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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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Label monoclonal antibodies site specifically with ETAC reagents

ETAC and labeling monoclonal antibodies

Monoclonal antibodies and their small fragments (Fabs, scFv, diabodies etc.) are intriguing objects for creation of protein-based medicines. These proteins can be site-specifically modified with ETAC-dPEG® ("ETAC" abbreviates "Equilibrium Transfer Alkylation Cross-link"; "dPEG®" is the registered trade name for "discrete Poly(Ethylene Glycol)") reagents. Using ETAC, a three-carbon bridge is formed linking the two cysteine sulfur atoms. The dPEG® attached to the ETAC reduces the protein's immunogenicity and prevents rapid clearance of the protein from the bloodstream. This, in turn, helps to maintain a desired therapeutic concentration between doses, thereby reducing the risk of loss of efficacy. The structure of ETAC-reagent and generation of the dPEG®-monosulfone which undergoes a site-specific conjugation with a Fab are outlined below in Figure 1. For details, see, for example, "Comparative binding of disulfide-bridged PEG-Fabs", Bioconjugate Chemistry (2012), 23, 2262-2277; and "Disulfide bridge based PEGylation of proteins", Advances in Drug Delivery Reviews (2008), 60, 3-12.

 

Monoclonal antibodies can be site specifically labeled using the ETAC-dPEG® reagent shown here, after generation of the monosulfone as depicted in the scheme.
Figure 1: Structure of the ETAC-dPEG® reagent and generation of the monosulfone to label monoclonal antibodies site specifically.

 

An accessible disulfide bond can be selectively reduced under mild conditions with DTT or TCEP (Quanta BioDesign product number PN10014) without destroying the tertiary structure of the monoclonal antibody or antibody fragment. Once an accessible disulfide is reduced, the two free cysteine sulfur atoms become available for reaction with the ETAC-reagent (See "Disulfide bridge based PEGylation of proteins"). The PEG-bis-sulfone eliminates the sulfinic anion and generates PEG-monosulfone which then undergoes conjugation, particularly with Fabs. Conjugation occurs by the formation of a three-carbon bridge linking the two cysteine sulfur atoms with PEG attached through the middle carbon of the bridge. Conjugation of PEG at this site normally has minimal impact on the binding properties of the monoclonal antibody or Fab. Figure 2, below, shows a generalized scheme for this process.

General scheme showing how monoclonal antibodies or other disulfide-containing proteins can be site-specifically labeled using an ETAC-dPEG® reagent.
Figure 2: General scheme showing how monoclonal antibodies or other disulfide-containing proteins can be site-specifically labeled using an ETAC-dPEG® reagent.

 

Potential problems with PEGylated monoclonal antibodies as drugs

Having high molecular weight dPEG® in solution can stabilize the native and compact structure of human albumin, does not obscure the protein’s active surface, and folds independently of the protein. The majority of clinically used PEGylated medicines are heterogeneous mixtures that have been produced by non-specific and inefficient PEG-conjugation reaction to different nucleophilic sites on the protein ( See "Comparative binding of disulfide-bridged PEG-Fabs").

Several monoclonal antibodies marketed as drugs use maleimide to conjugate PEG to thiol. Examples of PEGylated monoclonal antibodies manufactured this way include the drug certolizumab pegol (tradename Cimzia) and the now-withdrawn drug peginesatide, a PEGylated peptide.  Although, thiol conjugation is quite efficient, the addition of unpaired cysteine can result in disulfide scrambling and protein aggregation. In addition, there are data that maleimide derived reagents are labile to hydrolysis and can undergo exchange reactions in vivo. See Reversible maleimide-thiol adducts yield glutathione-sensitive poly(ethylene glycol)-heparin hydrogels. Polym Chem (2013), 4, 133-143; and Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjugate Chem (2011), 22(10), 1946-1953Therefore, development of site-specific approaches and novel reagents that address low conjugation efficiency and PEG-conjugate stability is important, especially in PEGylated monoclonal antibodies and Fab fragments that have potential to be used as therapeutic agents.

 

ETAC-dPEG® reagents offer advantages over traditional PEGylation reagents

Our novel heterobifunctional ETAC-dPEG® (linear or branched) reagents with active TFP- or NHS-groups (for example, ETAC-dPEG®24-NHS ester, PN11685, Figure 3, and ETAC-dPEG®36-TFP ester, PN11686, Figure 4) can be used for preparation of homogeneous, highly potent antibody drug conjugates. It allows to combine the unique targeting capabilities of monoclonal antibodies with therapeutic or diagnostic payload (cytotoxic drug, toxin), dPEG®- and cleavable or noncleavable linker. As a result, the targeted cell (e.g. cancerous) can be damaged either by the released cytotoxic drug, or by the complex of degraded antibody, linker, and drug.

 

Figure 3: PN11685, ETAC-dPEG®-NHS ester, can be used to label monoclonal antibodies or other proteins site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 3: PN11685, ETAC-dPEG®24-NHS ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies or other proteins site specifically. For more information, please contact Quanta BioDesign, Ltd.

Figure 4: PN11686, ETAC-dPEG®36-TFP ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 4: PN11686, ETAC-dPEG®36-TFP ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies site specifically. For more information, please contact Quanta BioDesign, Ltd.

 

It is important to note that a dPEG® is a single molecular species. (See "What is dPEG®?") Traditional PEGylation reagents are dispersed polymer mixtures. Working with a traditional, polydispersed PEG makes complete characterization of the final product (whether small molecules, peptides, monoclonal antibodies, or other proteins) difficult, because the complex heterogeneity of the polydispersed PEG makes it intractable to analysis.

 

PEGylation reagents for all applications

More than just ETAC reagents, Quanta BioDesign, Ltd., offers PEGylation reagents for almost every need in bioconjugation, therapeutics, diagnostics, theranostics, nanotechnology, bionanotechnology, and many other fields. Our functional groups are diverse, and our chemistry allows us to make a broad range of dPEG® linkers from 1-49 ethylene glycol units as single molecular entities. Look through our catalog today! If you cannot find the linker chemistry you want for your specific application, please feel free to call us and ask. We offer custom synthesis of dPEG® reagents to our customers. We will be happy to speak with you and discuss your needs.  Just call us today!

 

Links to additional PEGylation reagents of interest

TCEP - an effective reagent for reducing disulfides to thiols.

Biotinylation Reagents - biotinylate almost anything with one of our Biotin-dPEG® reagents.

Crosslinking reagents with a variety of chemistry options, including thiol-reactive maleimide and SPDP.

Fluorescent and dye labels - label monoclonal antibodies or other proteins.

 

Victor D. Sorokin, Ph.D. – Received his Ph.D. in Organic Chemistry from the Moscow State University in Russia and completed his postdoctoral training at the University of Texas (1993-1996) where he started his industrial career. He has recently joined our company as a Sr. Product Development Scientist. He has over 15 years of synthetic organic chemistry experience with chemical and pharmaceutical companies developing the synthesis of complex molecules (including natural chiral compounds) on mg/g scale, allowing scale-up to multi-kg quantities. For the last 5 years he has been working on the synthesis of various nucleosides (both DNA, RNA) and oligonucleotides in solution phase using H-phosphonate and phosphoramidate chemistry approach. You can contact Victor on LinkedIn at www.linkedin.com/pub/victor-sorokin/11/417/34b.

 

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