Ongoing research investigating new therapeutics continues to improve survival rates and quality of life for cancer patients. The use of therapeutic antibodies as immunotherapy for hematologic cancers and solid tumors has shown promise as an effective treatment strategy. This is due, in part, to the greater specificity of monoclonal antibodies (mAbs) compared to other treatments. There are hundreds of clinical trials underway to examine the efficacy of mAbs for treating various forms of cancer. Next-generation antibodies including antibody-drug conjugates (ADCs), engineered antibodies, and bispecific antibodies, are a key area of research and development, with potentially significant clinical implications. However, prior to entering the clinical setting, mAb development begins with significant pre-clinical research. Successful mAb discovery and development relies on information-rich technologies for rapid identification and characterization of candidate molecules.
Antibody-Drug Conjugates Deliver Potent Agents
Antibody-drug conjugates (ADCs) consist of a mAb attached to a biologically active drug with a linker that is stable in the circulation. The highly selective mAb targets tumor-associated antigens, enabling delivery of chemotherapeutic agents, some of which have high systemic toxicity. Upon binding, the ADC is internalized and the cytotoxic agents are released by lysosomal degradation. ADC development is complex, as it is crucial to ensure rapid internalization to prevent systemic release of cytotoxic agents, however this may dampen antibody-dependent cell-mediated cytotoxicity (ADCC). As such, it is necessary to evaluate internalization and ADCC, in addition to other cell health and phenotypic parameters during development.
Several ADCs have been approved by the Food and Drug Administration (FDA) for various indications. Among them, brentuximab vedotin is a CD30-specific mAb linked to an antimitotic agent. It is approved for classical Hodgkin lymphoma, systemic anaplastic large cell lymphoma, and other CD30-expressing T-cell lymphomas. For the treatment of HER2-positive metastatic breast cancer, ado-trastuzumab-emtansine targets HER2 and consists of the monoclonal antibody trastuzumab covalently linked to cytotoxic agent DM1.
Engineering for Enhanced Cytotoxicity
Monoclonal antibodies contain an antigen-binding fragment (Fab) and a crystallizable fragment (Fc). Many mAbs exert their effects through immune cell recruitment, which relies on the interaction between the Fc domain and its receptors. Fc engineering confers greater affinity for immune effector cells, resulting in greater cytotoxicity and phagocytosis. Glycoengineering and site mutagenesis methods modulate the Fc-FcγR (Fc region of IgG) and FcRn (neonatal Fc receptor) interactions. One such example is obintuzumab, a glycoengineered mAb FDA approved for use in combination with bendamustine and obinutuzumab monotherapy for patients with follicular lymphoma. It is a successor to the type I CD20 antibody, rituximab, and demonstrates enhanced binding affinity to the FcγRIII receptor on immune effector cells.
Antibody modifications achieved through Fc engineering can have significant effects on activity and specificity. Several assays are essential in the development of Fc engineered antibodies to assess antibodyreceptor binding affinity as well as Fc-mediated immune effector function. FcRn and FcγR binding are frequently assessed via ELISA and flow cytometry methods. ADCC and antibody-dependent cellular phagocytosis (ADCP) assays, along with complement-dependent cytotoxicity (CDC) assays provide insight into the mechanism of action.
Immune Cell Retargeting with Bispecific Mabs
The first description of bispecific antibodies dates back to the 1960s, when they were developed with the hybridoma technique. Unlike a monospecific antibody, that has two identical antigen-binding sites, bispecific antibodies have two unique antigen-binding sites. Bispecific mAbs are capable of binding a CD3 receptor to activate cytotoxic T lymphocytes on one antigen-binding site, and a tumor antigen (CD20, HER2, etc.) on the other, enabling cross-linking and simultaneous activation of tumor and effector cells. For example, catumaxomab, which now has FDA orphan drug status for EpCam positive gastric and ovarian tumors, binds EpCAM, CD3 on T cells, and the Fc portion recruits immune cells.
Bispecific antibodies pose several challenges for development. It is necessary to determine which Fab interfaces may be combined without compromising function or affinity of either arm, and their specificity toward multiple targets necessitates substantial characterization to ensure efficacy. In addition to binding affinity and specificity, assays are necessary to determine target cell lysis by effector cells activated by the mAb.
In-Depth Insights for Next-Generation Antibody Development
Developing novel next-generation antibodies is a complex process that requires in-depth analysis and characterization of candidate molecules to identify optimal candidates. Often, this is achieved with time-consuming assays to assess phenotype, activation, cytotoxicity, cell health, etc. New technology has improved the speed and accuracy of analyses to identify candidates with the desired biological outcome based on multiple parameters. The IntelliCyt iQue3 with ForeCyt® software enables simultaneous analysis of cell-mediated cytotoxicity, cell health, function, activation, and more in each microplate well. The ability to multiplex assays (such as ADCC and bead-based assays to measure secreted cytokines), as well as rapidly analyze antibody specificity and EC50 measurements, dramatically increases throughput. Offering improved speed and greater biological insights, Sartorius solutions accelerate next-generation mAb therapy discovery and development.
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