antibody.

In a response dated December 2, 2011, the applicant amended the claims and argued that one of skill in the art at the time of invention would not have been motivated to substitute the CNTO 95 antibody for the huMy9-6 monoclonal because the two antibodies are “very different.” The applicant also argued that an artisan would not have reasonably expected success in substituting one antibody with another antibody that is structurally and chemically very different. In addition, the applicant argued that the art did not suggest that conjugating an anti-alphaV antibody to a cytotoxic drug would provide an important improvement or advantage over the use of the unconjugated CNTO 95 antibody. In support of the arguments, the applicant submitted three declarations. In the first, a named inventor declared that the effectiveness of the CNTO 95-maytansinoid conjugate CNTO 365 in treating tumors was surprising. In the second, a scientist declared that an artisan would not have been motivated to substitute huMy9-6, a highly selective antibody, with CNTO 95, an antibody with high reactivity with normal tissue, and would not have had a reasonable expectation of success. In the third, another scientist provided results from a phase I clinical study using CNTO 365, which the applicant argued showed unexpected and surprisingly low toxicity.

On January 12, 2012, the USPTO examiner maintained the obviousness rejections of the then-pending claims over the same art. The examiner wrote that while CNTO 95 was unexpectedly well tolerated in human clinical trials, the unexpected results did not overcome clear and convincing evidence of obviousness.

In a response dated September 12, 2012, the applicant amended the claims to “closely encompass the CNTO 365 conjugate described and tested in the application,” and argued that the claimed conjugates demonstrated unexpected results because they had a more than four-fold lower EC50 in toxicity studies relative to even other CNTO 95 conjugates. The USPTO examiner issued a notice of allowance, and then the ’483 patent issued on December 10, 2013. The examiner wrote that the amended claims were allowed because CNTO 365 was shown to have superior efficacy.

Example 2: Arguments of No Motivation and Unexpected Results During the Prosecution of U.S. Patent No. 9,308,278 [7]

The USPTO issued the ’278 patent to Agensys, Inc. on April 12, 2016, with claims to ADCs and pharmaceutical compositions comprising the ADCs. For example, independent claim 1 is as follows:

1. An antibody drug conjugate obtained by a process comprising the step of:

conjugating an antibody or antigen binding fragment thereof to monomethyl auristatin F (MMAF), which antibody or antigen binding fragment thereof is expressed by a host cell comprising a nucleic acid sequence encoding an amino acid sequence of a VH region consisting of SEQ ID NO:7, from residues 20 to 142, and a nucleic acid sequence encoding an amino acid sequence of a VL region consisting of SEQ ID NO:8, from residues 20 to 127, thereby producing the antibody drug conjugate.

On July 2, 2015, the USPTO examiner rejected the then-pending claims for obviousness over combinations of five references. According to the examiner, four of the references taught cancer immunotherapy using anti-161P2F10B antibodies such as H16-7.8 conjugated to auristatins such as monomethyl auristatin E (MMAE) for use in treating cancer, and the fifth reference taught that MMAF is an antimitotic auristatin derivative with a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE. The examiner wrote that an artisan would have been motivated to replace MMAE with MMAF based on the fifth reference’s showing of improved therapeutic effects.

In a response dated September 23, 2015, the applicant argued that the first four references would not have motivated an artisan to conjugate the H16-7.8 antibody with MMAF or to target cells expressing 161P2F10B protein with the claimed ADC because the references broadly disclosed more than twenty different monoclonal antibodies and more than fifty different cytotoxic agents, not one of which was MMAF. The applicant also argued that the claimed ADC comprising the claimed H16-7.8 antibody conjugated to MMAF produced surprising results. In support of this argument, the applicant relied on data showing that the H16-7.8 MMAF conjugate inhibited tumor growth for sixty days, a result not obtained with either the H16-1.11 MMAF conjugate or the H16-7.8 MMAE conjugate. The USPTO withdrew the obviousness rejections, and then the ’278 patent issued on April 12, 2016. The examiner wrote that the applicant’s argument of unexpected results was persuasive.

Example 3: Arguments of New Components, No Motivation, and No Reasonable Expectation of Success During the Prosecution of U.S. Patent No. 9,850,312 [8]

The USPTO issued the ’312 patent to Daiichi Sankyo Company, Limited and Sapporo Medical University on December 26, 2017, with claims to ADCs, pharmaceutical compositions comprising the ADCs, antitumor drugs and anticancer drugs containing the ADCs, and methods of treating cancer using the ADCs. For example, independent claim 1 is as follows:

1. An antibody-drug conjugate, wherein a linker and an antitumor compound represented by the following formula and anti-TROP2 antibody are connected:

-(Succinimid-3-yl-N)—CH2CH2CH2CH2CH2—C(=O)-GGFG-NH—CH2—O—CH2—C(=O)—(NH-DX) . . .

wherein the anti-TROP2 antibody comprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 23, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 24 and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 25 in its heavy chain variable region and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 26, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 27 and CDRL3 consisting of the amino acid sequence of SEQ ID NO:28 in its light chain variable region.

On October 21, 2016, the USPTO examiner rejected the then-pending claims for obviousness over three references. According to the examiner, the first reference taught drug delivery systems in which exatecan is linked to a GGFG tetrapeptide, but not the ADC with anti-TROP2 antibody and the linkers in the then-pending claims. The examiner wrote that the second reference taught ADCs using maleimidocaproyl attached to an amino acid spacer attached to a maytansinoid drug moiety, and that the third reference taught ADCs having the anti-TROP2 antibody hRS7 with a drug. The examiner wrote that it would have been obvious to prepare the ADC using the first reference’s exatecan linked to a GGFG tetrapeptide composition with the maleimidocaproyl of the second reference and the anti-TROP2 antibody of the third reference.

In a response dated January 18, 2017, the applicant amended the claims and argued that the claimed ADC comprised a novel linker having a specific structure and a novel anti-TROP2 antibody. The applicant argued that even if exatecan was known in the art, its ability to maintain and exert antitumor activity in the claimed structure was “a totally new finding” and there was no expectation of success. The applicant also argued that the only cited reference that disclosed an anti-TROP2 antibody did not disclose one with the claimed CDR sequences. The applicant argued that the references did not teach or suggest the claimed antibody or provide the necessary motivation to arrive at the claimed antibody with a reasonable expectation of success. The examiner issued a notice of allowance, and then the ’302 patent issued on December 26, 2017.

Conclusion
Companies developing ADCs should strategically obtain patent protection for their products, keeping in mind that their ability to have a patent granted may hinge on the success of their arguments of nonobviousness of the invention. As seen from the examples above, applicants often use a combination of arguments and claim amendments when responding to an obviousness rejection. By considering how other companies have responded to obviousness rejections by the USPTO, companies can gain insight into how to obtain and preserve patent protection for their own ADC inventions.

How to cite:
Eaton J, Miller P, Cyr SK. Four Ways to Show Nonobviousness of ADC Inventions (2018),
DOI: 10.14229/jadc.2018.10.05.001.

Original manuscript received: August 25, 2018 | Manuscript accepted for Publication: August 21, 2018 | Published online September 27, 2018 | DOI: 10.14229/jadc.2018.10.05.001.

Last Editorial Review: October 5, 2018

Featured Image: Patent Concept button. Courtesy: © Fotolia. Used with permission.

This work is published by InPress Media Group, LLC (Four Ways to Show Nonobviousness of ADC Inventions) 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.

The post Four Ways to Show Nonobviousness of ADC Inventions appeared first on ADC Review.

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
Platform approaches

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).[3]

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…” [2]

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.[4][5] Another source states that 182 companies around the world have ADCs in their pipeline.[6] 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.[7][8]

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. [9][10][13]

**Figure 1.0:** Schematic showing the complexity and various components of an antibody drug conjugate.

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.[12]

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.[6]

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.[7]

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.[11] 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.”[10]

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.[13] Valliere- Douglass et al. suggest that conventional analytical methods used for standard biopharma characterization are not sufficient for ADCs.[14] 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.[15]

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.

Analysis Needed	Appropriate Analytical Technique
Primary Structure (Complete Sequence)	Peptide map-UPLC-UHR QToF
Disulfide linkage	Peptide map-UPLC/MS/MS
Secondary/tertiary structure	CD, Fluorescence, HDX-MS
Fragments Aggregates	SEC-MALS, MFI
Charge	icIEF
Glycosylation	Peptide map-UPLC/MS/MS or cleavage/labeling/UPLC
Ohter post translational modifications	Peptide map-UPLC-UHR-QToF
Antigen binding	ELISA, ECL, SPR
Biological activity, as appropriate	Cell bioassay (proliferation, cytotoxicity, affector)
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.[17]

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:[17]

Analytical Capabilities
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).[18]

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”[19]

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.[19][20]

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.[13] 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.[21]

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
Circular dichroism
Differential scanning calorimetry

CQAs (relating to safety and efficacy of the drug) for an ADC product also include the following additional assays:

Analysis Needed	Appropriate analytical techniques
Drug-to-antibody ratio (DAR)	HIC
Drug load distribution	Peptide map-UPLC-UHR QToF
Linkage sites	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
Antigen binding
Cytotoxic assays
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
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.

Cytotoxic Assays
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:

Dose formulation
Infusion set/syringe compatibility
In-use stability
Residual cytotoxins
Dose Formulation

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.

In-use Stability
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.” [22]

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:

DAR
ECL
Size Exclusion Chromatography (SEC)
Micro Flow Imaging (MFI)
Residual Cytotoxins

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:

DMA (Dimethylacetone)
DMF (Dimethylforamide)
THF (Tetrahydrofuran)
Palladium
Platinum

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.[23]

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. [24] 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.[24]

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:

Complete sequence
Post translational modifications
Glycan profiles
Payload linkage sites
Disulfide linkages

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.

Scientific expertise
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
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
Solution

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

Outcome
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.

7.0 Conclusion…
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

Abbreviations:
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.

Keywords:
ADCs, Antibody-drug Conjugates, Characterization, Chemistry Manufacturing and Controls (CMC)

August 1, 2017 | Corresponding Author:
* Glenn Petrie, Ph.D. gpetrie@eag.com

How to cite:
Petrie G, Antibody Drug Conjugate Development: Keys to Rapid IND Submission and Approval (2017), DOI: 10.14229/jadc.2017.08.04.002.

Original manuscript received: April 12, 2017 | Manuscript accepted for Publication: July 3, 2017 | Published online September 4, 2017 | DOI: 10.14229/jadc.2016.09.04.001.

Last Editorial Review: August 17, 2017

Featured Image: Capped vials on an analysis autosampler – selective focus. Courtesy: © Fotolia. Used with permission.

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Single-use technology, designed for the manufacturing of biopharmaceutical products, has made major inroads over the last 30 years. First introduced in the late 1970s in the form of disposable capsules and a range of filters, single-use technologies were revolutionized in the late 2000s with the introduction of single-use 2D and 3D process containers and filter assemblies for mixing and storage systems. Today, these technologies have been adopted across the upstream manufacturing process, downstream purification and fill-finish of entire classes of biologic drugs.

The adoption of single-use technology is especially growing in the development and manufacturing of biologics and complex drugs like Antibody-drug Conjugates (ADCs).

Antibody-drug conjugates
ADCs are highly potent biopharmaceutical drugs designed as a targeted therapy in the treatment of cancer. They are highly hazardous materials, often with occupational exposure limits (OEL) below 100ng/M³/8Hr work day.

The acute potency of ADCs creates a significant risk to personnel involved with the various manufacturing stages. The accepted method to counter the risk of exposure to ADCs is the implementation of so-called barrier isolation systems. These systems are recognized as the highest level of current containment technology, creating both respiratory and dermal protection.

While the use of glass or stainless-steel legacy systems may effectively protect operators, significant equipment decontamination is required. Since most ADCs are produced on a small (production) run/campaign with manufacturing typically taking days rather than months, the cleaning validation burden associated with a hard-shell glass or stainless-steel isolator can be an issue.

Alternative to traditional technology
“Single-use technologies are an alternative to traditional glass or stainless-steel manufacturing with the key difference in their materials of construction. Glass and stainless-steel equipment have decades of historical data and, as a result, their use is well characterized,” noted Karen Green, product manager for single-use assemblies at MilliporeSigma.

“Single-use systems are commonly composed of polymeric materials, which are not as well-known or characterized for biologics processing. These differences result in different approaches to validation and qualification,” she added.

Figure 1.0 A typical ADC process work flow. The input mAb is prepared into reaction conditions by simple dilution or through buffer exchange by ultrafiltration/diafiltration (UF/DF), a very economical, high-yield and robust separation process. In the processing equipment, the antibody will be further modified, then conjugated with the drug linker to form the crude ADC.

Benefits
In the manufacturing of highly complex active pharmaceutical ingredients (APIs) such as ADCs, single-use technologies offer specific benefits in the upstream manufacturing and production of monoclonal antibodies (mAbs) and downstream bioconjugation.

For example, single-use technology enables faster process changeover and facility flexibility that is not possible when traditional equipment is used.

“Since each single-use system is pre-sterilized and used only once, there is no need to sterilize or clean systems between batches, saving time and enabling manufacturers to produce multiple products within the same facility. Furthermore, single-use systems are often mobile, allowing them to be moved within the facility as needed, enabling additional facility flexibility,” explained Mary Robinette, principal project engineer at MilliporeSigma.

Contract development and manufacturing organizations
According to various reports, 70-80% of the manufacturing of ADCs is outsourced to contract development and manufacturing organizations (CDMOs).[1]

“Due to [this] increased outsourcing pattern, CDMOs entertain many different types of ADCs. The use of single-use technology by CDMOs will help speed up the product change over time, avoiding time spent in establishing cleaning methods for each product that is produced, and eliminating upfront investment for expensive capital equipment such as reactors for each product,” said Gang Yao, Ph.D., principal scientist, process & analytical development at MilliporeSigma.

“In the end, customers benefit from lower manufacturing costs and speed to market. The faster turnover will result in more batches made to meet the commercial demand,” he added.

Implementation of single-use technology
Single-use technologies have advanced in several ways over the past decade. Their materials of construction are better known and have established leachables and extractables profiles, and manufacturing techniques have evolved leading to cleaner and more robust films.
Due to these advancements, the adoption rate of single-use has steadily increased across the biopharmaceutical industry, including ADC manufacturing. However, ADC manufacturers will need to be assured of solvent compatibility with bag liners and other single-use components since the manufacturing of ADCs often involves either dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) for the conjugation process.

They also will need to trust that the potential for a leak during the conjugation process is extremely low and that they can successfully scale from a smaller development scale to large-scale GMP production.

Single-use technology suppliers, like MilliporeSigma, have recognized these concerns and have demonstrated that the materials in single-use technologies are indeed compatible with two commonly used solvents (DMSO and DMA) at the temperatures and duration typically used for ADC processing.

Addressing the aggressive conditions used during bioconjugation to ensure compatibility with the polymeric materials used in single-use assemblies and understanding extractables and leachables under these conditions are vital. MilliporeSigma provides supporting data to ensure that the use of solvent during the manufacturing process will not negatively impact the conjugate by demonstrating solvent compatibility as well as sharing representative leachable and extractable data.

MilliporeSigma also has demonstrated that small-scale development batches can be successfully scaled up to large-scale GMP batches using a completely single-use process, guaranteeing operator safety at all steps in the manufacturing. In this single-use process, the fluid contact materials do not change, only the size of the components of the process assemblies. “However, operator safety becomes very critical with the use of more potent linker payloads that typically demonstrate IC₅₀ values in the low-to-mid picomolar range,” Yao added.

Mobius® FlexReady Solution with Smart Flexware

Assemblies for Chromatography and TFF

Mobius® Mixer

Mobius® Single-use Bioreactors

Pellicon® Capsules with Ultracel® Membrane

Tangential flow filtration
Tangential flow filtration (TFF) is a common unit operation in ADC manufacturing and enables concentration and exchange to pre-formulation buffer.[2]

The presence of toxic linker-payloads following conjugation presents challenges in traditional TFF operations. The scale of TFF also can be a challenge.

“MilliporeSigma has developed a completely enclosed single-use TFF capsule. This device is shipped gamma sterilized with RO (reverse osmosis) water, which reduces flushing requirements and enables faster batch turnaround while utilizing the same Ultracel 30 kDa membrane found in our traditional flat-sheet devices,” noted Nicholas Landry, group product manager ultrafiltration at MilliporeSigma.

“The device was engineered with operator safety and containment in ADC processes as design principles,” he added.

Upstream and downstream processing
While single-use technology has generally been used in upstream processing in the manufacturing of mAbs, the technology is now also available in downstream bioconjugation.

One of the major benefits of single-use technology in downstream processing is bioburden control. Single-use technology offers a more closed processing opportunity compared to traditional glass or stainless-steel reactors, thus reducing the opportunities for bioburden growth.

Another significant benefit of single-use technology is that there is no cross-contamination from inefficient cleaning, allowing faster turnover between process changeovers in a biopharmaceutical manufacturing facility, while at the same time, reducing cleaning validation requirements.

In the final verdict, single-use technology has proven, compared to traditional methods, to be a flexible, cost-effective and efficient alternative that provides improved safety. There is no cross-contamination from inefficient cleaning and no cleaning required between batches, resulting in a quicker turnover of the facility.

Reference
[1] Roots Analysis, Antibody Drug Conjugates Market (2nd Edition), 2014 – 2024.
[2] Czapkowski B, Steen J, et al. “Trial of High Efficiency TFF Capsule Prototype for ADC Purification,” ADC Review, April 12, 2017. [Article]

Last Editorial Review: November 20, 2018

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1.0 Abstract
In our globalized world, human pharmaceutical residues and traces of other (chemical) down-the-drain contaminants have become an environmental concern. Following the detection of (pharmaceutical) drug residues in drinking and surface waters , regulatory agencies around the world, including the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), have developed detailed guidance on how pharmaceutical products should be assessed for possible adverse environmental effects.

Hence, an Environmental Risk Assessment or ERA is required as part of the clinical development, regulatory submission and marketing authorization of pharmaceuticals. This is mandatory for drugs both for the treatment of human diseases as well as veterinary use.

Using fate, exposure and effects data, an environmental risk assessment or ERA evaluates the potential risk of (new) medicinal compounds and the environmental impact they cause.

Despite the available guidance from regulatory agencies, regulatory policy is complex, and a number of aspects related to ERA remain unclear because they are not yet well defined. Furthermore, the specific requirements are not always straightforward. Moreover, while some types of chemicals are exempt (e.g., vitamins, electrolytes, peptides, proteins), such exemption may be overruled when a specific mode of action (MOA) involves endocrine disruption and modulation.

In this white paper, which focuses on human pharmaceuticals rather than veterinary pharmaceuticals, the author reviews topics ranging from regulations and environmental chemistry to exposure analysis and environmental toxicology. He also addresses key aspects of an ERA.

2.0 Introduction
The effective functioning of a modern, healthy society increasingly demands developing novel therapeutic agents for the treatment of human and veterinary disease as well as the new and emerging technologies that form the foundation for advancement. A proper understanding of environmental health and safety risks that may have been introduced into the environment as part of developing these new medicines is an important part of this process.

To understand these risks, Environmental Risks Assessments or ERAs are designed to systematically organize, evaluate and understand relevant scientific information. The purpose of such assessment is to ascertain if, and with what likelihood, individuals are directly or indirectly exposed to (novel) medicinal compounds, (bio) pharmaceutical products or active pharmaceutical ingredients in our immediate environment, as well as the consequences of such exposure. The information can then be used to assess if the use of these agents may result in unintended health-related impairment or harm as the result of such exposure, as well as the impact these agents may have on a globalized world. [1]

3.0 Exposure
Exposure may occur if humans come into contact with (novel) medicinal compounds, (bio) pharmaceutical products or active pharmaceutical ingredients. And while therapeutic agents may be intended to cause some measure of harm – for example, chemotherapeutic agents in the treatment of patients with various forms of cancer designed to “kill” malignant cells – unintended environmental exposure may, in turn, cause unintended serious adverse events. In many cases, such exposure may be limited to trace levels of the active pharmaceutical ingredient.

Over the past 30 years, the impact of such exposure, as well as its implications, have become clearer. Because early analytical equipment was not very sensitive, traces of (novel) therapeutic and medicinal compounds, (bio) pharmaceuticals and active pharmaceutical ingredients were not easily detected in the environment until the 1990s. The result was that the impact of these agents in the environment was generally considered nonexistent and unimportant.

However, since the late 1940s, scientists have been aware of the potential that a variety of chemicals are able to mimic endogenous estrogens and androgens. [2][3][4]

The first accounts indicating that hormones were not completely eliminated from municipal sewage, wastewater and surface water were not published until 1965, by scientists at Harvard University, [5][6] and it was not until 1970 that scientists, concerned with wastewater treatment, probed to what extent steroids are biodegradable, because hormones are physiologically active in very small amounts. [7]

However, the first report specifically addressing the discharge of medicinal compounds, pharmaceutical agents or active pharmaceutical ingredients into the environment was published in 1977 by scientists from the University of Kansas. [8]

Despite these and many other early findings, the subject of medicinal compounds such as steroids and other pharmaceutical residues in wastewater did not gain significant attention until the 1990s, when the occurrence of hermaphroditic fish was linked to natural and synthetic steroid hormones in wastewater. [9]

In numerous studies and reports, researchers hypothesized and confirmed that effluent discharge in the aquatic environment, such as municipal sewage, wastewater systems as well as surface waters, contained either a substance or (multiple) substances, including natural and synthetic hormones, that are estrogenic to fish, affecting their reproductive systems. [10]

In time, scientists confirmed that these adverse effects, and implications of endocrine disruption and modulation, were caused by residues of estrogenic human pharmaceuticals. [1]

After discovering hermaphroditic fish in and near water-treatment facilities, scientists identifying the estrogenic compounds that were most likely associated with this occurrence confirmed that substances such as ethynylestradiol, originating from pharmaceutical use, generated a similar effect in caged fish exposed to levels as low as 1 to 10 ng 1−1 and that positive responses may even arise at 0.1 to 0.5 ng 1−1. [9]

Although it was now recognized that the therapeutic agent or active pharmaceutical ingredient itself was biologically active, experts generally believed that there was only a limited environmental impact during manufacturing; and because these therapeutic agents were only manufactured in relatively small amounts, they were not concerned about the potential environmental risk of pharmaceutical residues and trace contaminants. [1]

4.0 Pharmacotherapy
Today, with pharmacotherapy a common part of our daily life, many concerned citizens realize that pharmaceutical residues and trace contaminants may represent an increased environmental risk with potential consequence for human and animal health. [1]

And although the concentrations of these residues rarely exceed the level of parts per billion (ppb), limiting acute toxicity, the emergence of these residues and traces in the environment fundamentally changed the way we look at the (potential) risk of these active pharmaceutical ingredients in the ecosystem. [1]

But regulators have also come to understand that environmental risk assessment developed for non-medicinal chemical containment cannot necessarily be applied to (novel) medicinal compounds, (bio) pharmaceutical products or active pharmaceutical ingredients. They understand that protecting the environment, while at the same time improving human and animal health, requires a better understanding of how to protect the environment (the ecosystem) as well as the active pharmaceutical ingredient in its own regulated environment.

5.0 Value for society
The issue of medicinal compounds, (bio) pharmaceutical agents and active pharmaceutical ingredients in our environment is complex. This complexity is, in part, derived from the medicinal value of these compounds and the general acceptance that patient use – and therefore the excretion of active pharmaceutical ingredients into the environment and, as a result, the potential of harmful effects to the ecosystem and human health – rather than other methods of release, is the primary reason why we find traces of these agents in our environment. [11]

There is no doubt that modern medicines developed by research-based pharmaceutical companies have brought tremendous value. For example, the development of antibiotics generated enormous gains in public health through the prevention and treatment of bacterial infections. In the 20th century, the use of antibiotics aided the unprecedented doubling of the human life span. [12][13]

Before the development of insulin in the late 1920s and early 1930s, people diagnosed with diabetes (type 1) were not expected to survive. In 1922, children with diabetes rarely lived a year after diagnosis. Five percent of adults died within two years, and less than 20% lived more than 10 years. But since insulin became available, the drug has become a daily routine for people with diabetes, creating a real survival benefit and making the difference between life and death. [14]

Pharmaceutical agents have also drastically impacted social life. The introduction of the pill in the early 1960s, for example, affected women’s health, fertility trends, laws and policies, religion, interpersonal relations, family roles, women’s careers, gender relations and premarital sexual practices, offering a host of contraceptive and non-contraceptive health benefits. [15]

It can be said that the emergence of the women’s rights movement of the late 1960s and 1970s is directly related to the availability of the pill and the control over fertility it enabled: It allowed women to make personal choices about life, family and work. [15]

The development of novel targeted anticancer agents, including antibody-drug conjugates or ADCs, have resulted in a new way of treating cancer and hematological malignancies with fewer adverse events, longer survival and better quality of life (QoL).
In the end, the economic impact of pharmaceutical agents, some hailed as true miracles, has been remarkable, contributing to our ability to cure and manage (human) disease and allowing people to live longer, healthier lives.
At the same time, the (clinical) use of (novel) medicinal or (bio) pharmaceutical agents and their underlying active ingredients can also harbor a number of risks for the environment.

6.0 Understanding environmental risk
In the development of novel therapeutic agents, intensive pre-clinical investigations yield a vast amount pharmacological and toxicological data. During the discovery and (early) development of therapeutic agents, researchers are paying close attention to target specificity and pathways to understand how an innovative drug compound may have beneficial efficacy in the treatment of human or veterinary diseases. Because adverse events are undesirable, drug developers often focus on therapeutics with a well-understood mechanism of action (MOA) and low toxicity (often measured in ng/L). [1]

As a result, only a small number of pharmaceuticals will be classified as highly and acutely toxic, requiring new approaches to identify pharmaceutical agents in robust environmental hazard and risk assessments. [16]

7.0 Pharmaceutical risk assessment
While non-medicinal and chemical entities produced in significant commercial quantities require an environmental risk assessment based on a minimum set of hazard data to assess and manage risks to humans and the environment, such an approach does not necessarily apply to (novel) therapeutic agents. One reason is that the health and wellbeing of humans should never be assessed and managed on the basis of risk alone. Regulators generally require drug developers or sponsors to undertake a comprehensive assessment of the potential risks and benefits of a proposed therapeutic agent, which may demonstrate significant risk to the patient. However, these risks are largely offset by the medicinal benefits of such agents.

Regulators around the world require a systematic and transparent assessment of the (potential) of environmental risk in addition to a (novel) medicinal agent’s quality, safety and efficacy, and relevance as part of regulatory decision-making. [17]

8.0 Environmental risk and regulatory requirements in the United States
The legal mandate of protecting the environment in the United States consists of the National Environmental Policy Act of 1969 (NEPA), which requires all federal agencies to assess the environmental impact of their actions and the impact on the environment, and the Federal Food, Drug and Cosmetic Act (FFDCA) of 1938 (amended in 1976).

This legal framework further determines that the regulation of pharmaceuticals in the environment is the responsibility of the United States Environmental Protection Agency or EPA and the United States Food and Drug Administration (FDA), which is required to consider the environmental impact of approving novel therapeutic agents and biologics applications as an integral part of the regulatory process.

The FDA has required environmental risk assessments for (novel) medicinal compounds, (bio) pharmaceutical agents and active pharmaceutical ingredients for veterinary use (since 1980) as well as the treatment of human diseases (since 1998).

As such, the FDA regulations in 21 CFR part 25 identify which Pharmaceutical Environmental Risk Assessment or PERA is required as part of a New Drug Application or NDA, abbreviated application, Investigational New Drug application or IND. [18]

The same regulations (21 CFR 25.30 or 25.31) identify categorical exclusions for a number of products and product categories – including vitamins, electrolytes, peptides, proteins, etc. – that do not require the preparation of an environmental risk assessment or ERA because, as a class, these agents, individually or cumulatively, do not significantly affect the quality of the (human) environment.

In addition, and in contrast to the categorical exclusion, these regulations also identify cases when such an exemption may be overruled as the result of a specific mode of action (MOA) involving endocrine disruption and modulation. [18]

9.0 Required ERA
Under the applicable regulations, NDAs, abbreviated applications and supplements to such applications do not qualify for a categorical exclusion if the FDA’s approval of the application results in an increased use of the active moiety or active pharmaceutical ingredient, as a result of higher dose levels, use of a longer duration, for a different indication than was previously approved, or if the medicinal agent or drug is a new molecular entity and the estimated concentration of the active therapeutic agent at the time of entry into the aquatic environment is expected to be 1 part per billion (ppb) or greater.

Furthermore, a categorical exclusion is not applicable when approval of an application results in a significantly altered concentration or distribution of a (novel) therapeutic agent, the active pharmaceutical ingredient, its metabolites or degradation products in the environment.

Regulations also refer to so-called extraordinary circumstances (stated in 21 CFR 25.21 and 40 CFR 1508.4) where a categorical exclusion does not exist. This may be the case when a specific product significantly affects the quality of the (human) environment and the available data establishes that there is a potential for serious harm. Such environmental harm may go beyond toxicity and may include lasting effects on ecological community dynamics. Hence, it includes adverse effects on species included in the United States Endangered Species Act (ESA) as well as other federal laws and international treaties to which the United States is a party. In these cases, considered extraordinary circumstances, an environmental risk assessment is required unless there are specific exemptions relating to the active pharmaceutical ingredient.

10.0 Naturally Occurring Substances
Based on the current regulations, a drug or biologic may be considered to be a “naturally occurring” substance if it comes from a natural source or is the result of a biological process. This applies even if such a product is chemically synthesized. The regulators consider the form in which an active ingredient or active pharmaceutical agent exists in the environment to determine if a medicinal compound or biologic is a naturally occurring substance. Biological and (bio) pharmaceutical compounds are also evaluated in this way.

According to the Guidance for Industry, a protein or DNA containing naturally occurring amino acids or nucleosides with a sequence different from that of a naturally occurring substance will, after consideration of metabolism, generally qualify as a naturally occurring substance. The same principle applies to synthetic peptides and oligonucleotides as well as living and dead cells and organisms. [18]

11.0 Preparing an Environmental Risk Assessment
If an environmental risk assessment is required, the FDA requires drug developers and/or sponsors to focus on characterizing the fate and effects of the active pharmaceutical ingredient in the environment as laid out in the Guidance for Industry, Environmental Assessment of Human Drugs and Biologics Applications (1998). [18]

This is generally the case if the estimated concentration of the active pharmaceutical ingredient being considered reaches, at the point of entry into the aquatic environment, a concentration ≥1 PPB; significantly alters the concentration or distribution of a naturally occurring substance, its metabolites or degradation products in the environment; or, based on available data, it can be expected that an increase of the level of exposure may, potentially, lead to serious harm to the environment. [18]

To guarantee that satisfactory information is available, the 1998 Guidance for Industry lays out a tiered approach for toxicity testing to be included in an environmental risk assessment. [Figure I] [18]

Furthermore, if potential adverse environmental impacts are identified, the environmental risk assessment should, in accordance with 21 CFR 25.40(a), include a discussion of reasonable alternatives designed to offer less environmental risk or mitigating actions that lower the environmental risk.

**Figure 1:** *Tiered Approach to Fate and Effect Testing (USA) [18]*

12.0 A Tiered Approach
The fate and effects testing is based on a tiered approach:
12.1 Tier 1
This step does not require acute ecotoxicity testing to be performed if the EC50 or LC50 divided by the maximum expected environmental concentration (MEEC) is ≥1,000, unless sublethal effects are observed at the MEEC. If sublethal effects are observed, chronic testing as indicated in tier 3 is required. [18]

12.2 Tier 2
In this step, acute ecotoxicity testing is required to be performed on a minimum of aquatic and/or terrestrial organisms. In this phase, acute ecotoxicity testing includes a fish acute toxicity test, an aquatic invertebrate acute toxicity test and analgal species bioassay.

Similar to tier 1, tier 2 does not require acute ecotoxicity testing to be performed if the EC50 or LC50 for the most sensitive organisms included in the base test, divided by the maximum expected environmental concentration (MEEC) is, in this tier, ≥100, unless sublethal effects are observed at the MEEC. However, as in the case of tier 1, if sublethal effects are observed, chronic testing as indicated in tier 3 is required. [18]

12.3 Tier 3
This tier requires chronic toxicity testing if the active pharmaceutical ingredient has the potential to bioaccumulate or bioconcentrate, or if such testing is required based on tier 1 or tier 2 test results. [18]

13.0 Bioaccumulation and Bioconcentration
Bioaccumulation and bioconcentration are complex and dynamic processes depending on the availability, persistence and physical/chemical properties of an active pharmaceutical ingredient in the environment. [18]

Bioaccumulation and bioconcentration refer to an increase in the concentration of the active pharmaceutical ingredient in a biological organism over time, compared with the concentration in the environment. In general, compounds accumulate in living organisms any time they are taken up and stored faster than they are metabolized or excreted. The understanding of this dynamic process is of key importance in protecting human beings and other organisms from the adverse effects of exposure to a (novel) medicinal compound, (bio) pharmaceutical agent or active pharmaceutical ingredient, and it is a critical consideration in the regulatory process. [21]

According to the definition in the Guidance for Industry, active pharmaceutical ingredients are generally not very lipophilic and are, in comparison to industrial chemicals, produced in relatively low quantities. Furthermore, the majority of active pharmaceutical ingredients generally metabolize to Slow Reacting Substances or SRSs that are more polar, less toxic and less pharmaceutically active than the original parent compound. This suggests a low potential for bioaccumulation or bioconcentration. [18]

Following a proper understanding of this process, tier 3 chronic toxicity testing is required if an active pharmaceutical ingredient has the potential to bioaccumulate or bioconcentrate. A primary indicator is the octanol/water partition coefficient (Kow). If, for example, the logarithm of the octanol/water partition coefficient (Kow) is high, the active pharmaceutical ingredient tends to be lipophilic. If the coefficient is ≥3.5 under relevant environmental conditions, such as a pH of 7, chronic toxicity testing is required.

Tier 3 does not require further testing if the EC50 or LC50 divided by the maximum expected environmental concentration (MEEC) is ≥10, unless sublethal effects are observed at the MEEC.

In accordance with the Guidance for Industry, a drug developer or sponsor should include a summary discussion of the environmental fate and effect of the active pharmaceutical ingredient in an environmental risk assessment. The environmental risk assessment should also include a discussion of the affected aquatic, terrestrial or atmospheric environments. [18]

14.0 Special Consideration: Environmental Impact Statement
Following the filing of an environmental risk assessment for gene therapies, vectored vaccines and related recombinant viral or microbial products, the FDA will evaluate the information and, based on the submitted data, determine whether the proposed (novel) medicinal compound, (bio) pharmaceutical agent or active pharmaceutical ingredient may significantly affect the environment and if an Environmental Impact Statement (EIS) is required. According to 21 CFR 25.52, if an EIS is required, it will be available at the time the product is approved. Furthermore, if required, an EIS includes, according to 40 CFR 1502.1, a fair discussion of the environmental impact as well as information to help decision-makers and the public find reasonable alternatives that help in avoiding or minimizing adverse impacts or enhance environmental quality. [19]

However, if the FDA determines that an EIS is not required, a Finding of No Significant Impact (FONSI) will, according to 21 CFR 25.41(a), explain why this is not required. This statement will include either the environmental risk assessment or a summary as well as reference to underlying documents supporting the decision. [19]

15.0 European requirements
In Europe, environmental risk assessments were, in accordance EU Directive 92/18/EEC and the corresponding note for guidance issued by the European Medicines Agency (EMA), first required for (novel) medicinal agents for veterinary use in 1998. The requirement for an environmental risk assessment for (novel) medicinal agents, (bio) pharmaceuticals and active pharmaceutical ingredients for the treatment of human disease was first described in 2001 in Directive 2001/83/EC.

Subsequent to an initial guiding document published in January 2005, the European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) issued its final guidance for the assessment of environmental risk of medicinal products for human use in 2006. [20]

After the discovery of pharmaceutical residues and trace contaminants in the environment, regulators in the European Union require that an application for marketing authorization of a (novel) medicinal or (bio) pharmaceutical agent is accompanied by an environmental risk assessment.

This requirement is spelled out in the revised European Framework Directive relating to medicinal products for human use. It applies for new registrations as well as repeat registrations for the same medicinal agent if the approval of such an extension or application leads to the risk of increased environmental exposure.

In Europe, the objective of the environmental risk assessment is to evaluate, in a step-wise, phased procedure, and as part of the Centralized Procedure by the European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP), the potential environmental risk of (novel) medicinal compounds, (bio) pharmaceutical agents and/or active pharmaceutical ingredients. Such an assessment will be executed on a case-by-case basis.

16.0 Phase I
In this process, Phase I estimates the exposure of the environment to the drug substance and is only focused on the active pharmaceutical ingredient or drug substance/active moiety, irrespective of the intended route of administration, pharmaceutical form, metabolism and excretion.
This phase excludes amino acids, proteins, peptides, carbohydrates, lipids, electrolytes, vaccines and herbal medicines, because regulators believe that these biologically derived products are unlikely to present a significant risk to the environment. [21]
The exemption for these biologically derived biopharmaceuticals is generally interpreted as an exemption for all biopharmaceutical agents manufactured via live organisms and that have an active ingredient that is biological in nature. [21]

Yet, not all biologically derived biopharmaceuticals are (easily) biodegradable, and scientists have detected modified natural products, including plasmids, in the environment. Furthermore, some protein structures, including prions, are very environmentally stable and resistant to degradation, allowing them to persist in the environment. [22] Hence, this approach requires future scientific justification.
In Phase I, following the directions included in the European Chemicals Bureau (2003) Technical Guidance Document, an active pharmaceutical ingredient or drug substance/active moiety with a logKow >4.5 requires further screening for persistence, bioaccumulation and toxicity, or a PBT assessment.

For example, based on the OSPAR Convention and REACH Technical Guidance, highly lipophilic agents and endocrine disruptors are referred to PBT assessments.
Phase I also includes the calculation of the Predicted Environmental Concentration or PEC of active pharmaceutical ingredients, which, in this phase, is restricted to the aquatic environment, and a so-called “action limit” requiring additional screening.
The “action limit” threshold for the PEC in surface water (PECsurface water), for example, is calculated by using the daily dose of an active pharmaceutical ingredient, the default values for wastewater production per capita, and the estimated sale and/or distribution of the active pharmaceutical ingredient if there is evidence of metabolism and no biodegradation or retention following sewage treatment is observed.

17.0 Phase II
Phase II, divided into two parts, tier A and tier B, assesses the fate and effects of novel medicinal compounds, (bio) pharmaceutical agents or active pharmaceutical ingredients in the environment.

Following the assessment of the PEC/PNEC ratio based on relevant environmental fate and effects data (Phase IIA), further testing may be needed to refine PEC and PNEC values in phase II tier B. A PEC/PNEC ratio of This process helps regulators to evaluate potential adverse effects independently of the benefit of the (novel) medicinal compound, (bio) pharmaceutical agent or active pharmaceutical ingredient, or the direct or indirect impact on the environment.

Stage in regulatory evaluation	Stage in risk assessment	Objective	Method	TEST / DATA REQUIREMENT
Phase I	Pre-screening	Estimation of exposure	Action limit	Consumption data, logKow
Phase II Tier A	Screening	Initial prediction of risk	Risk assessment	Base set aquatic toxicology and fate
Phase II Tier B	Extended	Substance and compartment-specific refinement and risk assessment	Risk assessment	Extended data set on emission, fate and effects

Table 1: The Phased Approach in Environmental Risk Assessment in Europe

18.0 Outcome of fate and effects analysis
In all cases, the medicinal benefit for patients has relative precedence over environmental risks. This means that even in the case of an unacceptable (residual) environmental risk caused by a novel medicinal compound, pharmaceutical agent or active pharmaceutical ingredient, after third-tier considerations, prohibition of a new active pharmaceutical ingredient is not taken into consideration.

If European regulators determine that the possibility of environmental risk cannot be excluded, mitigating, precautionary and safety measures may require the development of specific labeling designed to address the potential risk, as well as adding adequate information in the Summary of Product Characteristics (SPC), Package Leaflet (PL) for patient use, product storage and disposal. The information on the label, SPC and PL should also include information on how to minimize the discharge of the product into the environment and how to deal with disposal of unused product, such as in the case of shelf-life expiration.

In extreme cases, a recommendation may be included for restricted in-hospital or in-surgery administration under supervision only, a recommendation for environmental analytical monitoring, or a requirement for ecological field studies. [20] [23]

19.0 Combined effects
Often overlooked by regulators is the fact that the regulatory frameworks such as the European REACH Regulation, the Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD) mainly focus on toxicity assessment of individual chemicals or active pharmaceutical ingredients.

This poses a problem for the proper execution of environmental risk assessments and regulation because the effect of contaminant mixtures with multiple chemical agents and active pharmaceutical ingredients, irregardless of their source, is a matter of growing, and recognized, scientific concern. [24]

To solve this problem, scientists are working on experimental, modeling and predictive environmental risk assessment approaches using combined effect data, the involvement of biomarkers to characterize Mode of Action, and toxicity pathways and efforts to identify relevant risk scenarios related to combined effects of pharmaceutical residues, trace contaminants as well as non-medicinal (industrial) chemicals. [24]

20.0 International harmonization
Created in the 1990s, the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) was set up as an agreement between the European Union, the United States and Japan to harmonize different regional and national requirements for registering pharmaceutical agents in order to reduce the need to duplicate testing during the research and development phase of (novel) medicinal compounds, (bio) pharmaceutical agents and active pharmaceutical ingredients. However, to date, and partly as a result of the overlying differences in regulations and directives, environmental risk assessments have, so far, not been included in the harmonization procedures. [25]

In contrast, the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products, or VICH, similar to the ICH a trilateral set up in 1996 between the European Union, the Unites States and Japan, does include the assessment of ecotoxicity and the evaluation of environmental impact of veterinary medicinal products.

The VICH guideline, intended to provide a common basis for an Environmental Impact Assessment or EIA, offers guidance for the use of a single set of environmental fate and toxicity data and is designed to guide scientists to secure the type of information needed to protect the environment. The guideline, published in 2004 and recommended for implementation in 2005, was developed as a scientifically objective tool to help scientists and regulators extract the maximum amount of information from studies to achieve an understanding of the potential (risk) of specific Veterinary Medicinal Products to the environment. [26]

21.0 Impact of Environmental Risk Assessment
Although an environmental risk assessment is part of the regulatory approval and marketing authorization process in both the United States and Europe, the actual impact can be different.

In Europe, an adverse environmental risk assessment for (novel) medical compounds, (bio) pharmaceutical agents or active pharmaceutical ingredients for human use does not impact or influence the marketing approval application. EU Directive 2004/27/EC/Paragraph 18 stipulates that the environmental impact should be assessed and, on a case-by-case basis, specific arrangements to limit it should be envisaged. In any event, the impact should not lead to refusal of a marketing authorization.

However, a parallel directive pertaining to veterinary medicine, as laid out in EU Directive 2009/9/EC, stipulates that, in the case of veterinary medicine, an environmental impact assessment should be conducted to assess the potential harmful effects and the kind of harm the use of such a product may cause to the environment, as well as to identify any precautionary measures that may be necessary to reduce such risk.

Furthermore, the directive requires that, in the case of live vaccine strains which may be zoonotic, the risk to humans also needs to be assessed. In the case of veterinary medicine, an environmental impact assessment is part of the overall risk-benefit assessment, and, in the case of a negative result, may potentially lead to a refusal to approve the medicinal compound, (bio) pharmaceutical agent or active pharmaceutical ingredient.

In the United States, the FDA has eliminated environmental assessment requirements for certain types of veterinary drugs when they are not expected to significantly affect the environment. However, a negative assessment, based on unacceptable risk to “food” or “non-food” animals, can result in a refusal of a New Animal Drug Application (NADA) or a Supplemental New Animal Drug Application (SNADA). [26]

22.0 Conclusion
The central questions in the development of (novel) medicinal compounds, (bio) pharmaceutical products or active pharmaceutical ingredients for the treatment of human and veterinary disease is whether a novel agent will have an effect on the environment.

Regulators around the world, including in the United States and Europe, follow different assessment methodologies to ascertain these risks. However, all regulators use fate, exposure and effects data to help them understand if a (novel) medicinal compound, (bio) pharmaceutical agent or active pharmaceutical ingredient harbors a potential environmental risk, causing potential harmful effects on the ecosystem, and how this impacts human and veterinary health.

In all cases, environmental risk assessments are carried out based on scientifically sound premises, relying on established, accepted and universally known facts.

Overall, environmental risk assessments are useful analytical tools, providing critical information contributing to public health, as well as key instruments in guiding environmental policy decision-making.

As such, they play a key role in building a better, healthier world.

August 3, 2017 | Corresponding Authors: Duane Huggett, Ph.D | DOI: 10.14229/jadc.2017.29.08.001

Received: February 24, 2017 | Accepted for Publication: April 28, 2017 | Published online August 3, 2017

Last Editorial Review: August 3, 2017

The post Environmental Risk Assessment and New Drug Development appeared first on ADC Review.

Each structurally unique molecular entity is assigned its own corporate ID. Additionally each batch, or lot, of compound material is assigned a unique lot number.

The same number sequence is used by both software packages removing the possibility of identical PR- and A-numbers.

To minimize the impact on already established workflows for both antibodies and small molecules, a solution that leveraged both GBRS and A-coder was desired.

**Figure 3.0:** Step 1 of association process: structure of retrieval the linker-drug.

First, the structure of the linker-drug is retrieved using the A# (Figure 3.0). Next, the drug is identified either by modification of the retrieved linker-drug structure or using the A#.

**Figure 4.0:** Step 2 of association process: identification of the payload.

As the structure of both the linker-drug and drug are known, the linker is then automatically identified by the software (Figure 5).

**Figure 5.0:** Step 3 of association process: automatic identification of linker.

hydrolysis of the maleimide ring affords a stable attachment;
the environment surrounding the cysteine influences hydrolysis of the maleimide ring.

**Figure 7.0:** Association of ADC components enables SAR visualization, for example, ADCs with auristatin as the MOA.

**Figure 8.0:** X-combo is registered as the ring hydrolyzed maleimide structure (X represents the antibody).

Corresponding Author:
* adrian.hobson@abbvie.com

Author Contributions:
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Original manuscript received: July 24, 2017 | Manuscript accepted for Publication: August 3, 2017 | Published online August 14, 2017 | DOI: 10.14229/jadc.2017.14.08.002

Last Editorial Review: August 11, 2017

The post Registration of Antibody Drug Conjugates appeared first on ADC Review.


*Naked antibody concentration* (ng·mL-1)	Effective ADC EC₅₀ (ng·mL-1)
0	1,560
11.5	1,695
115.0	2,229
1,150.0	4,253
11,500.0	7,844
115,000.0	11,241
Table 2.1

2.1 In vitro cytotoxicity of ADC co-existed with naked antibody

2.2 In silico cytotoxicity of ADC co-existed with naked antibody

The bias from Schild model caused by last unsatisfied prerequisite can be modified by bystander effect.

The unmodified Schild equation predicts the effective ADC EC₅₀ as follows:

Co-existing naked antibody concentration [nkdAb] was set to 0-115000 ng/mL (0, 11.5 ng/mL, 115 ng/mL, 1150 ng/mL, 11500 ng/mL, 115000 ng/mL) and unmodified effective ADC EC₅₀ could be predicted.

2.2.2 Bystander effect modification
ADC incubation usually takes 3 to 7 days from binding to receptors to having targeted cell killed. [2]


Naked antibody concentration	Experimental Results	Schild equation results	Our model results
ng·mL-1	ng·mL-1	ng·mL-1	ng·mL-1
0	1,560	1,560	*
11.5	1,695	1,816	1,485
115.0	2,229	4,123	2,886
1,150.0	4,253	27,189	4,286
11,500.0	7,844	257,846	5,686
115,000.0	1,1241	2,564,417	7,087
Table 2.2

In another word, the drug-response logistic curve was shifted by logarithm of naked antibody concentration when naked antibody existed (Fig. 2.2).

The modified equation predicts the effective ADC EC₅₀ as follows:

The comparison of all data points amongst three results is shown in Fig. 2.4.

The training algorithm is presented later.

3.0 Discussion
Previous study neglected the impact of naked antibody on ADC efficacy due to its small percentage of total antibody (5%)[1].

Because bystander effect varied from cell to cell and ADC to ADC, we generated the model by experimental data, which was universal for all cytotoxicity modeling using the same type of cell and ADC. In our model, the bystander constant was the base of logarithm relation, which might be meaningful elsewhere in the field of ADC.

Abbreviations:
ADC, antibody drug conjugate; DAR, drug-to-antibody ratio; T-DM1, ado-trastuzumab emtansine; nkdAb, naked antibody.

Keywords:
drug-detached naked antibody, ADCs efficacy impairment, antagonism, Schild model, bystander effect.

August 14, 2017 | Authors: Nanfang Hong [1], Jianmin Fang * [1]
[1] School of Life Science and Technology, Tongji University, Shanghai, China

Corresponding Author:
* jfang@tongji.edu.cn

Author Contributions:
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

How to cite:
Hong N, Fang J, Drug-detached Naked Antibody Impairs ADC Efficacy (2017),
DOI: 10.14229/jadc.2016.09.04.001.

Original manuscript received: May 12, 2017 | Manuscript accepted for Publication: August 3, 2017 | Published online September 4, 2017 | DOI: 10.14229/jadc.2016.09.04.001.

Last Editorial Review: September 1, 2017

The post Drug-detached Naked Antibody Impairs ADC Efficacy appeared first on ADC Review.

Most recruited respected academic-clinician leaders from NCI-designated comprehensive cancer centers.
They were established at large, well-respected, financially secure not-for-profit regional community hospital systems interested in increasing quality of care and standards of practice.
They sought to infuse greater academic rigor into their programs, develop translational research programs, enroll more patients from their respective communities into clinical trials, and provide the best cancer care to populations not served by traditional academic centers.
They often adapted existing resources through collaborative efforts rather than reinventing the wheel.

Baptist Health South Florida’s Miami Cancer Institute, Miami, Fla., and West Cancer Center, Memphis, Tenn., were identified in later articles.

3.0 The Conference Objectives included

Recognizing and discussing the emerging presence of hybrid academic-community cancer centers.
Reviewing similarities and differences between existing models that vary according to individual centers’ strengths and needs.
Identifying and recommending new concepts inspired by models and practices of other centers.
Formulating a plan for future and ongoing collaboration in support of further development of hybrid academic-community cancer centers.