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Towards translational ImmunoPET/MR imaging of invasive Pulmonary Aspergillosis: The Humanized Monoclonal Antibody JF5 detects Aspergillus Lung Infections in Vivo
Antibody Humanization service
Fusion Antibodies has the knowledge and expertise to build and deliver a tailored package of the services required to achieve outstanding results. With over >110 humanization projects successfully completed to date (from Rabbit, Rat, Mouse, Chicken, Llama), including multiple antibodies in clinical trials.
In this case study example Fusion Antibodies provided a tailored package of Antibody Humanization services to successfully humanize murine mJF5 for the University of Exeter spin-out company ISCA Diagnostics Ltd. (www.iscadiagnostics.com).
The Humanized Monoclonal Antibody JF5 detects Aspergillus Lung Infections in Vivo
Invasive pulmonary aspergillosis (IPA)is a frequently fatal lung disease of neutropenic patients caused by the ubiquitous airborne fungusAspergillus fumigatus. As a leading cause of death in haematological malignancy and hematopoietic stem cell transplant patients, the fungus accounts for the majority of the >200,000 life threatening infections annually with an associated mortality rate of 30–90%.
Diagnosis of IPA is a major challenge as clinical manifestations of the disease are nonspecific, and methods for the detection of circulating biomarkers such as β-D-glucan or galactomannan (GM) in the bloodstream lack specificity or sensitivity. For this reason, culture of the fungus from lung biopsy tissues remains the gold standard test for IPA diagnosis, but this invasive procedure lacks sensitivity, delays diagnosis, and is frequently not possible in already critically ill patients.
In a previous study (Rolle et al.  Proceedings of the National Academy of Sciences of the United States of America 113 (8): E1026-E1033), ISCA Diagnostics and co-workers developed a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (ImmunoPET/MRI) using a [64Cu]DOTA-labelled mouse monoclonal antibody (mAb), mJF5. The highly specific tracer allows repeated imaging of A. fumigatus lung infections and differentiation of IPA from pulmonary inflammation and from infections caused by bacteria.
To enable translation of the tracer to a clinical setting, ISCA Diagnostics’ aim was to develop a humanised version of the antibody JF5 (hJF5), and to evaluate its performance in pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer (Davies et al.  Theranostics7 (14): 3398-3414).
Fusion Antibodies developed a humanized version of JF5 (hJF5), using CDR grafting and applied its proprietary CDRx™ platform. Fusion Antibodies delivered the following package of Antibody Humanization services:
- Antibody humanization design
- Plasmid synthesis and cloning
- Transient expression
- Stable cell line development
Fusion Antibodies designed humanized variants by CDR grafting into mature human frameworks selected by homology and position of key residues identified as critical for optimal retention of CDR-loop confirmation. Humanized variants were analysed for sequence liabilities including T-cell epitopes, glycosylation and deamidation sites. Humanized heavy and light chains were transiently expressed in CHO cells to determine expression yield and to select a lead candidate. A stable pool of CHO cells was developed expressing the lead candidate.
ELISA tests investigated the immunoreactivity of the hJF5 antibody following labelling with chelators DOTAGA and NODAGA. PET/MR imaging, ex vivo bio distribution and autoradiography examined the uptake of [64Cu]NODAGA labelled hJF5 antibody in the lungs of mice infected with A. fumigatus. Results were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.
Results and conclusion
The pre-clinical studies conducted using the 64Cu-labeled hJF5 antibody in a neutropenic mouse model of IPA, have demonstrated improved uptake of the hJF5-based PET tracer in infected lung tissues compared to its murine counterpart.
The results show both mJF5 and hJF5 bind to the antigenic determinant β1,5-galactofuranose (Galf) present in the target mannoprotein antigen. ELISA tests reveal unlabelled humanized hJF5 and hJF5-NODAGA antibodies exhibit increased binding with the purified mannoprotein antigen compared to murine mJF5 and mJF5-NODAGA (Figure 1). Furthermore, uptake of humanized hJF5-NODAGA in the lungs of infected mice was significantly higher compared to murine mJF5 (Figure 2). This trend in uptake was further evidenced by ex vivoautoradiography of the lungs of infected mice (Figure 3).
In summary, Fusion Antibodies successfully developed a humanized version of the Aspergillus-specific mAb JF5. The improved imaging capabilities of hJF5 provides an excellent platform for clinical studies of IPA detection using antibody-guided molecular imaging (Thornton  Frontiers in Microbiology 9: 691).
The JF5 humanization project represents another successful project for Fusion Antibodies and is a case study example of the robust nature of Fusion Antibodies’ CDRxTM Humanization platform.
Figure 1. ELISA of mJF5, hJF5, human IgG1 isotype control antibody ET901, and non-radiolabelled DOTAGA- or NODAGA-conjugated antibody derivatives, using microtiter plates coated with 1 mg/mL of purified Aspergillus mannoprotein antigen. No notable reductions in binding of the mJF5 and hJF5 antibodies are apparent as a result of conjugation to the chelators DOTAGA or NODAGA. Note, however, the increase in binding of the hJF5 and NODAGA-hJF5 antibodies compared to the mJF5 and NODAGA-mJF5 counterparts at equivalent antibody protein concentrations.
Figure 2. Quantification of the PET images 48 h p.i. for lung tissues. Uptake of the various tracers in the lungs of infected animals compared to PBS controls at 48 h p.i. is shown in groups of n=4-5 mice. Significantly higher uptake of [64Cu]NODAGA-hJF5 in the lungs of A. fumigatus infected animals was obtained compared to all other tracers. Data are expressed as the mean ± SD %ID/cc. Group differences were examined using one-way ANOVA, followed by post hoc Tukey–Kramer, *P < 0.05.
Figure 3. Representative autoradiographic images with subsequent H&E staining of the lung tissue 48 h p.i. Autoradiographs of lung sections (right) with the corresponding H&E staining (left). Ex vivo autoradiography shows higher tracer accumulation in the lungs of infected animals receiving the JF5 antibody tracers compared to the respective PBS controls. Note, there is a higher uptake of [64Cu]NODAGA-hJF5 in the lungs of infected animals compared to the other infected groups.
Non-phosphorylated and Phosphorylated Signal Transduction Peptide (STP) of Tissue Factor (TF) in Urine are Potential Biomarkers for Bladder and Colon Cancer.
Tissue factor (TF, CD142) is commonly associated with the initiation of blood clotting. Many studies have shown that solid cancers including colon, breast, prostate, bladder and pancreas express high levels of TF and there is evidence that TF plays an integral role in the carcinogenic process as well as in metastasis.
The objective of this study was to develop immunoassay methods to investigate the TF-STP (signal transduction peptide) domain in urinary microvesicles from healthy individuals and patients with various tumour types. Using immunological methods to quantitate unphosphorylated TF and TF phosphorylated at ser253 and ser258.
Fusion Antibodies generated monoclonal antibodies (Anti-TF2 Mab and Anti-TF2-PSer253 Mab) to specifically target two peptides in the STP region of TF (Table 1).
Both polyclonal and monoclonal antibodies directed against various phosphorylated and unphosphorylated peptides were used to construct three ELISAs that specifically recognise unphosphorylated TF and TF phosphorylated at ser253 or at ser258. Urine samples were obtained from healthy individuals and from patients with various solid tumour types.
The results were presented as ng/ml of urine for each of the 3 anayltes in each sample. ROC curves were plotted for each diagnostic measurement and the area under the curve calculated. Optimal cut-off points were calculated from the ROC curves using the Youden Index. Sensitivities, specificities, positive predictive value (PPV) and negative predictive values (NPV) were calculated for each of the diagnostic measurements.
Results and conclusion
In summary, three highly sensitive ELISA assays were developed by Fusion Antibodies to quantitate unphosphorylated TF, TF-Pser253 and TF-Pser258 in urine. TF-Pser258 represents a potential new urinary biomarker for bladder cancer including early disease. Unphosphorylated TF is also a novel marker for advanced bladder cancer and their concurrent use may allow a nuanced approach to the diagnostics of bladder cancer from the outset. TF-Pser253 had a sensitivity of 71% and specificity of 69% at 0.55 ng/ml cut-off determined by ROC analysis. Suggesting that TF-Pser253 could be a promising urinary marker for early diagnosis of colon cancer.
Fsn1006 is a dual targeting antibody targeting both AREG (Amphiregulin) and HB-EGF (Heparin-binding EGF-like growth factor. The objective of this study was to humanize Fsn1006 while retaining the affinities.
Turning murine research MAbs into humanized drug candidates. Fsn1006: An antibody with Dual Specificity
Fsn1006 is a dual targeting antibody targeting both AREG (Amphiregulin) and HB-EGF (Heparin-binding EGF-like growth factor), two ligands in the EGFR pathway (Figure 1). From animal models Fusion Antibodies has shown that dual-targeting of 2 ligands in the pathway resulted in improved tumour cell killing effect. Fsn1006 has low nM affinity for both targets and the objective of this study was to humanize Fsn1006 while retaining the affinities.
Figure 1: Fsn1006 targets two ligands in the EGFR pathway (AREG and HB-EFGF). Fsn1006 has the potential to reach more patients than currently approved therapies and be more effective because of the novel targeted approach, multiple modes of action, efficacy in multiple cancer types and an ability to enhance current therapies.
Fsn1006 was humanized using Fusion Antibodies’ modernized CDR grafting technique. The CDRs were identified using both Kabat and IMGT definitions of CDRs (Figure 2) and grafted into mature frameworks with somatic hypermutation (not germline frameworks). Mature human frameworks were screened and selected as they have greater variability than germline sequences. A T-cell epitope screen was included in the design process and immunogenic constructs were avoided. Three recipient vH and three recipient vL sequences were selected and CDR grafting was performed. The panels of nine humanized antibody combinations were screened by biacore/SPR analysis to assess affinity (Figure 3).
Figure 2: CDRs were identified by a combination of Kabat and IMGT definitions to ensure all residues critical for binding were maintained.
Results and Conclusion
Fusion Antibodies was successfully able to humanize Fsn1006 while also retaining the affinity characteristics shown in the parental antibody. Fusion Antibodies’ modernized CDR grafting humanization process is capable of maintaining (and in this case improving) the measured affinity of therapeutic antibodies.
Figure 3: Biacore analysis showed that the parental antibody had a KD of 8.9nM and humanized variants v4, v5 and v6 showed improved affinities of 5.1nM, 5.2nM and 7.0nM respectfully.