title: Functionality-Enhanced Antibodies for Precision Cancer Therapy
Creators: name: Schoenfeld, Katrin
Publisher: name: UNSPECIFIED
Year: 2024

description: In recent decades, monoclonal antibodies have emerged as powerful biologics that have revolutionized the treatment of various diseases, including cancer. Nevertheless, ongoing research efforts are being made to enhance antibody functionality, enabling the generation of more precise and effective antibody therapeutics. 
The first study within this cumulative thesis focused on the development of a conditionally activated anti-IgM antibody-drug conjugate for precise targeting of B cell lymphoma. To this end, a chicken-derived IgM-specific antibody (aIgM) was isolated and fused to the epitope-bearing IgM domain CH2 as an affinity-based masking unit through a tumor protease-sensitive linker. The designed CH2-masked aIgM antibody displayed no interaction with IgM from human serum, while protease treatment of the CH2-aIgM restored the antigen binding functionality. On the cellular level, CH2-aIgM was also inert to interactions with IgM-positive lymphoma B cells, whereas the protease-cleaved variant demonstrated excellent on-cell affinities in the single-digit nanomolar range, comparable to the parental unmasked antibody. The aIgM antibody variants were subsequently coupled to the cytotoxic drug monomethyl auristatin E (MMAE) under generation of antibody-drug conjugates. Specific and effective receptor-mediated cellular uptake of the aIgM antibody was closely associated with the induction of apoptotic lymphoma cell death. Hence, cytotoxicity of the inactive CH2-aIgM antibody-drug conjugate was reduced to a minimum. As off-target effects limit the therapeutic potency of antibodies, the masked aIgM antibody-drug conjugate design ensures diffusion in the systemic blood circulation without being captured by secreted, pentameric IgM molecules or affecting non-malignant IgM-positive B cells. Upon reaching the tumor site, protease-mediated linker hydrolysis results in antibody-drug conjugate activation followed by cancer cell death, thereby expanding the therapeutic index determined by drug toxicity relative to drug effectivity.
The objective of the second project involved the generation of T cell receptor-directed antibody-drug conjugates for the treatment of T cell-derived cancers. Given the unfeasibility of targeting pan-T cell antigens as this is associated with the risk of life-threatening T cell aplasia, a tumor-specific antibody was devised by addressing the clonally rearranged T cell receptor (TCR) of a malignant T cell population. The isolation of an anti-TCR (aTCR) idiotype antibody from a chicken immune library, was followed by conjugation to the cytotoxic microtubule-inhibitor MMAE via a cleavable linker, resulting in the generation of antibody-drug conjugates with drug-to-antibody ratios of two and five on average, respectively. The unconjugated antibody exhibited exceptional specificity and affinity in the single digit nanomolar range for the target TCR on molecular and cellular level. Primarily based on the induction of apoptotic cell death, the aTCR antibody-drug conjugates revealed potent in vitro anti-tumor activity. Following the cellular release of cytotoxin, anti-proliferative bystander effects were observed on non-tumor cells, which may further contribute to aTCR antibody-drug conjugates’ tumoricidal properties. This novel, tailored approach facilitates efficient elimination of lymphoma/leukemia T cells, while preserving the T cell repertoire responsible for an intact cellular immunity.

The third part of this thesis explored the development of bispecific killer cell engagers (BiKEs), which employ species cross-reactive NKG2D binders to redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies. Recruitment of natural killer (NK) cells via the activating natural-killer group 2, member D (NKG2D) receptor represents an emerging strategy for cancer therapy, harnessing the cytotoxic immune effector function of NK cells. The generation of cross-reactive antibodies to human and murine NKG2D was achieved through a series of consecutive chicken immunizations with the receptors derived from both species, followed by antibody library screening. Subsequently, bispecific tetravalent antibodies were assembled from a panel of four isolated anti-NKG2D single-chain variable fragments (scFvs) combined with a previously described tumor-targeting anti-ErbB2/HER2 moiety. The four devised BiKE molecules (termed scNKAB-ErbB2) demonstrated binding specificity and affinity to HER2/ErbB2-expressing cancer cells as well as NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D, and NKG2D-positive primary human and murine lymphocytes. Competition of the scNKAB-ErbB2 with the natural NKG2D ligand MICA was investigated, and two of the entities were found to behave competitively to MICA, thereby preventing immune escape mechanisms of tumors. Ultimately, all four BiKEs proved effective in specifically redirecting the cytotoxic activity of human and murine NKG2D CAR-engineered NK-92 cells and primary human and murine lymphocytes to ErbB2-positive cancer cells. The designed BiKEs represent novel molecules with the potential to effectively mediate NKG2D-dependent NK cell cytotoxicity against tumor cells, and with implemented immune receptor species cross-reactivity facilitating the preclinical development in immunocompetent mouse tumor models. 
In summary, the projects presented within this thesis intended to devise functionality-enhanced antibodies for precision cancer therapy. The incorporation of novel, advanced modifications into antibodies, such as conditionally released masking units, highly potent cytotoxic drugs, custom-made specificities as well as additional specificities effectively recruiting immune effector cell mechanisms, may pave the way for the next generation of antibody-based cancer therapeutics.