IgA

Summary

During the last few decades, the number of patients newly diagnosed with cancer, has increased steadily. Cancers are broadly classified into solid and hematological tumors. Solid tumors grow as a mass within a particular type of tissue (e.g. breast cancer), whereas hematological tumors begin in blood-forming tissue or immune cells (e.g. leukemia/lymphoma). Depending on the type of cancer, patients have access to a set of treatment options which include radiotherapy, surgery and chemotherapy. However, systemic treatment approaches like chemotherapy lead to severe side effects, as not only cancerous cells are targeted but also healthy tissue. To circumvent this, a more cancer cell specific therapy is needed.

The specific recognition and elimination of pathogens like viruses and bacteria, is a task of our own immune system. Our immune system is divided into two components: the innate and the adaptive immune system.
The major functions of the innate immune system includes: (a) the recruitment of immune cells (such as natural killer cells, macrophages, and T cells) to the site of infection, which specifically eliminate of foreign organisms; and (b) the activation of the complement cascade, which contributes to the identification and removal of the foreign pathogen, and helps in activating the recruited immune cell. Eventually, it forms a bridge to the adaptive immune system, which is crucial for the formation of a memory (long-lasting response against a particular foreign organism). After engulfment of the pathogen by the recruited immune cells of the innate immune system, they present small molecules (peptides) on their cell surface to another important cellular component of the immune system, namely the B cells. Only B cells which express a B cell receptor specifically recognizing those presented peptides get activated. A B cell receptor is basically an antibody anchored within the cell membrane and displays unique specific to only one particular antigen (peptide). This process known as antigen presentation, results finally in the generation of antibodies by the B cells which are specifically recognizing the small peptide presented by the cells.

Antibodies are commonly described as Y-shaped molecules. Their antigen specificity is encoded by the 2 Fab-regions, which allows the recognition, binding and thus labeling of the invading pathogen. The second functional structure of an antibody is the Fc-fragment. Depending on the isotype of the antibody (in humans 5 different have been described: IgM, IgA, IgG, IgE, and IgD) different effector mechanisms get elicited by the Fc fragment upon binding of an antibody to the antigen (foreign molecule). Generally, they include the engagement of effector mechanisms described for the innate immune system to eliminate the pathogen-antibody complexes: (1) activation of immune effector cells by binding to so-called Fc receptors (which are specific for each antibody isotype, i.e. IgM-Fcµ, IgA-Fcα, IgG-Fcγ, IgE-Fcε, IgD-Fcδ receptor), and (2) activation of the complement system.
The establishment of cancer immunotherapy exploits the specificity found within the immune system’s defense mechanisms. Here, immune cells (e.g. T cells) and antibodies are designed to recognize structures on tumor cells, known as tumor-associated antigens.

Chapter 1 is a general introduction into the field of antibody therapy, in which structure, mechanisms-of-action and the recent developments are described. Several monoclonal antibodies (mAbs) have been approved for the treatment of both, solid and hematological tumors. The net advantage of mAbs is their multi-functionality. On the one hand they specifically bind to tumor-associated antigens which can result in e.g. blockage of a signaling pathway, and on the other hand they engage the patient’s own immune system which results in the eradication of cancerous cells by e.g. immune cells. Currently, all approved mAbs for the treatment of cancer are of the IgG isotype. Observed anti-tumor responses are attributed to (a) the activation of the complement system, and (b) the engagement of effector cells which express activating Fc gamma receptors (e.g. NK cell and macrophages). In Chapter 2 we outlined the complement cascade, which leads to the formation of membrane attack complexes and subsequent tumor cell lysis. We further elucidated mechanisms which regulate the activation of the complement system and thoroughly reviewed approaches taken to enhance complement-mediated tumor cell killing. Chapter 3 focuses on the interaction of a mAb with Fc receptors. The binding and subsequent response of antibody-opsonized particles to FcR is a process referred to as outside-in signaling. Some Fc receptors have also been implicated in inside-out signaling, a process in which cytokines in the surrounding can increase the receptors avidity and thus the resulting anti-tumor response. We further summarized the available data on how Fc receptor modulation by e.g. cytokines influences mAb mediated immunotherapy. 

However, complete responses with IgG-mAb therapy are rare, resistance occurs and many patients relapse. Therefore, good alternatives for the currently approved IgG mAb format are needed. Recent research indicated that IgA mAbs, another isotype, induce a potent anti-tumor response in mice. Compared to IgG antibodies, IgA antibodies are more T-shaped molecules. The anti-tumor response has been shown to be mediated by the engagement of FcaRI expressing effector cells. This receptor is expressed by cells of mainly the myeloid lineage, including neutrophils, monocytes, several macrophage subsets. 

The overall goal of this thesis was to further evaluate the potential of IgA mAb therapy. 
The first part of this thesis focuses on extending the in vivo half-life of IgA mAbs. Preclinical studies to evaluate the potential of new therapies are predominantly performed in mice. Compared to IgG mAbs, IgA mAbs have a shorter half-life in mice. This impedes the direct comparison of in vivo results obtained with the two different isotypes as very frequent dosing of the IgA mAbs is required to achieve a comparable exposure. 
In Chapter 4 we describe the design of IgA-Her2 mAbs indirectly targeting the neonatal Fc receptor mediated recycling pathway by attaching an albumin-binding domain (ABD). With this approach we were able to significantly enhance the serum exposure and extend the molecules half-life in mice. This study compared for the first time the efficacy of an IgA1 and IgA2 anti-tumor mAb in vivo. This research project finally showed that engaging the FcRn recycling pathway, which contributes to the exaggerated long half-life of IgG and albumin in humans, is a promising approach. 

In a study which assessed the in vivo potential of anti-tumor IgA2 mAbs for the first time, it was shown that IgA mAbs are quickly cleared from circulation by the asialoglycoprotein receptor. This receptor recognizes galactose residuse. Antibodies carry glycosylation sites, thus by altering the glycosylation pattern by e.g. reducing glycosylation sites or actually attaching the terminal sialyl residue to the sugar chain, could influence the half-life. In this thesis, two studies are described which aimed at the extension of the IgA half-life by modifying the glycosylation pattern. In Chapter 5, a molecular engineering approach is outlined, resulting in the generation of an IgA1/IgA2 hybrid molecule, here called IgA2.0. Based on rational protein design, three major modifications within an IgA2-EGFR mAb were introduced, which resulted in the stabilization of the molecule, enhanced production yields and an alteration of the glycosylation pattern. In Chapter 6, modification of the glycosylation pattern was achieved by producing the mAbs in cells which are overexpressing a2,3-sialyltransferase, an enzyme which attaches terminal sialyl residues to glycan structures. For both studies we were able to show that the engineered versions exhibited an enhanced in vivo half-life in mice compared to the unmodified parental mAbs. 

In conclusion, the presented studies nicely show that the half-life of IgA mAbs can be altered by (A) targeting the FcRn recycling pathway, and (B) reducing the ASGP-R mediated clearance by modifying the glycosylation pattern. The combination of these findings will facilitate the design of an optimally half-life extended IgA molecule.

The second part of this thesis describes the generation of unique IgA antibodies targeting hematological tumors. CD20 is an established target for mAb-mediated immunotherapy. CD20 mAb treatment of patients with e.g. B cell lymphomas results in the removal of cancerous and healthy B cells. However, as CD20 is not expressed on hematopoetic stem cells, the precursors of all kind of blood cells, the formation of new blood cells is not impacted. We first raised CD20 specific antibodies using an in-house immunization technology. In Chapter 7a, the extensive characterization of this new panel of mouse IgG mAbs is described and gives more insight into the possible relationships between Ab properties and effector functions. In vitro and in vivo killing capacity of a subpanel was assessed, and promising candidates were chimerized (a method to reduce the immunogenicity of the molecule by replacing big parts of the mouse sequences with the human counterpart). Chapter 7b summarizes their functional characterization. Here, tumor cell killing by complement activation and immune cell engagement was assessed. The functionality between the newly generated IgG1-CD20 mAbs and the parental mouse mAbs differed. Based on this observation we concluded that the selection of Ab candidates for further development can be based on properties like affinity obtained from the mouse Ab, however anti-tumor responses have to be evaluated after each modification. Using the knowledge obtained in Chapter 7a and 7b, we selected our lead candidates for the generation of unique IgA-CD20 mAbs (Chapter 8). The evaluation of preliminary in vitro characterization highlights the promising therapeutic potential of these unique IgA-CD20 mAbs. 

In the final chapter, Chapter 9, we discuss the results and conclusions obtained during the course of these research projects and propose future perspectives. The content of this stresses largely the need to thoroughly evaluate every step in the process of antibody development, from basic engineering approaches to in vitro characterization and testing in in vivo models suitable to answer the research question. Further, the results obtained here represent an excellent basis for future research in the field of IgA-mediated tumor therapy.

Why we think IgA antibodies are a good alternative to IgG antibodies for cancer therapy:
- Engagement of a unique subset of effector cells which express the Fcα receptor = neutrophils
- No Fc receptors known which reduce the immune response mediated by the effector cells (i.e. no inhibitory receptors; known within the Fcγ receptor family)
- Within the Fcα receptor no genetic variations (polymorphisms) have been described which alter the interaction between antibody and Fc receptor (known for Fcγ receptors)