Use Of Monoclonal Antibodies Research Articles

Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticles.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.

Advances in Monoclonal Antibody Therapies for Triple-Negative Breast Cancer: Immunotherapeutic and Targeted Strategies.

Triple-negative breast cancer (TNBC) presents considerable obstacles because of its highly aggressive characteristics and limited availability of specific therapeutic interventions. The utilization of monoclonal antibody (mAb)-based immunotherapy is a viable approach to tackle these difficulties. This review aims to examine the present state of mAb-based immunotherapy in TNBC, focusing on the underlying mechanisms of action, clinical applications, and existing challenges. The effectiveness of mAbs in reducing tumor development, regulating immune responses, and changing the tumor microenvironment has been demonstrated in many clinical investigations. The challenges encompass several aspects such as the discovery of biomarkers, understanding resistance mechanisms, managing toxicity, considering costs, and ensuring accessibility. The future is poised to bring forth significant advancements in the field of biomedicine, particularly in the areas of new mAbs, personalized medicine, and precision immunotherapy. In conclusion, mAb-based immunotherapy has promise in revolutionizing the treatment of TNBC, hence providing a possible avenue for enhanced patient outcomes and quality of life.

Phage Antibodies for Kanamycin Detection

Recombinant antibodies specific to kanamycin were obtained using a sheep display library of scFv fragments (Griffin.1) and the possibility of their use for the determination of kanamycin by dot-immunoassay was demonstrated. The minimum detectable concentration of kanamycin is 1 μg/mL (distinguishable label binding other than background). It has been shown that anti-kanamycin phage antibodies are specific for kanamycin and do not interact with other antibiotics (neomycin, tetracycline, ampicillin, gentamicin). Anti-kanamycin phage antibodies are a promising alternative to monoclonal antibodies for use in the determination of kanamycin.

A review on emerging targeted therapies for the management of metastatic colorectal cancers

Colorectal cancers are among the most commonly found cancers over the world. In spite of the recent advancements in diagnosis and prognosis, the management of this metastatic condition remains a challenge. The utility of monoclonal antibodies in the healing of patients with colorectal cancer has opened a new chapter in the quest for newer therapies. The resistance to the standard treatment regimen made it mandatory to search for newer targets. Mutagenic alterations in the gene engaged in cellular differentiation and growth pathway have been the reason for resistance to treatment. The newer therapies target the various proteins and receptors involved in the signal transduction and down streaming pathways leading to cell proliferation. This review presents an insight into the newer targeted therapies for colorectal cancer involving tyrosine kinase blockers, epidermal growth factor receptors, vascular endothelial growth factor, immune checkpoint therapy, and BRAF inhibitors.

Expression of plant-produced anti-PD-L1 antibody with anoikis sensitizing activity in human lung cancer cells via., suppression on epithelial-mesenchymal transition.

Immune checkpoint antibodies in cancer treatment are receptor-ligand pairs that modulate cancer immunity. PD-1/PD-L1 pathway has emerged as one of the major targets in cancer immunotherapy. Atezolizumab, the first anti-PD-L1 antibody approved for the treatment of metastatic urothelial, non-small cell lung, small cell lung and triple-negative breast cancers, is produced in Chinese Hamster Ovary (CHO) cells with several limitations i.e., high-production costs, low-capacity yields, and contamination risks. Due to the rapid scalability and low production costs, the transient expression in Nicotiana benthamiana leaves was investigated by co-infiltration of Agrobacterium tumefaciens GV3101 cultures harboring the nucleic acid sequences encoding for Atezolizumab heavy chain and light chain in this study. The transient expression of Atezolizumab in transformed N. benthamiana accumulated up to 86.76 μg/g fresh leaf weight after 6 days of agroinfiltration (OD 600 nm: 0.4) with 1:1 ratio of heavy chain to light chain. The structural and functional characteristics of plant-produced Atezolizumab was compared with commercially available Tecentriq® from CHO cells with similar binding efficacies to PD-L1 receptor. The direct anti-cancer effect of plant-produced anti-PD-L1 was further performed in human lung metastatic cancer cells H460 cultured under detachment condition, demonstrating the activity of anti-PD-L1-antibody on sensitizing anoikis as well as the suppression on anti-apoptosis proteins (Bcl-2 and Mcl-1) and modulation of epithelial to mesenchymal regulating proteins (E-cadherin, N-cadherin, Snail and Slug). In conclusion, this study manifests plants as an alternative cost-effective platform for the production of functional monoclonal antibodies for use in cancer therapy.

Inebilizumab: A Review in Neuromyelitis Optica Spectrum Disorder.

Inebilizumab (Uplizna®) is a recently approved monoclonal antibody for use in adults with neuromyelitis optica spectrum disorder (NMOSD) who are anti-aquaporin-4 (AQP4) antibody seropositive. Inebilizumab targets the B cell antigen CD19 and effectively depletes circulating B cells, thus suppressing inflammatory NMOSD attacks that are potentially disabling or life-threatening. It is approved as an intravenous infusion in several countries. In the pivotal phase 2/3 N-MOmentum trial, inebilizumab reduced the risk of NMOSD attacks compared with placebo, including in AQP4-antibody seropositive patients. Inebilizumab also significantly reduced the risk of disability score worsening, the number of NMOSD-related hospitalisations and MRI lesion count, but had no significant effect on low-contrast binocular vision. The treatment effect on relapse risk and disability scores was sustained in inebilizumab-treated patients for ≥ 4 years during the open-label extension. Inebilizumab was generally well tolerated, with the most common adverse events being urinary tract infection and arthralgia. Thus, inebilizumab is an effective treatment option for adults with AQP4-antibody seropositive NMOSD.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40263-022-00949-7.

Clonal Wars: Monoclonal Antibodies Against Infectious Pathogens.

Monoclonal antibodies are coming of age as powerful tools for the prevention of infectious diseases. In recent years, the rate of antibody discovery has accelerated, and the coronavirus disease 2019 (COVID-19) pandemic has shone a spotlight on the role of these antibodies in combating pathogens. However, questions remain about the utility of monoclonal antibodies, especially when effective vaccines are also available. In this article, I discuss the role of monoclonal antibodies and briefly describe the effort to identify potent human monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including our study on bispecific antibodies that neutralize SARS-CoV-2 variants of concern.

Evaluation of PD-1 and PD-L1 expression in canine urothelial carcinoma cell lines

Urothelial carcinoma (UC) is the most common urinary tumor in dogs and despite combinational therapies, only modest improvements in survival have been achieved in recent years. Given the utility of monoclonal antibodies against PD-1 and PD-L1 in human UC, we evaluated the protein and mRNA expression in three established canine urothelial carcinoma cell lines. Flow cytometry and western blot analysis confirmed cell line expression of both molecules in varying degrees. Reverse transcription PCR (RT-PCR) documented mRNA expression in all three cell lines for both PD-1 and PD-L1. Fluorescence microscopy was consistent with strong PD-1 and PD-L1 expression in the canine cell lines and was in line with previous human literature. Importantly, the flow cytometry work described in this study revealed higher cell intrinsic PD-1 expression in these cell lines which may have implications for tumor behavior and potential treatment opportunities in the future. Further work is necessary to determine the expression patterns in canine UC and potential for benefit with immunotherapy directed against PD-1 and PD-L1.

Safety and Tolerability of Sars-Cov-2 Vaccination and Natural History of Infection Among Patients with Castleman Disease

Castleman Disease (CD) represents a group of rare and heterogeneous hematologic disorders that have common lymph node histopathology. Patients with CD are often immunosuppressed as a consequence of immunemodulating therapy or possibly due to an underlying immunologic dysfunction attributable to B-cell dysfunction. The most severe CD cases experience a cytokine storm disorder, a life-threatening exacerbation of circulating cytokines and immune-cell hyperactivation. Infection with SARS-CoV-2 progresses to a severe cytokine storm in the most severe cases of COVID-19. Interleukin-6 (IL-6) is central to the pathogenesis of CD, and increased IL-6 often accompanies severe COVID-19 cases; inhibition of IL-6 with monoclonal antibodies has been shown to be effective therapy for both CD and severe COVID-19. We therefore sought to understand the impact of COVID-19 infection on the natural history of CD and also examined the safety and tolerability of COVID-19 vaccines in this vulnerable patient population.Patients enrolled in a longitudinal natural history study of CD (N=298) were invited to participate in a survey designed to characterize their experience with COVID-19 disease and vaccination. Surveys were emailed to all eligible patients, and reminders were sent up to 3 times. All data is self-reported; descriptive analyses are reported herein.Of the 298 patients who received a survey, 101 (33.9%) completed it. Sixty-nine (68%) had been tested for SARS-CoV-2 at least once, and 10 (14.5%) reported testing positive - including 6 UCD, 3 iMCD, and 1 HHV8+ MCD patients. The reported prevalence of SARS-CoV-2 infection in the US compares at 10.5%. Two of the 10 patients reported asymptomatic disease (both UCD), 7 reported mild disease (4 UCD, 1 iMCD, 1 HHV8+MCD), and 1 reported moderate disease requiring hospitalization but not a ventilator or intensive care (iMCD). This severity distribution suggests that these potentially immunocompromised patients experience a range of disease severity consistent with SARS-CoV-2 infection in the broader US population. The most commonly-reported symptoms included fevers/chills, headaches, and loss of taste or smell (N=7 each), as well as shortness of breath/difficulty breathing, muscle and body aches, and cough (N=5 each). Rarer symptoms were also noted among the iMCD patients, including discoloration of skin, lips, or nailbeds (N=1) and newfound confusion (N=2). Two of the 10 patients reported stopping siltuximab treatment during their COVID-19 diagnosis; both subsequently resumed treatment. No other treatment changes were reported.Of the 101 respondents, 87 (86%) had received at least 1 vaccine dose. Treatments, such as immunosuppressants and immunomodulators, were paused for 7 of these patients including, during the vaccination period; this was presumably done to increase the likelihood of a robust response to the vaccine. Fifty-one patients (59%) reported side effects to either dose 1 or 2. Side effects were generally mild, and none required hospitalization. Side effects were more commonly reported after dose 2, with the most common being arm pain (N=34), fatigue (N=30), and headache (N=26). Of those who reported not receiving the vaccine, 2 intend to receive it in the future, 5 reported being unsure about receiving it, and 7 do not intend to receive the vaccine. Common concerns include potential interaction with CD (N=9) and limited safety data (N=8).This study represents the first investigation into the experience of CD patients with SARS-CoV-2 testing, diagnosis, and vaccination. We did not observe a markedly increased inflammatory response to SARS-CoV-2 infection, and vaccination was well-tolerated. A limitation is self-selection survey bias; it is possible that those who chose to participate represent those who had a milder reaction in general. However, it is noteworthy that there were no reports of severe disease in this sample. The prevalence of confirmed SARS-CoV-2 infection in this cohort (14.5%) is marginally higher than reported in the US population (10.5%) but statistical comparisons were not performed given that this study does not provide a general epidemiological estimate. However, the distribution of symptoms and vaccine adverse effects in this sample were comparable to the general population. Though additional follow-up is planned for the future, these data are an important basis for understanding the interaction of SARS-CoV-2 and CD. DisclosuresCasper: EUSA Pharma: Consultancy. Fajgenbaum: Pfizer: Other: Study drug for clinical trial of sirolimus; N/A: Other: Holds pending provisional patents for ‘Methods of treating idiopathic multicentric Castleman disease with JAK1/2 inhibition’ and ‘Discovery and validation of a novel subgroup and therapeutic target in idiopathic multicentric Castleman disease’; EUSA Pharma: Research Funding. OffLabel Disclosure:Our abstract makes reference to the following: “Interleukin-6 (IL-6) is central to the pathogenesis of CD, and increased IL-6 often accompanies severe COVID-19 cases; inhibition of IL-6 with monoclonal antibodies has been shown to be effective therapy for both CD and severe COVID-19.” Inhibition of IL-6 with monoclonal antibodies for use in COVID-19 is off-label.

Monoclonal Antibodies against SARS-CoV-2: Potential Game-Changer Still Underused.

Even several months after the start of a massive vaccination campaign against COVID-19, mortality and hospital admission are still high in many countries. Monoclonal antibodies against SARS-CoV-2 are the ideal complement to vaccination in infected subjects who are at high risk for progression to severe disease. Based on data of the Italian Ministry of Health, in the period April–August 2021, monoclonal antibodies were prescribed to 6322 patients. In the same period, 70,022 patients over 70 years old became infected with SARS-CoV-2. Even considering that all monoclonal antibodies were prescribed to this category of patients, we calculated that only 9% of these subjects received the treatment. Moreover, using efficacy data provided by clinal trials, we estimated the potential benefit in terms of reduction of hospital admissions and deaths. Considering utilisation of monoclonal antibodies in half infected patients over 70 years, we estimated that hospital admissions and deaths might have been reduced by 7666 and 3507, respectively. Finally, we calculated the economic benefit of monoclonal use. In the same scenario (50% use of monoclonal antibodies to patients over 70), we estimated potential savings of USD 117,410,105. In conclusion, monoclonal antibodies were used in a small proportion of patients over 70 in Italy. A more extensive use might have resulted in a marked decrease in hospital admissions, deaths and in conspicuous saving for the health system.

Evolving Antibody Therapies for the Treatment of Type 1 Diabetes.

Type 1 diabetes (T1D) is widely considered to be a T cell driven autoimmune disease resulting in reduced insulin production due to dysfunction/destruction of pancreatic β cells. Currently, there continues to be a need for immunotherapies that selectively reestablish persistent β cell-specific self-tolerance for the prevention and remission of T1D in the clinic. The utilization of monoclonal antibodies (mAb) is one strategy to target specific immune cell populations inducing autoimmune-driven pathology. Several mAb have proven to be clinically safe and exhibit varying degrees of efficacy in modulating autoimmunity, including T1D. Traditionally, mAb therapies have been used to deplete a targeted cell population regardless of antigenic specificity. However, this treatment strategy can prove detrimental resulting in the loss of acquired protective immunity. Nondepleting mAb have also been applied to modulate the function of immune effector cells. Recent studies have begun to define novel mechanisms associated with mAb-based immunotherapy that alter the function of targeted effector cell pools. These results suggest short course mAb therapies may have persistent effects for regaining and maintaining self-tolerance. Furthermore, the flexibility to manipulate mAb properties permits the development of novel strategies to target multiple antigens and/or deliver therapeutic drugs by a single mAb molecule. Here, we discuss current and potential future therapeutic mAb treatment strategies for T1D, and T cell-mediated autoimmunity.

Early clinical experience with a monoclonal antibody against the calcitonin gene-related peptide receptor in adolescents with migraine: A case series

Management of migraine in adolescents poses a great challenge, as many of the approved pharmacological migraine preventive agents have age restrictions. Following favorable safety and efficacy reports of the new class agent calcitonin gene-related peptide (CGRP) monoclonal antibody for use in migraine prevention, there is growing interest in its application in pediatric migraine. We present here a case series detailing our experience of using erenumab, a CGRP monoclonal antibody, in six adolescent patients. Two patients had a reduction of at least 50% in the mean number of monthly migraine days, one patient reported subjective improvement, while three patients did not respond to the first dose of erenumab and discontinued treatment. One patient reported constipation associated with erenumab use. We speculate that CGRP monoclonal antibody could potentially be a viable option in adolescent patients with migraine. Further evidences that support efficacy and safety of erenumab in this group is needed.

Immunotherapy against Systemic Fungal Infections Based on Monoclonal Antibodies.

The increasing incidence in systemic fungal infections in humans has increased focus for the development of fungal vaccines and use of monoclonal antibodies. Invasive mycoses are generally difficult to treat, as most occur in vulnerable individuals, with compromised innate and adaptive immune responses. Mortality rates in the setting of our current antifungal drugs remain excessively high. Moreover, systemic mycoses require prolonged durations of antifungal treatment and side effects frequently occur, particularly drug-induced liver and/or kidney injury. The use of monoclonal antibodies with or without concomitant administration of antifungal drugs emerges as a potentially efficient treatment modality to improve outcomes and reduce chemotherapy toxicities. In this review, we focus on the use of monoclonal antibodies with experimental evidence on the reduction of fungal burden and prolongation of survival in in vivo disease models. Presently, there are no licensed monoclonal antibodies for use in the treatment of systemic mycoses, although the potential of such a vaccine is very high as indicated by the substantial promising results from several experimental models.

A Review on The Development, Production Strategies, and Utilization of Monoclonal Antibodies

A Review on The Development, Production Strategies, and Utilization of Monoclonal Antibodies

Implications of Fc Neonatal Receptor (FcRn) Manipulations for Transplant Immunotherapeutics.

Alloimmune injury to allografts is mediated by pathogenic donor-specific alloantibodies, usually of the IgG isotype. Currently, strategies used to reduce donor-specific alloantibodies are collectively called desensitization. Despite successes, these treatments have limited efficacy and can be associated with adverse events, infectious complications, and high cost. Fc neonatal receptor (FcRn) was originally discovered as a transport mechanism for IgG from maternal circulation to fetus. FcRn receptors are now known to be widely distributed in virtually all tissues. IgG and albumin binding to FcRn is pH-dependent, which results in a significant prolongation their half-life. Structural analysis shows FcRn is a nonclassical major histocompatibility complex Class I receptor, which is emerging as a novel target to significantly reduce the half-life of pathogenic antibodies or extend the half-life of therapeutic monoclonals. Manipulation of IgG-Fc/FcRn interactions has implications for treatment of virtually all IgG-mediated diseases. The use of monoclonals directed at the FcRn can rapidly enhance the turnover of total IgG, including pathogenic IgG. In this review, we highlight the aspects of FcRn biology responsible for development of FcRn targeted therapeutics aimed at pathogenic autoantibodies and alloantibodies. We also explore the novel modifications of therapeutic monoclonals that exploit FcRn functions to enhance therapeutic efficacy.

Generation and characterization of D-dimer specific monoclonal antibodies for use in latex agglutination test.

The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather unsatisfactory. Our aim was to develop a new monoclonal antibody against D-dimer with a proper specificity, and estimating its suitability using in a latex agglutination diagnostic test. Monoclonal antibodies were generated using hybridoma technology. Their titer was determined by a self-developed ELISA method. The cross-reactions of the antibodies were tested. Characterization of the epitope specificity of a selected antibody was performed through digestion of D-dimer followed by Western blotting. The amino acid sequences of the active antigen fragments were determined. According to the ELISA results, 38 cell groups were constated as antibody-producing hybridomas, among them 7 gave raised titer of antibody and were cloned. Based on the cross-reaction analysis, none of the antibodies gave cross-reaction with fibrin-E and fibrinogen-E fragments but reacted with fibrin D and fibrinogen D fragments. A low cross-reaction was showed with fibrinogen and fibrin X and Y. Contrary to the others, antibody 2B9 gave no cross-reaction with fibrinogen and reacted weakly with fibrin X and Y fragments. According to the epitope analysis the antibody 2B9 binds to amino acids 94–99 and to amino acids 140–147 on the beta chain and it recognizes the amino acids 23–32 and 93–98 on the gamma chain of D-dimer. Considering the characteristics of the above mentioned monoclonal antibody 2B9, we found that it is suitable to be a basis for a D-dimer diagnostic test with proper specificity.

Risk-benefit Balance Evaluation of New Monoclonal Antibodies for Use in Pregnant Women

Risk-benefit Balance Evaluation of New Monoclonal Antibodies for Use in Pregnant Women

A Quantitative Method for Detecting Ara h 2 by Generation and Utilization of Monoclonal Antibodies.

Peanut (Arachis hypogaea) is one of the most common food allergens that can induce fatal anaphylaxis, and Ara h 2 is one of the major allergen components involved in peanut allergy. The aim of this study was to develop a quantitative method for detecting peanut allergen using monoclonal antibodies against Ara h 2. The splenocytes of immunized mice were fused with myeloma cells (SP2/0), and stable mAb-producing clones were obtained by limiting dilution. mAbs against Ara h 2 were isolated from mouse ascites, and specificity was confirmed by immunoblotting. Five mAbs with high purity and specific reactivity were obtained, which were referred to as 1-2E10, 2-1D5, 3-1C5, 4-1C2, and 5-1G4, respectively. After screening different mAb combinations for development of a sandwich ELISA, we selected 5-1G4 as the capture antibody and 1-2E10 as the detection antibody for the measurement of Ara h 2 from which an optimal correlation between the Ara h 2 concentration and the OD value was obtained. This sandwich ELISA could specifically detect Ara h 2 in peanut extract at concentrations as low as 5 ng/mL and up to 10 μg/mL. These mAbs can, therefore, serve as quantitative diagnostic reagents for peanut and peanut product risk assessment.

DARATUMUMAB FOR THE TREATMENT OF MULTIPLE MYELOMA

The use of proteasome inhibitors and immunomodulatory drugs in the clinical practice has contributed to the significant improvement in survival for patients with multiple myeloma over the past decades. Alongside this, due to the recurrent course of the disease, there is a need to introduce new classes of drugs to clinical practice. In 2015, the FDA (USA) approved two monoclonal antibodies for use in patients with relapsed multiple myeloma, and immunotherapy has rapidly become indispensable in the management of such patients. The article presents an analysis of the published data regarding the mechanism of action, safety and clinical efficacy of daratumumab, a human monoclonal antibody that targets CD38 tumor protein, for the treatment of patients with multiple myeloma. In Russia, daratumumab is registered (RU LP-004367 of 07.07.2017) and is indicated as monotherapy for patients with relapsed or refractory multiple myeloma, who have received prior therapies, incuding proteasome inhibitors and immunomodulatory drugs. Daratumumab demonstrated an excellent safety profile. In the context of daratumumab therapy, the moderate-grade infusion-related reactions occurring mostly during the first infusion are the main adverse events. Daratumumab-based combination therapies are currently under active evaluation in patients with relapsed and newly diagnosed myeloma.

722. AAV Capsid Evolution for Enhanced Antibody Delivery to Human Skeletal Muscle for Use in Next-Generation HIV Vaccines and Muscle Gene Therapies

Our goal is to develop an AAV vector that can transduce human skeletal muscle after intramuscular administration at levels sufficient to express therapeutic levels of antibodies with broad-spectrum protection against HIV. AAV has begun being used in passive vaccines for HIV and influenza. Yet for many indications, greater human skeletal muscle transduction is needed than has been achieved with existing AAV serotypes. To bioengineer novel capsids with unparalleled human skeletal muscle transduction, we utilized primary human skeletal muscle cells from surgical resections to screen libraries of replication-competent shuffled AAV capsids. Two screens were performed in pools of primary human skeletal muscle stem cells or myotubes from six patients. Six rounds of replicating selection were carried out and the five most highly selected variants from each screen were vectorized and validated against existing serotypes with muscle tropism (AAV1, 6 and 8). In primary human muscle stem cells, variants NP22, NP66 and NP94 had significantly increased transduction that ranged from a 65 to 284-fold improvement over AAV1, 27 to 118-fold over AAV6, and 10 to 45-fold over AAV8. In primary human myotubes, NP22, NP66 and NP94 again showed significantly increased transduction that ranged from a 13 to 464-fold improvement over AAV1, 25 to 871-fold over AAV8, and NP94 showed a 15-fold improvement over AAV6. To assess human skeletal muscle transduction capabilities in vivo, we injected xenografted humanized muscle NSG mice with shuffled or control capsids expressing luciferase by intramuscular injection (1E9/leg) and assessed transduction weekly by live imaging in time course studies over two months. AAV6 was the first to uncoat but NP66 produced the highest sustained transduction levels. To control for species-specific transduction, we performed the same time course experiment in strain- and gender-matched non-transplanted mice. Strikingly, AAV6 outperformed shuffled variants when no human muscle fibers were present, highlighting the importance of performing preclinical studies in human cells and xenograft systems whenever possible to prevent misleading results which are mouse-specific. To further demonstrate the superiority of our shuffled variants and more accurately predict eventual muscle transduction in humans, we transduced human skeletal muscle explants from surgical resections ex vivo. Four adult patients (two male and two female) had healthy skeletal muscle tissue removed for ex vivo transduction analyses with luciferase. In all 4 patients, NP22 and NP66 (and NP94 in 3/4 patients) had significantly increased transduction by live fiber luciferase imaging as well as luciferase assays on lysed fibers that ranged from a 4 to 116-fold improvement over all control serotypes. To support preclinical vaccine testing in non-human primates, we assessed transduction in rhesus macaque skeletal muscle explants ex vivo. Here again we showed that NP22 and NP66 showed a 30 to 57-fold improvement in transduction over control serotypes. Taken together, our results demonstrate that capsid variants NP22 and NP66 (and in some cases NP94), have highly significant increased human skeletal muscle fiber transduction when assessed in vitro, in vivo and ex vivo. These capsids represent powerful tools to express therapeutic quantities of human monoclonal antibodies for use in passive vaccines, or transgenes for various muscle disorders in gene therapy, specifically in humans.