ABSTRACT
Commercially available human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, gamma irradiated hPL offers an additional safety measure in the clinic. Chimeric Antigen Receptor (CAR) expressing T-cells have demonstrated potent clinical efficacy in patients with hematological malignancies. In addition, there are several phase I clinical trials evaluating the use of CAR-T-cells for targeting of solid tumorassociated antigens. Some of the challenging issues found during production of CAR-T cells are the efficiency of T cell transduction to generate CAR-T cells, the expansion of T cells to clinically relevant numbers and the long-term survival in vivo of the therapeutic cells. The use of human platelet lysate has been demonstrated to improve these issues. Our data from experiments performed using human CD3+ from donors demonstrates that human platelet lysates offer an improved performance on T cell expansion versus serum derived products. hPL efficiently promotes T cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, we efficiently developed a protocol for suspension culture of T cells, which could facilitate the large-scale expansion of allogeneic CAR-T cells.
ABSTRACT
Remdesivir-loaded liposomes for inhalation were prepared and the in vitro properties were evaluated. Firstly, preparation methods of remdesivir-loaded liposomes were screened, and single-factor experiments were conducted to optimize the prescription and preparation process. Then the physical property, deposition ratio and aerodynamic particle size distribution of remdesivir-loaded liposomes suspension for inhalation were comprehensively evaluated. As a result, the optimal liposomes were prepared by the thin-film dispersion method with pH 6. 5 phosphate-buffered saline as the hydration medium. In the prescription, the ratio of drug to DPPC was 1:20;the cholesterol accounted for 10% of total lipids;and 20% DSPE-mPEG 2000 was added as stabilizer. 4% trehalose was added as lyoprotectant when lyophilizing to obtain ideal appearance, good stability and a small particle size change after reconstitution. Remdesivir-loaded liposomes were spherical with smooth surface and uniform particle size distribution under transmission electron microscope. In vitro release tests showed no significant change for release curves of remdesivir-loaded liposomes suspension before and after nebulization. Deposition experiments indicated that the fine particles fraction of liposomes was 51. 4%, and the mass median aerodynamic diameter was less than 5 μm measured by next generation impactor. To sum up, remdesivir-loaded liposomes for inhalation with high encapsulation efficiency and stability can achieve a suitable particle size distribution to effectively deposit in the lung after nebulization, which provides a new approach for the treatment of COVID-19.
ABSTRACT
Background: The 3CL protease (3CLpro) of coronaviruses (CoV) is responsible for essential & early steps of viral replication. Early treatment of SARS-CoV2 infection with a 3CLpro inhibitor has shown to substantially reduce the rate of hospitalization & death from COVID-19. There is a need for a protease inhibitor that can be used as a stand-alone agent to treat and prevent SARS-CoV-2 infection globally, in the setting of remote testing & healthcare delivery, and as unsupervised outpatient use by a significant number of people who take other medications. Methods: PBI-0451 was assessed in cultures of inducible pluripotent stem cell-derived alveolar type II (iPS-AT2) cells, in nonclinical PK and toxicity studies, and an ongoing randomized, double-blind first-in-human (FIH) study evaluating the tolerability, safety, and PK of single and multiple doses administered as an oral suspension to healthy adult subjects. The effect of food and the potential for a drug-drug interaction (DDI) with ritonavir were also explored. Results: PBI-0451 potently inhibited SARS-CoV-2 replication in iPS-AT2 cells with multi-log reductions in viral titer and mean (SD) IC50 & EC90 values of 32 (25) & 106 (90) nM, respectively. No clinically relevant adverse effects of PBI-0451 were observed in 14-day GLP toxicity studies in mice and dogs, including on the cardiovascular, CNS, or respiratory systems. PBI-0451 was not genotoxic in Ames and micronucleus tests. In the ongoing FIH study to date, study treatments were generally well tolerated with no study drug or study discontinuations. No Grade 2, 3, 4, or severe adverse events were reported. Preliminary single-dose concentration-time profile of PBI-0451 following administration with food demonstrated a 2-compartment PK profile with a median terminal elimination t1/2 ranging from 11-14 hours. PBI-0451 demonstrated good oral bioavailability and a linear increase in exposure over a 10-fold dose range when administered with food, achieving concentrations >1-, 3-& 10-fold the plasma protein binding-adjusted EC90 value (374 ng/mL) against SARS-CoV-2 at doses of 100, 300 & 1050 mg, respectively. The PK of PBI-0451 was unaffected by coadministration with ritonavir. Conclusion: PBI-0451 has shown favorable nonclinical properties and early clinical safety & PK that supports its continued evaluation as a stand-alone agent. Ongoing multiple-dose evaluation will further elucidate its clinical profile and inform the dose & dosing regimen selection for potential Phase II/III studies.
ABSTRACT
Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.
ABSTRACT
Background: COVID-19 is a disease caused by the SARS-CoV-2 virus that is often associated with pneumonia and acute respiratory distress syndrome (ARDS). Pathogenesis of COVID-19 is closely related to the host ' s response to the virus. Impaired regulation of immunity has been observed in patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs), having multipotency, ability to selfrenew, and ability to evade immune response may be useful in in the treatment of COVID-19 pneumonia. MSCs have an immunomodulatory effect on almost all types of immunocompetent cells: T-and B-lymphocytes, natural killer cells, monocytes and macrophages, dendritic cells, neutrophils. This study assesses the safety and tolerability of pooled allogeneic mesenchymal stem cells (poolMSC) in COVID 19 pneumonia. Method: The study included 5 patients with PCR confirmed COVID-19 pneumonia-4 men and 1 woman, average age 62.4 years. Pooled MSC (pMSC) was a mixture of 3 cultures of olfactory mucosa-derived MSCs (OM-MSCs) obtained from the mucous membrane of the middle nasal passage of healthy volunteers. MSCs were tested for viability (>95%), immunophenotype CD90 + CD105 + CD73 + CD31-CD45-HLA-DR-, and sterility. pMSC at a dosage of 1×10 6 cells per kg of body weight were suspended in 100 ml of saline and injected intravenously over 60 minutes. Results: All patients were carefully examined at baseline before pMSC infusion. Clinical examination was done on the day of infusion of pMSC with a skin test to pMSC was performed. In the absence of systemic and local allergic reactions pMSC were injected. Three patients received one pMSC infusion, and two patients received two pMSC infusions at an interval of 4 days. None of the patients had any adverse reactions to the pMSC skin test or infusion. Conclusion: Assessment of pMSC infusion demonstrated good tolerance and safety of intravenous use in patients with severe pneumonia caused by the SARS-CoV-2 and complicated by ARDS.