High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.

Systemic chemotherapy is a major therapeutic approach for nearly all types and stages of cancer. Success of this treatment depends not only on the efficacy of the therapeutics but also on the transport of the drug to all tumor cells in sufficient concentrations. Intratumoral drug transport is limited by characteristics of the tumor microenvironment such as elevated interstitial pressure and sparse, irregular vascularization. Moreover, poor tumor selectivity, leads to systemic toxicity. Bacteria possess a host of characteristics that address the aforementioned challenges in conventional drug delivery approaches including tumor selectivity, preferential tumor colonization, effective tumor penetration, which can be augmented via genetic engineering. However, in clinical trials conducted to date, bacteria have rarely been able to inhibit tumor growth solely by their presence in the tumor. The overall goal of this doctoral dissertation is to develop a novel tumor treatment system based on Salmonella Typhimurium VNP20009 (genetically modified for preferential tumor colonization and attenuation) coupled with biodegradable copolymer, poly(lactic-co-glycolic acid) nanoparticles, hereafter referred to as NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery System). To this end, a NanoBEADS fabrication procedure that is robust and repeatable was established and a microfluidic chemotaxis-based sorting platform for the separation NanoBEADS from unattached nanoparticles was developed. The transport efficacy of NanoBEADS compared to the commonly used passively-diffusing nanoparticle was investigated in vitro and in vivo and the intratumoral penetration of the therapeutic vectors was quantified using a custom image processing algorithm. The mechanism of intratumoral penetration was elucidated through 2D and 3D invasion assays. Lastly, we developed a biophysical model of intratumoral transport of NanoBEADS based on the intratumoral penetration experimental results towards the theoretical evaluation of the drug transport profile following the administration of NanoBEADS. / PHD

The lack of efficacy of existing chemotherapeutic treatments of solid tumors is partially attributed to the limited diffusion distance of therapeutics and the low selectivity of anti-cancer drugs with respect to cancerous tissue, which also leads to high levels of systemic toxicity in patients. Thus, chemotherapy can be enhanced through improving anti-cancer drug carrier selectivity and transport properties. Several strains of gram positive (e.g. Clostridium and Bifidobacterium) and gram-negative (e.g. Salmonella Typhimurium and Escherichia coli) bacteria have been shown to possess the innate ability to preferentially colonize tumor tissues. The overall goal of this dissertation is to characterize the transport and sensing of Bacteria-Enabled Drug Delivery Systems (BEADS) in select relevant environments and to investigate the associated underlying principles. BEADS consist of an engineered abiotic load (i.e. drug-laden micro or nano-particles) and a living component (i.e. bacteria) for sensing and actuation purposes. Findings of this dissertation work are culminated in experimental demonstration of deeper penetration of the NanoBEADS within tumor tissue when compared to passively diffusing chemotherapeutic nanoparticles. Lastly, the transport mechanisms that Salmonella Typhimurium VNP20009 utilize to preferentially colonize solid tumors are also examined with the ultimate goal of engineering intelligent and more efficacious drug delivery vehicles for cancer therapy. / Ph. D.

To personalize treatment for cancer, correct staging of the primary tumor, nodal disease and metastatic disease is of essence. By targeting tumor specific receptors with radiolabeled antibodies, specificity and accuracy of imaging may be improved. Radio-immunodiagnostics can potentially detect small volume disease, occult metastasis and recurrent cancer in treated tissue. This thesis focuses on evaluation of radio-immunoconjugates directed towards CD44v6, which is a surface receptor overexpressed in many head and neck squamous cell carcinomas. At the outset, the monoclonal chimeric antibody cMab U36 and its cleavage products Fab’ and F(ab’)2 were labeled with 125I and assessed in vitro and in vivo (paper I). The best distribution pattern and tumor to organ ratio was achieved with F(ab’)2. Due to the immunological responses humans can develop towards chimeric antibodies, they are not optimal for clinical use, and subsequently fully human antibody fragments were developed. AbD15179, which is a monovalent fragment, was labeled with 111In and 125I and evaluated in vitro and in mice bearing CD44v6-expressing tumors. Tumor to organ ratios were improved compared to cMab U36 derived fragments, and 111In-AbD15179 displayed a more favorable distribution compared to 125I-AbD15179 (Paper II). A bivalent Fab-dHXL, AbD19384 derived from AbD15179, was then constructed and labeled with 125I and evaluated in cell- and biodistribution studies. Furthermore, an imaging study in a small animal PET was performed with 124I-AbD19384 (Paper III). Uptake in kidneys was reduced and liver uptake increased compared to AbD15179 reflecting the larger molecule. The high CD44v6 expressing tumor was clearly visualized with maximum uptake at 48 hours post injection.In paper IV human single chain fragments towards CD44v6v were selected, and the top candidates A11 and H12 were further evaluated in vitro and in vivo. Single chain fragments are small molecules exhibiting fast clearance and high affinity to the target. The study proved this by demonstrating superior tumor to blood ratios of radiolabeled A11 and H12 compared to previously studied molecules.

<p>Conventional cancer treatment based on radiotherapy or chemotherapy affects all dividing cells. By directing the therapy specifically to the tumor cells, normal cells can be spared. Tumor targeting molecules carrying a cytotoxic moiety is then an attractive approach. </p><p>In this thesis, an affibody molecule with high affinity for the protein HER-2, that is strongly associated with aggressive forms of breast cancer, was selected. After radiolabeling with ¹²⁵I, the affibody molecule, in monovalent and bivalent form, was tested <i>in vitro</i> in HER-2 overexpressing tumor cells and in transplanted tumors in mice. </p><p>It was shown that the HER-2 targeting affibody molecule bound its target in a specific manner, both <i>in vitro</i> and <i>in vivo</i>. The small size of the affibody molecule resulted in fast clearance through the kidneys. An impressive tumor-to-blood ratio of 10 eight hours post injection was achieved and the tumors could easily be visualized in a gamma camera. </p><p>The biologic effects of the bivalent affibody molecule and a monovalent affinity maturated version was measured and compared with the effects of the monoclonal antibody trastuzumab. It was found that although all molecules target the same protein, the effects differed greatly.</p><p>The affibody molecule was also labeled with the alpha-emitting radionuclide ²¹¹At. Specific decrease in survival was seen in HER-2 overexpressing cells receiving the ²¹¹At labeled affibody molecule. The sensitivity to the treatment differed between cell lines, probably as a result of differences between the cell lines in internalization and nuclear size. The ²¹¹At labeled affibody molecules were also tested <i>in vivo</i>, where stability of the ²¹¹At label was a problem. To circumvent this problem, more stable conjugation chemistry was tested, as well as strategies to prevent uptake of released ²¹¹At by normal organs.</p><p>This thesis describes the selection and optimization of affibody molecules for medical use for the first time.</p>

<p>The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered.</p><p>The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented.</p><p>These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.</p>

Conventional cancer treatment based on radiotherapy or chemotherapy affects all dividing cells. By directing the therapy specifically to the tumor cells, normal cells can be spared. Tumor targeting molecules carrying a cytotoxic moiety is then an attractive approach. In this thesis, an affibody molecule with high affinity for the protein HER-2, that is strongly associated with aggressive forms of breast cancer, was selected. After radiolabeling with 125I, the affibody molecule, in monovalent and bivalent form, was tested in vitro in HER-2 overexpressing tumor cells and in transplanted tumors in mice. It was shown that the HER-2 targeting affibody molecule bound its target in a specific manner, both in vitro and in vivo. The small size of the affibody molecule resulted in fast clearance through the kidneys. An impressive tumor-to-blood ratio of 10 eight hours post injection was achieved and the tumors could easily be visualized in a gamma camera. The biologic effects of the bivalent affibody molecule and a monovalent affinity maturated version was measured and compared with the effects of the monoclonal antibody trastuzumab. It was found that although all molecules target the same protein, the effects differed greatly. The affibody molecule was also labeled with the alpha-emitting radionuclide 211At. Specific decrease in survival was seen in HER-2 overexpressing cells receiving the 211At labeled affibody molecule. The sensitivity to the treatment differed between cell lines, probably as a result of differences between the cell lines in internalization and nuclear size. The 211At labeled affibody molecules were also tested in vivo, where stability of the 211At label was a problem. To circumvent this problem, more stable conjugation chemistry was tested, as well as strategies to prevent uptake of released 211At by normal organs. This thesis describes the selection and optimization of affibody molecules for medical use for the first time.

The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered. The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented. These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.

Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A. In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging. Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients. / QC 20100723

Effective detection and treatment of tumor malignancies depends upon identifying targets – molecular markers that differentiate cancer cells from healthy cells. Current cancer therapies involve targeting overexpressed specific gene products. An alternative approach is proposed here: to specifically target combinations of cell-surface receptors using heteromultivalent ligands (htMVLs). There are about 2500 genes encoding for cellsurface proteins in the human genome that can potentially be targeted. Taken as sets, there can be ~ 10⁶ two-receptor combinations and ~ 10⁹ three-receptor combinations available. Our group envisions that using cell-surface protein combinations that are expressed on a cancer cell but not on a normal cell, multivalent constructs displaying complementary ligands of weak affinities can be assembled. These multivalent ligands should bind with high avidity to cancer populations in vivo, and provide a degree of specificity not seen with current approaches. As a proof-of-concept, a series of multivalent ligands were designed and synthesized for a model system consisting of the human Melanocortin subtype 4 receptor (hMC4R) and the Cholecystokinin subtype 2 receptor (CCK-2R). Modeling studies on GPCR dimers predicted that a minimum linker span of 20 - 50 Å would be required to non-covalently crosslink these two receptors. The multivalent ligands were assembled using a modular parallel synthesis approach and using solidphase chemistries. A variety of linkers were explored ranging from highly rigid to highly flexible, and using natural and/or synthetic building blocks. Ligand binding affinities were evaluated using a lanthanide based competitive binding assay in cells that expressed both receptors (bivalent binding) vs those that expressed only one of the receptors (monovalent binding), and were demonstrated to have enhanced binding affinities of up to nearly two orders of magnitude. The promising ligands were further explored by synthesizing fluorescently labeled and/or lanthanide chelate labeled monovalent and heterobivalent ligands designed for in vitro and in vivo studies. More explorative work using these labeled constructs is in progress. To the best of our knowledge, the author believes this is the first such demonstration of a 'synthetic htMVL' directed recruitment and crosslinking of two heterologous cell-surface receptors. This receptor combination approach opens up new possibilities for single cell imaging, cancer detection and therapeutic intervention, and can provide a revolutionary new platform technology with which to direct therapeutics to defined cell populations.