Probody therapeutic design of 89Zr-CX-072 promotes accumulation in PD-L1 expressing tumors compared to normal murine lymphoid tissue

PURPOSE: Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti-PD-L1-mediated toxicities. To study its targeting of PD-L1-expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 (89Zr). EXPERIMENTAL DESIGN: 89Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis. RESULTS: PET imaging revealed 89Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89Zr-PbCtrl. Tumor tissue autoradiography showed high 89Zr-CX-072 uptake in high PD-L1-expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89Zr-CX-075, which lacks the Probody design. CONCLUSIONS: 89Zr-CX-072 accumulates specifically in PD-L1-expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491)

Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis

Understanding the role of dicer in astrocyte development.

The Dicer1 allele is used to show that microRNAs (miRNAs) play important roles in astrocyte development and functions. While it is known that astrocytes that lack miRNAs are dysregulated, the in vivo phenotypes of these astrocytes are not well understood. In this study, we use Aldh1l1-EGFP transgene, a marker of astrocytes, to characterize mouse models with conditional Dicer1 ablation (via either human or mouse GFAP-Cre). This transgene revealed novel features of the defective astrocytes from the absence of miRNA. Although astrocyte miRNAs were depleted in both lines, we found histological and molecular differences in the Aldh1l1-EGFP cells between the two Cre lines. Aldh1l1-EGFP cells from hGFAP-Cre mutant lines displayed up-regulation of Aldh1l1-EGFP with increased proliferation and a genomic profile that acquired many features of wildtype primary astrocyte cultures. In the young mGFAP-Cre mutant lines we found that Aldh1l1-EGFP cells were disorganized and hyperproliferative in the developing cerebellum. Using the Aldh1l1-EGFP transgene, our work provides new insights into the roles of miRNAs in astrocyte development and the features of astrocytes in these two mouse models

Understanding the Role of Dicer in Astrocyte Development

<div><p>The <i>Dicer1</i> allele is used to show that microRNAs (miRNAs) play important roles in astrocyte development and functions. While it is known that astrocytes that lack miRNAs are dysregulated, the <i>in vivo</i> phenotypes of these astrocytes are not well understood. In this study, we use Aldh1l1-EGFP transgene, a marker of astrocytes, to characterize mouse models with conditional <i>Dicer1</i> ablation (via either human or mouse GFAP-Cre). This transgene revealed novel features of the defective astrocytes from the absence of miRNA. Although astrocyte miRNAs were depleted in both lines, we found histological and molecular differences in the Aldh1l1-EGFP cells between the two Cre lines. Aldh1l1-EGFP cells from hGFAP-Cre mutant lines displayed up-regulation of Aldh1l1-EGFP with increased proliferation and a genomic profile that acquired many features of wildtype primary astrocyte cultures. In the young mGFAP-Cre mutant lines we found that Aldh1l1-EGFP cells were disorganized and hyperproliferative in the developing cerebellum. Using the Aldh1l1-EGFP transgene, our work provides new insights into the roles of miRNAs in astrocyte development and the features of astrocytes in these two mouse models.</p></div

Analysis of hGFAP:CKO mice using the Aldh1l1-EGFP transgene shows presence of dysregulated astrocytes with increase EGFP signals.

<p>(a and b) P16 hGFAP:CKO forebrains have decreased sizes. (c,d, and e) P16 cortex and (f, g, and h) spinal cord sections indicate the presence of Aldh1l1-EGFP positive cells with higher EGFP signal in the hGFAP:CKO CNS. (i) The increase in EGFP signals can be visualized using FACS (blue represents P16 wildtype and dotted black represents P16 hGFAP:CKO Aldh1l1-EGFP positive cells). X axis in i represents EGFP levels in log2 scale. Y axis represents cell numbers. Scale bars in a and b represent 5 mm. Scale bars in c, d, and e represent 50 μm. Scale bars in f, g, and h represent 100 μm. FACS data for panel i is representative of 3 experiments. All histological experiments are representative data based on 3 independent observations.</p

Pathway enriched in wildtype FACS cells compare to hGFAP:CKO FACS cells.

<p>Pathway enriched in wildtype FACS cells compare to hGFAP:CKO FACS cells.</p

Genes with >2-fold down-regulation in hGFAP:CKO Aldh1l1-EGFP that are also down-regulated in primary cultures.

<p>Genes with >2-fold down-regulation in hGFAP:CKO Aldh1l1-EGFP that are also down-regulated in primary cultures.</p

EGFP positive cells are disorganized and have increased EGFP signals in mGFAP:CKO cerebellums.

<p>(a) In symptomatic 2 month old mGFAP:CKO mice, massive cell loss was observed in mutant cerebellum (arrows indicate missing cells in cerebellar lobes). (b-d) EGFP positive cells had up-regulated signals and appeared in the molecular layers in the mutant cerebellums. (e) qRT-PCR also indicated up-regulated levels of <i>Aldh1l1</i> transcripts (p-value< 0.01, n = 3). (e) <i>Gfap</i> showed an upward trend suggesting astrogliosis (p-value = 0.05, n = 3). (e) <i>Aqp4</i> levels were similar between wildtype and mutant animals (p-value = 0.14, n = 3). (e) <i>Glast</i> level was decreased in mutant cerebellums (p-value < 0.01, n = 3). (f-h) In P16 mGFAP:CKO, defects with Aldh1l1-EGFP could already be observed in the cerebellum. Aldh1l1-EGFP cells were present in mutant molecular layer. (i-k) While ki67 positive cells were limited in wildtype cerebellum, we saw a noticeable increase in mutant cerebellum (ki67 cells are red). (l-m) These ki67 positive cells co-localized predominately with EGFP cells. <i>Aldh1l1</i> and <i>Gfap</i> transcripts were up-regulated while <i>Glast</i> had a small decrease expression level in mutant cerebellum. (o) No change was detected in <i>Aqp4</i> (<i>Aldh1l1</i> p-value< 0.01, <i>Aqp4</i> p-value = 0.16, <i>Gfap</i> p-value< 0.05, and <i>Glast</i> p-value< 0.05, n = 3). Scale bars in a-d and f-n represent 50 μm. Gray bars represent wildtype mice and yellow bars represent mGFAP:CKO mice in e and o. Asterisks in e and o indicate significant level (** represents p< 0.01 and * represents p< 0.05). Y axes in e and o represent the levels for gene of interest normalized to <i>Gapdh</i>. The average expression level of wildtype samples were normalized to 1. Unpaired t-test was used to determine p-values.</p

Genes with >2-fold up-regulation in hGFAPCKO Aldh1l1-EGFP that are also up- regulated in primary cultures.

<p>Genes with >2-fold up-regulation in hGFAPCKO Aldh1l1-EGFP that are also up- regulated in primary cultures.</p

qRT-PCR indicates that FACS isolated hGFAP:CKO Aldh1l1-EGFP cells exhibit immature molecular phenotype.

<p>qRT-PCR indicated mutant cells have significantly down-regulated expression of <i>Aqp4</i> and <i>Glast (p-value< 0</i>.<i>05)</i>. The expression of <i>Gfap</i> did not reach significant down-regulation (p-value of <i>Gfap</i> is 0.09). <i>Fgfr3</i> levels appeared similar and <i>Nes</i> levels were up-regulated (p-value < 0.05). As expected, an amplicon that detected exon 23 of <i>Dicer1</i> had dramatically reduced levels in the mutant cells (p-values < 0.01). Unpaired t-test was used to determine p-values. Gray bars represent wildtype and yellow bars represent hGFAP:CKO. Asterisks indicate significant level (** represents p< 0.01 and * represents p< 0.05). Y axis represents expression levels for the gene of interest normalized to <i>Gapdh</i>. The average expression level of wildtype samples were normalized to 1. N = 3</p