Reducing target binding affinity improves the therapeutic index of anti-MET antibody-drug conjugate in tumor bearing animals.

Many oncology antibody-drug conjugates (ADCs) have failed to demonstrate efficacy in clinic because of dose-limiting toxicity caused by uptake into healthy tissues. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI) using two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) conjugated to monomethyl auristatin E (MMAE). The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV- and HAV-ADCs showed similar levels of anti-tumor activity in the xenograft model, while the 111In-DTPA studies showed similar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has ~2-fold slower blood clearance than the HAV-ADC, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In- and 124I- DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, intravital microscopy (IVM) studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. These results demonstrated that lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the target mediated drug disposition (TMDD) to MET expressed in normal tissues while maintaining uptake/delivery to the tumor. This approach can be applied to multiple ADCs to improve the clinical outcomes.

Canadian Anatomic Kidney Score: Quantitative Macroscopic Assessment of Donor Kidney Quality for Transplantation.

Background: The Canadian Anatomic Kidney Score (CAKS) is a novel 6-point grading system that standardizes the gross description of a donor kidney across 3 components-vessels, anatomy, and sticky fat. We hypothesized that the CAKS predicts allograft functional outcomes and provides additional information to the Kidney Donor Profile Index (KDPI) and histologic assessment of the donor kidney. Methods: Single-center cohort of 145 patients who underwent renal transplantation with CAKS analysis between 2018 and 2021. CAKS was prospectively determined before transplantation. Preimplantation core biopsies were assessed according to the Remuzzi score (RS). The primary outcome was 1-y allograft function represented by an estimated glomerular filtration rate (eGFR). Results: Linear regression without adjustment for KDPI or RS showed a significant association between the CAKS and 1-y eGFR (-8.7 mL/min/1.73 m2 per point increase in CAKS; 95% CI, -13.0 to -4.4; P < 0.001). Most of that association was attributed to the vessel component (-12.1; -19.4 to -4.8; P = 0.002). Adjustment for KDPI and RS attenuated the relationship between 1-y function and CAKS (-4.6; -9.5 to 0.3; P = 0.065) and vessel component (-7.4; -15.2 to 0.5; P = 0.068). Conclusions: Anatomic assessment of donor kidneys at the time of transplantation associates with allograft function at 1 y. Vascular assessment appears to make the dominant contribution.

Choosing the larger kidney on CT volumetry: a study on the early post-donation kidney function of living donors.

BACKGROUND: Selecting the smaller kidney for donation has been advocated if there is a size difference of > 10% between the 2 kidneys but has never been prospectively evaluated. With increase in donor nephrectomies, it is important to evaluate this to minimize loss of renal function to donors. METHODS: 75 consecutive donor nephrectomy patients were included in our longitudinal study. The Split Renal Volume (SRV) of bilateral kidneys were measured using contrasted computer tomography scans and patients segregated into 2 groups depending on donated kidney having more (Group 1) or less than (Group 2) 52.5% of SRV. RESULTS: Patients in Group 1 (n = 19) and 2 (n = 56) were of similar age (43.8 vs. 48.3), BMI (22.4 vs. 25.2), sex (57.9 vs. 55.4% women), respectively. Although total kidney volumes were similar in both groups, Group 1 had significantly smaller right kidney volumes (120.4 ± 24.9 vs. 142.7 ± 28.4 mls, p = 0.003). EGFR pre-operatively (116.3 ± 20.8 vs. 106.3 ± 23.8 mL/min/1.73 m2) and at 6-months (65.7 ± 13.3 vs. 66.9 ± 15.5 mL/min/1.73 m2) were not different between groups. However, patients in Group 1 had significantly greater absolute (50.6 ± 14.9 vs. 39.5 ± 14.7 mL/min/1.73 m2) and relative decline (43.0 ± 8.6 vs. 36.3 ± 10.6%) in eGFR at 6 months (p = 0.06, 0.009). CONCLUSION: With a SRV difference of 5% between the 2 sides, removal of the larger kidney for living kidney donation resulted in greater early decline of renal function than kidney donors whose larger or equivalent kidney is preserved.

An in vitro Comparative Assessment of Single-Use Flexible Ureteroscopes Using a Standardized Ureteroscopy Training Model.

INTRODUCTION: Perceived benefits like decreased contamination rates and reduced postoperative incidence of complications after urolithiasis surgery have led to increased adoption of single-use flexible ureteroscopes (su-fURS). Using a validated, standardized simulator model with enhanced "fluoroscopic" capabilities, we performed an in vitro comparative assessment of four commercially available models of su-fURS. Both objective and subjective parameters were assessed in this study. METHODS: Two standardized tasks, (1) exploration of the model's kidney collecting system and (2) repositioning of a stone fragment from the upper renal to lower renal pole were assigned to participants, who performed these tasks on all four scopes. Four models of su-fURS (Boston LithoVue, PUSEN PU3033A, REDPINE, INNOVEX EU-ScopeTM) were assessed, with task timings as end-points for objective analysis. Cumulative "fluoroscopic" time was also recorded as a novel feature of our enhanced model. Post-task questionnaires evaluating specific components of the scopes were distributed to document subjective ratings. RESULTS: Both subjective and objective performances (except stone repositioning time) across all four su-fURS demonstrated significant differences. However, objective performance (task timings) did not reflect subjective scope ratings by the participants (Rs < 0.6). Upon Kruskal-Wallis H test with post hoc analyses, REDPINE and INNOVEX EU-ScopeTM were the preferred su-fURS as rated by the participants, with overall scope scores of 9.00/10 and 9.57/10. CONCLUSIONS: Using a standardized in vitro simulation model with enhanced fluoroscopic capabilities, we demonstrated both objective and subjective differences between models of su-fURS. However, variations in perception of scope features (visibility, image quality, deflection, maneuverability, ease of stone retrieval) did not translate into actual technical performance. Eventually, the optimal choice of su-fURS fundamentally lies in individual surgeon preference, as well as cost-related factors.

Multi-photon microscopy for the evaluation of interstitial fibrosis in extended criteria donor kidneys: A proof-of-concept study.

INTRODUCTION: To evaluate the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence in multiphoton microscopy (MPM) for the quantification of collagen/fibrosis on preimplantation biopsies of extended criteria donors (ECD). MATERIALS AND METHODS: Twenty preimplantation core biopsies were extracted from 10 donor kidney samples, of which originated from seven donors. Kidney Donor Profile Index (KDPI) and Remuzzi scores of biopsies were calculated. Collagen parameters measured included quantification by the Collagen Area Ratio in Total Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI). RESULTS: Biopsies classified with > 85% KDPI scores had significantly higher CART (p = .011) and lower CRI values (p = .025) than biopsies with ≤ 85% KDPI scores. Increase in CRI values correlated significantly with rise in recipient creatinine levels 1-year post-transplant (p = .027; 95% CI: 4.635-66.797). CONCLUSION: MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained preimplantation biopsies of donor kidneys stratified by KDPI scores. This initial evaluation found significant differences in both parameters between donor kidneys with more or less than 85% KDPI.

Validation of laparoscopy and flexible ureteroscopy tasks in inanimate simulation training models at a large-scale conference setting.

OBJECTIVE: Simulation based training with training models is being increasingly used as a tool to help trainees mount the learning curve. However, validation studies of surgical simulators are often limited by small numbers. We aim to evaluate the feasibility of validating simulation-training tasks in laparoscopy and flexible ureteroscopy (FURS) rapidly at a large-scale conference setting for residents. METHODS: Seventy-six urology residents from various Asian countries were assessed on their laparoscopic and FURS skills during the 14th Urological Association of Asia Congress 2016. Residents performed the peg transfer task from the fundamentals of laparoscopic surgery (FLS) and completed inspection of calyces and stone retrieval using a flexible ureteroscope in an endourological model. Each participant's experience (no experience, 1-30 or >30 procedures) in laparoscopy, rigid ureteroscopy (RURS) and FURS was self-reported. RESULTS: Median time taken to complete the laparoscopic task decreased with increasing laparoscopic experience (209 s vs. 177 s vs. 145 s, p=0.008) whereas median time taken to complete the FURS tasks reduced with increasing FURS experience (405 s vs. 250 s vs. 163 s, p=0.003) but not with RURS experience (400.5 s vs. 397 s vs. 331 s, p=0.143), demonstrating construct validity. Positive educational impact of both tasks was high, with mean ratings of 4.16/5 and 4.10/5 respectively, demonstrating face validity. CONCLUSION: Our study demonstrates construct and face validities of laparoscopy and FURS simulation tasks among residents at a conference setting. Validation studies at a conference setting can be an effective avenue for evaluating simulation models and curriculum in the future.

Implementation of a Ureteric Colic Telemedicine Service: A Mixed Methods Quality Improvement Study.

OBJECTIVE: To assess the effectiveness of a telemedicine service for ureteric colic patients in reducing the number of unnecessary face-to-face consultations and shortening waiting time for appointments. METHODS: A telemedicine workflow was implemented as a quality improvement study using the Plan-Do-Study-Act method. All patients presenting with ureteric colic without high-risk features of fever, severe pain, and hydronephrosis, were recruited, and face-to-face appointments to review scan results were replaced with phone consultations. Data were prospectively collected over 3 years (January 2017 to December 2019). Patient outcomes including the reduction in face-to-face review visits, time to review, reattendance and intervention rates, were tracked in an interrupted time-series analysis, and qualitative feedback was obtained from patients and clinicians. RESULTS: Around 53.2% of patients presenting with ureteric colic were recruited into the telemedicine workflow. A total of 465 patients (46.2%) had normal scan results and 250 patients (24.9%) did not attend their scan appointments, hence reducing the number of face-to-face consultations by 71.1%. A total of 230 patients (22.9%) required subsequent follow-up with urology, while 61 patients (6.1%) were referred to other specialties. Mean (SD) time to review was 30.0 (6.2) days, 6-month intervention rate was 3.4% (n = 34) and unplanned reattendance rate was 3.2% (n = 32). Around 93.1% of patients reported satisfaction with the service. CONCLUSION: The ureteric colic telemedicine service successfully and sustainably reduced the number of face-to-face consultations and time to review without compromising on patient safety. The availability of this telemedicine service has become even more important in helping us provide care to patients with ureteric colic in the current COVID-19 pandemic.

Influence of physiochemical properties on the subcutaneous absorption and bioavailability of monoclonal antibodies.

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease.

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.

Systemic immune-checkpoint blockade with anti-PD1 antibodies does not alter cerebral amyloid-ß burden in several amyloid transgenic mouse models.

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death-1 (PD1) checkpoint inhibition produces an IFN-γ-dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., (): Nature Medicine, 22:135-137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid-ß pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti-PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte-derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., (): Nature Medicine, 22:135-137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.

Reconstruction of 3D structures of MET antibodies from electron microscopy 2D class averages.

Dynamics of three MET antibody constructs (IgG1, IgG2, and IgG4) and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 Å precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models.

Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A.

Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.

Therapeutic Antibody Engineering To Improve Viscosity and Phase Separation Guided by Crystal Structure.

Antibodies at high concentrations often reveal unanticipated biophysical properties suboptimal for therapeutic development. The purpose of this work was to explore the use of point mutations based on crystal structure information to improve antibody physical properties such as viscosity and phase separation (LLPS) at high concentrations. An IgG4 monoclonal antibody (Mab4) that exhibited high viscosity and phase separation at high concentration was used as a model system. Guided by the crystal structure, four CDR point mutants were made to evaluate the role of hydrophobic and charge interactions on solution behavior. Surprisingly and unpredictably, two of the charge mutants, R33G and N35E, showed a reduction in viscosity and a lower propensity to form LLPS at high concentration compared to the wild-type (WT), while a third charge mutant S28K showed an increased propensity to form LLPS compared to the WT. A fourth mutant, F102H, had reduced hydrophobicity, but unchanged viscosity and phase separation behavior. We further evaluated the correlation of various biophysical measurements including second virial coefficient (A2), interaction parameter (kD), weight-average molecular weight (WAMW), and hydrodynamic diameters (DH), at relatively low protein concentration (4 to 15 mg/mL) to physical properties, such as viscosity and liquid-liquid phase separation (LLPS), at high concentration. Surprisingly, kD measured using dynamic light scattering (DLS) at low antibody concentration correlated better with viscosity and phase separation than did A2 for Mab4. Our results suggest that the high viscosity and phase separation observed at high concentration for Mab4 are mainly driven by charge and not hydrophobicity.

Training improves laparoscopic tasks performance and decreases operator workload.

BACKGROUND: It has been postulated that increased operator workload during task performance may increase fatigue and surgical errors. The National Aeronautics and Space Administration-Task Load Index (NASA-TLX) is a validated tool for self-assessment for workload. Our study aims to assess the relationship of workload and performance of novices in simulated laparoscopic tasks of different complexity levels before and after training. METHODS: Forty-seven novices without prior laparoscopic experience were recruited in a trial to investigate whether training improves task performance as well as mental workload. The participants were tested on three standard tasks (ring transfer, precision cutting and intracorporeal suturing) in increasing complexity based on the Fundamentals of Laparoscopic Surgery (FLS) curriculum. Following a period of training and rest, participants were tested again. Test scores were computed from time taken and time penalties for precision errors. Test scores and NASA-TLX scores were recorded pre- and post-training and analysed using paired t tests. One-way repeated measures ANOVA was used to analyse differences in NASA-TLX scores between the three tasks. RESULTS: NASA-TLX score was lowest with ring transfer and highest with intracorporeal suturing. This was statistically significant in both pre-training (p < 0.001) and post-training (p < 0.001). NASA-TLX scores mirror the changes in test scores for the three tasks. Workload scores decreased significantly after training for all three tasks (ring transfer = 2.93, p < 0.001, precision cutting = 3.74, p < 0.001, intracorporeal suturing = 2.98, p < 0.001). CONCLUSION: NASA-TLX score is an accurate reflection of the complexity of simulated laparoscopic tasks in the FLS curriculum. This also correlates with the relationship of test scores between the three tasks. Simulation training improves both performance score and workload score across the tasks.

The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies.

The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application.

Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF.

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.