Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

An inhibitory antibody targeting carbonic anhydrase XII abrogates chemoresistance and significantly reduces lung metastases in an orthotopic breast cancer model in vivo.

Carbonic anhydrase XII (CAXII) is a membrane-tethered ectoenzyme involved in intracellular pH regulation and overexpressed across various types of human cancer. Because CAXII inhibition shows antitumor activity in vitro, it is thought that the enzyme is mandatory for maximum tumor growth, above all under hypoxic conditions. Recently, it has been shown that CAXII is co-expressed along with the P-glycoprotein (P-GP) on many tumor cells and that both proteins physically interact. Of interest, blocking CAXII activity also decreases P-GP activity in cancer cells both in vitro and in vivo. Previously, we have reported on the development of a monoclonal antibody, termed 6A10, which specifically and efficiently blocks human CAXII activity. Here, we demonstrate that 6A10 also indirectly reduces P-GP activity in CAXII/P-GP double-positive chemoresistant cancer cells, resulting in enhanced chemosensitivity as revealed by enhanced accumulation of anthracyclines and increased cell death in vitro. Even more important, we show that mice carrying human triple-negative breast cancer xenografts co-treated with doxorubicin (DOX) and 6A10 show a significantly reduced number of metastases. Collectively, our data provide evidence that the inhibition of CAXII with 6A10 is an attractive way to reduce chemoresistance of cancer cells and to interfere with the metastatic process in a clinical setting.

A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape.

CD73 catalyzes the conversion of ATP to adenosine, which is involved in various physiological and pathological processes, including tumor immune escape. Because CD73 expression and activity are particularly high on cancer cells and contribute to the immunosuppressive properties of the tumor environment, it is considered an attractive target molecule for specific cancer therapies. In line, several studies demonstrated that CD73 inhibition has a significant antitumor effect. However, complete blocking of CD73 activity can evoke autoimmune phenomena and adverse side effects. We developed a CD73-specific antibody, 22E6, that specifically inhibits the enzymatic activity of membrane-tethered CD73 present in high concentrations on cancer cells and cancer cell-derived extracellular vesicles but has no inhibitory effect on soluble CD73. Inhibition of CD73 on tumor cells with 22E6 resulted in multiple effects on tumor cells in vitro, including increased apoptosis and interference with chemoresistance. Intriguingly, in a xenograft mouse model of acute lymphocytic leukemia (ALL), 22E6 treatment resulted in an initial tumor growth delay in some animals, followed by a complete loss of CD73 expression on ALL cells in all 22E6 treated animals, indicating tumor immune escape. Taken together, 22E6 shows great potential for cancer therapy, favorably in combination with other drugs.

Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA.

Autoantibodies against double-stranded DNA (anti-dsDNA) are a hallmark of systemic lupus erythematosus (SLE). It is well documented that anti-dsDNA reactive B lymphocytes are normally controlled by immune self-tolerance mechanisms operating at several levels. The evolution of high levels of IgG anti-dsDNA in SLE is dependent on somatic hypermutation and clonal selection, presumably in germinal centers from non-autoreactive B cells. Twin studies as well as genetic studies in mice indicate a very strong genetic contribution for the development of anti-dsDNA as well as SLE. Only few single gene defects with a monogenic Mendelian inheritance have been described so far that are directly responsible for the development of anti-dsDNA and SLE. Recently, among other mutations, rare null-alleles for the deoxyribonuclease 1 like 3 (DNASE1L3) and the Fc gamma receptor IIB (FCGR2B) have been described in SLE patients and genetic mouse models. Here, we demonstrate that double Dnase1l3- and FcgR2b-deficient mice in the C57BL/6 background exhibit a very early and massive IgG anti-dsDNA production. Already at 10 weeks of age, autoantibody production in double-deficient mice exceeds autoantibody levels of diseased 9-month-old NZB/W mice, a long established multigenic SLE mouse model. In single gene-deficient mice, autoantibody levels were moderately elevated at early age of the mice. Premature autoantibody production was accompanied by a spontaneous hyperactivation of germinal centers, early expansions of T follicular helper cells, and elevated plasmablasts in the spleen. Anti-dsDNA hybridomas generated from double-deficient mice show significantly elevated numbers of arginines in the CDR3 regions of the heavy-chain as well as clonal expansions and diversification of B cell clones with moderate numbers of somatic mutations. Our findings show a strong epistatic interaction of two SLE-alleles which prevent early and high-level anti-dsDNA autoantibody production. Both genes apparently synergize to keep in check excessive germinal center reactions evolving into IgG anti-dsDNA antibody producing B cells.

Tumor-derived extracellular vesicles activate primary monocytes.

Tumor cells educate immune effector cells in their vicinity by releasing factors that manipulate their phenotype and function. In fact, the thus generated immunosuppressive tumor microenvironment constitutes an integral part and a hallmark of solid tumors and contributes significantly to tumor development and immune escape. It has long been thought that soluble factors like prostaglandin E2 and TGF-ß are the main mediators of these effects. But tumor cells also constantly release large number of extracellular vesicles (EVs), which are important conveyors of immune responses. We show here that tumor-derived EVs interact with primary monocytes and induce an activated phenotype, which is also observed in tumor-associated macrophages. Thus, both tumor-derived EVs and soluble factors together collaborate to form the immunosuppressive milieu of the tumor environment.