Humanization of an anti-mucin antibody for breast and ovarian cancer therapy.

Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product.

Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the zeta-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcgamma or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the zeta-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with zeta-chain intracellular signaling domains only. IL-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3'-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.