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Graph showing the relationship of dPEG® linker length in relation to potency and specificity in extracellular drug conjugates.

Extracellular Drug Conjugates Therapeutically Exploit Protein Proximity

Pharmaceutical company Centrose, founded by James R. Prudent, Ph.D., developed a new class of antibody drug conjugates called extracellular drug conjugates. Nature Publishing Group published the research as a open access paper in its Molecular Therapy journal.1 Apart from the interesting and important development of a new class of antibody drug conjugate (ADC), the research also showed how important linker length 2 is to the potency and specificity of the EDC.

How Extracellular Drug Conjugates Work

Extracellular drug conjugates (EDCs) are designed similarly to ADCs. That is, EDCs consist of a monoclonal antibody (mAb), a linker, and a cytotoxic agent. Antibody drug conjugates require internalization into a diseased cell where the cytotoxic agent can then be released to act on its target.3 The cytotoxic agent may require intracellular modification or degradation to act on its target molecule.

By contrast, extracellular drug conjugates require no internalization. Rather, EDCs target cell surface proteins that are expressed on a target (i.e., cancerous) cell. Moreover, the cytotoxic agent that is linked to the mAb does not target the same protein targeted by the mAb. Rather, the cytotoxic agent kills the targeted cells by affecting a protein or enzyme that is different from the protein or enzyme bound by the mAb but that is closely associated with the target protein or enzyme. Click here to see Centrose's explanation of how EDCs work, including a three-minute video, which you can also view on Centrose's website.

In this particular study, the research team observed that some proteins that are overexpressed on the surface of cancer cells are closely associated with the sodium potassium ATPase1 (NKA, and also known as the sodium potassium pump; see Figure 1). Cardiac glycosides such as digoxin or ouabain inhibit the NKA. Cells affected by these or other cardiac glycosides swell, then undergo necrotic cell death.1,4,5 The team reasoned that by (1) combining antibodies to proteins that are both (a) overexpressed on the cell surface of cancer cells and (b) closely associated with the NKA (2) with cardiac glycosides that strongly inhibit the NKA (3) would create cancer therapeutic antibody drug conjugates that were localized to the extracellular space, hence, Extracellular Drug Conjugates.


Creating the Extracellular Drug Conjugates in this Study

Through prior testing (not in this paper) the team found that the cardiac glycoside scillarenin β-L-aminoxyloside (Figure 1) highly inhibited the NKA. This was chosen as the drug for conjugation to the antibody.

Chemical structure of Scillarenin β-L-aminoxyloside, the cardiac glycoside used in this study as the cytotoxic agent for the extracellular drug conjugates.
Figure 1: Scillarenin β-L-aminoxyloside, the cardiac glycoside used in this study as the cytotoxic agent for the extracellular drug conjugates.

The team also developed or acquired nine (9) mAbs. For directly testing the EDCs, the mAbs had to meet one of the following three criteria:

  1. was a marker for metastatic cancer commonly known to associate with the NKA;
  2. was cancer related and thought to associate with the NKA; or
  3. was found by the current study to associate with the NKA and was a current cancer antibody drug target.

As controls, the research team selected mAbs to proteins that were expressed on the cell surface but did not associate with the NKA or that were not expressed on any cell surface.

The researchers also investigated the effect of linker length between the mAb and the drug (abbreviated CG1) using Quanta BioDesign's MAL-dPEG®n-NHS esters. These versatile heterobifunctional linkers come in a variety of specific lengths and are single molecular weight PEG derivatives (i.e., they have no dispersity). The maleimidopropyl group on one end reacts with free sulfhydryl groups forming a thioether linkage, while the NHS ester group on the other end will react with free amines to form a peptide bond. Four lengths of PEG — n = 2, 12, 24, and 36 dPEG® units (27, 56, 105, 144 Angstroms) — were chosen to connect CG1 to the EDC. See Figure 1. Although Figure 1 shows a single CG1-dPEG®n conjugated to the mAb, calculations by Centrose showed that the average EDC had a DAR of four (4). See reference 1, page 5.


Chemical structures of the MAL-dPEG®n-NHS-ester linkers used to construct the extracellular drug conjugates used in this study.
Figure 2: MAL-dPEG®n-NHS-ester linkers used to construct the extracellular drug conjugates used in this study.

Extracellular Drug Conjugates Demonstrate In Vitro Efficacy...

The research team examined the efficacy of the extracellular drug conjugates after conjugating CG1 (the cardiac glycoside) to the mAb for dysadherin (a protein marker associated with metastatic cancer). They also measured the efficacy and toxicity of CG1 by itself or conjugated to one of the dPEG® linkers but not conjugated to the EDC.

For the EDC-dPEG®n-CG1 conjugates, the Centrose team first measured antibody binding on the surface of the different cell lines. Then, by monitoring cell viability, they tested the cells' sensitivity to the EDC-dPEG®n-CG1 conjugates at concentrations from 1 to 200,000 pmol/L. The dose-response curve in Figure 3, below, shows that increasing the linker length in EDC-dPEG®n-CG1 conjugates improved target specificity and potency. However, decreasing linker length in dPEG®n-CG1 constructs that were not conjugated to mAb increased toxicity and reduced specificity.


Dose-Response Curve and Potency-Specificity Graph for the Extracellular Drug Conjugates Based on Anti-dysadherin. Note that the potency and specificity increase with linker length for the mAb-dPEG®n-CG1 conjugates, but in the dPEG®n-CG1 constructs not conjugated to mAb, the specificity and toxicity increase as the linker length decreases.
Figure 3: Dose-Response Curve and Potency-Specificity Graph for the Extracellular Drug Conjugates Based on Anti-dysadherin. Note that the potency and specificity increase with linker length for the mAb-dPEG®n-CG1 conjugates, but in the dPEG®n-CG1 constructs not conjugated to mAb, the specificity and toxicity increase as the linker length decreases.

Similar dose-response curves were obtained for some of the other tested cell lines. Cell lines expressing a cell surface antigen closely associated with the NKA were particularly sensitive to the EDC-dPEG®n-CG1 conjugates, but control cell lines (those either not expressing a cell surface antigen or expressing a cell surface antigen not associated with the NKA) were relatively insensitive to the EDC-dPEG®n-CG1 conjugates.


...And They Work In Vivo Also

The in vitro results also translated to in vivo studies in mice. In xenograft studies in mice bearing human pancreatic cancer tumors, EDC-DYS (EDC specific to dysadherin conjugated to dPEG®-CG1, with a DAR of 4) was compared to the standard dosing regimen of gemcitabine. EDC-DYS outperformed gemcitabine in a dose-dependent manner. Similarly, EDC-CD38 (a marker for various lymphomas and multiple myeloma) beat CHOP, a chemotherapy cocktail used as a standard treatment for Ramos B-cell lymphoma. Likewise, EDC-CD20 (another lymphoma marker) exceeded Rituximab's performance. The control experiments showed that EDC-CONTROL conjugates (mAb targeted to antigens not expressed on the cell surface) did not reduce tumor size in mice.


Extracellular Drug Conjugates Offer New Therapeutic Options

EDCs are a new class of antibody drug conjugate, and they offer new, and potentially superior, therapeutic options for patients. Though similar in design and construction to a standard ADC, an EDC is different. The EDC always resides in the extracellular space, and it targets two cell surface proteins. These two features define the EDC. Neither the mAb nor the cytotoxic drug need to be internalized, released, or broken down in order to act. Many cells evolve to evade chemotherapy by rapidly exporting or neutralizing drugs that are released intracellularly. This unique EDC feature impedes cells in evolving resistance to the EDC.

These results show that EDCs are potentially useful in killing cancers that are resistant to multiple drugs, metastatic, and/or aggressive. Thus, in the future, EDCs may offer new therapeutic options for cancers that are otherwise rather difficult to treat.


Quanta BioDesign's dPEG® Reagents Were Important to the Success of This Research

Quanta BioDesign's maleimido-dPEG®n-NHS ester products were important to the success of this research on extracellular drug conjugates. Unlike traditional PEG derivatives, our dPEG® derivatives are single molecular weight compounds. We manufacture all of our products entirely in the USA by a patented, proprietary process. Our dPEG®s have no dispersity. Consequently, standard analytical techniques suffice for analyzing them to determine their purity. With traditional, dispersed PEGs, "purity" becomes a much more elusive term and is more difficult to measure.

Whether you are developing a new ADC or something else, Quanta BioDesign can help. We have reagents for many different types of conjugation chemistry, and we are open to custom syntheses. We manufacture products on scales from milligrams to multiple kilograms. Our responsive customer service works hard to get you what you need when you need it. If you want to learn more about us, visit our website, or contact us directly. You will be glad that you did.



  1. David J. Marshall, Scott C. Harried, John L. Murphy, et al. Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins, Molecular Therapy advance online publication 19 July 2016; doi: 10.1038/mt.2016.119
  1. A linker is the component of the antibody drug conjugate that joins a monoclonal antibody (mAb) to a cytotoxic drug. An ideal linker is stable in circulation but should release the cytotoxic drug when the mAb reaches the target. "Linker length" refers to the distance between the mAb and the cytotoxic drug. Distance may be expressed in number of atoms or in units of Angstroms.
  1. For more information on how monoclonal antibodies and antibody drug conjugates work in cancer therapy go here and/or here.
  1. Menger, L, Vacchelli, E, Adjemian, S, Martins, I, Ma, Y, Shen, S et al. (2012). Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med 4: 143ra99.

Do you have questions or comments about this post? Please leave a comment below. Also, be sure and check out our list of related products below.


About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at, on Twitter at @RobertHWoodman and @QuantaBioDesign. Feel free to contact him via social media.


Product Pages for the Quanta BioDesign Products Used in This Research

PN10266, MAL-dPEG®2-NHS ester

PN10284, MAL-dPEG®12-NHS ester

PN10314, MAL-dPEG®24-NHS ester

PN10904, MAL-dPEG®36-NHS ester (contact us for information and pricing)


Related Products

PN10549, MAL-dPEG®2-TFP ester

PN10553, MAL-dPEG®12-TFP ester

PN10554, MAL-dPEG®24-TFP ester

PN10555, MAL-dPEG®36-TFP ester


TFP esters are superior to NHS esters. Click here to learn why.

To see all of our crosslinking reagents, click here.


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Thiol Reactive Crosslinkers for Bioconjugation

Thiol reactive crosslinkers are one of the most common classes of crosslinkers in bioconjugation (1). The popularity of conjugation to a thiol is due in part to its presence in many proteins, but they are not as prevalent as amines, which are another site for conjugation. This will allow for greater control of the conjugation. Even greater control of the conjugation process is afforded if a thiol reactive compound is combined with an amine reactive compound to create a heterobifunctional crosslinker.

Thiol reactive crosslinkers from Quanta BioDesign, Ltd.


1. Maleimide crosslinkers

Quanta BioDesign offers a variety of homo- and heterobifunctional thiol reactive crosslinkers for bioconjugation with dPEG® spacers of several different lengths. Among those we offer are maleimide crosslinkers. Near neutral pH, the double bond of the maleimide reacts preferentially and very rapidly with a thiol to form a thioether bond that is not susceptible to reduction (2). Quanta BioDesign offers homobifunctional bis-maleimides as well as a few heterobifunctional maleimide bioconjugation crosslinkers with the other end being an amine reactive active ester. One of our most popular products is Mal-dPEG®4-NHS ester, product number 10214 (shown in Figure 1). It contains a 22 atom (24.8 Å) tetraethylene glycol spacer functionalized on one end with a thiol reactive maleimidopropyl group and on the other end with an amine reactive propionic acid-N-hydroxysuccinimide (NHS) ester.


Thiol reactive PN10214, MAL-dPEG®4-NHS ester
Figure 1: Thiol reactive crosslinker PN10214, one of Quanta BioDesign's most popular PEGylation reagents, has a thiol reactive maleimide group on one end and an amine reactive NHS ester on the other end of a tetraethylene glycol linker.


Quanta BioDesign also offers this product with dPEG®2, dPEG®6, dPEG®8, dPEG®12, dPEG®24, and longer dPEG® linkers. You can view them all on our website. They are listed below in this post.  Another version of this product is PN10551, where the NHS ester is replaced by the 2,3,5,6-tetrafluorophenyl (TFP) ester. In-house research by Quanta BioDesign, Ltd., demonstrates that the TFP ester is much less susceptible than the NHS ester to hydrolysis.


2. Pyridyl disulfide crosslinkers

Quanta BioDesign also offers pyridyl disulfide (SPDP) bioconjugation crosslinkers (see also here), and with these, the thiol reacts with the SPDP moiety to produce a new disulfide bond, as illustrated in Reaction 1. Pyridine-2-thione is generated, but it cannot react with any remaining SPDP crosslinker because it does not contain a thiol (3, 4). If desired, the newly-formed disulfide bond can be cleaved with a reducing agent. It can also be oxidized back to the disulfide bond, which provides a flexibility not available with the maleimide crosslinkers. Like the maleimides, the SPDP crosslinkers are also offered as the NHS and TFP esters.


The thiol reactive pyridyl disulfide (SPDP) group is used in bioconjugation.
Reaction Scheme for the thiol reactive pyridyl disulfide (SPDP) group in bioconjugation


Thiol reactive crosslinkers are available now from Quanta BioDesign

Both the maleimide and SPDP crosslinkers with dPEG® are available from Quanta BioDesign with a variety of PEG spacer lengths, ranging from four to twenty-four ethylene oxide units (and in some cases, even longer) Whatever the length and functionality you need for your thiol reactive crosslinking PEGylation reagent, Quanta BioDesign can provide it for you. If you do not see what you want in our catalog, contact us about a custom synthesis. We can provide you with what you are looking for.



1.  Hermanson, Greg T. Bioconjugate Techniques, 3rd Edition. Waltham, MA: Elsevier (Academic Press), copyright 2013, 1146 pages. (A copy of the 2nd edition of Greg's phenomenal work is available from Quanta BioDesign, Ltd., for $75 plus shipping, or for free with any order of $500 or more, excluding tax and shipping. Look here for more details.)

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2.  Smyth, Derek G., Blumenfeld, O. O., and Konigsberg, W. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. (1964), 91, 589-595.

Click here to return to the text.

3.  Carlsson, J., Håkan, D., and Axén, R. Protein thiolation and reversible protein-protein conjugation. Biochem J. (1978), 173, 723-737.

Click here to return to the text.

4.  Myers, D. A., Murdoch, W. J., and Villemez, C. L. Protein-peptide conjugation by a two-phase reaction, Biochem J. (1985), 227(1), 343.

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Additional Products from Quanta BioDesign, Ltd.

MAL-dPEG®2-NHS ester

MAL-dPEG®4-NHS ester

MAL-dPEG®6-NHS ester

MAL-dPEG®8-NHS ester

MAL-dPEG®12-NHS ester

MAL-dPEG®24-NHS ester

Please call or email us and ask about our MAL-dPEG®x-TFP ester derivatives. If you want a longer length dPEG® spacer than you see here, please call or email us about that also. We will be glad to discuss them with you!

Dan Dawson, M.S. received his BS in Chemistry from the University of Indianapolis in 2006, and his M.S. in Organic Chemistry from the University of Michigan in 2008. Dan is a Process Development Chemist involved in process development and scale-up activities. You can connect with Dan on LinkedIn at

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