A Bispecific γδ T-cell Engager Targeting EGFR Activates a Potent Vγ9Vδ2 T cell-Mediated Immune Response against EGFR-Expressing Tumors.

Vγ9Vδ2 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing Vγ9Vδ2 T cells to EGFR-expressing tumors. An EGFR-Vδ2 bispecific T-cell engager (bsTCE) was generated, and its capacity to activate Vγ9Vδ2 T cells and trigger antitumor activity was tested in multiple in vitro, in vivo, and ex vivo models. Studies to explore safety were conducted using cross-reactive surrogate engagers in nonhuman primates (NHP). We found that Vγ9Vδ2 T cells from peripheral blood and tumor specimens of patients with EGFR+ cancers had a distinct immune checkpoint expression profile characterized by low levels of PD-1, LAG-3, and TIM-3. Vγ9Vδ2 T cells could be activated by EGFR-Vδ2 bsTCEs to mediate lysis of various EGFR+ patient-derived tumor samples, and substantial tumor growth inhibition and improved survival were observed in in vivo xenograft mouse models using peripheral blood mononuclear cells (PBMC) as effector cells. EGFR-Vδ2 bsTCEs exerted preferential activity toward EGFR+ tumor cells and induced downstream activation of CD4+ and CD8+ T cells and natural killer (NK) cells without concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs. Administration of fully cross-reactive and half-life extended surrogate engagers to NHPs did not trigger signals in the safety parameters that were assessed. Considering the effector and immune-activating properties of Vγ9Vδ2 T cells, the preclinical efficacy data and acceptable safety profile reported here provide a solid basis for testing EGFR-Vδ2 bsTCEs in patients with EGFR+ malignancies.

A bispecific T cell engager recruits both type 1 NKT and Vγ9Vδ2-T cells for the treatment of CD1d-expressing hematological malignancies.

Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory T cells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proinflammatory cytokine production, effector cell expansion, and target cell lysis in vitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.

High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe.

Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon internalization into the acidic environment of endocytic organelles, whereas background fluorescence of noninternalized CypHer5E is minimal. The pH-dependency of the CypHer5E signal enables robust discrimination of antibody internalization from surface binding. The favorable signal-over-background ratio allows a homogeneous assay design with high-throughput fluorescence imaging in 384- and 1536-well formats. The biophysical readout of the primary internalization event substantially shortens incubation times compared to killing assays using toxin internalization. The assay was validated with tumor-relevant targets, including receptor tyrosine kinases (EGFR and HER2) and a class II cytokine receptor (TF) expressed by A431, AU565, and SKOV-3 cells and transient expression systems (CHO-S). Our method enables functional screening of large antibody libraries to identify therapeutic antibody candidates with internalization characteristics favorable for the development of ADCs.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling.

BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

Sequential recruitment of the repair factors during NER: the role of XPG in initiating the resynthesis step.

To address the biochemical mechanisms underlying the coordination between the various proteins required for nucleotide excision repair (NER), we employed the immobilized template system. Using either wild-type or mutated recombinant proteins, we identified the factors involved in the NER process and showed the sequential comings and goings of these factors to the immobilized damaged DNA. Firstly, we found that PCNA and RF-C arrival requires XPF 5' incision. Moreover, the positioning of RF-C is facilitated by RPA and induces XPF release. Concomitantly, XPG leads to PCNA recruitment and stabilization. Our data strongly suggest that this interaction with XPG protects PCNA and Pol delta from the effect of inhibitors such as p21. XPG and RPA are released as soon as Pol delta is recruited by the RF-C/PCNA complex. Finally, a ligation system composed of FEN1 and Ligase I can be recruited to fully restore the DNA. In addition, using XP or trichothiodystrophy patient-derived cell extracts, we were able to diagnose the biochemical defect that may prove to be important for therapeutic purposes.

Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.

Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.

Fate of RNA polymerase II stalled at a cisplatin lesion.

Elongating RNA polymerase II blocked by DNA damage in the transcribed DNA strand is thought to initiate the transcription-coupled repair process. The objective of this study is to better understand the sequence of events that occurs during repair from the time RNA polymerase II first encounters the lesion. This study establishes that an immobilized DNA template containing a unique cisplatin lesion can serve as an in vitro substrate for both transcription and DNA repair. RNA polymerase II is quantitatively stalled at the cisplatin lesion during transcription and can be released from the template, along with the nascent transcript, in an ATP-dependent manner. RNA polymerase II stalled at a lesion and containing a dephosphorylated repetitive carboxyl-terminal domain (CTD) appears to be more sensitive toward release. However, a dephosphorylated CTD can become readily phosphorylated in front of the lesion by CTD kinases in the presence of ATP. The observation that RNA polymerase II and transcript release occurs in a TFIIH-deficient repair extract but not in a reconstituted repair system demonstrates that disassembly of the elongation complex can occur independently of the repair process and vice versa. Indeed, the presence of RNA polymerase II at the lesion does not prevent dual incision from occurring. Finally, we also propose that the Cockayne's syndrome B protein factor, believed to be the mammalian transcription repair coupling factor, is neither involved in transcript release nor required for dual incision in the presence of lesionstalled RNA polymerase II in vitro. More likely, it prevents RNA polymerase from backing up when it encounters the lesion. The ability to transcribe and repair the same damaged DNA molecule fixed on beads, along with the fact that the reaction conditions can be freely altered, provides a powerful tool to study the fate of RNA polymerase II blocked on the cisplatin lesion.

The comings and goings of nucleotide excision repair factors on damaged DNA.

To understand the mechanism of nucleotide excision repair (NER), one of the major human DNA repair pathways, we have set up a DNA repair system in which a linear damaged DNA substrate is immobilized by its terminus. By isolating functionally active intermediate complexes, our data dissect the ordered arrival and displacement of NER factors in the progress of the dual incision step. We describe (i) the role of ATP in remodelling the NER-initiating complex of XPC/TFIIH/damaged DNA as a prerequisite for the recruitment of the next NER factors; (ii) the coordination between damage removal and DNA resynthesis and the release of XPC-HR23B, TFIIH and XPA upon arrival of XPG and XPF-ERCC1, respectively; (iii) how RPA remains associated with the excised DNA initiating the assembly of resynthesis factors such as PCNA; (iv) the recycling of XPC-HR23B, TFIIH and XPA in the NER; and the shuttling of TFIIH between NER and transcription. Thus, our findings define multiple functions of NER factors to explain the molecular basis of human NER disorders.