Dendritic Cell‐Mediated Cross‐Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS‐CoV‐2

SARS-CoV-2 B.1.351 and B.1.167.2 viruses used in this study were obtained through the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 653316. SARS-CoV-2 B.1 (MAD6 isolate) was kindly provided by José M. Honrubia and Luis Enjuanes (CNB-CSIC, Madrid, Spain). The authors thank Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. Research in LAV laboratory was funded by the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 y COVID19); the MCIN/AEI/10.13039/501100011033 (PID2020-117323RB-I00 and PDC2021-121711-I00), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (DTS20/00089), partially supported by the ERDF, the Spanish Association Against Cancer (AECC 19084); the CRIS Cancer Foundation (FCRISIFI-2018 and FCRIS-2021-0090), the Fundación Caixa-Health Research (HR21-00761 project IL7R_LungCan), and the Comunidad de Madrid (P2022/BMD-7225 NEXT_GEN_CART_MAD-CM). Work in the DS laboratory was funded by the CNIC; the European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091; MCIN/AEI/10.13039/501100011033 (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); Fondo Solidario Juntos (Banco Santander); and “La Caixa” Foundation (LCF/PR/HR20/00075). The CNIC was supported by the ISCIII, the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020- 001041-S funded by MCIN/AEI/10.13039/501100011033). Research in RD laboratory was supported by the ISCIII (PI2100989) and CIBERINFEC; the European Commission Horizon 2020 Framework Programme (grant numbers 731868 project VIRUSCAN FETPROACT-2016, and 101046084 project EPIC-CROWN-2); and the Fundación CaixaHealth Research (grant number HR18-00469 project StopEbola). Research in CNB-CSIC laboratory was funded by Fondo Supera COVID19 (Crue Universidades-Banco Santander) grant, CIBERINFEC, and Spanish Research Council (CSIC) grant 202120E079 (to J.G.-A.), CSIC grant 2020E84 (to M.E.), MCIN/AEI/10.13039/501100011033 (PID2020- 114481RB-I00 to J.G-A. and M.E.), and by the European CommissionNextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) to J.G.-A. and M.E. Work in the CIB-CSIC laboratory was supported by MCIN/AEI/10.13039/501100011033 (PID2019-104544GB-I00 and 2023AEP105 to CA, and PID2020-113225GB-I00 to F.J.B.). Cryo-EM data were collected at the Maryland Center for Advanced Molecular Analyses which was supported by MPOWER (The University of Maryland Strategic Partnership). I.H.-M. receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 71367. L.R.-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck.S

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.L.A-V. was supported by grants from the MCIN/AEI/10.13039/ 501100011033 (PID2020-117323RB-100 and PDC2021-121711-100), the Instituto de Salud Carlos III (DTS20/00089), the CRIS Cancer Foundation (FCRIS-2021-0090), the Spanish Association Against Cancer (PROYE19084ALVA), the Fundación ‘‘La Caixa’’ (HR21-00761 project IL7R_LungCan) and the Fundación de Investigación Biomédica 12 de Octubre Programa Investiga (2022-0082). B.B and L.S. were supported by grants PI20/01030 and PI19/00132 from the Instituto de Salud Carlos III (PI20/01030). FJB and MF-G were supported by grants PID2020- 113225GB-I00 and PRE2018-085788 funded by MCIN/AEI/10.13039/ 501100011033. L.R-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. C. D-A. was supported by a predoctoral fellowship from the MCIN/AEI/ 10.13039/501100011033 (PRE2018-083445). L.D-A. was supported by a Rio Hortega fellowship from the Instituto de Salud Carlos III (CM20/ 00004). O.H. was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Instituto de Salud Carlos III (IFI18/ 00045)Peer reviewe

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2

17 p.-4 fig.Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT, are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT, the bispecific trimerbody TNTDNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNTDNGR-1, but not TNT, protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.SARS-CoV-2 B.1.351 and B.1.167.2 viruses used in this study were obtained through the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 653316. SARS-CoV-2 B.1 (MAD6 isolate) was kindly provided by José M. Honrubia and Luis Enjuanes (CNB-CSIC, Madrid, Spain). The authors thank Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. Research in LA-V laboratory was funded by the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 y COVID-19); the MCIN/AEI/10.13039/501100011033 (PID2020-117323RB-I00 and PDC2021-121711-I00), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (DTS20/00089), partially supported by the ERDF, the Spanish Association Against Cancer (AECC 19084); the CRIS Cancer Foundation (FCRIS-IFI-2018 and FCRIS-2021-0090), the Fundación Caixa-Health Research (HR21-00761 project IL7R_LungCan), and the Comunidad de Madrid (P2022/BMD-7225 NEXT_GEN_CART_MAD-CM). Work in the DS laboratory was funded by the CNIC; the European Union's Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091; MCIN/AEI/10.13039/501100011033 (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); Fondo Solidario Juntos (Banco Santander); and “La Caixa” Foundation (LCF/PR/HR20/00075). The CNIC was supported by the ISCIII, the MCIN and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033). Research in RD laboratory was supported by the ISCIII (PI2100989) and CIBERINFEC; the European Commission Horizon 2020 Framework Programme (grant numbers 731868 project VIRUSCAN FETPROACT-2016, and 101046084 project EPIC-CROWN-2); and the Fundación Caixa-Health Research (grant number HR18-00469 project StopEbola). Research in CNB-CSIC laboratory was funded by Fondo Supera COVID-19 (Crue Universidades-Banco Santander) grant, CIBERINFEC, and Spanish Research Council (CSIC) grant 202120E079 (to J.G.-A.), CSIC grant 2020E84 (to M.E.), MCIN/AEI/10.13039/501100011033 (PID2020-114481RB-I00 to J.G-A. and M.E.), and by the European Commission-NextGenerationEU, through CSIC's Global Health Platform (PTI Salud Global) to J.G.-A. and M.E. Work in the CIB-CSIC laboratory was supported by MCIN/AEI/10.13039/501100011033 (PID2019-104544GB-I00 and 2023AEP105 to CA, and PID2020-113225GB-I00 to F.J.B.). Cryo-EM data were collected at the Maryland Center for Advanced Molecular Analyses which was supported by MPOWER (The University of Maryland Strategic Partnership). I.H.-M. receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 71367. L.R.-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck.Peer reviewe

Towards a Change in the Diagnostic Algorithm of Autism Spectrum Disorders: Evidence Supporting Whole Exome Sequencing as a First-Tier Test

Autism spectrum disorder (ASD) is a prevalent and extremely heterogeneous neurodevelopmental disorder (NDD) with a strong genetic component. In recent years, the clinical relevance of de novo mutations to the aetiology of ASD has been demonstrated. Current guidelines recommend chromosomal microarray (CMA) and a FMR1 testing as first-tier tests, but there is increasing evidence that support the use of NGS for the diagnosis of NDDs. Specifically in ASD, it has not been extensively evaluated and, thus, we performed and compared the clinical utility of CMA, FMR1 testing, and/or whole exome sequencing (WES) in a cohort of 343 ASD patients. We achieved a global diagnostic rate of 12.8% (44/343), the majority of them being characterised by WES (33/44; 75%) compared to CMA (9/44; 20.4%) or FMR1 testing (2/44; 4.5%). Taking into account the age at which genetic testing was carried out, we identified a causal genetic alteration in 22.5% (37/164) of patients over 5 years old, but only in 3.9% (7/179) of patients under this age. Our data evidence the higher diagnostic power of WES compared to CMA in the study of ASD and support the implementation of WES as a first-tier test for the genetic diagnosis of this disorder, when there is no suspicion of fragile X syndrome