NetForum uses cookies to ensure that we give you the best experience on our website. If you continue to use the site, we'll assume that you are happy to receive these cookies on the NetForum website. Read about our cookies.
NetForum Community
Learn. Share. Optimize.
Log in | Sign up now | Submit content | Contact
Go to similar content

In vivo chemistry for pre-targeted tumor imaging and therapy in mice

White Paper
Philips NM Marketing Philips Healthcare USA

M.S. Robillard, R. Rossin
Department of Biomolecular Engineering, Philips Research, Eindhoven, the Netherlands.

Note: This contribution is partly based on an article previously published in Angewandte Chemie Internationali.

Monoclonal antibodies (mAbs) have long been recognized as effective tumor-targeting vehicles. Targeted delivery of radionuclides to tumor-associated antigens for imaging and therapy of cancer is an active research area in the preclinical as well as the clinical domainii. The U.S. Food and Drug Administration's (FDA) approval at the beginning of this century of the first two radio-labeled monoclonal antibodies (Zevalin® and Bexxar®) for the treatment of non-Hodgkin's lymphoma patients is a landmark event in the history of radioimmunotherapy (RIT, Figures 1 and 2). Targeted radiopharmaceuticals are well-suited for the therapy of this disease, as the tumors are very accessible to antibodies, and the lymphocytes are very sensitive to radiation.
 
The absorbed dose in the tumor vs. that in normal tissue is an important predictor for therapeutic success and the occurrence of side effects in RIT. In contrast to lymphohematopoietic cancers, solid tumors such as colorectal cancer have been much less responsive to RIT. Solid tumors show a very low uptake (0.001-0.01% injected dose per gram tumor) of the targeting antibody, resulting in a cumulative tumor dose of 1500 cGy, while typically 5000 cGy is needed to achieve therapeutic responses in most cancers. When the tumor uptake is limited, the dose needed to achieve a therapeutic response cannot be administered because of the need to restrict the dose to other, normal tissue. For example, the slow plasma clearance properties of radio-labeled antibodies result in prolonged exposure of the highly radio-sensitive bone marrow to ionizing radiation.

Despite the high clinical need and large potential markets, solid tumors have remained out of reach of RIT. This challenge has led to a number of approaches to improving the tumor-tonontumor radiation dose ratios to a level that will allow effective tackling of the tumor while sparing normal tissues.

The tumor-to-nontumor dose ratio can be boosted by a two-step targeting approach known as pretargeting (see Figure 1), which essentially separates the delivery of the radioactive isotope from that of the antibodyiii. In this approach, a non-radio-labeled antibody modified with a tag is injected and allowed to reach maximum tumor uptake and sufficient clearance from blood. The clearance from blood may be accelerated by injecting a clearing agent, also known as a chase, which binds to the residual antibodies in blood and rapidly directs these to the liver. Then, a relatively small and fast-clearing molecule carrying a radionuclide is administered that quickly binds to the tumor-localized antibody via the tag, while non-bound probe is rapidly cleared from the body.
Figure 1 Principles of conventional RIT and pretargeted RIT.
Figure 1
Principles of conventional RIT and pretargeted RIT.

Pre-targeting combines the excellent tumor-seeking properties of antibodies with the fast distribution and clearance of low molecular weight probe, and has demonstrated an ability to provide substantially higher tumor-tonontumor ratios of radioactivity than those achievable through the standard one-step radioimmunotherapy approachiii.

Two pre-targeting approaches have been evaluated in the clinic:
  • Procedures that use the interaction between biotin-streptavidin
  • Procedures that use bispecific antibodies with affinity for the tumor and for a radio-labeled small moleculeiii.

A phase I pre-targeting study in 15 non-Hodgkin's lymphoma patients using an anti-CD20/ streptavidin fusion protein and an 90Y-labeled biotin probe afforded a high mean tumor-to-whole- body radiation dose ratio of 49 and two complete responsesiv. A similar approach in nine colorectal cancer patients gave an 8- to 11- fold increase of tumor-to-normal organ dose ratios compared to conventional RITv.

However, to date not one pre-targeting technology has won FDA approval. The superior image contrast and the ability to administer higher (therapeutic) radiation doses compared to directly labeled antibodies is offset by the drawbacks of the current biological pre-targeting systems, such as immunogenicity (streptavidin systems) or the need to reengineer the parent antibody (bispecific antibodies). To address this, we sought to design a pre-targeting system that combines a straightforward, rapid and cost efficient antibody modification with only very limited perturbation of its in vivo properties and a low likelihood of immunogenicity. For this we turned towards a special class of organic reactions known as bio-orthogonal reactionsvi. These precious and rare reactions have the unique characteristic that they occur in water and biological environments (e.g. pH 7, 37ºC) and are unperturbed by the wide range of constructs that are present in such complex mixtures.

The exquisite selectivity of these reactions has been exploited for the tagging and visualization of biomolecules in cells, zebrafish and mice for chemical biology research purposesvii-x. However, the reactivity of the reactions used requires a high dose of probe to achieve detectable binding. Applications in molecular imaging and medicine that require low doses and high reaction yields have therefore remained unrealized.

Faster reactions with retained selectivity could extend in vivo chemistry to clinically relevant procedures in mammalian disease models, by allowing the intravenous administration of a small amount of probe to non-invasively delineate low-abundance species. The rapid blood clearance and excretion typical of most small to medium-sized molecules necessitates that the reaction occurs within minutes. Such selectivity and effective equimolar reactivity has so far never been demonstrated in a living animal.

The fast reaction kinetics and selectivity in vitro of a recently developed reaction, the inverse electron- demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) derivatives, suggested potential for effective use at the low concentrations of tumor antigensxi. It was, however, unknown whether the Diels-Alder components could also contend with the more demanding conditions in vivo, such as side reactions and metabolism, as well as prolonged residence times and the corresponding required stability. We therefore designed a novel tumor pre-targeting approach based on the Diels-Alder reaction between tetrazine-DOTA derivative 1 radio-labeled with 111In and anti-TAG72 antibody CC49 (2) conjugated to TCO through the lysine residue (Figure 2). The TAG72 antigen was selected because of its limited internalization and shedding as well as its over-expression in a wide range of solid tumors, including colorectal cancerxii.
Figure 2 Principle of pre-targeted RIT using the inverse-electron-demand Diels–Alder reaction.
Figure 2
Principle of pre-targeted RIT using the inverse-electron-demand Diels–Alder reaction.

Methods
All animal experiments were approved by the ethical review committee of the Maastricht University Hospital (the Netherlands), and were performed according to the principles of laboratory animal care (NIH publication 85-23, revised 1985), and the Dutch national law "Wet op de Dierproeven" (Stb 1985, 336).

Imaging experiments
Tumor-bearing mice were injected with 111In-tetrazine (21 µg/75 µL, 20-50 MBq) 24 h after administration of 100 µg mAb (CC49- TCO, CC49 or Rtx-TCO). Single photon emission computed tomography (SPECT) was performed two hours after the tetrazine injection with a four-headed multi-pinhole small animal SPECT/CT imaging system (NanoSPECT, Bioscan Inc., Figure 3).
Figure 3 Bioscan NanoSPECT/CT scanner.
Figure 3
Bioscan NanoSPECT/CT scanner.

SPECT acquisitions on live mice (1 hour total) were performed with 1.4 mm diameter pinholes. Post-mortem high resolution scans were conducted three hours after the tetrazine injection with 1.0 mm diameter pinholes. Prior to each SPECT session a CT scan was performed to obtain anatomical information on radioactivity distribution. After image reconstruction, regions of interest were drawn manually in triplicate for tumor, liver, kidney and thigh muscle. A phantom filled with a known amount of 111In was used to calibrate the scanner for tissue radioactivity quantification.

Biodistribution experiments
Dual isotope biodistribution experiments were performed by injecting tumor-bearing mice (n = 3) intravenously with 125I-labeled mAbs (CC49-TCO, CC49 or Rtx-TCO, 100 µg/100 µL, ca. 0.2 MBq) and, 24 h later, with 111Intetrazine (21 µg/75 µL, ca. 0.8 MBq). Three hours after tetrazine administration, the animals were anesthetized with isoflurane and sacrificed by cervical dislocation. Blood was withdrawn by heart puncture and organs and tissues of interest were harvested, blotted dry and weighed. The radioactivity of the samples was measured in a ?-counter along with standards to determine the %ID/g.

Results and discussion
The investigation commenced with determination of the stability and reactivity of the two components in biological media. In vitro assays in phosphate buffered saline (PBS), serum, and blood (Table 1) showed that 111In-1 should be stable for the duration of its presence in mice (blood clearance half-life of 9.8 minutes, see Figure 4). 111In-1 and CC49-TCO displayed high reactivity towards one another in vitro in semi-equimolar conditions and at low concentration (3.3 µM) within 10 minutes in PBS, serum and blood (Table 2). During the completion of our studies, a lower in vitro reactivity for a more stable system was reported, see Devaraj et al.xiii.
Table 1 In vitro stability of <sup>111</sup>In-1. Data normalized to 100% at t = 0 and presented as mean ± SD.Table 2 In vitro reactions to determine the stoichiometry between <sup>111</sup>In-1 (three concentrations)
and CC49-TCO (3.3 μM) after 10 min incubation at 37ºC; reaction yields in % based on <sup>111</sup>In-1.
Equivalents <sup>111</sup>In-1 with respect to CC49. Parentheses: number of tetrazine probe molecules
bound per CC49. Data are presented as mean ± SD.Figure 4 Blood clearance of
<sup>111</sup>In-tetrazine (0.04±0.02 %ID/g at
24 h). The data represent the mean
%ID/g±SD (n = 4).
Table 1
Table 2
Figure 4
In vitro stability of 111In-1. Data normalized to 100% at t = 0 and presented as mean ± SD.
In vitro reactions to determine the stoichiometry between 111In-1 (three concentrations) and CC49-TCO (3.3 μM) after 10 min incubation at 37ºC; reaction yields in % based on 111In-1. Equivalents 111In-1 with respect to CC49. Parentheses: number of tetrazine probe molecules bound per CC49. Data are presented as mean ± SD.
Blood clearance of 111In-tetrazine (0.04±0.02 %ID/g at 24 h). The data represent the mean %ID/g±SD (n = 4).

The yields obtained from the reactions with 10 and 15 equivalents of 111In-1 indicated the presence of an average of 7.4 reactive TCO moieties per antibody. Furthermore, the nonspecific binding of 111In-1 to unmodified CC49 antibody or other media constituents was found to be very low. To assess the stability of the antibody-bound TCO moiety in vivo mice were treated with CC49-TCO and the extracted blood samples were then reacted ex vivo with an excess of tetrazine 1. The reaction yield, corrected for CC49-TCO blood clearance, revealed that 75% of the CC49-bound TCO present in blood was still reactive after 24 hours circulation, thus indicating good in vivo stability.

To test the Diels-Alder reaction in living animals we administered CC49-TCO to mice bearing colon cancer xenografts, followed one day later with injection of 3.4 equivalents of 111In-1 with respect to TCO. The chemically tagged tumors reacted rapidly with 111In-1, resulting in pronounced localization of radioactivity in the tumor, as demonstrated by single photon emission computed tomography/computed tomography (SPECT/CT) imaging of live mice three hours after injection (Figure 5a). The SPECT quantification of the tumor gave 4.2% injected dose per gram (%ID/g) and a tumor-to-muscle ratio (T/M) of 13.1. We also observed limited uptake in blood and non-target tissues, such as the liver, which we attributed to reaction with residual circulating CC49-TCO.
Figure 5
Figure 5
Small animal SPECT/CT imaging of live mice bearing colon carcinoma xenografts: posterior projections of mice pre-injected with a) CC49-TCO (100 µg) followed 1 day later by 111In-1 (25 eq. to CC49; 3.4 eq. to TCO, 42 MBq), b) CC49 (100 µg) followed 1 day later by 111In-1 (same amount as in a), 20 MBq), and c) Rtx-TCO (100 µg) followed 1 day later by 111In-1 (same amount as in a), 50 MBq); d)-f) single transverse slices (2 mm) passing through the tumors in a-c).

Besides the tumor, the bulk of the radioactivity was found in the bladder, and some residual activity was visible in the kidney (Figures 5a and 6a). Importantly, the tumor could not be discriminated from the surrounding tissue in mice treated with unmodified CC49 (Figure 5b, e; 0.3%ID/g, T/M = 0.5). Almost no radioactivity was retained in the blood and non-target organs, as the probe was again rapidly eliminated through the urinary tract, thus signifying its bio-orthogonality.

Mice treated with TCO-modified rituximab (Rtx), which lacks specificity for TAG72, showed the expected retention of 111In-1 in blood and non-target organs, and a significantly reduced accumulation in the tumor (Figure 5c, f; 1.0%ID/g, T/M = 2.1). The high-resolution postmortem image (Figure 6b) shows 111In activity in the aorta and carotid arteries as well as in the retro-orbital regions. The rim of the tumor is also visible, because of extensive vascularization, whereas the rest of the tumor is devoid of radioactivity.
Figure 6 Small animal SPECT/CT high resolution
postmortem images of mice from Figure 6;
Left mouse) treated with CC49-TCO and <sup>111</sup>In-1;
Right mouse) treated with Rtx-TCO and <sup>111</sup>In-1.
Figure 6
Small animal SPECT/CT high resolution postmortem images of mice from Figure 6; Left mouse) treated with CC49-TCO and 111In-1; Right mouse) treated with Rtx-TCO and 111In-1.

Next, we studied the distribution and specific co-localization of both pre-targeting components through corresponding dual isotope biodistribution experiments with 125I-labeled antibodies (CC49-TCO, CC49, or Rtx-TCO) and 111In-1 (Figure 7 ). Residual 125I-antibodies were detectable in blood and in blood-rich organs such as the heart and lung 27 hours after injection, and showed the typical distribution pattern of long-circulating antibodies (Figure 7a)xiv.

Both CC49 and CC49-TCO accumulated efficiently in tumors, with tumor-to-blood ratios (T/B) around 3 (Figure 7c) and high T/M ratios (Figure 7d). Considerably lower accumulation in the tumor, accompanied by a T/B ratio lower than 1 was found for Rtx-TCO (Figure 8a), thereby supporting the antigen-specific binding of CC49-TCO. The 111In-1 biodistribution data confirmed the 111In-1 SPECT images.

In the mice pretreated with CC49-TCO or Rtx-TCO, the distribution of 111In-1 mirrored that of the antibodies (Figure 7a-d). For example, a fivefold higher uptake of 111In-1 was found in the tumors containing 125ICC49-TCO compared to 125I-Rtx-TCO. Almost no 111In uptake was detected in the blood and in most tissues of the group pretreated with unmodified 125I-CC49.
Figure 7 Dual isotope biodistribution experiment.
Figure 7
Dual isotope biodistribution experiment.

Figure 7a. Uptake of 125I-CC49-TCO, 125I-CC49 and 125I-Rtx-TCO in selected organs 27 h after intravenous injection.

Figure 7b. Uptake of 111In-1 3 h post injection and 27 h after injection of 125I- antibodies (green bars: 125I-CC49-TCO + 111In-1; red bars: 125I-CC49 + 111In-1; blue bars: 125I-Rtx-TCO + 111In-1)

Figure 7c,d. Tumor-to-blood (7c) and tumor-to-muscle (7d) ratios in mice administered with 125I-CC49- TCO or 125I-Rtx-TCO followed 1 day later by 111In-1 (green bars: 125I-data; blue bars: 111In-data).

Figure 7e. Reaction yields between TCO and tetrazine 1 in blood and tumor in living mice (green bars: CC49-TCO + tetrazine; blue bars: Rtx-TCO + tetrazine). Data are presented as mean ± SD (n = 3).

Importantly, while the uptake of 125I-CC49 by the tumors was the highest among the three groups, the 111In uptake by the tumors in this same group was 16 times lower than that in the 125I-CC49-TCO group. In all mice, the kidney exhibited a relatively high uptake of 111In as a consequence of 111In-1 excretion. The finding that the tetrazine accumulated only in tissues containing a TCO-modified species indicated that a chemical reaction between these two entities occurred in vivo.

Calculation of the absolute amounts of TCO and tetrazine present in tissues from the %ID/g values of 125I and 111In revealed a remarkable 57% (in blood) and 52% (in tumor) reaction yield between TCO and tetrazine moieties* at TCO concentrations of 0.4 nmol/g (blood) up to 0.9 nmol/g (in tumor), and max 2.1 nmol/g for the tetrazine (Figure 7e). It is noteworthy that this high reaction yield and corresponding tumor contrast were achieved in the complex interior of a mouse in a reaction time of minutes, limited by the 9.8 minute circulation half-life of 111In-1.

Conclusion
We have demonstrated the first use of a bioorthogonal chemical reaction between two exogenous moieties in living animals for the non-invasive imaging of low abundance targets under clinically relevant conditions. Intravenous administration of a small, semi-equimolar amount (nanomol) of a rapidly excreted probe convincingly delineated a tumor-bound antibody in a high chemical yield, despite the challenging pharmacokinetic constraints of a mammalian disease model. The inverse-electron-demand Diels-Alder reaction, therefore, has the potential to improve the state of the art of pretargeting, because it circumvents the use of immunogenic streptavidin systems and the protein engineering techniques used for bispecific antibodies. This pretargeting system can be applied to a range of antibodies (and fragments) due to its universal and straightforward conjugation chemistry. In addition, it combines this rapid and cost-efficient antibody modification with only very limited perturbation of the in vivo properties and a low likelihood of immunogenicity. The latter would potentially allow for repeat treatments, resulting in a higher efficacy.

Current efforts are directed towards reducing the amount of free circulating CC49-TCO to increase the tumor-to-blood ratio of 111In-1 in preparation for radiotherapy studies with tumor-bearing mice. Finally, this approach may re-introduce full length antibodies to nuclear imaging.

References

  1. Rossin R, Verkerk PR, Van den Bosch SM, Vulders RCM, Verel I, Lub J, et al. In vivo chemistry for pretargeted tumor imaging in live mice. Angew Chem Int Ed. 2010; 49: 3375-378.
  2. Milenic DE, Brady ED, Brechbiel MW. Antibody-targeted radiation cancer therapy. Nat Rev Drug Disc. 2004; 3(6): 488-498.
  3. Goldenberg DM, Sharkey RM, Paganelli G, Barbet J, Chatal JF. Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy. J Clin Oncol. 2006; 24(5): 823- 834.
  4. Forero A, Weiden PL, Vose JM, Knox SJ, LoBuglio AF, Hankins J, et al. Phase 1 trial of a novel anti-CD20 fusion protein in pretargeted radioimmunotherapy for B-cell non-Hodgkin lymphoma. Blood. 2004; 104(1): 227-236. Epub 2004 Mar 2.
  5. Shen S, Forero A, LoBuglio AF, Breitz H, Khazaeli MB, Fisher DR, et al. Patient-specific dosimetry of pretargeted radioimmunotherapy using CC49 fusion protein in patients with gastrointestinal malignancies. J Nucl Med. 2005; 46(4): 642-651.
  6. Best MD. Click chemistry and bioorthogonal reactions: Unprecedented selectivity in the labeling of biological molecules. Biochemistry. 2009; 48(28): 6571-6584.
  7. Prescher JA, Dube DH, Bertozzi CR. Chemical remodelling of cell surfaces in living animals. Nature. 2004; 430(7002): 873-877.
  8. Dube DH, Prescher JA, Quang CN, Bertozzi CR. Probing mucin-type O-linked glycosylation in living animals. Proc Natl Acad Sci USA. 2006; 103(13): 4819-4824. Epub 2006 Mar 20.
  9. Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, et al. Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci USA. 2007; 104(43): 16793-16797. Epub 2007 Oct 17.
  10. Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR. In vivo imaging of membrane-associated glycans in developing zebrafish. Science. 2008; 320(5876): 664-667. 
  11. Blackman ML, Royzen M, Fox JM. Tetrazine ligation: Fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J Am Chem Soc. 2008; 130(41): 13518-13519. Epub 2008 Sep 18.
  12. Schlom J, Eggensperger D, Colcher D, Molinolo A, Houchens D, Miller LS, et al. Therapeutic advantage of high-affinity anticarcinoma radioimmunoconjugates. Cancer Res. 1992; 52(5): 1067-1072.
  13. Devaraj NK, Upadhyay R, Haun JB, Hilderbrand SA, Weissleder R. Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition. Angew Chem. 2009; 121: 7147-7150; Angew Chem Int Ed. 2009; 48(38): 7013-7016.
  14. Boswell CA, Brechbiel MW. Development of radioimmunotherapeutic and diagnostic antibodies: An inside-out view. Nucl Med Biol. 2007; 34(7): 757-778. Epub 2007 Jun 8.


This content has been made possible by NetForum Community.
Share this on: Share your link in twitter Share your link in facebook Share your link on LinkedIn Print Rate this article: Log in to vote

 
Rating:
Votes:
4
Views:
40060
Added:
Oct 25, 2010

Rate this:
Log in to vote
 

White Paper
Oncology, pre-targeted tumor imaging
 

Clinical News
Best Practices
Case Studies
Publications and Abstracts
White Papers
Web seminars and Presentations
ExamCards
Protocols
Application Tips and FAQ
Training
Try an Application
Business News
Case Studies
White Papers
Web Seminars and Presentations
Utilization Services
Contributing Professionals
Contributing Institutions
Become a Contributor