No abstract

The antibody-mediated delivery of therapeutic agents to sites of angiogenesis is an attractive strategy for anticancer therapy, but is largely unexplored in hematologic malignancies. In the present study, we show that the extra domain B (EDB) of fibronectin, a marker of angiogenesis, is expressed in B-cell non-Hodgkin lymphoma (NHL) and that the human monoclonal anti-EDB antibody L19 can selectively localize to the lymphoma-associated subendothelial extracellular matrix. In vivo, the preferential accumulation of the antibody at the tumor site was confirmed by quantitative biodistribution analyses with radioiodinated antibody preparations. The fusion protein L19-IL2, which mediates the delivery of interleukin-2 (IL-2) to the neovasculature, displayed a superior antilymphoma activity compared with unconjugated IL-2 in localized and systemic xenograft models of NHL. When coadministered with rituximab, L19-IL2 induced complete remissions of estab- IntroductionConventional cytotoxic therapies of cancer often do not discriminate between tumor and normal tissues. To achieve therapeutically relevant concentrations in the tumor mass, large drug doses have to be administered to the patient, leading to a poor therapeutic index and unacceptable toxicities to healthy tissues. The selective delivery of therapeutic agents to the tumor site using antibodies against tumor-associated antigens represents a promising strategy to overcome the disadvantages of conventional cancer therapies. 1-3 Antigens expressed in the tumoral neovasculature are especially attractive targets for antibody-based pharmacodelivery applications due to their inherent accessibility for blood-borne agents. [4][5][6] The efficacy of targeting either tumor endothelial cells or the modified subendothelial extracellular matrix has been demonstrated in a variety of animal models of solid cancers using antibodies functionalized with different effector moieties, 4,[7][8][9][10][11][12][13][14] leading to the clinical development of immunocytokines and radioimmunoconjugates for the therapy of solid tumors. 6,15 Tumortargeting strategies based on the preferential accumulation of biopharmaceuticals around new blood vessels could also be conceivable for the therapy of leukemias and lymphomas, since the dependence of hematologic malignancies on a functional neovasculature has been highlighted already a decade ago. 16,17 Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy, with now more than 60 000 new cases being diagnosed each year in the United States. 18 The approval of rituximab, a chimeric monoclonal immunoglobulin G1 (IgG1) antibody specific to CD20, represented a major step toward a more selective and effective therapy of NHLs of B cell origin. While first shown to be effective in relapsed follicular lymphoma, anti-CD20 immunotherapy is nowadays incorporated in front-line therapy schemes of follicular and diffuse large B-cell lymphoma. 18 However, in spite of the unquestionable clinical effectiveness of rituximab, a high percentage of patients ...

We describe a method that enables specific and efficient conjugation of hydrazide-moieties to an IgG targeting the tumor neovasculature. The resulting chemically defined, homogeneous hydrazone-linked IgG conjugates remain immunoreactive and have a half-life of approximately 18 hours at physiological pH and temperature suitable for localized delivery of toxic drugs.

The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering research.

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding causes uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.