Functional relevance of <i>in vivo</i> half antibody exchange of an IgG4 therapeutic antibody-drug conjugate

<div><p>An increasing number of monoclonal antibodies and derivatives such as antibody-drug conjugates (ADC) are of the IgG1 and IgG4 isotype with distinct structural and functional properties. In cases where antibody-mediated cytotoxicity is not desired, IgG4 is often used, as its Fc region is relatively poor at inducing antibody-dependent cell-mediated or complement-dependent cytotoxicity. IgG4 ADCs with highly cytotoxic drugs against proliferating target cells but which lack or have diminished antibody effector functions against quiescent cells may have a favorable safety profile compared to IgG1. Another unique property of the IgG4 subclass is the capability to exchange half antibodies <i>in vivo</i> creating randomly bispecific antibodies. To investigate the functional properties of process-derived antibody species, and determine the influence of shuffling on the therapeutic efficacy, several model antibodies on the basis of the anti-CD138 antibody-drug conjugate BT062 (Indatuximab ravtansine) were generated: (I) A wild type nBT062, (II) a stable nBT062 comprising mutations to prevent half-antibody exchange, (III) a half nBT062 lacking covalent binding between two heavy chains and (IV) a stabilized, bispecific nBT062-natalizumab antibody with a second, monovalent specificity against CD49d. All nBT062 model variants were capable of CD138-specific binding and antigen-mediated internalization into cells. Furthermore, all nBT062 models inhibited tumor growth <i>in vitro</i> after conjugation with the maytansinoid DM4. The <i>in vivo</i> effects of the different molecular variants were assessed in the MAXF1322 xenograft model. The bispecific nBT062-natalizumab-DM4 demonstrated the least efficacy and was only moderately active even without the co-administration of a human IgG preparation. Wild type, stable and half nBT062-DM4 models demonstrated great anti-tumor activities. The efficacy of wild type and half nBT062-DM4 was reduced in the presence of IgG, while stable nBT062-DM4 was only marginally influenced. These pre-clinical data demonstrate the advantage of introducing half-antibody exchange-preventing mutations into therapeutic IgG4-based antibody drug-conjugates.</p></div

Targeting the Biophysical Properties of the Myeloma Initiating Cell Niches: A Pharmaceutical Synergism Analysis Using Multi-Scale Agent-Based Modeling

<div><p>Multiple myeloma, the second most common hematological cancer, is currently incurable due to refractory disease relapse and development of multiple drug resistance. We and others recently established the biophysical model that myeloma initiating (stem) cells (MICs) trigger the stiffening of their niches <i>via</i> SDF-1/CXCR4 paracrine; The stiffened niches then promote the colonogenesis of MICs and protect them from drug treatment. In this work we examined <i>in silico</i> the pharmaceutical potential of targeting MIC niche stiffness to facilitate cytotoxic chemotherapies. We first established a multi-scale agent-based model using the Markov Chain Monte Carlo approach to recapitulate the niche stiffness centric, pro-oncogenetic positive feedback loop between MICs and myeloma-associated bone marrow stromal cells (MBMSCs), and investigated the effects of such intercellular chemo-physical communications on myeloma development. Then we used AMD3100 (to interrupt the interactions between MICs and their stroma) and Bortezomib (a recently developed novel therapeutic agent) as representative drugs to examine if the biophysical properties of myeloma niches are drugable. Results showed that our model recaptured the key experimental observation that the MBMSCs were more sensitive to SDF-1 secreted by MICs, and provided stiffer niches for these initiating cells and promoted their proliferation and drug resistance. Drug synergism analysis suggested that AMD3100 treatment undermined the capability of MICs to modulate the bone marrow microenvironment, and thus re-sensitized myeloma to Bortezomib treatments. This work is also the first attempt to virtually visualize in 3D the dynamics of the bone marrow stiffness during myeloma development. In summary, we established a multi-scale model to facilitate the translation of the niche-stiffness centric myeloma model as well as experimental observations to possible clinical applications. We concluded that targeting the biophysical properties of stem cell niches is of high clinical potential since it may re-sensitize tumor initiating cells to chemotherapies and reduce risks of cancer relapse.</p></div

Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis

Despite the fundamental importance of proteasomal degradation in cells, little is known about whether and how the 26S proteasome itself is regulated in coordination with various physiological processes. Here we show that the proteasome is dynamically phosphorylated during the cell cycle at Thr 25 of the 19S subunit Rpt3. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrate that blocking Rpt3-Thr25 phosphorylation markedly impairs proteasome activity and impedes cell proliferation. Through a kinome-wide screen, we have identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates Rpt3-Thr25, leading to enhanced substrate translocation and degradation. Importantly, loss of the single phosphorylation of Rpt3-Thr25 or knockout of DYRK2 significantly inhibits tumour formation by proteasome-addicted human breast cancer cells in mice. These findings define an important mechanism for proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis