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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.

 

References/Endnotes

  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 https://www.linkedin.com/in/roberthwoodman, 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)

 

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PN10553, MAL-dPEG®12-TFP ester

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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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Amphotericin B conjugated to a discrete PEG (dPEG®) linker with a free amine have higher water solubility and lower toxicity than the parent compound.

Amphotericin B+dPEG®: Water-Soluble, Less Toxic, Potent

Amphotericin B conjugated to a discrete PEG (dPEG®) linker with a free amine have higher water solubility and lower toxicity than the parent compound.
Amphotericin B conjugated to a discrete PEG (dPEG®) linker with a free amine has higher water solubility and lower toxicity than the parent compound. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

 

About Amphotericin B

Structure of Amphotericin B. Used with permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.
Figure 1: Structure of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

Amphotericin B (Figure 1) is the "gold standard" treatment for systemic fungal infections and diseases caused by the parasite Leishmania. Sometimes it is the only effective treatment because drug resistance renders other treatments useless. Systemic fungal infections are an increasingly serious, widespread problem in medicine. Patients with weakened or suppressed immune systems (caused by HIV/AIDS, diabetes, organ transplants, some cancer treatments) are especially at risk. An estimated 1.5-2 million people die each year from systemic fungal infections (1). Despite its "gold standard" label, Amphotericin B has several well-known difficulties.

A polyene macrolide antifungal drug, Amphotericin B was first discovered in 1953 from the fermentation culture of the bacterium Streptomyces nodosus. It entered the pharmaceutical market in 1958 (2)(3)(4). Nearly insoluble in water, the drug is not absorbed from the human gastrointestinal tract and must be administered intravenously. Amphotericin B was first formulated as a suspension in sodium deoxycholate solution called Fungizone-Squibb (2)(7). Novel, less toxic lipid formulations have been developed recently (5)(6)(7), but these formulations cost more and require high doses to be effective, thus limiting their use (4)(7).

This drug is also toxic. Its side effects are well known, serious, and sometimes fatal (7)(8). Common side effects from taking this drug include high fever coupled with shaking chills ("shake and bake"), headache, kidney problems, low blood pressure, nausea, vomiting, unusual bruising or bleeding, and irregular heartbeat. Anaphylactic shock sometimes occurs with this drug. A 1997 report from the Center for Drug Evaluation and Research, a division of the United States Food and Drug Administration, stated that the toxicity of Amphotericin B was 1.5 mg/Kg in rats. Other reports reach similar conclusions (4)(5)(6)(7)(8).

Since its discovery, numerous researchers have attempted to modify the properties of Amphotericin B to make it more water-soluble, less toxic, or both. PEGylation is one obvious strategy for improving the water solubility of a water-insoluble drug. Greenwald, et al. (9), and Sedlak, et al. (10)(11) created PEGylated prodrug versions of the drug, but these constructs used large, dispersed, polymeric PEGs that are heterogeneous in size and, hence, difficult to characterize.

 

A Different Approach to Water-Soluble Amphotericin B

In a paper published in January 2016 in the Journal of Medicinal Chemistry, Assaf Halperin, Yana Shadkchan, Evgeni Pisarevsky, Alex Szpilman, Hani Sandovsky, Nir Osherov, and Itai Benhar, from Tel-Aviv University and the Israel Institute of Technology chose to work with short PEG chains of defined length and molecular weight. Specifically, they chose to create a stable amide bond on the mycosamine ring and expose a free amine at the opposite end of the linker. After testing PEG linkers with 4, 8, and 44 ethylene oxide units, they settled on the PEG8 linker, because it offered significant improvement in water solubility with little loss in efficacy. By contrast, the PEG4 linker improved water solubility, but less well than the PEG8 and PEG44 linkers. The PEG44 linker improved water solubility greatly, but the derivative's efficacy dropped thirty-fold compared to the parent drug.

 

Synthetic scheme used by Halperin, et al., to create the two PEGylated derivatives of Amphotericin B. Image used with permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.
Figure 2: Synthetic scheme used by Halperin, et al., to create the two PEGylated derivatives of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

For the construction of two different PEGylated derivatives, the research group used Quanta BioDesign's Fmoc-N-amido-dPEG®8-NHS ester, product number 10995, purchased from our German distributor, Iris Biotech, GmbH. (Click here for a complete list of our distributors.) The authors stated the reason for their choice in this way:

This approach creates a well-defined medium-molecular-weight product that can be easily separated and characterized by NMR and other common techniques .... The short and specific synthesis and well-defined structural nature of the antifungal conjugate is expected to be an advantage in clinical approval as well. (4, page 1201).

After the dPEG® was conjugated to Amphotericin B, the Fmoc protecting group was removed, exposing the free amine on the dPEG® linker. The synthetic scheme used by the authors is shown in Figure 2.

 

Amphotericin B-dPEG®8 Derivatives Show Promise as Clinical Replacements for the Parent Compound

In testing, both dPEG®ylated derivatives (AB1, the free acid; AM2, the methyl ester; see Figure 3) increased dramatically in water solubility, decreased considerably in toxicity, and maintained (with only a moderate decrease) efficacy.

The solubility of Amphotericin B in water is less than 1 µg/mL (<0.001 mg/mL). The AM2 derivative's water solubility increased to 700 µg/mL (0.7 mg/mL), while AB1 increased to 5,500 µg/mL (5.5 mg/mL). Previous efforts to make useful PEGylated derivatives of Amphotericin B used carbamate linkages between the drug and the PEG. These linkages hydrolyzed readily in phosphate-buffered saline (PBS). By contrast, neither AB1 nor AM2 showed any hydrolysis in PBS after 4 hours, and both hydrolyzed less than 1% after 24 hours in PBS, demonstrating that they are stable. Furthermore, pharmacokinetic studies of AM2 in mice showed no free Amphotericin B after 24 hours, indicating that the derivatives are stable.

Structures of the dPEG®8-amine derivatives of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.
Figure 3: Structures of the dPEG®8-amine derivatives of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

The authors tested the toxicity of AB1 and AM2 against the parent drug in three ways. First, they evaluated nonspecific toxicity of all three compounds by suspending fresh human red blood cells (hRBCs) in PBS and measuring lysis of the cells (hemolysis) in the presence of Amphotericin B or the derivatives. Second, they assayed the viability of mouse embryonic fibroblast cells using an MTT assay (for a detailed, technical discussion of the MTT assay plus procedures for conducting the assay, click here). Third, they measured the LD50 of the three compounds by intravenous injection in mice.

In the first test, Amphotericin B was highly toxic as expected. AB1 was less toxic to hRBCs than the parent compound, while AM2 caused very little hemolysis and thus demonstrated little non-specific toxicity.

MTT Assay comparing Amphotericin B to the AB1 and AM2 derivatives.
Figure 4: MTT Assay comparing Amphotericin B to the AB1 and AM2 derivatives. A color version of this image is at the beginning of this post. AM1 (should be AB1) and AM2 are the derivatives shown in Figure 3. AMB is Amphotericin B (see Figure 1 for structure). Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

The MTT assay (see Figure 4) showed dramatic differences between Amphotericin B and the derivatives. The MTT assay measures the half-maximal inhibitory concentration, also known as the IC50 (see also here), of a compound against cells in culture. In the assay, AB1 was forty times less toxic to the mouse embryonic fibroblasts than Amphotericin B, while AM2 was at least 600 times less toxic.

The in vivo toxicity testing showed dramatically different results among Amphotericin B, AB1, and AM2. The LD50 of Amphotericin B was 1.1 mg/Kg. Mice injected with twice the LD50 died instantly. By contrast, AB1 was not toxic to mice at the maximum injected dose of 22 mg/Kg, and AM2 was not toxic to mice at the maximum injected dose of 42 mg/Kg.

Efficacy (a measure of the potency of antifungal activity) was assessed both in vitro and in vivo. Both AB1 and AM2 retained activity against a broad spectrum of fungi but were 2-16 times less potent than Amphotericin B. When the length of time needed to kill all fungal cells was measured, AM2 eliminated all fungi in vitro within 2 hours, similar to Amphotericin B. In in vivo testing, Amphotericin B at 1 mg/Kg (near the LD50) cured 10 of 12 mice of fungal infection, compared to AM2, which achieved the same result using 3.5 mg/Kg. AM2, though, cured all mice of fungal infections at a dose of 7 mg/Kg, something that non-PEGylated parent drug could not do, because it was too toxic.

Interestingly, AM2 had a shorter serum half-life than Amphotericin B. This result seems odd at first glance because PEGylation is one of the primary means by which the serum half-life of therapeutic agents is extended. The reason for the difference is that Amphotericin B binds serum proteins, which lengthens the drug's serum half-life. Testing revealed that AM2 does not bind strongly to serum proteins. This allows it to be removed much more quickly from the body.

AB1 and AM2 both show promise as clinical antifungal agents. The increased water solubility and lower toxicity compared to Amphotericin B are important, but it is also important that the efficacy of the derivatives is not greatly diminished from the parent compound. The discrete PEGylation (dPEG®) reagent simplifies analysis of the products, and the specific dPEG® reagent chosen, PN10995, is ideal for conjugating the Amphotericin B derivatives to other molecules after removal of the Fmoc group protecting the terminal amine.

This work also highlights the remarkable power of a relatively small dPEG® linker. Many research groups tend to think that large, dispersed PEGs are the solution to their water solubility problems. This research shows that a dPEG®8 linker imparts significant water solubility and reduces toxicity, with only a modest impact on efficacy. This is truly important for understanding the power and ability of discrete PEGylation.

 

References

  1. Denning, D. W.; Bromley, M. J. Infectious disease: how to bolster the antifungal pipeline. Science (27 March 2015), 347(6229). 1414-1416.
  1. Dutcher, James D. The discovery and development of Amphotericin B. Dis. Chest (October 1968), 54(supplement 1), 40-42.
  1. Gallis, Harry A.; Drew, Richard H.; and Pickard, William W. Amphotericin B: 30 years of clinical experience. Clin. Infect. Dis. (1990), 12(2), 308-329.
  1. Halperin, Assaf; Shadkchan, Yana; Pisarevsky, Evgeni; Szpilman, Alex M.; Sandovsky, Hani; Osherov, Nir; and Benhar, Itai. Novel water-soluble Amphotericin B-PEG conjugates with low toxicity and potent in vivo efficacy. J. Med. Chem. (2016), 59, 1197-1206.
  1. Larabi, M.; Pages, N.; Pons, F.; Appel, M.; Gulik, A.; Schlatter, J.; Bouvet, S.; and Barrat, G. Study of the toxicity of a new lipid complex formulation of amphotericin B. J. Antimicrob. Chemother. (2004), 53, 81-88.
  1. Souza, L. C.; Campa, A. Pharmacological parameters of intravenously administered amphotericin B in rats: comparison of the conventional formulation with amphotericin B associated with a triglyceride-rich emulsion. J. Antimicrob. Chemother. (July 1999) 44(1): 77-84.
  1. Hamill, Richard J. Amphotericin B Formulations: A Comparative Review of Efficacy and Toxicity. Drugs (2013), 73: 919-934
  1. Laniado-Laborín, Rafael; and Cabrales-Vargas, Maria Noemí. Amphotericin B: side effects and toxicity. Rev Iberoam Micol. (2009) 26(4):223–227.

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.

 

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PN10994, Fmoc-N-amido-dPEG®4-NHS ester

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

PN11000, Fmoc-N-amido-dPEG®4-TFP ester

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PN10053, Fmoc-N-amido-dPEG®5-acid

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PN10273, Fmoc-N-amido-dPEG®8-acid

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Chart showing the analgesic activities of various galanin analogues used in a formalin pain assay.

PEGylated Galanin Shows Enhanced Analgesic Effects in PNS

Galanin is a naturally occurring neuropeptide in the human body that facilitates communication between cells to balance a myriad of physiological functions. Neuropeptides are biosynthesized molecules used by the human body for everything from neurogenesis to cell communication.  Galanin’s main receptor sites reside in the central nervous system (CNS), and it normally crosses the blood brain barrier; however, the peripheral nervous system (PNS) also reacts directly to galanin and its receptors in sites of pain mediation.1 Galanin’s role as a pain inhibitor is a peripheral one, which means that, as a drug, its analgesic role would be secondary.  Peripheral effects of peptides are overshadowed by their effectiveness in carrying out primary functions and limiting concentration in low receptor site areas. Peripheral roles of neuropeptides can play just as important of a role as the primary roles in regulating changes in the nervous system.

 

Structure of the galanin neuropeptide found in humans. The peptide sequence is GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS
Figure 1: Structure of the galanin neuropeptide found in humans. The peptide sequence is GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS

 

Common methods of altering drug permeability include lipidization, counterionization, poly-disperse PEGylation, carrier protein conjugation, and many more. The modification is a means to justify an end, in this case the efficacy of the drug. A research team from the University of Utah’s College of Pharmacology and Toxicology and NeuroAdjuvants, Inc., in Salt Lake City studied PEGylation of galanin in response to their research done on other methods of alteration to the neuropeptide. Results of previous research by the group showed increased penetration across the blood brain barrier by galanin. The analgesic properties of galanin are restricted when allowed to permeate unrestricted from blood to brain. The group hypothesized that “dPEGylation will restrict penetration of the neuropeptide analogues into the brain, while their activities in the PNS would be retained”2.

 

Figure 2: Octanol/Water Partition Coefficients (logD) of Galanin Analogues. Y axis: LogD=Log([peptide in octanol)/(peptide in water)]
Figure 2: Octanol/Water Partition Coefficients (logD) of Galanin Analogues. Y axis: LogD=Log([peptide in octanol)/(peptide in water)]
 

Figure 2 shows data for the availability of an altered galanin (Gal-B2) with a discrete PEG in solution between octane and water2. The data shows that the PEGylated Gal-B2 favors the aqueous phase instead, which hypothetically limits its permeability across the blood brain barrier.
The testing of the peptides was carried out by studying the effects both in vitro and in vivo testing methods. The in vitro method included testing the effectiveness of the peptide to bind to its receptor by measuring fluorescence in a competitive binding assay. After PEGylation the neuropeptides retained nanomolar affinities for their receptor sites2. The in vivo testing included formalin injections into the dermal layer of mice paws to trigger a pain response. The mouse’s response to the pain, quantified by licking the wound, was compared across a series of unmodified and modified peptides and a control.

 

Figure 3: Chart showing the analgesic activities of various galanin analogues used in a formalin pain assay.
Figure 3: Analgesic activities of various galanin analogues used in a formalin pain assay.

 

Figure 4: Table showing the anticonvulsant activity of various galanin analogues.
Figure 4: Anticonvulsant activity of various galanin analogues.

Figures 3 and 4 show some surprising results relative to the group's hypothesis. The PEGylated peptide retained similar analgesic activity as the unmodified peptide for acute pain (Figure 3, phase I), and was better than the unmodified peptide in controlling inflammatory pain (Figure 3, phase II). Figure 4 shows that the PEGylated peptides (Gal-B2-dPEG24 and Gal-R2-dPEG24) had reduced anticonvulsant protective effects when administered to mice via intracerebroventricular (icv) injections and had no anticonvulsant protective effects when administered via intraperitoneal (ip) injections. This is most clearly shown by comparing the area under the curve (AUC) of the unmodified Gal-B2 against the AUC of each modified peptide.

This research is significant in that it shows that PEGylation of a biologically active peptide can change both the biodistribution and bioactivity profile of the peptide. This gives promise for “further exploration of monodisperse oligoethyleneglycol residues toward more neuroactive peptides that mediate their actions in the central and peripheral nervous systems”2. As research into this field continues, access to high quality, diverse PEGylation reagents will be a cornerstone.

Our Role in This Research

In this research the role of discrete PEG was important, because polydisperse PEGylation forms a diverse array of peptides3. Quanta BioDesign's patented discrete PEG (dPEG®) technology produces PEGylation reagents that are single molecules, not an intractable mixture of different PEG linker lengths each with a different molecular weight. Using our m-dPEG®24-acid reagent to modify Gal-B2 through a lysine side chain resulted in specific single molecules (Gal-B2-dPEG24 and Gal-R2-dPEG24) that could be fully characterized.

An amide bond between the dPEG® and the peptide was the end goal and we offer myriad products with free acids, active esters, and amines to help create that bond. To create other types of linkages, we offer products with different functional groups, including hydrazides (to form hydrazone bonds), oxyamines (oxime bonds), aldehydes (secondary amines, oximes), thiols (disulfides), and many more. In addition to our linear dPEG® products, Quanta Biodesign also offers dPEG® reagents with multiple arms to better modify your small molecule, peptide, or protein without the cost of extra reaction sites.

 

References on Galanin and PEGylation:

1. Lang, R.; Gundlach, A. L.; Kofler, B. The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease. Pharmacol. Ther. 2007, 115, 177−207.

2. Zhang L, Klein BD, Metcalf CS, Smith MD, McDougle DR, Lee HK, White HS, Bulaj G. Incorporation of monodisperse oligoethyleneglycol amino acids into anticonvulsant analogues of galanin and neuropeptide Y provides peripherally acting analgesics. Mol. Pharm. (2013), 10(2): 574-85

3. Gaberc-Porekar, V.; Zore, I.; Podobnik, B.; Menart, V. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr. Opin. Drug Discovery Dev. 2008, 11, 242−50.

 

Example Linear, Amine-Reactive dPEG® Products (also available as NHS esters and 2,3,5,6-tetrafluorophenyl esters)

m-dPEG®2-acid

m-dPEG®4-acid

m-dPEG®12-acid

m-dPEG®24-acid (used in this study to modify galanin)

m-dPEG®37-acid

m-dPEG®48-CO(CH2)3-acid

 

Example Branched Amine-Reactive dPEG® Products (also available as NHS esters and 2,3,5,6-tetrafluorophenyl esters)

Carboxyl-dPEG®₄-(m-dPEG®₄)₃

Carboxyl-dPEG®₄ -(m-dPEG®₈)₃

Carboxyl-dPEG®₄-(m-dPEG®₁₁)₃

 

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dPEG®-Modified Diabodies Improve Tumor Imaging

Researchers in Australia and the United States have shown that dPEG®-modified diabodies improve positron emission tomography (PET) imaging of tumors by reducing kidney uptake of diabody and extending diabody half-life in the bloodstream, which thus allows more diabody to be taken up by the tumor.1 These findings suggest that better tumor imaging can be achieved using less material, because more of the diabody that targets the tumor gets to the tumor and less of it is excreted by the kidney.

Tumor Imaging and Diabodies

Tumor imaging in mammals requires matching blood clearance and tumor uptake with the half-life of the engineered antibody. Whole antibodies with molecular weights greater than 70,000 Daltons (abbreviated 70KDa) are blocked by the glomerular filtration barrier. Smaller antibodies have lower molecular weights; thus, they pass through the barrier into the kidney and are excreted. Whole antibodies, though, can be much more immunogenic than smaller antibody fragments, and the Fc region of a whole antibody can sometimes cause mis-targeting of antibodies. For this reason, antibody fragments are preferred over whole antibodies for applications such as tumor imaging.

Dimeric antibody fragments, known as "diabodies," are designed single-chain Fv (scFv) fragments consisting  of a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) by a peptide linker that is too short to permit the VH and VL fragments to associate with each other, thus forcing them to associate with complementary domains of another chain, creating two antigen binding sites. See the original paper here. For more information, see here (note: some of the information at this link is behind a paywall). Diabodies can be designed to be bivalent and bispecific. For reviews on bispecific antibodies clichere and here.

In this paper,1 diabodies against tumor-associated glycoprotein 72 (TAG-72) were used. TAG-72 is a mucin-like glycoprotein that is found on the surfaces of breast, ovarian, colon, and pancreatic cancer cells. The anti-TAG-72 diabodies that were studied clear rapidly from blood (t1/2=30-60 minutes), and they have good uptake by tumors expressing TAG-72, due in part to the bivalent nature of the diabodies. Because these diabodies have a molecular weight of about 50 KDa, they are also rapidly cleared (t1/2=12 hours) from the body through kidney uptake of the diabody, which is problematic for diagnostic imaging and therapeutic applications. If the diabodies could be kept from passing through the glomerular filtration barrier, they would stay in the bloodstream longer, thus giving them more time to find and bind tumor-associated TAG-72. (Please click here for more information on glomerular filtration.)

One way to keep smaller molecules such as diabodies from passing through the glomerular filtration barrier is to increase the hydrodynamic volume of the molecule and, hence, increase the apparent molecular weight of the molecule. Molecules with molecular weights greater than 70KDa do not pass easily, if at all, through the barrier. PEGylation increases the hydrodynamic volume of peptides and proteins. (Click here for more information.) PEG molecules are amphiphilic, and in aqueous solution, PEG will hydrogen bond extensively with water, increasing the hydrodynamic volume and causing the molecule to which the PEG is attached to behave as if it is a molecule with a much higher molecular weight. This makes the PEGylated molecule much more difficult to remove from the body by glomerular filtration

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Diabodies Have Improved Biodistribution When dPEG®ylated

Research by Li Lin et. al., affiliated with Beckman Research Institute of City of Hope, Duarte, California and Avipep Pty Ltd. of Parkville Victoria, Australia shows that conjugating diabodies to discrete polyethylene glycol (dPEG®) derivatives increases the apparent molecular weight of the diabody disproportionately to the size of the dPEG® derivative, thus reducing kidney uptake of the diabody, without adversely affecting the tumor-to-blood (T/B) ratio of diabody. Starting with Quanta BioDesign's Fmoc-NH-dPEG®n-COOH derivatives, Lin and coworkers synthesized DOTA-NH-PEGn-Cys-VS derivatives, where n=0, 12, 24, or 48 ethylene oxide repeats in the dPEG® linker (VS stands for vinyl sulfone). The synthetic scheme is shown in Figure 1.

 

Figure 1: Synthetic Scheme for the dPEG® DOTA derivatives used to modify diabodies.

Figure 1: Synthetic Scheme for the dPEG® DOTA derivatives used to modify diabodies.

 

These compounds were then site-specifically conjugated to an anti-TAG-72 diabody that had been engineered to have a surface disulfide moiety that could be reduced to form a free thiol for conjugation. Using 111In labeling of the DOTA-PEG constructs, the biodistribution of the derivatived diabodies was mapped in athymic mice. The results (see Figure 2, below) showed that as the PEG linker increased in length the rate of kidney uptake and blood clearance of the diabody decreased, while at the same time the amount of labeled diabody on the tumor increased. With the n=0 construct conjugated to the anti-TAG-72 diabody, the apparent molecular weight of the diabody was about 50KDa, and the blood clearance half-life was 30 minutes. With increasing PEG linker length, the apparent molecular weight of the diabodies (as determined by size exclusion chromatography) increased to  60kDa (n=12), 70kDa (n=24), and 80kDa (n=48). For the largest (n=48) derivative, the blood clearance half-life had increased to 6 hours. This increase in apparent molecular weight was much larger than the weight of the linker added to the diabody.

 

Biodistribution over time of DOTA-PEGn-AVP04-50 diabody conjugates in mice bearing LS174-T xeonografts.
Figure 2: Biodistribution over time of DOTA-PEGn-AVP04-50 diabody conjugates in mice bearing LS174-T xeonografts. Yellow=kidney; dark blue=tumor; red=blood; green=liver.

 

Labeling the DOTA-NH-PEGn-Cys-avibody derivatives with 64Cu (half life of 12 hours), the researchers used positron emission tomography (PET) to image tumors expressing TAG-72 antigen in athymic mice at different time points. The results are shown in Figure 3.

 

Tumor Imaging using PET with 64Cu-labeled DOTA-NH-PEGn-AVP04-50
Figure 3: PET imaging of LS174-T xenografts in athymic mice with 64Cu-labeled DOTA-NH-PEGn-AVP04-50. Red=blood; green=liver; yellow=kidney; turquoise=tumor. Pow A shows the PEG0 construct. Row B is the PEG12 construct. Row C is the PEG24 construct. Row D is the PEG48 construct.

 

Figure 2 shows that as the dPEG® linker length grows from 0 to 48 tumor uptake increases by approximately 20%, while the kidney uptake drops by about 90%. Figure 3 provides visual imaging of tumors within the test animals using 64Cu-labeled DOTA. It is clear that as the dPEG® increases, the kidney uptake decreases, and the tumor uptake increases.  The authors note, however, that the T/B ratios of the n=48 conjugate vary and are not ideal for imaging. One way to possibly avoid this problem is to have more than one DOTA on the dPEG® itself. Figure 4 shows one of our novel side-arm loaded dPEG® constructs using a tyrosine core. This could prove useful for conjugating more than one DOTA and one diabody onto the dPEG®.

 

PN11567; MAL-(NH-dPEG4-Tyr(CH2-CO2H))3-NH-m-dPEG24
Figure 4: PN11567; MAL-(NH-dPEG®4-Tyr(CH2-CO2H))3-NH-m-dPEG®24

 

One of the interesting findings in this paper is that the authors of this paper were able to achieve a large effect (an increase of hydrodynamic volume equivalent to a 30KDa increase in mass) with a relatively small PEG linker (48 ethylene oxide units is about 2.1KDa). Moreover, the linker used is a discrete molecule, not a dispersed polymer with mixture of sizes, nor a monodispersed molecule that was purified out of a dispersed mixture. With Quanta BioDesign's technology, we synthesize discrete PEG molecules starting with very high purity materials and build the linker in progressive steps. A dPEG® provides increased water solubility, greater hydrodynamic volume, decreased immunogenicity, ease of characterization (because it is a single molecule, not a mixture), prevention of aggregation, longer circulation time in the body, and so forth. The disadvantages of polymer PEGs, including difficulty characterizing the final product from a dispersed mixture of sizes and tremendous loss of activity when very large PEGs are attached to small molecules, proteins, or peptides, are not found in dPEG® products.

Quanta BioDesign invented dPEG® technology, and with it, we are reinventing PEGylation. We are always creating new dPEG® constructs. In addition, if you look through our catalog and do not see what you want, we can synthesize the dPEG® construct specific to your needs. You can also call or E-mail us with any questions that you may have about our products!

 

References

1 Li L, Crow D, et al. Site-specific Conjugation of Monodispersed DOTA-PEGn to a Thiolated Diabody Reveals the Effect of Increasing PEG size on Kidney Clearance and Tumor Uptake with Improved 64-copper PET Imaging. Bioconjugate Chemistry (2011), 4, 709–716. doi: 10.1021/bc100464e.

2 Virginia G. Johnson, et al. Analysis of a Human Tumor-associated Glycoprotein (TAG-72) Identified by Monoclonal Antibody B72.3. Cancer Research (1986), 46, 850-857.

3 Donald G. Sheer, Jeffrey Schlom, and Herbert L. Cooper.  Purification and Composition of the Human Tumor-associated Glycoprotein (TAG-72) Defined by Monoclonal Antibodies CC49 and B72.3. Cancer Research (1988), 48. 6811-6818.

 

About the Author

Ian Hotham, B.S., received his Bachelor of Science degree in Chemistry from The Pennsylvania State University in Spring of 2013. Ian is a Process Development Chemist involved in synthesizing new dPEG® and in improving and scaling up synthetic processes. You can connect with Ian on LinkedIn at www.linkedin.com/pub/ian-hotham/51/b28/a08

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A dPEG®ylated Avibody provides an example of the discrete nature of Quanta BioDesign's dPEG® reagents..

Noise control and removal through dPEG®ylation

Noise control and removal is important in any discipline where clean, clear signals are critical in measurement and data collection. For this reason, whilst the saying 'Beauty is in the eyes of the beholder' may be true in many instances, scientific research cannot permit subjective, qualitative thinking to trump objective data.

Quality data are the basis of all good research. Poor data give an erroneous understanding of the problem and the solution. For data to be useful, that is, for data to be trustworthy as to make sound business or economic decisions (or any significant decision for that matter), it must meet certain criteria of reproducibility, accuracy, validity and reliability.

However, it takes a village to raise a child (as the saying goes), and similarly it takes more than good instruments to get quality data. The accuracy does not come from the data itself, but in the instrumentation, methods and materials used to generate it. This is where the “d” in dPEG® becomes critical in bioconjugation or any other application that can benefit from PEGylation.

 

The dPEG® Difference

Discrete PEGs are single molecular weight compounds that are made step-wise like small molecules. Whilst they share many of the advantages of polymeric PEGs such as conferring stability, immunogenicity (1), improved solubility, extended serum half-lives (2) and improved pharmacokinetics among others. Quanta BioDesign, Ltd. dPEG®s are like driving an all-wheel drive vehicle equipped with snow tires on a bad snow day. Subaru says of its All-Wheel Drive system, "Confidence in Motion." At Quanta BioDesign we say, "Confidence in Science." There is confidence because of the control that dPEG®s give.

The discrete nature of our PEGs makes site specific conjugation very reliable, and make characterization and quantitation easier. Because the exact size of the dPEG® is known, characterization is made simpler, and the researcher can tell what fragment is responsible for the results they are seeing.

 

Noise Control Advantages with dPEG® Reagents

Over the years there have been great improvements in analytical instrumentation that have removed much noise in measurement. However, there is still noise that cannot be removed by instruments no matter how good it is. So, the options for the researcher are to either wear “big ear muffs” to cut out the noise or to engineer the noise out with a "good muffler." At Quanta BioDesign, we have opted for the latter solution because we understand the problem, seeing that the bigger polymeric mixtures become cumbersome to the instruments and cloud the data.

As an example, Figure 1, below, shows an example of how noise control can be obtained with Quanta BioDesign's dPEG® reagents. The figure shows the mass spectrum of an Avibody (Avipep Pty Ltd.) PEGylated specifically and cleanly with one of our dPEG® reagents.

 

A dPEG®ylated Avibody provides an example of the discrete nature of Quanta BioDesign's dPEG® reagents..
Figure 1: An example of noise control: a dPEG®ylated Avibody using Quanta BioDesign's dPEG® reagents.

By contrast, Figure 2, below, shows the MALDI-TOF mass spectrum of polydispersed PEG-1500. Noise control is impossible using this PEG! Yet this mass spectrum is typical of many PEGylated products that use the older, polydispersed PEG technology.

 

MALDI-TOF Mass Spectrum of PEG-1500. Noise control is not possible with a polydispersed PEG. Source: http://www.rzuser.uni-heidelberg.de/~bl5/ency/maldi2.html
Figure 2: MALDI-TOF Mass Spectrum of PEG-1500. Noise control is not possible with a polydispersed PEG. Source: http://www.rzuser.uni-heidelberg.de/~bl5/ency/maldi2.html

This noise control and removal also has many other advantages in validating processes and setting specifications. From a regulatory standpoint of drug development and marketing noise reduction improves the chance of success and mitigate against many legal liabilities that can arise from unstable products and data collection systems.

Here at Quanta BioDesign we do not simply make and sell dPEG®s. We also give confidence in results. Part of that confidence comes from the ability of our dPEG® reagents to engineer noise control and noise removal into our customers' products.

We believe that we are writing a brief history of the future of science and are confident that the early birds to dPEG® technology shall surely catch the worm.

For products and ordering visit us at www.quantabiodesign.com

 

 References:

1) A. Abuchowski, T. van Es, N. C. Palczuk, F. F. Davis, J. Biol.Chem. 1977, 252, 3578. http://www.ncbi.nlm.nih.gov/pubmed/16907

2) P. Caliceti, F. M. Veronese, Adv. Drug Delivery Rev. 2003, 55,1261. http://www.ncbi.nlm.nih.gov/pubmed/14499706

 

Quanta BioDesign dPEG® Reagents

Click here to see our peptide modification reagents

Click here to see our chemical modification reagents, including our branched serum half-life modifiers.

Click here to see our chemical crosslinking reagents.

Click here to see our biotinylation reagents.

 

About the author

James Guyo, MS, MBA earned his Bachelor of Science degree in Chemistry and Biochemistry from the University of Zimbabwe in 1996. Coming to the United States, James then earned his Master of Business Administration degree in 2002 and his Master of Science degree in Chemistry in 2004, both from Wright State University. As Director of Chemistry Operations he is involved in process development and optimization, research and development and scale-up activities among other things. He also has extensive experience doing contract cGMP. You can contact James at Quanta BioDesign, Ltd. or connect with him on LinkedIn.

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