Open-label phase I/II clinical trial of SARS-CoV-2 receptor binding domain-tetanus toxoid conjugate vaccine (FINLAY-FR-2) in combination with receptor binding domain-protein vaccine (FINLAY-FR-1A) in children.

OBJECTIVES: To evaluate a heterologous vaccination scheme in children 3-18 years old (y/o) combining two SARS-CoV-2r- receptor binding domain (RBD)protein vaccines. METHODS: A phase I/II open-label, adaptive, and multicenter trial evaluated the safety and immunogenicity of two doses of FINLAY-FR-2 (subsequently called SOBERANA 02) and the third heterologous dose of FINLAY-FR-1A (subsequently called SOBERANA Plus) in 350 children 3-18 y/o in Havana Cuba. Primary outcomes were safety (phase I) and safety/immunogenicity (phase II) measured by anti-RBD immunoglobulin (Ig)G enzyme-linked immunoassay (ELISA), molecular and live-virus neutralization titers, and specific T-cells response. A comparison with adult immunogenicity and predictions of efficacy were made based on immunological results. RESULTS: Local pain was the unique adverse event with frequency >10%, and none was serious neither severe. Two doses of FINLAY-FR-2 elicited a humoral immune response similar to natural infection; the third dose with FINLAY-FR-1A increased the response in all children, similar to that achieved in vaccinated young adults. The geometric mean (GMT) neutralizing titer was 173.8 (95% confidence interval [CI] 131.7; 229.5) vs Alpha, 142 (95% CI 101.3; 198.9) vs Delta, 24.8 (95% CI 16.8; 36.6) vs Beta and 99.2 (95% CI 67.8; 145.4) vs Omicron. CONCLUSION: The heterologous scheme was safe and immunogenic in children 3-18 y/o. TRIAL REGISTRY: https://rpcec.sld.cu/trials/RPCEC00000374.

Safety and immunogenicity of anti-SARS-CoV-2 heterologous scheme with SOBERANA 02 and SOBERANA Plus vaccines: Phase IIb clinical trial in adults.

BACKGROUND: SOBERANA 02 has been evaluated in phase I and IIa studies comparing homologous versus heterologous schedule (this one, including SOBERANA Plus). Here, we report results of immunogenicity, safety, and reactogenicity of SOBERANA 02 in a two- or three-dose heterologous scheme in adults. METHOD: Phase IIb was a parallel, multicenter, adaptive, double-blind, randomized, and placebo-controlled trial. Subjects (n = 810) aged 19-80 years were randomized to receive two doses of SARS-CoV-2 RBD conjugated to tetanus toxoid (SOBERANA 02) and a third dose of dimeric RBD (SOBERANA Plus) 28 days apart; two production batches of active ingredients of SOBERANA 02 were evaluated. Primary outcome was the percentage of seroconverted subjects with ≥4-fold the anti-RBD immunoglobulin G (IgG) concentration. Secondary outcomes were safety, reactogenicity, and neutralizing antibodies. FINDINGS: Seroconversion rate in vaccinees was 76.3% after two doses and 96.8% after the third dose of SOBERANA Plus (7.3% in the placebo group). Neutralizing IgG antibodies were detected against D614G and variants of concern (VOCs) Alpha, Beta, Delta, and Omicron. Specific, functional antibodies were detected 7-8 months after the third dose. The frequency of serious adverse events (AEs) associated with vaccination was very low (0.1%). Local pain was the most frequent AE. CONCLUSIONS: Two doses of SOBERANA 02 were safe and immunogenic in adults. The heterologous combination with SOBERANA Plus increased neutralizing antibodies, detectable 7-8 months after the third dose. TRIAL REGISTRY: https://rpcec.sld.cu/trials/RPCEC00000347 FUNDING: This work was supported by Finlay Vaccine Institute, BioCubaFarma, and the Fondo Nacional de Ciencia y Técnica (FONCI-CITMA-Cuba, contract 2020-20).

Safety and immunogenicity of the FINLAY-FR-1A vaccine in COVID-19 convalescent participants: an open-label phase 2a and double-blind, randomised, placebo-controlled, phase 2b, seamless, clinical trial.

BACKGROUND: A phase 1, clinical trial to evaluate FINLAY-FR-1A vaccine in COVID-19 convalescent individuals was completed. Here, we report results of the phase 2, clinical trial. METHODS: We studied 450 convalescent participants with a history of asymptomatic, mild, or moderate COVID-19 at the National Institute of Hematology and Immunology and the National Centre for Sexual Education in Havana, Cuba. The study included adults aged 19-78 years who had recovered from COVID-19 and had had a negative PCR test at least 2 months before the initiation of the study. Phase 2 was done sequentially in two stages. The first stage to assess safety comprised an open, non-controlled phase 2a study in participants aged 60-78 years who received a single dose of the FINLAY-FR-1A vaccine (50 µg of recombinant dimeric receptor binding domain [RBD]). The second stage comprised the placebo-controlled, double-blind, phase 2b trial in participants aged 19-78 years, where participants were randomly assigned (4:1) into two groups: an experimental group vaccinated with a single dose of the FINLAY-FR-1A vaccine, and a control (placebo) group injected with vaccine excipient. The primary outcomes were safety, evaluated 28 days after vaccination by the occurrence of serious adverse events in all participants, and successful immune response, assessed by neutralising antibody ELISA, and defined as half-maximal surrogate virus neutralisation titres of 250 or more. Secondary endpoints included vaccine immunogenicity assessed by ELISA anti-RBD and live-virus neutralisation test. All randomly assigned participants were included in the safety analysis (safety population), and immunogenicity was evaluated in participants without study interruptions (per-protocol population). The trial is registered with the Cuban Public Registry of Clinical Trials, RPCEC00000366-En and WHO-ICTRP and is complete. FINDINGS: From April 9, 2021, to April 17, 2021, 663 COVID-19 convalescent participants were enrolled in the study; 213 participants did not meet the selection criteria and 450 volunteers were recruited. 20 participants aged 60-78 years were included in the open, single-group, phase 2a study and 430 participants were randomly assigned to the experimental (n=344) or control groups (n=86) in the phase 2b study of participants aged 19-78 years. 19 (95%) of 20 phase 2a volunteers achieved a successful immune response after vaccination. No vaccine-associated serious adverse events were reported in the whole study population. Minor adverse events were found, the most common being pain at the injection site (105 [29%] of 364 in the intervention group; 13 [15%] of 86 in the placebo group). A successful immune response was found in 289 (81%) of 358 participants 28 days after vaccination. The vaccine elicited a greater than 31-times increase in anti-RBD-IgG antibodies compared with prevaccination rates, and the seroconversion rate was 302 (84%) of 358 on day 28 after vaccination; the geometric mean titres of live-virus neutralisation test increased from 15·4 (95% CI 10·3-23·2) to 400·3 (272·4-588·1) and high response was found against alpha, beta, and delta variants of concern. INTERPRETATION: A single dose of the FINLAY-FR-1A vaccine against SARS-CoV-2 strengthened the pre-existing natural immunity, with excellent safety profile. FUNDING: Cuba's Ministry of Science, Technology, and Environment.

Safety and immunogenicity of anti-SARS CoV-2 vaccine SOBERANA 02 in homologous or heterologous scheme: Open label phase I and phase IIa clinical trials.

BACKGROUND: SOBERANA 02 is a COVID-19 vaccine based on SARS-CoV-2 recombinant RBD conjugated to tetanus toxoid (TT). SOBERANA Plus antigen is dimeric-RBD. Here we report safety and immunogenicity from phase I and IIa clinical trials using two-doses of SOBERANA 02 and three-doses (homologous) or heterologous (with SOBERANA Plus) protocols. METHOD: We performed an open-label, sequential and adaptive phase I to evaluate safety and explore the immunogenicity of SOBERANA 02 in two formulations (15 or 25 µg RBD-conjugated to 20 µg of TT) in 40 subjects, 19-59-years-old. Phase IIa was open-label including 100 volunteers 19-80-years, receiving two doses of SOBERANA 02-25 µg. In both trials, half of volunteers were selected to receive a third dose of the corresponding SOBERANA 02 and half received a heterologous dose of SOBERANA Plus. Primary outcome was safety. The secondary outcome was immunogenicity evaluated by anti-RBD IgG ELISA, molecular neutralization of RBD:hACE2 interaction, live-virus-neutralization and specific T-cells response. RESULTS: The most frequent adverse event (AE) was local pain, other AEs had frequencies ≤ 5%. No serious related-AEs were reported. Phase IIa confirmed the safety in 60 to 80-years-old subjects. In phase-I SOBERANA 02-25 µg elicited higher immune response than SOBERANA 02-15 µg and progressed to phase IIa. Phase IIa results confirmed the immunogenicity of SOBERANA 02-25 µg even in 60-80-years. Two doses of SOBERANA02-25 µg elicited an immune response similar to that of the Cuban Convalescent Serum Panel and it was higher after the homologous and heterologous third doses. The heterologous scheme showed a higher immunological response. Anti-RBD IgG neutralized the delta variant in molecular assay, with a 2.5-fold reduction compared to D614G neutralization. CONCLUSIONS: SOBERANA 02 was safe and immunogenic in persons aged 19-80 years, eliciting neutralizing antibodies and specific T-cell response. Highest immune responses were obtained in the heterologous three doses protocol. TRIAL REGISTRY: https://rpcec.sld.cu/trials/RPCEC00000340, https://rpcec.sld.cu/trials/RPCEC00000347.

A randomized, double-blind phase I clinical trial of two recombinant dimeric RBD COVID-19 vaccine candidates: Safety, reactogenicity and immunogenicity.

BACKGROUND: The Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein is the target for many COVID-19 vaccines. Here we report results for phase I clinical trial of two COVID-19 vaccine candidates based on recombinant dimeric RBD (d-RBD). METHODS: We performed a randomized, double-blind, phase I clinical trial in the National Centre of Toxicology in Havana. Sixty Cuban volunteers aged 19-59 years were randomized into three groups (20 subjects each): 1) FINLAY-FR-1 (50 µg d-RBD plus outer membrane vesicles from N. meningitidis); 2) FINLAY-FR-1A-50 (50 µg d-RBD, three doses); 3) FINLAY-FR-1A-25 (25 µg d-RDB, three doses). The FINLAY-FR-1 group was randomly divided to receive a third dose of the same vaccine candidate (homologous schedule) or FINLAY-FR-1A-50 (heterologous schedule). The primary outcomes were safety and reactogenicity. The secondary outcome was vaccine immunogenicity. Humoral response at baseline and following each vaccination was evaluated using live-virus neutralization test, anti-RBD IgG ELISA and in-vitro neutralization test of RBD:hACE2 interaction. RESULTS: Most adverse events were of mild intensity (63.5%), solicited (58.8%), and local (61.8%); 69.4% with causal association with vaccination. Serious adverse events were not found. The FINLAY-FR-1 group reported more subjects with adverse events than the other two groups. After the third dose, anti-RBD seroconversion was 100%, 94.4% and 90% for the FINLAY-FR-1, FINLAY-FR-1A-50 and FINLAY-FR-1A-25 respectively. The in-vitro inhibition of RBD:hACE2 interaction increased after the second dose in all formulations. The geometric mean neutralizing titres after the third dose rose significantly in the group vaccinated with FINLAY-FR-1 with respect to the other formulations and the COVID-19 Convalescent Serum Panel. No differences were found between FINLAY-FR-1 homologous or heterologous schedules. CONCLUSIONS: Vaccine candidates were safe and immunogenic, and induced live-virus neutralizing antibodies against SARS-CoV-2. The highest values were obtained when outer membrane vesicles were used as adjuvant. TRIAL REGISTRY: https://rpcec.sld.cu/en/trials/RPCEC00000338-En.

A single dose of SARS-CoV-2 FINLAY-FR-1A vaccine enhances neutralization response in COVID-19 convalescents, with a very good safety profile: An open-label phase 1 clinical trial.

BACKGROUND: As a first step towards a vaccine protecting COVID-19 convalescents from reinfection, we evaluated FINLAY-FR-1A vaccine in a clinical trial. METHODS: Thirty COVID-19 convalescents aged 22-57 years were studied: convalescents of mild COVID-19, asymptomatic convalescents, both with PCR-positive at the moment of diagnosis; and individuals with subclinical infection detected by viral-specific IgG. They received a single intramuscular injection of the FINLAY-FR-1A vaccine (50 µg of the recombinant dimeric receptor binding domain). The primary outcomes were safety and reactogenicity, assessed over 28 days after vaccination. The secondary outcome was vaccine immunogenicity. Humoral response at baseline and following vaccination was evaluated by ELISA and live-virus neutralization test. The effector T cellular response was also assessed. Cuban Public Registry of Clinical Trials, WHO-ICTRP: https://rpcec.sld.cu/en/trials/RPCEC00000349-En. FINDINGS: No serious adverse events were reported. Minor adverse events were found, the most common, local pain: 3 (10%) and redness: 2 (6·7%). The vaccine elicited a >21 fold increase in IgG anti-RBD antibodies 28 days after vaccination. The median of inhibitory antibody titres (94·0%) was three times greater than that of the COVID-19 convalescent panel. Virus neutralization titres higher than 1:160 were found in 24 (80%) participants. There was also an increase in RBD-specific T cells producing IFN-γ and TNF-α. INTERPRETATION: A single dose of the FINLAY-FR-1A vaccine against SARS-CoV-2 was an efficient booster of pre-existing natural immunity, with excellent safety profile. FUNDING: Partial funding for this study was received from the Project-2020-20, Fondo de Ciencia e Innovación (FONCI), Ministry of Science, Technology and the Environment, Cuba.   RESUMEN. ANTECEDENTES: Como un primer paso hacia una vacuna que proteja a los convalecientes de COVID-19 de la reinfección, evaluamos la vacuna FINLAY-FR-1A en un ensayo clínico. MÉTODOS: Se estudiaron treinta convalecientes de COVID-19 de 22 a 57 años: convalecientes de COVID-19 leve y convalecientes asintomáticos, ambos con prueba PCR positiva al momento del diagnóstico; e individuos con infección subclínica detectada por IgG específica viral. Los participantes recibieron una dosis única por vía intramuscular de la vacuna FINLAY-FR-1A (50 µg del dominio de unión al receptor recombinante dimérico del SARS CoV-2). Las variables de medida primarias fueron la seguridad y la reactogenicidad, evaluadas durante 28 días después de la vacunación. La variable secundaria, la inmunogenicidad. La respuesta humoral, al inicio del estudio y después de la vacunación, se evaluó por ELISA y mediante la prueba de neutralización del virus vivo. También se evaluó la respuesta de células T efectoras. Registro Público Cubano de Ensayos Clínicos, WHO-ICTRP: https://rpcec.sld.cu/en/trials/RPCEC00000349-En. RESULTADOS: No se reportaron eventos adversos graves. Se encontraron eventos adversos leves, los más comunes, dolor local: 3 (10%) y enrojecimiento: 2 (6·7%). La vacuna estimuló un incremento >21 veces de los anticuerpos IgG anti-RBD 28 días después de la vacunación. La mediana de los títulos de anticuerpos inhibidores (94·0%) fue aproximadamente tres veces mayor que la del panel de convalecientes de COVID-19. Se encontraron títulos de neutralización viral superiores a 1:160 en 24 (80%) de los participantes. También hubo un aumento en las células T específicas de RBD que producen IFN-γ y TNF-α. INTERPRETACIÓN: Una sola dosis de la vacuna FINLAY-FR-1A contra el SARS-CoV-2 reforzó eficazmente la inmunidad natural preexistente, con un excelente perfil de seguridad. FINANCIAMIENTO: Se recibió un financiamiento parcial del Proyecto-2020-20, Fondo de Ciencia e Innovación (FONCI), Ministerio de Ciencia, Tecnología y Medio Ambiente, Cuba.

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines.

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Obtención de una versión IgG1 de ratón del rituximab para detectar la molécula CD20 humana / Obtaining a mouse IgG1 version of rituximab to detect the human CD20 molecule

Introducción: El rituximab, anticuerpo quimérico que reconoce la molécula CD20 humana, se ha utilizado en el tratamiento de diversos trastornos linfoproliferativos de células B. Para la selección de los potenciales beneficiarios del tratamiento con rituximab se han desarrollado técnicas que, mediante el uso de anticuerpos monoclonales, detectan la presencia del CD20 en los linfocitos de estos pacientes. Objetivo: Obtener y caracterizar un anticuerpo recombinante IgG1 de ratón específico para la molécula CD20 humana, que contenga las regiones variables del anticuerpo rituximab. Métodos: Para la expresión estable del anticuerpo recombinante se empleó la transducción lentiviral de células de embrión de riñón humano (HEK293). La caracterización inmunoquímica del anticuerpo se realizó por la técnica de Western Blot y su capacidad de reconocimiento de la molécula CD20 humana se evaluó por citometría de flujo e inmunohistoquímica. Resultados: Se obtuvo el anticuerpo 1F5 que reconoce, por citometría de flujo, la molécula CD20 en líneas celulares humanas de origen linfoide, así como en células de sangre periférica de humanos sanos y pacientes con trstornos linfoproliferativos de células B. Sin embargo, la técnica de inmunohistoquímica solo permitió detectar con este anticuerpo la molécula CD20 en tejidos frescos, no así en los embebidos en parafina. Conclusiones: Este trabajo sugiere las potencialidades del uso del anticuerpo 1F5 para las mediciones de la expresión de CD20 por citometría de flujo en pacientes con leucemias B o linfomas B avanzados en fase de leucemización. Esto complementaría los estudios para la selección apropiada de pacientes para el tratamiento con el rituximab(AU)

Introduction: Rituximab, chimeric antibody specific for human CD20 molecule, has been widely used in the treatment of several B-cell linfoproliferative disorders. For the selection of patients with the greatest potential to benefit from the therapy with rituximab, a number of techniques using monoclonal antibodies have been developed to detect the CD20 molecule. Objective: To obtain and to characterize a mouse IgG1 recombinant antibody, specific for human CD20, that contains the variable regions of rituximab. Methods: The lentiviral transduction of human embryonic kidney cells (HEK293) was used for the stable expression of the recombinant antibody. The immunochemical characterization of the antibody was performed by Western Blot and the recognition of CD20 was evaluated by immunohistochemistry and flow cytometry. Results: We generated the antibody 1F5, able to recognize by flow cytometry the CD20 molecule expressed on lymphoid human cell lines, as well as peripheral blood mononuclear cells from healthy donors and patients with B-cell lymphoproliferative disorders. However, 1F5 antibody detected the CD20 molecule on fresh tissues, but not on formalin-fixed paraffin embedded tissues,by immunohistochemistry. Conclusions: This work suggests the potential use of 1F5 antibody for the measurement of CD20 expression by flow cytometry in patients with B-cell leukemias or B-cell lymphomas in phase of leukemization. This could complement the studies to ensure the appropriate selection of patients for the treatment with rituximab(AU)