<p>The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.</p><p>This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of ¹²⁵I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).</p><p>In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (Z_EGFR:955)₂ was selected and analysed both <i>in vitro</i> and <i>in vivo</i>. In papers II, III and V, the results obtained when using (Z_EGFR:955)₂ were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, <i>in vivo</i> studies were made, demonstrating that [¹¹¹In](Z_EGFR:955)₂ gave a tumour-specific ¹¹¹In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. </p><p>The results of these studies indicated that the affibody molecule (Z_EGFR:955)₂ is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (Z_EGFR:955)₂ might be of interest for therapy applications.</p>

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.

The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications. This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of 125I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR). In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (ZEGFR:955)2 was selected and analysed both in vitro and in vivo. In papers II, III and V, the results obtained when using (ZEGFR:955)2 were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, in vivo studies were made, demonstrating that [111In](ZEGFR:955)2 gave a tumour-specific 111In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. The results of these studies indicated that the affibody molecule (ZEGFR:955)2 is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (ZEGFR:955)2 might be of interest for therapy applications.

<p>Targeted radiotherapy (TRT) of cancer is a promising approach that enables selective treatment of tumour cells, while sparing normal tissue. The humanized monoclonal antibody A33 (huA33) is a potential targeting agent for TRT of colorectal cancer, since its antigen is expressed in more than 95 % of all colorectal carcinomas. The aim of this thesis was to evaluate the therapeutic potential of the two huA33-based TRT-conjugates, ¹⁷⁷Lu-huA33, and ²¹¹At-huA33.</p><p>The conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, bound specifically to colorectal cancer cells, both <i>in vitro</i> and <i>in vivo</i>. A dose dependent cytotoxic effect of ²¹¹At-huA33 was also demonstrated <i>in vitro</i>. From a therapeutic perspective, both conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of an expected thyroid uptake of ²¹¹At). After injection of ²¹¹At-huA33, the blood absorbed a slightly higher dose than the tumour, but for ¹⁷⁷Lu-huA33, the tumour received a 12 times higher dose than blood. Two days after intravenous injection of ¹⁷⁷Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, also performed in tumour-bearing mice, there was an excellent therapeutic effect of ¹⁷⁷Lu-huA33. About 50 % of the treated animals were tumour free 140 days after injection of ¹⁷⁷Lu-huA33, while none of the non-radioactive controls survived beyond 20 days after injection of treatment substances.</p><p>In conclusion, this thesis demonstrates that the therapeutic conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, are promising targeting agents that might help improve therapy of colorectal cancer.</p>

<p>Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. </p><p>In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate.</p><p>In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36.</p><p>In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice.</p><p>In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.</p>

<p>Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. </p><p>In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with ²¹¹At and ¹³¹I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using ¹²⁴I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies.</p><p>These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.</p>

Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with 211At and 131I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using 124I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies. These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.

Targeted radiotherapy (TRT) of cancer is a promising approach that enables selective treatment of tumour cells, while sparing normal tissue. The humanized monoclonal antibody A33 (huA33) is a potential targeting agent for TRT of colorectal cancer, since its antigen is expressed in more than 95 % of all colorectal carcinomas. The aim of this thesis was to evaluate the therapeutic potential of the two huA33-based TRT-conjugates, 177Lu-huA33, and 211At-huA33. The conjugates 177Lu-huA33, and 211At-huA33, bound specifically to colorectal cancer cells, both in vitro and in vivo. A dose dependent cytotoxic effect of 211At-huA33 was also demonstrated in vitro. From a therapeutic perspective, both conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of an expected thyroid uptake of 211At). After injection of 211At-huA33, the blood absorbed a slightly higher dose than the tumour, but for 177Lu-huA33, the tumour received a 12 times higher dose than blood. Two days after intravenous injection of 177Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, also performed in tumour-bearing mice, there was an excellent therapeutic effect of 177Lu-huA33. About 50 % of the treated animals were tumour free 140 days after injection of 177Lu-huA33, while none of the non-radioactive controls survived beyond 20 days after injection of treatment substances. In conclusion, this thesis demonstrates that the therapeutic conjugates 177Lu-huA33, and 211At-huA33, are promising targeting agents that might help improve therapy of colorectal cancer.