Identification and Characterization

Pancreatic cancer (PC) ranks as the fourth leading cause of cancer death in both men and women in the United States, leading to the loss of over 40,000 lives a year. PC is a highly heterogenous group of diseases, as malignancies can develop from both the endocrine and exocrine pancreas. Pancreatic ductal adenocarcinoma (PDAC), an exocrine tumor, accounts for approximately 85% of PC cases. While it only occurs rarely -- 2% of all malignant tumors--, it is devastating as the average survival after diagnosis is only one year. The low survival rates partly arise from the fact that PDACs do not manifest with specific symptoms and therefore are often diagnosed at a late stage. On average, patients are diagnosed at age 71, but the risk of developing PDAC rises with age.

While a lot of research is being conducted, major clinical advances have not been achieved due to several unique obstacles. PDACs dynamically interact with their stroma, producing a dense fibrous tissue around the tumour that functions as a barrier to systemic drug delivery and penetration. Currently, the lack of specific serological biomarkers is hindering the development of screening programs to enable earlier diagnosis. Additionally, metastatic spread often occurs early, even before the primary tumour can be detected by current methods. Because of these complexities, there is an enormous need for new detection methods and treatment options.

In the last decades, monoclonal antibody-based strategies have been widely utilized in the field of oncology. The affinity and antigen-specificity of antibodies make them attractive for therapeutic and diagnostic applications, and render them excellent tools to study cellular and molecular processes. Antibody binding may have different effects, as it can cause the neutralization, relocation or even degradation of its targets. These characteristics have earned them the name 'magic bullets of medicine'. In the clinic, antibodies against PDL1 and CTL4 are currently widely used in the treatment of hematological cancers and show great succes. Several currently available antibody therapies have been tested in PDAC, but almost none were found to be effective (PMC4714146). New attempts to generate customized antibodies with specialized binding properties, half-lives and functions through structural modifications, have opened up new avenues for the exploitation and application of antibodies. Major advances in clinical efficacy have been made through the modification of Fc regions, resulting in the optimization of anti-tumor immune responses in what tumor?. In the in vitro setting, the addition of localization signals onto antibody fragments allows for subcellular targeting. This strategy is limited for full-size antibodies which, due to their reliance on intrachain disulfide bonds and pairing of heavy and light chain variable regions, cannot be properly assembled in the reducing environment of the cytoplasm. Full size antibodies can be shrunk down to single-chain variable fragments (scFv) (image 1b), the variable regions of the heavy and light chain, connected by a glycine or glycine-serine linker.

The discovery of heavy chain only antibodies in camelids opened up a new avenue for antibody-based strategies. The antibodies within this unique subset of IgG-type immunoglobulins, are composed of two identical heavy chains bearing a variable domain (VHH), but lack lightchains and CH1 domains. The variable domain consists of four framework regions, which connect its three hypervariable loops (CDR). They are derived from a single cluster of D and J segments that serve both conventional and heavy chain only antibodies. There is strong sequence homology to human V region, particularly to the Vh3 family (PMID: 19010777). The paratope of the variable domain is determined by its three CDRs, but interaction with the antigen occurs primarily via CDR3. The CDR3 loops are longer than in conventional antibodies, enabling them to reach sites which are inaccessible to conventional Vh-Vl pairs, such as clefts or depressions on the proteins surface (PMID: 22886243) . Consequently, the range of epitopes probed by heavy chain only antibodies may differ from conventional antibodies. Promising in the identification of targets for which no regular antibodies are available.

Heavy chain only antibodies can be shrunk into variable region fragments, which are often referred to as nanobodies or VHH. Nanobodies come with an unique set of advantages and characteristics, including small size (15kD), higher tissue penetration rate compared to conventional antibodies, high thermostability, good solubility, low immunogenicity and ease of production. Furthermore, the singular nature of VHHs allows them to be used for intracellular expression. While conventional antibodies cannot be properly assembled in the reducing intracellular environment, VHHs are ideally suited for cytosolic expression because of their reduced reliance on disulfide bond formation. This trait has been productively exploited for in vivo cellular imaging and to confer new phenotypic traits (e.g. viral resistance). Lastly, the pharmacokinetic behaviour of VHHs, specifically their short half-life, is well suited for applications where fast clearance is required such as tumor imaging, or delivery of toxins and radioisotopes. This can be modulated by formatting, pegylation or fusion to serum albumin.

Installation of any payload onto a VHH requires site specific, chemical modification, which can be realized via enzymatic methods, such as sortase mediated transpeptidation. Sortase recognizes a specific aminoacid motif (LPXTG), which can be installed on the C-terminus of a VHH, and ligates it with an oligoglycine nucleophile of choice. This method combines the precision of a genetically encoded tag with the specificity of enzymatic reactions, and affords construction of proteins conjugated with non-genetically encoded functional groups or non-natural moieties. Through sortase mediated installation of radioisotopes, the in vivo distribution of the labeled VHH can be tracked with positron emission tomography (PET). This can be applied to follow immune responses over time in a non-invasive manner in live animals, or to image any target structure for which specific probes are available.

The protein family of integrins has been implicated in many processes within the context of cancer, including proliferation, migration and invasion (PMId: 20029421), and have been discussed as putative biomarkers and targets for therapy. Integrins, consisting of a and b subunits, are unique amongst transmembrane receptors because of their capacity to signal bi-directionally, enabling them to sense, modulate and respond to extracellular stimuli. Several integrins have been found to be differentially expressed in PDAC, including a2b1 and avb6. The AvB6 integrin is only expressed on epithelia during embryogenesis, wound healing and neoplasia, but it does not occur on healthy, adult tissue. Additionally, its expression during cancer can act as prognostic factor and is associated with poor patient survival. The A2B1 integrin Nanobodies that recognize these surface receptors could potentially be used to image and treat pancreatic cancers, and would be important additions to the research toolbox.

Previously, alpaca immunizations have been carried out with purified integrins and pancreas cancer organoids of human and mouse origin. From this immunization campaign, nanobodies against ?±V??6, ?±4??1 and ?±5??1 have been isolated via yeast display technology, which have yet to be biochemically characterized. Additionally, a new immunization with a2b1 has been completed, for which a phagemid VHH library has been constructed which was screened via phage display. Specificity of the identified VHHs has to be established. In this project, the individual nanobodies will be expressed with C-terminal sortase recognition motif to enable site-specific installation of fluorophores, metal chelators and other payloads. The final products will be used for flow cytometry, to establish antigen specificity and for biochemical characterization.

• A2b1
• AVB6
• A4b1
• A5B1</li

Regarding cancer cells, integrins serve roles in numerous aspects, including proliferation, survival, migration and invasion (1).

The protein family of integrins, cell bound adhesion molecules, could serve as tumour specific targets.

a2b1 - has shown to be involved in the malignant phenotype of pancreatic cancer.

a5b1 - MM - induction and/or upregulation of expression associated with progression in patient Bx 1 OvCa - expression significantly correlates with higher clinical stage 48

avb6 - Itegrin avb6, a member of the integrin protein family, is overexpressed in numerous types of carcinomas, such as colon, lung, cervical, ovarian, and pancreatic cancers, but is expressed at low or undetectable levels in healthy organs (3). Pancreatic ductal adenocarcinomas exhibit the highest integrin avb6 expression among gastroenteropancreatic adenocarcinomas (4). Moreover, the high expression of integrin avb6 in carcinomas is a prognostic factor of the disease and is correlated with poor patient survival (5,6). Thus, molecular imaging agents that target integrin avb6 would be highly useful in the receptor-targeted detection of pancreatic cancer and in the noninvasive monitoring of tumor prognosis

5. Bates RC, Bellovin DI, Brown C, et al. Transcriptional activation of integrin beta6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma. J Clin Invest. 2005;115:339“347. 6. Hazelbag S, Kenter GG, Gorter A, et al. Overexpression of the avb6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. J Pathol. 2007;212:316“324.

Overexpressed:

66. Nieberler, M. et al. Exploring the Role of RGD-Recognizing Integrins in Cancer. Cancers (Basel) 9 (2017).

95. Sipos, B. et al. Immunohistochemical screening for beta6-integrin subunit expression in adenocarcinomas using a novel monoclonal antibody reveals strong up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro. Histopathology 45, 226- 236 (2004).

a4b1 -

MM - expression low to absent in early disease, emergent and upregulated during disease progression in patient Bx 1, 25, 46, 47

Through this bidirectional linkage, integrins provide spatially restricted communication lines between the cell and their microenvironment, act as key mechanosensing and force-transducing units and coordinate actin cytoskeletal polymerisation to regulate important biological functions including proliferation, gene expression, cell survival and cell motility (Legate et al, 2009). In the setting of cancer, these same integrin-mediated processes are exploited by tumour cells to promote invasiveness and oncogenic survival and to engineer a host microenvironment that is conducive to tumour growth and metastatic spread.

ANTIBODY blockade references

20. Callahan MK, Wolchok JD. At the bedside: CTLA-4- and PD-1-blocking antibodies in cancer immunotherapy. J Leukoc Biol. 2013;94(1):41“53. [PMC free article] [PubMed]

21. Hamid O, et al. Safety and Tumor Responses with Lambrolizumab (Anti-PD-1) in Melanoma. N Engl J Med. 2013 [PMC free article] [PubMed]

22. Topalian SL, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366(26):2443“54. [PMC free article] [PubMed]

23. Brahmer JR, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol. 2010;28(19):3167“75. [PMC free article] [PubMed]

24. Ohaegbulam KC, et al. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends Mol Med. 2015;21(1):24“33. [PMC free article] [PubMed]

Key characteristics of VHHs include their high affinity and specificity (equivalent to conventional antibodies), high thermostability, good solubility and strictly monomeric behavior, small size (2.5‰nm in diameter and about 4‰nm in length; ~15‰kDa), relatively low production cost, ease of genetic engineering, format flexibility or modularity, low immunogenicity, and a higher penetration rate into tissues (3, 41“44). The short half-life of VHHs in blood circulation is well suited to certain applications such as tumor imaging or delivery of toxin or radioisotopes to diseased tissues where rapid clearance is required. However, the pharmacokinetic behavior of VHHs can also be improved by extending their half-lives using different formatting options, including PEGylation or fusion to serum albumin or an anti-serum albumin moiety (43, 45, 46).

As intrabodies, VHHs are also ideally suited for cytosolic expression due to their ability to fold in the reducing intracellular environment. This feature likely reflects the single disulfide linkage present in the VHH domain, as compared to the multi-domain structure and multiple disulfide linkages of conventional antibodies, and may not be completely general to all VHHs but appears to be quite common; intracellular expression of VHHs has been widely and productively exploited for in vivo cellular imaging (5, 57) as well as to inhibit the function of viral proteins (58, 59)

a sngle cluster of D and J segments that serves the heavy chain only antibody variable regions as well as the variable regions of convntional IgGs. Later genomic studies shed light on the origin of HCAbs in dromedary camels and alpacas. It is now established that HCAbs are produced from the same igh locus as conventional antibodies but with distinct sets of genes for the generation of HCAbs. (18641337)

the paratope of the VHH is generated by its three CDRs, to achivie the requisite buried surface area when contacting antigen, the cdr3 loops are longer than in conventional antibodies. VHHs typically bind in clefts or depressions on the protein surface, or at domain-domain and subunit-subunit interfaces.

While interaction with the antigen occurs primarily via CDR3, eamples of extensive involvement of CDR2 or the framework have been identified as well. Consequently the range of epitopes sampled by VHHs may overlap but are not identical. A striking example is a VHH directed against HIV gp120, believed to bind to and protrude into its CD4- binding pocket, which may explain the broad neutralizing potential oft this particular llama derived VHH.

with particular emphasis

a2b1 als marker

CA-19-9

Integrins background

Monoclonal antibody-based treatment of cancer has been established as one of the most successful therapeutic strategies for both hematologic malignancies and solid tumors in the last 20 years. The initial combining of serological techniques for cancer cell surface antigen discovery with hybridoma technology led to a series of landmark clinical trials that paved the way for new generation antibodies and subsequent clinical success. The modulation of immune system interplay with tumor cells through targeting of T cell receptors has emerged as a powerful new therapeutic strategy for tumor therapy and to enhance cancer vaccine efficacy.

Clinical trials demonstrated that PD-1 blockade is an attractive way to reinstate host's immune function in lymphoid neoplasms, particularly classical Hodgkin lymphoma. PD-1 blockade as a single therapy or in combination with other immune checkpoint inhibitors are explored in other hematologic cancers.

aims of this project

Material & methods

VHH sequences from phage panning library

readily available from ploegh lab & collaborators

Sequence analysis

Results

A. Phage based panning

Figure 1. A. Sequence analysis. Flow diagram, (B) sequence alignment 5 sequences

(A)

(B)

Figure 2. (A) Alexa coupling strategy, (B.) LCMS

Figure 3. (A) Flowcytometry results (B) KD determination

B Previously identified integrin specific VHH

Figure 1. Sequences

Figure 2. FACS data

Figure 3. Competition assays?

B. PVDF membrane based panning

Figure 3. Flow diagram amounts

Figure 4. immunoprecipitation

Figure 5. Mass spec results

Figure 6. Targets identified?

Discussion

Recap

Broad context

Critical perspective on own work

perhaps too many rounds of phage panning?

Future direction

Coupling of radioactive particles to as therapeutic

coupling of dyeing agent to it to enhance tumour visibility surger

Near infrared photoimmunotherapie moiety

Resistance to therapy is a characteristic feature of PDAC, and the extent of resistance is greater than in many other human tumors. This could be due to inefficient drug delivery, intrinsic and acquired resistance of the tumor, tumor hypoxia, or the insensitivity of cancer stem-like cells to currently used agents.

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