Antibody engineering for tumor immunotherapy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2002.Vita.Includes bibliographical references (leaves 130-140).Antibodies have been used as cancer therapeutics for several decades. One area in which this therapy may be improved is the retention time of antibody in the tumor relative to normal tissue. In this Thesis, we have attempted to elucidate the mechanisms that are most influential to improving antibodies as cancer therapeutics. Carcinoembryonic antigen (CEA) has long been identified as a tumor-associated antigen. CEA is also quite stable, with a cell-surface shedding half-life of approximately 7 days. Directed evolution methodology has been utilized to design an antibody fragment with properties that would improve tumor retention. Specifically, antibody engineering methods were used to produce a humanized, extremely high affinity and stable single chain antibody fragment (scFv) against CEA. Several mutant scFv libraries were constructed and screened against soluble CEA with yeast surface display and fluorescent activated cell sorting (FACS). A series of antibodies were engineered that span three orders of magnitude in off-rate improvement. These antibody fragments show excellent stability at physiologically relevant temperatures. In addition, soluble protein expression levels were greatly improved. The final product has a dissociation half-life of approximately 7 days, currently the longest engineered half-life of an scFv against a tumor-associated antigen. Binding and diffusion in micrometastases was also modeled to gain an improved understanding of the quantitative interplay among the rate processes of diffusion, binding, degradation, and plasma clearance in tumor microspheroids.(cont.) Modeling studies illuminated the importance of targeting stable tumor-associated antigens. The elimination rate of the antigen was of critical importance to the change in the therapeutic effect of antibodies with increasing affinity. The significance of this result in the context of previous experimental studies will be discussed. By affinity maturing an antibody with a dissociation half-life equal to the turnover half-life of the antigen, we have engineered an antibody with effectively irreversible binding to CEA. Differences in retention for the series of scFvs will thus be dominated by the off-rate of the antibody and not the half-life of CEA. With this in mind, the molecules designed in this study can be used to reconcile the issue of affinity's impact on efficacy in tumor therapy.by Christilyn Paula Graff.Ph.D

Rodent EAE model for the study of axon integrity and remyelination

Myelin oligodendrocyte glycoprotein (MOG)-induced murine experimental autoimmune encephalomyelitis (EAE) is a widely accepted model for studying the clinical and pathological features of multiple sclerosis (MS). The model has previously been used to demonstrate that fostering remyelination can be effective in moderating disease progression. Approaches to enhance myelination include the transplantation of OPCs, Schwann cells, olfactory ensheathing cells, or neural stem cells into the primary demyelinated lesions1, or the promotion of oligodendrocyte precursor cell (OPC) differentiation2-5, such as by LINGO-1 antagonism6,7. Functional recovery from demyelination can be assessed via approaches for the histological preparation of tissues for light microscopy, magnetic resonance DTI imaging, and electron microscopy, as presented in this protocol

LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis

Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current treatments therefore target anti-inflammatory mechanisms to impede or slow disease progression. The identification of a means to enhance axon myelination would present new therapeutic approaches to inhibit and possibly reverse disease progression. Previously, LRR and Ig domain-containing, Nogo receptor-interacting protein (LINGO-1) has been identified as an in vitro and in vivo negative regulator of oligodendrocyte differentiation and myelination. Here we show that loss of LINGO-1 function by Lingo1 gene knockout or by treatment with an antibody antagonist of LINGO-1 function leads to functional recovery from experimental autoimmune encephalomyelitis. This is reflected biologically by improved axonal integrity, as confirmed by magnetic resonance diffusion tensor imaging, and by newly formed myelin sheaths, as determined by electron microscopy. Antagonism of LINGO-1 or its pathway is therefore a promising approach for the treatment of demyelinating diseases of the CNS. © 2007 Nature Publishing Group.link_to_subscribed_fulltex

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub-nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors

Characterization of Inhibitory Anti-insulin-like Growth Factor Receptor Antibodies with Different Epitope Specificity and Ligand-blocking Properties: IMPLICATIONS FOR MECHANISM OF ACTION IN VIVOS⃞

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 μm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy