The RNA-binding protein La/SSB associates with radiation-induced DNA double-strand breaks in lung cancer cell lines

First published: 12 October 2021Abstract Background: Platinum-based chemotherapy and radiotherapy are standard treatments for non-small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment-induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies. Aim: In this study, we examined radiation-induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB. Method: Three lung cancer lines (A549, H460 and LL2) were irradiated with different X-ray doses or X-radiated with a 5 Gy dose and examined at different time-points postirradiation for DNA DSB in the form of γ-H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ-H2AX co-localise and performed proximity ligation assay (PLA) and co-immunoprecipitation to confirm the interaction of these proteins. Results: We found that the radio-resistant A549 cell line compared to the radiosensitive H460 cell line showed faster resolution of radiation-induced γ-H2AX foci over time. Conversely, we found more co-localised γ-H2AX and La/SSB foci by PLA in irradiated A549 cells. Conclusion: The co-localisation of La/SSB with radiation-induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate thisAlexander H. Staudacher, Yanrui Li, Vasilios Liapis, Michael P. Brow

Improved non-invasive positron emission tomographic imaging of chemotherapy-induced tumor cell death using Zirconium-89-labeled APOMAB®

Purpose: The chimeric monoclonal antibody (mAb) chDAB4 (APOMAB®) targets the Lupus associated (La)/Sjögren Syndrome-B (SSB) antigen, which is over-expressed in tumors but only becomes available for antibody binding in dead tumor cells. Hence, chDAB4 may be used as a novel theranostic tool to distinguish between responders and nonresponders early after chemotherapy. Here, we aimed to ascertain which positron emitter, Zirconium-89 ([⁸⁹Zr]Zr(IV)) or Iodine-124 ([¹²⁴I]I), was best suited to label chDAB4 for post-chemotherapy PET imaging of tumor-bearing mice and to determine which of two different bifunctional chelators provided optimal tumor imaging by PET using [⁸⁹Zr]Zr(IV)-labeled chDAB4. Methods: C57BL/6 J mice bearing subcutaneous syngeneic tumors of EL4 lymphoma were either untreated or given chemotherapy, then administered radiolabeled chDAB4 after 24 h with its biodistribution examined using PET and organ assay. We compared chDAB4 radiolabeled with [⁸⁹Zr] Zr(IV) or [¹²⁴I] I, or [⁸⁹Zr]Zr-chDAB4 using either DFO-NCS or DFOSq as a chelator. Results: After chemotherapy, [⁸⁹Zr]Zr-chDAB4 showed higher and prolonged mean (± SD) tumor uptake of 29.5 ± 5.9 compared to 7.8 ± 1.2 for [¹²⁴I] I -chDAB4. In contrast, antibody uptake in healthy tissues was not affected. Compared to DFO-NCS, DFOSq did not result in significant differences in tumor uptake of [⁸⁹Zr]Zr-chDAB4 but did alter the tumor:liver ratio in treated mice 3 days after injection in favour of DFOSq (8.0 ± 1.1) compared to DFO-NCS (4.2 ± 0.7). Conclusion: ImmunoPET using chDAB4 radiolabeled with residualizing [⁸⁹Zr] Zr(IV) rather than [¹²⁴I] I optimized post-chemotherapy tumor uptake. Further, PET imaging characteristics were improved by DFOSq rather than DFO-NCS. Therefore, the radionuclide/chelator combination of [⁸⁹Zr] Zr(IV) and DFOSq is preferred for the imminent clinical evaluation of chDAB4 as a selective tumor cell death radioligand.Vasilios Liapis, William Tieu, Stacey E. Rudd, Paul S. Donnelly, Nicole L. Wittwer, Michael P. Brown, and Alexander H. Staudache

Fc gamma receptor is not required for in vivo processing of radio- and drug-conjugates of the dead tumor cell-targeting monoclonal antibody, APOMAB®

The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anti- cancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug con- jugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of 89Zr-labeled chDAB4. Similarly, no differences were observed between LL2 tumor-bearing wild-type and Fcγ KO mice in post-chemotherapy uptake of 89Zr-chDAB4. Also, the post-chemotherapy activity of a chDAB4-antibody drug conjugate (ADC) directed against LL2 tumors did not differ among tumor-bearing wild-type, Fcγ KO and NOTAM mice, nor did the proportions and characteristics of the LL2 tumor immune cell infiltrates differ significantly among these mice. In conclusion, Fc-FcγR interactions are not essential for the diagnostic or therapeutic applications of chDAB4 conjugates because the tumor-associated macrophages, which engulf the chDAB4-labelled dead cells, respond to endogenous ‘eat me’ signals rather than depend on functional FcγR expression for phagocytosis.Alexander H. Staudacher, Vasilios Liapis, Nicole L. Wittwer, William Tieu, Hiu Chun Lam, Jeanette Leusen, Michael P. Brow

Integrable Spin Chains with U(1)^3 symmetry and generalized Lunin-Maldacena backgrounds

We consider the most general three-state spin chain with U(1)^3 symmetry and nearest neighbour interaction. Our model contains as a special case the spin chain describing the holomorphic three scalar sector of the three parameter complex deformation of N=4 SYM, dual to type IIB string theory in the generalized Lunin-Maldacena backgrounds discovered by Frolov. We formulate the coordinate space Bethe ansatz, calculate the S-matrix and determine for which choices of parameters the S-matrix fulfills the Yang-Baxter equations. For these choices of parameters we furthermore write down the R-matrix. We find in total four classes of integrable models. In particular, each already known model of the above type is nothing but one in a family of such models.Comment: 16 pages, 3 figures, references correcte

Positron emission tomographic imaging of tumor cell death using zirconium-89-labeled APOMAB(R) following cisplatin chemotherapy in lung and ovarian cancer xenograft models

Published online 06 July 2021Purpose Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. Procedures In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [⁸⁹Zr]ZrIV. Tumor responses were monitored using bioluminescence imaging and caliper measurements. [⁸⁹Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. Results Tumor uptake of [⁸⁹Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. Conclusion Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.Vasilios Liapis, William Tieu, Nicole L. Wittwer, Tessa Gargett, Andreas Evdokiou, Prab Takhar, Stacey E. Rudd, Paul S. Donnelly, Michael P. Brown, Alexander H. Staudache

Tumour-associated macrophages process drug and radio-conjugates of the dead tumour cell-targeting APOMAB® antibody

APOMAB (chDAB4) is a dead tumour cell-targeting antibody which has been used preclinically as a diagnostic agent and therapeutically as a radioimmunotherapy and antibody drug conjugate (ADC). However, little is known of the intra-tumour processing of chDAB4 when bound to dead tumour cells. In this study we examine the role of macrophages in the in vitro and in vivo processing of radiolabelled chDAB4 and a chDAB4 ADC. We found that chDAB4 binds to macrophages in vitro, resulting in the killing of macrophages when using the ADC, chDAB4-SG3249. Free drug released by the macrophage processing of chDAB4-SG3249 could result in killing of 'bystander' Lewis lung (LL2) carcinoma cells. Furthermore, macrophages phagocytosed chDAB4-bound dead LL2 cells and were killed when they phagocytosed chDAB4-SG3249-bound dead LL2 cells in vitro. In vivo, we found markedly different tumour retention of chDAB4 in the LL2 tumour model depending on whether it was radiolabelled with a residualising radionuclide (⁸⁹Zr), which is retained intracellularly, or a non-residualising radionuclide (¹²⁴I), which can diffuse out of the cell. This prolonged retention of ⁸⁹Zr vs¹²⁴I indicated intra-tumoral processing of chDAB4 in vivo. The tumour uptake of 89Zr-chDAB4 was reduced after macrophage depletion, which also reduced the efficacy of the chDAB4 ADC in vivo. This study shows that macrophages can process chDAB4 and chDAB4 ADC in vitro and shows the importance of tumour-associated macrophages in the tumour retention of chDAB4 and the efficacy of chDAB4 ADC in vivo.Alexander H. Staudacher, Vasilios Liapis, William Tieu, Nicole L. Wittwer, Michael P. Brow

APOMAB antibody-drug conjugates targeting dead tumor cells are effective in vivo

"Published first November 9, 2018."Antibody-drug conjugates (ADC) have revolutionized the field of cancer therapy. ADCs combine the high specificity of tumor-targeting monoclonal antibodies with potent cytotoxic drugs, which cannot be used alone because of their high toxicity. Till date, all ADCs have either targeted cell membrane proteins on tumors or the tumor vasculature and microenvironment. Here, we investigate ADCs of APOMAB (DAB4, or its chimeric derivative, chDAB4), which is a mAb targeting the La/SSB protein, which is only accessible for binding in dying or dead cancer cells. We show that DAB4-labeled dead cells are phagocytosed by macrophages, and that the apoptotic/necrotic areas within lung tumor xenografts are bound by DAB4 and are infiltrated with macrophages. We show that only DAB4-ADCs with a cleavable linker and diffusible drug are effective in two lung cancer models, particularly when given after chemotherapy. These results are consistent with other recent studies showing that direct internalization of ADCs by target cells is not essential for ADC activity because the linker can be cleaved extracellularly or through other mechanisms. Rather than targeting a tumor cell type specific antigen, DAB4-ADCs have the advantage of targeting a common trait in most solid tumors: an excess of post-apoptotic, necrotic cells either adjacent to hypoxic tumor regions or distributed more generally after cytotoxic therapy. Consequently, any antitumor effects are solely the result of bystander killing, either through internalization of the dead, ADC-bound tumor cells by macrophages, or extracellular cleavage of the ADC in the tumor microenvironment.Alexander H. Staudacher, Yanrui Li, Vasilios Liapis, Jeff Jia Cheng Hou, David Chin, Olan Dolezal, Timothy E. Adams, Patrick H. van Berkel and Michael P. Brow