Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67%–93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative reverse transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence.W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV A mutatTranslation into human clinical trials requires validation of vaccine formulations in preclinical studies. First, W614A-3S were conjugated to a carrier protein, Cross-Reacting Material 197 (CRM197). CRM197 is a genetically detoxified form of diphtheria toxin. A single mutation at position 52, substituting glutamic acid for glycine, causes the ADP-ribosyltransferase activity of the native toxin to be lost . CRM197 BALB/cByJ female mice were purchased from Janvier Labs , housed at a free animal facility (Centre d’Experimentation Fonctionnelle (CEF) of Sorbonne University, France) under specified pathogen-free conditions, and used for experiments at 6–10 weeks of age. New Zealand rabbits were housed at Covalab company .Rabbits received 50 μg of the peptide W614A-3S coupled with CRM197 by the intramuscular route at Week 0 (W0), W2, W4, and W10. Mice received 10 μg of peptide coupled with keyhole limpet hemocyanin  carrier protein or 11.7 μg of W614A-3S peptide coupled with CRM197  by intramuscular injections (in both quadriceps of left and right thighs) at the same kinetic (or W18 for mouse CRM197 experiment). W614A-3S peptide (NH2-PWNASASNKSLDDIW-COOH) is a mutated peptide of HIV-1 gp41  of each well was monitored at 450 nm with a FlexStation 3 ELISA reader  and Ascent Software version 2.6. EC50 was calculated by sigmoidal dose response (Prism 9 software). For the NAb assay, serum-purified W614A-3S-specific Abs were obtained as described  was added to measure luciferase activity with a Mithras luminometer . Positive controls were monoclonal Abs with known neutralizing titers. Neutralization titers were defined as the sample dilution at which RLU were reduced by 50% compared to virus control wells after subtraction of background RLU in control wells with only cells. Inhibitory concentrations (IC) 50 were calculated with a linear interpolation method using the mean of the duplicate responses  and stored at -80°C prior to evaluation of W614A-3S-specific IgG titration by ELISA. MaxiSorp plates  were coated overnight with 50 ng/well of W614A-3S peptide . For W614A-3S-monoclonal Ab evaluation, plates were coated with 50 ng/well of W614A-3S-KLH vaccine. Wells were saturated with PBS1X + 1% bovine serum albumin (Life Technologies) prior to serum serial dilutions of 1/10 or different dilutions of W614A-3S-monoclonal Abs . All samples were tested in duplicate. Peptide-specific Abs were revealed by horseradish peroxidase (HRP)-conjugated anti-mouse IgG  or detection antibody biotinylated anti-rabbit IgG  followed by HRP-streptavidin . Enzymatic activity was measured by adding tetramethylbenzidine  and stopped by 1 N Hescribed . Brieflyesponses . Neutral+ GC (GL7+IgDLow) and NGC (GL7-IgD+) B cell isolations [chromium single cell V(D)J assay].Draining lymph nodes  were harvested 7 days after each immunization. Cells were stained with W614A-3S coupled with biotinylated ovalbumin (Ova) protein  for 30 min at room temperature before cell surface antigen staining with a standard method after receptor Fc blocking with CD16/CD32 , and the following anti-mouse Abs: CD3e , CD45R/B220 (clone RA3-6B2), CD19 (clone 1D3), IgG1 (clone A85-1), IgD (clone 11-26c.2a), T- and B-cell activation antigen (clone GL7), and streptavidin (BD Biosciences). For cell analysis, dead cells were excluded by using the LIVE/DEAD fixable kit . Cells were analyzed by BD LSRFortessa flow cytometry or isolated by BD FACSAria II sorter. A pool of five mice per condition at W3 and W5 was used for W614A-3S-specific B cell isolations (BioMark Dynamic array). A pool of 25 mice per condition at W11 was used for W614A-3S-specific IgG1A total of 747 single cells were analyzed from Alum conditions (n = 375 cells) and SQE conditions (n = 372 cells) across W3 and W5. Single-cell gene expression analysis was performed using the BioMark 96.96 Dynamic Array IFCs and the Biomark HD System from Fluidigm. Two-step single-cell gene delta gene expression was performed using EvaGreen Supermix according to Fluidigm real-time PCR protocol. Briefly, reverse transcription using a 2-step VILO cDNA Synthesis Kit  was performed directly on single cells prior to specific target cDNA amplification.Probes for 96 genes were selected from the catalog of delta gene expression assay J reagent kits (10x Genomics) to attain between 500 and 10,000 cells per condition. Library preparation for V(D)J sequencing was performed according to the manufacturer’s protocol  prior to sequencing on the Illumina MiSeq. Quality of raw reads was assessed using the FastQC 0.11.8 quality control tool, and clonotype quantification was performed with the Cell Ranger 3.1.0 V(D)J pipeline. Only 433 clonotypes defined as IgG protein sequences with exactly one heavy and one light chain were considered in the analysis. CDRH3 and CDRL3 sequence alignments were performed with Unipro UGENE software.W614A-3S-specific IgG1V(D)J sequences of W614A-3S-specific GC and NGC B cells were chosen from squalene conditions. Sixteen monoclonal antibodies were produced by the ProteoGenix company. An endotoxin-free DNA preparation was made for the pTXs1 expression constructions prior to XtenCHO™ transient expression. The recombinant Abs were then purified by affinity vs. protein G. After production, specificity tests by ELISA and a neutralization assay were performed.Statistical analyses and graphical representation were performed using either GraphPad Prism 9, FlowJo X, OMIQ, Unipro UGENE software, or R 3.6.2. A two-way ANOVA test was used for kinetic peptide-specific IgG evaluation. Mann–Whitney tests were used to compare SQE and Alum conditions.All experimental protocols were approved by the French animal experimentation and ethics committee and validated by Service Protection et Santé Animales, Environnement, with the numbers 00954.02 and APAFIS#5863–2016062710255883 v3 for mouse experiments.To evaluate the efficacy of W614A-3S-CRM197-conjugated vaccine, formulated in SQE and Alum, in inducing bNAbs, rabbits were immunized intramuscularly at week 0 (W0), W2, W4, and W10 in two independent experiments (n = 5 per condition) .In order to further elucidate the immune mechanism of induction of NAbs versus non-NAbs using these two formulations, we validated our vaccine strategy using W614A-3S peptide, coupled with either the KLH or the CRM197 carrier with SQE or Alum formulation in the mouse model. The immunization protocol is shown in We hypothesized a differential B cell maturation following vaccination with different adjuvant formulations. In order to study antigen-specific B cells, we measured W614A-3S-specific B cells in draining lymph nodes (dLNs), using W614A-3S-biotinylated ovalbumin (Ova). Flow cytometry analysis of W614A-3S-biotinylated Ova staining of B cells in either PBS or W614A-3S formulated in SQE or Alum is shown in In conclusion, we found that SQE formulation, W614A-3S conjugated with a carrier protein, induced bNAbs while the Alum formulation did not with a similar amount of W614A-3S-specific B cells. Here, we aim to elucidate the mechanism of B cell immune responses.We used a single-cell quantitative reverse transcription-PCR (qRT-PCR) approach to compare between the two formulations the quality of W614A-3S-specific B cell populations isolated from dLNs, 1 week after the 2nd and 3rd immunizations  and non-GC  B cells for single-cell V(D)J sequencing  and light (L) IgG1 chain sequences, the third complementarity-determining region (CDR3), was carried out for both conditions and in cell types with a total of 433 distinct sequences, regardless of their frequencies in samples. Clonotypes with exactly one heavy and one light chain were considered in the analysis. The proportions of cells with different lengths of CDRH3 were relatively similar in Alum and SQE conditions for GC or NGC B lymphocytes . We cloned and produced recombinant monoclonal IgG1 with completed V(D)J sequences among the most frequent ones in SQE conditions. We confirmed that selected monoclonal Abs were specific to the W614A-3S peptide (+) were detected following injection of both formulations, gene expression, B cell differentiation, and BCR selection were distinct.Altogether, both approaches using single-cell analysis of gene expression and BCR sequencing revealed that even though W614A-3S-specific B cells . We demonstrated that the formulation of W614A-3S-carrier protein with either Alum or SQE impacts the quantity and quality of B lymphocytes. These differences result in 1) the induction of non-NAbs for the Alum formulation versus the production of NAbs for the SQE formulation, which were capable of neutralizing a wide range of HIV-1 strains including Tier 1 and Tier 2 viruses (73%–93% breadth); 2) major differences in the proportion of differentiated B cells expressing GC B-cell-associated genes; and 3) a distinct amino acid sequence of the CDR3 region. One limitation is the paucity of these bNAbs in the serum of animals. Indeed, rare neutralizing activities were detected using the whole sera of rabbits or mice; a purification step of peptide-specific IgG1 was necessary to detect virus neutralization. The dichotomy between Alum and SQE formulation is intriguing and strengthens our results.BNAbs generally arise late in the course of HIV infection. The most mature bNAbs revealed one or more unusual features, such as high levels of somatic hypermutation, unusually long complementary-determining regions like CDRH3, and polyreactivity for non-HIV-1 antigens –20. Whil® that has been licensed in Europe for adjuvanted flu vaccines J sequencing of W614A-3S-specific BCR validates the dichotomy between SQE and Alum formulations. Significant diversity of the B cell repertoire of GC and NGC-B cells was observed following W614A-3S vaccination using SQE compared to Alum formulations. We did not find any neutralizing activities among cloned Abs according to the defined sequence. There are some limitations in our study, notably in regard to BCR sequencing for detection of NAbs. Indeed, we used W614A-3S peptide conjugated with biotinylated ovalbumin in order to purify antigen-specific B cells that might impact the selection of certain BCR during this step. Of note, vaccination of animals with a W614A-3S peptide in IFA induces neutralizing anti-HIV-1 Abs, among which we found a unique clone, F8, by hybridoma generation , suggestWe found a dichotomy in the induction of bNAbs, B-cell gene expression, and BCR sequence after immunization using W614A-3S formulated in SQE compared to Alum. Our study challenges HIV vaccine development and discovery of formulation that could program B-cell differentiation favoring bNAbs. These data have been used in Go/No-go criteria for a clinical study. Indeed, an experimental medicine study using W614A-3S-CRM197 vaccine candidate  is ongoing with the hope to speed up HIV vaccine research .www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198883; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198952.The original contributions presented in the study are publicly available. This data can be found here: The animal study was reviewed and approved by Service Protection et Santé Animales, Environnement.BC and VV designed the study. OB, CC, MT, MB, LB, and VV carried out the experimental work and contributed to the data acquisition, analysis, and interpretation. SB, KA, and JN compiled and analyzed the single-cell repertoire data. ES and DK were responsible for adjuvant production and quality control. MB and GS were responsible for the neutralization assays and analysis. BC, PD, GS, DK, and VV provided financial support. BC and OB wrote the manuscript. All authors contributed to the article and approved the submitted version.This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 681137. BC’s laboratory has received funding from the Fondation pour la Recherche Médicale “Equipe FRM 2020” award. GC’s laboratory thanks the Fondation Dormeur, Vaduz, for funding laboratory instruments relevant to this project. High-throughput sequencing has been performed by the ICGex NGS platform of the Institut Curie supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the Agence Nationale de la Recherche (“Investissements d’Avenir” program), by the Canceropole Ile-de-France, and by the SiRIC-Curie program – SiRIC Grant “INCa-DGOS-4654”.VV is a member of the scientific advisory board of Minka Therapeutics. ES and DK were employed by Polymun Scientific Immunbiologische Forschung GmbH. SB, KA, and JN were employed by Altrabio.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


When tested in a 5-day-old Pekin duck model, Y and PS induced comparable levels of neutralizing antibody, whereas Y elicited significantly stronger cellular immune response relative to PS. Using a duck adoptive transfer model, we showed that both CD4+ and CD8+ T cells provided significant protection from TMUV-related disease, with CD8+ T cell conferring more robust protection to recipient ducklings. For TMUV, CD4+ T cells mainly provided help for neutralizing antibody response, whereas CD8+ T cells mainly mediated viral clearance from infected tissues. The difference in T cell immunity between Y and PS was primarily attributed to CD4+ T cells; adoptive transfer of Y-specific CD4+ T cells resulted in significantly enhanced protective ability, neutralizing antibody response, and viral clearance from the brain relative to PS-specific CD4+ T cells. Further investigations with chimeric viruses, mutant viruses, and their parental viruses identified two mutations (T151A and R304M) in the envelope (E) protein that contributed significantly to TMUV-specific CD4+ T cell-mediated protective ability and neutralizing antibody response, with more beneficial effects being conferred by R304M. These data indicate T cell-mediated immunity is important for protection from disease, for viral clearance from tissues, and for the production of neutralizing antibodies, and that the difference in CD4+T cell immunity between high- and low-virulence TMUV strains is primarily related to residues 151 and 304 in the E protein.Tembusu virus (TMUV) can result in a severe disease affecting domestic ducks. The role of T cells in protection from TMUV infection and the molecular basis of T cell-mediated protection against TMUV remain largely uncharacterized. Here, we used the high-virulence TMUV strain Y and the low-virulence TMUV strain PS to investigate the protective role for TMUV-specific CD4 Tembusu virus (TMUV) is an enveloped mosquito-borne flavivirus with a positive-sense, single-stranded RNA genome of approximately 11 kb. The RNA genome contains one large open reading frame (ORF), which is preceded by a 5’ untranslated region (UTR) and followed by a 3’ UTR. The ORF encodes ten polypeptides: three structural proteins  that form the viral particle and seven nonstructural proteins  that are required for viral replication (Anas platyrhynchos domesticus) less than 7 weeks of age have shown that the severity of TMUV-caused disease is associated with multiple factors, such as the age of birds, the route of application, the strain of virus, and the infectivity titer. In ducklings less than 9 days of age, TMUV generally causes fatal infection, with mortality ranging from 18% to 100% , TMUV has been shown to elicit high, long-lasting neutralizing antibodies after natural infection and vaccination with attenuated vaccine . Previou+ and CD8+ T cells at 5 days pi  , 28, and days pi . The E,  vaccine , 22, 27.To date, the role of T cells in protection from TMUV infection and the molecular basis of T cell-mediated protection against TMUV remain largely uncharacterized. Earlier works in our laboratory showed that the natural isolate Y and the plaque-purified strain PS, which exhibited distinct virulence in a 2-day-old Pekin duckling model, elicited comparable levels of neutralizing antibody . In thisNewly hatched Pekin ducklings were derived from Peking duck breeding farm, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. Their parents had never received TMUV vaccine. All ducklings were confirmed to be free of TMUV infection by testing serum samples using PRNT for antibodies to TMUV  and TMUVThe BHK-21 cell-derived Y and PS strains of TMUV were isolated and propagated previously, which were shown to display high- and low-virulence for 2-day-old Pekin ducklings respectively . ParentaTo obtain working stocks, viruses were propagated in BHK-21 cells as described previously . For str3 PFU, respectively. The mock-infected control group received 0.2 ml of supernatant prepared from uninfected BHK-21 cells by im inoculation at 3 days of age. The ducklings were monitored for 15 days for mortality.The first experiment was conducted to investigate the pathogenicity of strain Y in Pekin ducklings with different ages, employing strain PS as a control virus. A total of 140 newly hatched ducklings were divided into seven groups (n=20). When the ducklings grew to 3, 5, and 7 days of age, they were inoculated with virus by intramuscular (im) route at a dose of 2×10+ and CD8+ T cells respectively. Subsequently, the selected ducks were euthanized, tissues  were collected for measurement of TMUV burden and expression of cytokines and T cell markers, and spleens were weighed. Tissues collected at 5 days pi were also used for examination of histopathological changes and viral antigens. At 9, 11, 13, and 15 days pi, serum samples of three ducks were collected from each group. These sera, together with those collected between 1 and 7 days pi, were used for detection of neutralizing antibodies. For isolation of Y- and PS-specific CD4+ and CD8+ T cells and naïve CD4+ and CD8+ T cells, the EDTA-anticoagulated blood samples were collected at 9 day pi from infected and uninfected ducklings.To systematically compare the virulence of Y and PS in 5-day-old Pekin ducklings, experimental infections were performed as described above. In each group (n=60), 20 ducklings were monitored for 15 days for signs of encephalitis and weight loss as well as mortality, and 40 ducklings were used for examination of gross lesions and sample collection. Signs of encephalitis were divided into 5-grade severities of no change, very mild, mild, moderate, and marked, giving scores 0, 1, 2, 3, and 4, respectively. The ducklings were weighed once every 2 days between 1 and 15 days pi. Three ducks were randomly selected from each group at 1, 3, 5, and 7 days pi, and sera and ethylenediamine tetraacetic acid (EDTA)-anticoagulated bloods were sampled for detection of viremia and counting of CD4+ and CD8+ T cells, the EDTA-anticoagulated blood samples were collected at 9 days pi from ducklings infected with mutant viruses and their parental viruses.To identify the protein associated with the difference in cellular immune response between Y and PS, chimeric viruses (rPS-YE and rPS-YNS1-3’UTR) and their parental viruses (rY and rPS) were used to infect 5-day-old ducklings (n=23) as described above. To identify the residues associated with the difference in cellular immune response between Y and PS, mutant viruses  and their parental viruses (rY and rPS) were used to infect 5-day-old ducklings (n=23). In each case, a mock-infected control (n=23) was included, and tissues (brain and thymus) were sampled from three ducks in each group at 7 days pi for measurement of the expression of cytokines and T cell markers. For isolation of R38K-, T151A-, R304M-, rY-, and rPS-specific CD42O. The RT-qPCR was performed using the conditions reported previously  and reverse transcribed using a M-MLV Reverse Transcriptase kit , according to the manufacturer’s instructions. 5 μl of cDNA was mixed with 1 μl of each of forward and reverse primers [eviously .The tissue samples were fixed in 4% formalin at room temperature for 24h. Five-μm-thick paraffin-embedded sections were prepared by a standard protocol . After sTen-μm-thick paraffin-embedded sections were prepared by a standard protocol . The sec2 incubator 1 h for adsorption. Finally, the cell cultures were covered with overlay medium consisting of DMEM containing 1% low melting-point agarose  and 2% FBS, and the plaque assay was conducted. Antibody titer is expressed as 50% end point titer .Neutralizing antibodies in sera were detected using a previously reported PRNT . First, 2O. Duck glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an endogenous control. RT-qPCR was performed for GAPDH and each cytokine and T cell marker as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s and 60°C for 30 s, and extension at 72° for 60s. Relative expression was calculated for each cytokine using a 2-△△ct method , IL-17, gamma interferon (IFN-γ), and tumor necrosis factor-beta (TNF-β), and T cell markers, including CD4 and CD8. Sample processing, RNA extraction, and cDNA synthesis were the same as described above. 5 μl of cDNA was mixed with 1 μl of each of forward and reverse primers Table 1 t method .+ (or CD8+) T cells in PBMCs (3 ml/duck) were analyzed on a BD Arial Fusion flow cytometry , using mouse anti-duck mAb MCA2478 (or mouse anti-duck CD8 mAb MCA2479)  and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG . Frequencies of CD4+ and CD8+ T lymphocytes in PBMCs were presented with FlowJo 7.6 software . To prepare T cells for adoptive transfer, we conducted a fluorescence-activated cell sorting (FACS) on the BD Arial Fusion flow cytometry. CD4+ and CD8+ T cells were sorted from PBMCs.Peripheral blood mononuclear cells (PBMCs) were isolated from the EDTA-anticoagulant blood samples (3ml/duck) using a duck lymphocyte isolation kit , according to the manufacturer’s instructions. Following sucrose density gradient centrifugation, PBMCs that were located in a layer with relative density of 1.050–1.078 g/ml were harvested. The cells were washed three times and resuspended in 5 ml of PBS. CD4+, Y-specific CD8+ T cells, PS-specific CD4+, PS-specific CD8+ T cells, naïve CD4+ T cells, naïve CD8+ T cells (1×107/duck), or PBS (2 ml/duck). 12 h later, the recipients were challenged by im route with TMUV Y at a dose of 2×103 PFU. A mock-transferred, non-challenged group (control) was included, which was inoculated twice with PBS (2 ml/duck). The ducklings were monitored for signs of encephalitis, weight loss, and mortality. Serum samples of three ducklings were collected from each group between 1 and 15 days after challenge for measurement of neutralizing antibodies and viral RNA levels. At 7 days after challenge, three ducklings in each group were euthanized for measurement of spleen weight, and their tissues  were sampled for measurement of viral RNA levels and examination of histopathological changes.To compare the T cell-mediated immunity between Y and PS viruses, adoptive T cell transfer protocol was conducted using 3-day-old Pekin ducklings as recipients. Groups of 23 ducklings were inoculated by intravenous (iv) route with Y-specific CD4+ and CD8+ T cells were transferred into 3-day-old ducklings as described above. The ducklings in each group (n=23) were monitored for mortality. The serum samples of three ducklings were collected from each of groups receiving CD4+ T cells between 1 and 15 days after challenge for detection of neutralizing antibodies. The brain samples of three ducklings were collected from each of groups receiving CD4+ T cells at 7 days after challenge for measurement of viral RNA levels.To compare the T cell-mediated immunity between mutant viruses and their parental viruses, rY-, rPS-, T151A-, and R304M-specific CD4Log-rank test. Viral RNA levels, neutralizing antibody titers, and cytokine and T cell marker mRNA levels were analyzed by two-tailed Student t test. Body weight, spleen weight, and frequencies of CD4+ and CD8+ T cells in PBMCs were analyzed by two-way analysis of variance (ANOVA).All data were analyzed using GraphPad Prism software (version 5.0) . Survival curves were analyzed by the 4 PFU of Y or its rescued virus rY by intracerebral or subcutaneous routes, mortality as high as 90–100% occurred between 4 and 7 days pi (3 PFU). All of the 20 3-day-old infected ducklings died between 4 and 7 days pi., 10 of the 20 5-day-old infected ducklings died between 7 and 15 days pi., and two of the 20 7-day-old infected ducklings died at 7 days pi . This suggests that Y exhibits moderate pathogenicity in 5-day-old Pekin duckling model relative to those observed in 3- and 7-day-old Pekin duckling models. PS retained low pathogenicity, similar to previously reported pathogenicity in 2-day-old Pekin duck model , tremor (6/20), and paralysis (5/20). In ducklings inoculated with PS, small portion (3/20) displayed signs of listlessness within 7 to 9 days pi , relative to infection with PS. These data indicate that strain Y induces stronger cellular immune response in brain and thymus than strain PS. Infection with Y induced 2- to 4-fold decreases in expression of IL-2 at 7 days pi, IFN-γ at 3 and 5 days pi, TNF-β at 5 and 7 days pi, and CD8 at 3 dpi (P<0.05) and 2- to 4-fold increases in CD8 expression at 5 and 7 dpi (P<0.05) in spleen, as compared to infection with PS, suggesting that strain Y exerts an inhibitory effect on expression of cytokines in spleen. Significant differences in frequency of CD8+ T cells between Y- and PS-infected ducklings were observed. Infection with Y resulted in more significant decrease in frequency of CD8+ T cells at 3 days pi , and more significant increase in frequency of CD8+ T cells at 9 and 11 days pi , relative to those observed for PS. These data reveal a negative correlation between TMUV virulence and frequency of CD8+ T cells at the peak of viremia and a positive correlation between TMUV virulence and frequency of CD8+ T cells after virus clearance from the circulation. Altogether, these data suggest that strain Y induces higher magnitude of cellular immune responses than PS, which can be reflected by measurement of expression of cytokines and T cell markers in brain and thymus of infected ducklings.The marked differences in virulence and the similarity in neutralizing antibody responses between Y and PS suggest that there may be a link between the magnitude of cellular immune responses and TMUV virulence. To confirm the hypothesis, we measured the expression of cytokines  and T cell markers (CD4 and CD8) in brain, thymus, and spleen (+ T cells (90%), and naïve CD8+ T cells (100%), transfer of Y- and PS-specific CD8+ T cells and Y-specific CD4+ T cells provided significant protection  against lethal infection. Whereas PS-specific CD4+ T cells conferred only 30% protection to recipient ducklings , naïve CD4+ T cells between 1 and 7 days after challenge, as compared to those derived from ducklings received PBS and naïve CD4+ T cells. The neutralizing antibodies in ducklings received Y- and PS-specific CD4+ T cells peaked at 5 and 7 days after challenge, respectively. By comparison, the neutralizing antibodies in ducklings received naïve CD4+ T cells peaked at 11 days after challenge. In addition, significantly higher levels of neutralizing antibody were detected in ducklings received Y-specific CD4+ T cells between 3 and 5 days after challenge than in ducklings received PS-specific CD4+ T cells , and had no significant effect on viral RNA levels in spleen, thymus, and serum. By comparison, transferred Y- and PS-specific CD8+ T cells reduced viral RNA levels in brain , spleen , and thymus  and in serum  between 1 and 5 days after challenge, and cleared virus from the blood at 7 days after challenge. There was no significant difference in viral RNA levels in brain, spleen, thymus, and serum between groups received Y- and PS-specific CD8+ T cells. Overall, our data suggest that virus-specific CD8+ T cells play an important role in clearing infection from tissues and preventing virus persistence, with Y- and PS-specific CD8+ T cells being similar in this function.To compare the effect of Y- and PS-specific T cells on viral load, we measured viral RNA levels in tissues  at 7 days after challenge and in serum samples between 1 and 9 days after challenge (P<0.05). These data indicate that the E protein plays a major role in determining the differences in cellular immune response between Y and PS.To determine which protein is responsible for the differences in cellular immune response between Y and PS, 5-day-old Pekin ducks were infected with chimeric viruses rPS-YE and rPS-YNS1-3’UTR and control viruses rY and rPS. We measured the expression of IL-2, IL-17, IFN-γ, TNF-β, CD4, and CD8 mRNAs in brain and thymus at 7 days pi (P<0.05). Thus, both T151A and R304M induced mRNAs of these cytokines and T cell makers to similar levels as measured for rY. Infection with R38K had little or no effect on expression of the tested cytokines (except TNF-β in thymus) and T cell markers in brain and thymus (P<0.05). Our data indicate that mutations T151A and R304M in the E protein contribute to the differences in cellular immune response between Y and PS.We then identify E protein residues responsible for the differences in cellular immune response between Y and PS. 5-day-old Pekin ducklings were infected with the R38K, T151A, and R304M mutant viruses and the rY and rPS control viruses, and the levels of IL-2, IL-17, IFN-γ, TNF-β, CD4, and CD8 mRNAs in brain and thymus were measured at 7 days after infection  were comparable to those in group received rPS-specific CD8+ T cells (75%), but lower than those in group received rY-specific CD8+T cells (100%). The result indicates that the R304M and T151A mutations have little or no contribution to CD8+ T cell-mediated protection.We evaluated adoptive transfer of CD8+ T cells from ducklings infected with mutant viruses T151A and R304M for its effects on protective capacity, neutralizing antibody response, and TMUV burden in brain , as compared to that in group received rPS-specific CD4+T cells (20%). The data indicate that both T151A and R304M mutations contribute to markedly enhanced CD4+ T cell-induced protective efficacy, with the R304M mutation contributing more to protection against lethality. Neutralizing antibodies in ducklings received rPS-specific CD4+T cells peaked at 7 days after challenge. By comparison, neutralizing antibodies in ducklings received T151A- and R304M-specific CD4+T cells peaked 2 days earlier, like that observed for rY-specific CD4+T cells. The levels of neutralizing antibody detected in ducklings received T151A-, R304M-, and rY-specific CD4+T cells between 1 and 5 days after challenge were similar, all of which were higher than those detected in ducklings received rPS-specific CD4+T cells, with significant higher antibody titers occurring at 5 days after challenge. These data indicate that both R304M and T151A mutations contribute to significantly increased CD4+ T cell-mediated neutralizing antibody response. The levels of viral RNA detected in brain of ducklings received T151A- and R304M-specific CD4+T cells at 7 days after challenge were similar to those derived from ducklings received rPS-specific CD4+T cells, indicating that both R304M and T151A mutations have no contribution to CD4+ T cell-mediated viral clearance from the brain.We assessed adoptive transfer of CD4in brain D. Varyi+ and CD8+ T cells by using the high-virulence TMUV strain Y and the low-virulence TMUV strain PS. Because strain Y caused very high mortality in recently reported 2-day-old Pekin duckling model  and the virulence of TMUV.The present observation confirmed recent findings in which TMUV strains Y and PS were shown to elicit comparable levels of neutralizing antibody in 2-day-old Pekin duck model . This inWe observed that the viral RNA levels in brain, thymus, spleen, and blood in both Y- and PS-infected ducklings tended to decline from 3 to 7 days pi, indicating that virus-induced immune responses have exerted their functions in inhibiting virus replication, limiting virus dissemination, clearing infection from tissues, and preventing viral persistence. The levels of viral RNA in Y-infected duckling were reduced to those in PS-infected ducklings at 7 days pi, suggesting a role of the excess of Y-specific T cell immunity over PS-specific T cell immunity in controlling TMUV burden. It is likely that more rapid clearance of virus from the blood of PS-infected ducklings than from Y-infected ducklings might be attributed to the role of glycosaminoglycan-binding motif at residue 304 in the E protein of PS. TMUV burden in brain of both Y- and PS-infected ducklings were reduced more slowly as compared to those in the extraneural tissues, which might be associated with immune responses in the CNS involve recruitment of peripheral immune cells to the CNS –38.+ T cells and Y- and PS-specific CD8+ T cells provided significant protection against a lethal infection with TMUV Y, indicating a crucial role of T cells in the protective immune response to TMUV. CD8+ T cells were shown to confer more robust protection to recipient ducklings than CD4+ T cells, suggesting that for TMUV, similarly to dengue virus (DENV)  , 40, CD8+ T cells primarily made contribution to the production of neutralizing antibodies, and CD8+ T cells primarily mediated viral clearance for brain, thymus, spleen, and blood, similar to other flaviviruses   , 41–45.  T cells . Whereas+ T cells. We observed that the contribution of both T151A- and R304M-specific CD4+ T cells to protection and antibody production failed to achieve the levels conferred by rY-specific CD4+ T cells, suggesting that a combination of the T151A and R304M mutations might synergistically enhance virus-specific CD4+ T cell immunity.From studies on expression of cytokines and T cell markers in ducklings infected with chimeric and mutant viruses we conclude that E protein residues 151 and 304 are the key determinants of the magnitude of TMUV-specific T cell immune response. Based on adoptive transfer of mutant virus-specific T cells, this work has also shown that E protein residues 151 and 304 are the key determinants of protection against lethal infection and neutralizing antibody production mediated by TMUV-specific CD4+ T cells mainly target the E, C, and NS1 proteins, whereas CD8+ T cells preferentially recognize the NS3, NS4B, and NS5 proteins  have shown that CD4proteins –52. We sY and PS , and thaY and PS . Further+ and CD8+ T cells in the protective immune response and the control of TMUV infection. A positive correlation exists between the virulence of TMUV and T cell immunity, including CD8+ T cell-mediated protection and CD4+ T cell-mediated protection, neutralizing antibody response, and viral clearance from the brain. We have also demonstrated that the difference in CD4+ T cell-mediated immunity is mainly related to residues 151 and 304 in the E protein. Our studies contribute to the better understanding of the role of T cell immunity in the protective immune response and the molecular basis of TMUV-induced CD4+ T cell immunity.Taken together, our studies demonstrate a critical role of CD4The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Welfare and Ethics Committee of China Agricultural University.RM conducted experiments, analysis, and writing of the original manuscript draft. BY, CF, and JH were involved in sample collection, sample processing, analysis, and data collection. XW was involved in the analysis and interpretation of data. DZ performed analysis, writing, and reviewing. All authors contributed to the article and approved the submitted version.This project was supported by grants from China Agriculture Research System of MOF and MARA for DZ and the National Key Research and Development Program of China (2016YFD0500107) for DZ.We thank Lixin Yang  for advice and suggestions in duck experiments; Duo Peng, Qiong Li, Jiaying Wang, and Zixin Feng  for their support in sample collection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide–HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy. Significance StatementIntracellular peptides complexed to human leukocyte antigen Class I (HLA-I) molecules are critical for directing appropriate cellular immunity mediated by T lymphocytes. This study describes additional conserved features of HLA-I molecules that drive binding, stability, and diversity of the presented peptide repertoire. Nonpocket interactions between specific prevalent HLA-I residues and peptide positions 1 and 4 make unexpectedly strong contributions to peptide stabilization and antigen presentation. These interactions are shared across the HLA-I system and other species, and may constitute a subdominant anchor motif. Better understanding of the role of subdominant interactions and the individual contributions of nonpocket residues toward peptide binding, as described here, can be leveraged to create more accurate and generalizable methods for identifying targets of T-cell mediated immune responses.HLA-IHuman leukocyte antigen class IMHC-IMajor histocompatibility complex class ITCRT-cell receptorCTLCytotoxic T lymphocytesMSMass spectrometryp4Peptide position 4 amino acidpΩPeptide C-terminal amino acidMDMolecular dynamicsβ2mBeta-2 microglobulinMajor histocompatibility complex class I (MHC-I) molecules are expressed at the surface of nearly all nucleated vertebrate cells, where they each present a diverse array of peptides of 8 to 13 amino acids in length sampled from recently synthesized cellular proteins , 3. Peptα-helices that form the binding cleft (Mass spectrometric (MS) analysis of HLA-I peptide ligands eluted directly from human cell lines and tissue samples has enabled the identification of peptide-binding motifs for >100 HLA-I molecules, which has facilitated the development of peptide binding prediction algorithms with very good accuracy and demonstrated clinical utility . HLA-I png cleft . HoweverTo identify such secondary interactions, we conducted a comprehensive survey of HLA-I peptide ligands and observed significantly elevated frequencies of negatively charged amino acids aspartic acid (D) and glutamic acid (E) at peptide position 4 (p4), with the highest prevalence found in the HLA-A2 family. Crystal structures of A*02:01, the most prevalent HLA-A2 allotype, revealed that D/E4 was located near the top of the binding cleft adjacent to two positively charged HLA-I residues, indicating a potential for ionic interactions . This agTo identify potential secondary interactions mediating HLA-I-mediated antigen presentation, we performed a comprehensive search of MS-eluted peptides from 108 HLA-I allotypes to assess whether specific amino acid residues were preferred in the middle positions of peptide ligands (i.e. positions p4 to p-1). We found a striking preference for the negatively charged residues aspartic acid (D) and glutamic acid (E) at peptide position 4 (p4) for most HLA-I allotypes Fig. . FurtherBased on these observations, we hypothesized that the high prevalence of D/E at p4 is driven by specific interactions with residues in the HLA peptide-binding cleft. The superposition of multiple crystal structures of A*02:01-restricted D/E4-containing peptides revealed that D/E4 interacted primarily with the basic arginine residue R65 of A*02:01, though some interactions also occurred with the basic lysine residue K66 . By contα1 helix residues than to residues on the opposite α2 helix. In A*02:01/GLKEGIPAL, R65 showed extensive interactions solely with E4 at the top of the peptide binding cleft while K66, located on the inner side of the cleft, interacted in a network with peptide residues L2 and E4 in addition to HLA residues E63 and H70  or the three mutant A*02:01 alleles with green fluorescent protein (GFP) tagged onto the C-terminus were used to transduce the lung cancer cell line H1975. Transduced cells demonstrating comparable levels of total GFP expression  showed sP < 0.001; Fig. Based on the A*02:01/peptide crystal structure-based evidence, we hypothesized that the loss of the interaction between peptide D/E4 residues and R65/K66 in A*02:01 may have been responsible for the loss of peptide repertoire and cell surface expression observed for the A*02:01 mutants. Analysis of the frequency of D/E4 residues in peptides eluted from the A*02:01 mutants indeed showed a significant loss of presented D4 and E4 peptides . The impact of these mutations on pΩ was particularly surprising since p65/66 residues are relatively distant from the F pocket which harbors pΩ residues. Nonetheless, peptides eluted from both A2-R65G and A2-DM showed a significant preference for valine residues at pΩ with a corresponding decreased preference for leucine (LΩ) compared to A2-WT. Fig. . VΩ is mIn peptides eluted from the A2-K66N and A2-DM mutants, we also observed alterations to amino acid frequencies in peptide position 2 (p2). These changes were not surprising since several A*02:01/peptide crystal structures have shown K66 consistently forming hydrogen bonds with the backbone of p2 residues . PeptideWe next determined the impact of basic residue alterations at p65/66 for HLA-A*24:02. Since A*24:02 naturally contains only one basic residue at K66, two different A*24:02 mutant alleles were created: the lysine in p66 was substituted with asparagine to create A*24:02-K66N (A24-K66N), resulting in the G65/N66 pairing that was a feature of the A2-DM mutant described above, and that was also identical in sequence to the known allele HLA-A*24:145. The second A*24:02 mutant changed the glycine at p65 to arginine to create A*24:02-G65R (A24-G65R), recapitulating the R65/K66 pairing found in WT A*02:01, and identical in sequence to HLA-A*24:29 Fig.  (4).Recombinant lentiviruses expressing WT HLA-A*24:02 (A24-WT) or the two A*24:02 mutants were used to transduce the lung cancer cell line H1975. Similar to HLA-A*02:01, the A*24:02 mutants consistently demonstrated distinct cell surface expression levels with the following hierarchy: A24-G65R > A24-WT > A24-K66N Fig.   . We nextThe proportion of D/E4 peptides eluted from the different A24 variants also followed a similar pattern, with A24-G65R binding to a significantly higher percentage of D/E4 peptides and A24-K66N binding a significantly lower frequency of D/E4 peptides compared to A24-WT Fig. . We nextTo gain further insights, MD simulations of A*24:02/QFKDNVILL peptide complexes were run for A24-WT, A24-G65R, and A24-K66N . CoulombSince the observed changes in eluted D/E4 peptide frequencies between the different A*24:02 variants did not fully account for the striking changes in peptide repertoire diversity, we next examined the amino acid frequencies at other peptide positions of MS-eluted peptides. This analysis revealed that while peptides eluted from A24-WT and A24-G65R showed very similar characteristics, peptides eluted from A24-K66N exhibited frequency alterations at multiple peptide positions Fig. . For exaWe next investigated the contribution of p4 residues to the binding and stability of A*24:02 peptides with or without dominant primary anchors. The native QFKDNVILL peptide containing two subdominant primary anchor residues (F2 and LΩ), demonstrated relatively poor binding to HLA-A*24:02 Fig. . SubstitP < 0.01)  Fig. . These eP < 0.001; Fig. Our initial survey of eluted peptide ligands revealed a preference for D/E4 across almost all HLA-I allotypes analyzed . All majP < 0.01). Notably, similar trends were also observed for HLA-B, -C, and -G molecules  and multiple HLA-I residues , many of which were disrupted with the N66 substitution. R65, by contrast, is located at the top of the detached . ConsideOne of the more surprising findings of our study was the starkly different impacts that the K66N substitution had on HLA-A*02:01 and A*24:02. Although K66 loss in A*24:02 resulted in a dramatic decrease in surface expression and peptide diversity, peptide N-terminal amino acid frequencies showed minimal alterations. By contrast, K66N substitution of A*02:01 in both the A2-K66N and A2-DM mutants resulted in a striking increase in the frequency of positively charged amino acids arginine or lysine in position 1 of eluted peptides. A similar phenotype was previously reported for peptides eluted from the naturally occurring A*02:20 allotype , in accordance with our findings . Our surIt is important to note that a number of studies have addressed the importance of R65 and K66 for TCR engagement and CD8+ T-cell activation . TCRs reThe findings reported here advance our fundamental understanding of peptide ligand binding and MHC-I-mediated antigen presentation and raise several implications. For example, peptide ligands restricted to HLA-A*02:01 and A*24:02 that contain D/E in peptide position 4 could be of particular interest as targets for vaccines or T cell-based immunotherapies against viral diseases or cancer, especially for peptides with subdominant primary anchors. This same rationale may also apply to other HLA-I allotypes containing R65 and/or K66, although further studies will be required to confirm this. Based on their frequent co-occurrence throughout the HLA-I system, ligands containing both K/R1 and D/E4 may show further enhanced stability in the binding cleft, as the intrapeptide interactions between them would suggest; it will be of great interest to assess how TCR binding might impact these interactions. More broadly, the rules uncovered by this study may be leveraged to improve peptide binding predictions for the many HLA-I allotypes lacking well-defined binding motifs. Furthermore, it is probable that other shared MHC-I/peptide interactions exist throughout the HLA-I system that remain to be identified. The approach used here, combining site-directed mutagenesis, global immunopeptidome analysis, quantitative wet-bench assays, and computational simulations provides a road map for discovering such interactions, which will ultimately facilitate improved accuracy of immune targeting in many different human disease contexts.Crystal structures of selected HLA-I/peptide complexes were obtained from the Protein Data Bank  and minimally revised using the PyMOL Viewer  to make Additional HLA-I/peptide structures were modeled to account for peptide variants or HLA-I mutations of interest to our study, and for which no crystal structures were available on PDB. These additional structures mostly related to single amino acid substitutions, or very limited combinations of substitutions, in relation to an available reference crystal structure. These substitutions were performed with the mutagenesis wizard of PyMOL Viewer, based on the best ranked rotamer for each substitution. Note that these modified HLA-I/peptide structures were used as input for MD , which should further resolve any issues related to suboptimal positioning of the mutated amino acids.https://getcontacts.github.io/), and ggplot2 (Input structures for MD simulations were preprocessed with the PDB2PQ webserver . Structu ggplot2 . AdditioH1975 lung cancer cells were purchased from the ATCC (CRL-5908). Site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit  was performed on WT HLA-A*02:01 and HLA-A*24:02 cDNAs subcloned into pDONR221 . Creation of the A*02:01  and A*24:02 (G65R and K66N) mutant constructs were confirmed by Sanger sequencing. WT and mutated A*02:01 and A*24:02 cDNAs were subcloned into destination lentiviral expression vector pLV460 using LR reaction-based methodology .293METR cells were plated in 10 cm plates  and transfected with 10 μg pCMVdelta8.91, 1 μg VSV-g, 10 μg pLV460 with A*02:01 or A*24:02 variant cDNAs, and 50 μl lipofectamine 2000 . Viral supernatant was collected after 2 and 3 days, centrifuged and concentrated using Lenti-X . The lung cancer cell line H1975  was transduced with lentiviral vectors expressing WT or mutated HLA-A*02:01 or HLA-A*24:02 in 4 μg/ml polybrene , and surface expression was analyzed by staining with HLA-A*24:02-specific  or HLA-A*02:01-specific  antibodies and flow cytometric analysis (BD FACS Canto II).Peptides were eluted from immunoprecipitated HLA-I molecules as described previously . In briePeptides eluted from HLA-I molecules on H1975 cells were desalted by Zip-Tip, and split in half with one half analyzed by the Proteomics Facility at U.T. MD Anderson Cancer Center and the other half analyzed by the ThermoFisher facility in San Jose, CA. Analysis of peptides eluted from HLA-I molecules at the Proteomics Facility at U.T. MD Anderson Cancer Center was similar to a previous report . In brieIndividual peptide matches underwent quality assessment by reference to multiple orthogonal parameters, including Mascot Ion score (minimum score 25), MS1 measured differential to the calculated peptide mass (delta mass), and predicted binding to the relevant HLA-I allotypes as determined by high-resolution genetic sequencing and the NetMHCpan4.1 algorithm . PeptideAll HLA-A*02:01- and A*24:02-restricted peptides used in the study were purchased from Genscript at purity of > 80% with quality control by reverse-phase HPLC and mass spectrometry (SC1208). Recombinant HLA-A*02:01 and HLA-A*24:02 molecules were made and re-folded as described elsewhere . ConformDifferences between WT and mutated amino acid positional frequencies of eluted peptides were analyzed using the 2-sample test (Z test) for equality of proportions without continuity correction. Comparisons of two means with unequal variances were analyzed using unpaired t tests with Welch’s correction. Comparisons of multiple means with unequal variances were performed using Brown–Forsythe ANOVA and multiple pairwise comparisons were then performed using Dunnett’s T3 test.pgac124_Supplemental_FilesClick here for additional data file.


S492R- or EGFRG465R-driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFRS492R, which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFRG465R). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFRS492R and EGFRG465R as well as EGFRWT. The evolved cetuximab variants  with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFR Acquired resistance to cetuximab can be mediated by generation of mutations in the EGFR ectodomain. Here the authors report a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse resistance without altering the binding epitope or undermining antibody efficacy. However, the emergence of primary (de novo) or secondary (acquired) resistance with different underlying mechanisms, mainly but not limited to antigen point mutation-mediated drug resistance7, aberrancies in downstream signaling pathways10 and upregulated expression of drug resistance-related proteins12, undermines the application of therapeutic antibodies for targeted cancer therapy.Since Muromonab-CD3, marketed under the name Orthoclone OKT3, was approved by the FDA in 1986 for the treatment of acute organ transplant rejection13. Panitumumab, a fully human IgG2 mAb, is another EGFR-targeted therapeutic mAb for mCRC14. Both cetuximab and panitumumab compete with epidermal growth factor (EGF) for binding to perturb the downstream signaling of EGFR, thus inhibiting the proliferation of tumor cells16. The point mutation T790M, an acquired mutation located in the intracellular domain of EGFR, is the most commonly associated with intrinsic resistance as for acquired resistance to EGFR tyrosine kinase inhibitors (TKIs)18, whereas point mutations in the EGFR ectodomain (ECD) are inclined to disrupt the specific targeting of therapeutic antibodies19. Analysis of clinical cell-free DNA (cfDNA) from mCRC patients with a history of anti-EGFR mAb treatment revealed a cluster of EGFR ECD mutations that mediate acquired resistance by attenuating anti-EGFR antibody binding, for example, EGFR ECD mutations at positions V441, S442, R451, I462, S464, G465, K467, K489, I491 and S49226. All the above residues except R451 are located in the binding site of cetuximab and/or panitumumab24. S492R and G465R accounted for approximately 20% of clinical acquired resistance cases, and are among the most common point mutations conferring resistance to anti-EGFR antibodies21. Strategies for reversing point mutation-driven resistance to anti-EGFR antibodies have focused on the employment of new antibody combinations, e.g., MM-151 and Sym004, to target nonoverlapping epitopes, a time-consuming and labor-intensive approach27.Cetuximab, a human-mouse chimeric IgG1 mAb targeting epidermal growth factor receptor (EGFR), is the first mAb drug approved for treating patients with irinotecan-refractory and/or oxaliplatin-refractory metastatic colorectal cancer (mCRC)S492R or EGFRG465R, the two most common antibody-resistance point mutations in cetuximab treatment. We use the SGAPAE approach to determine the energy difference between the bound and unbound states of the interface using the Rosetta platform, developing a semirationally designed library of cetuximab mutants with a restricted epitope. The focused library allows us to efficiently identify three cetuximab variants with minimal mutations  to reverse EGFRS492R or EGFRG465R-driven resistance to cetuximab, while maintaining the optimized physical and chemical properties of cetuximab to a great extent and thus ensuring their druggability. The SGAPAE approach thus constitutes an efficient and viable strategy to overcome single mutation-driven drug resistance while maintaining the druggability of the parental antibodies, and should be a promising approach for the evolution of other biologics.In this study, we provide a structure-guided and phage-assisted evolution (SGAPAE) approach for the evolution of the parental antibody cetuximab to reverse resistance mediated by EGFR26.Recent studies have shown that a number of EGFR ECD mutations may be acquired in patients with metastatic colorectal cancer (mCRC) after cetuximab and/or panitumumab treatment. These EGFR ECD mutations occurred in EGFR domain III (amino acids 334–504) which is the binding epitope for cetuximab and panitumumab Fig. , mediati21. A retrospective analysis of plasma samples from a phase III trial (ASPECCT) in a large number of colorectal cancer patients revealed that the EGFR S492R mutation developed in 16% of patients in the cetuximab arm  with NIH3T3 cells expressing wild-type EGFR or mutant EGFR  on EGFR. We performed homology modeling to predict the effect of the S492R and G465R mutations on cetuximab binding to EGFR  with a large side chain (guanidino group), however, could not fit into the small central cavity in the cetuximab CDR, thus resulting in steric clashes between cetuximab and EGFRS492R or EGFRG465R.High-resolution EGFR/cetuximab structures show a large, flat binding interface primarily comprising CDRL3, CDRH3, and CDRH2 of cetuximabGFR Fig. . The cryS492R- or EGFRG465R-mediated resistance  interface analysis, to narrow down the list of residues critical for restoring cetuximab binding with the EGFR mutants. Among the multiple indicators , dG_separated, which represents the energy difference between protein interface separation and binding, was chosen to evaluate the impact of mutations on cetuximab affinity. EGFR S492R or G465R mutation significantly decreased the binding affinity of cetuximab to EGFR, resulting from an increased free energy of binding. The predicted ∆∆G values for 100 models of EGFRS492R/cetuximab and EGFRG465R/cetuximab complexes were consistent with the clinical data (∆∆G > 0)  and EGFRG465R/cetuximab  complexes, respectively  in the three clusters  and Ctx-Y104D-scFv    Fig. . AnotherH3) Fig. . For furH3) Fig. . The SDSH3) Fig. . On thisH3) Fig. .Fig. 4IdS492R-expressing stable NIH3T3 cells (S492R-EGFR-NIH3T3), while Ctx-VY, Ctx-Y104D, Ctx-DD and Ctx-VWY exhibited successfully restoration of a strong interaction with S492R-EGFR-NIH3T3 cells similar to that of panitumumab, an anti-EGFR antibody with a binding site different from that of cetuximab. Intriguingly, Y104D resulted in a higher Emax with EGFRS492R-expressing stable NIH3T3 cells and S492R-EGFR-ECD-Fc than panitumumab, Ctx-VY, Ctx-DD and Ctx-VWY. Among all the cetuximab variants, only Ctx-W52D exhibited restoration of strong binding to EGFR-expressing stable cells and for EGFR-ECD-Fc with the G465R mutation, which abolished the binding of both cetuximab and panitumumab. In addition, we tested the binding affinity of Ctx-W52D in the EGFRG465E model. However, Ctx-W52D could not reverse G465E-driven resistance to cetuximab  is alteredin both Ctx-VY and Ctx-Y104D. Hence, we performed site saturation mutagenesis for VH-Y104 and Ctx-Y104X  to thoroughly investigate the impact of this residue on the binding affinity of cetuximab for EGFRKD values of 0.40 ± 0.28, 2.25 ± 1.57, 3.76 ± 2.42, and 0.79 ± 0.17 nM, respectively  compared to that of cetuximab , which accounted for the incomplete restoration of binding affinity for EGFRWT. The interaction of cetuximab with EGFRS492R or EGFRG465R was not detectable by SPR; in contrast, Ctx-VY, Ctx-Y104D, and Ctx-W52D, exhibited major restoration of binding with EGFRS492R  and EGFRG465R (KD = 4.56 ± 1.13 nM) with respect to the cetuximab/EGFRWT interaction  or Y104D mutation (Ctx-Y104D) and possible strong charge accumulation through the Asp-Arg (D-R) salt bridge (Ctx-Y104D versus EGFRS492R)  salt bridge-mediated reconnection and reduction in steric hindrance probably contributed simultaneously to restoration of the binding ability, since the large indolyl side chain of tryptophan (W) was downsized to the carboxyl group of aspartate (D). We also compared the paratopes of cetuximab and the cetuximab variants in surface mode  Fig. . Regardiode Fig. . The siz65R Fig. .Fig. 5Th36. To evaluate the inhibitory activities of the cetuximab variants, we established stable NIH3T3 cells expressing full-length EGFRWT or EGFRMut as an in vitro cell model. Robust phosphorylation of EGFR and activation of Akt and Erk in the downstream pathway were induced in stable WT-EGFR-NIH3T3, S492R-EGFR-NIH3T3 and G465R-EGFR-NIH3T3 cells upon EGF stimulation, according to the western blot results  in a concentration-dependent manner, with EC50 values of 6.2, 6.3, 9.7, 8.3 and 8.1 nM, respectively , whereas panitumumab, Ctx-VY and Ctx-Y104D induced the degradation of S492R-mutated EGFR with EC50 values of 10.7, 4.0, and 11.8 nM, respectively , with an EC50 of 15 nM in G465R-EGFR-eGFP-HEK293T cells; However, cetuximab and panitumumab did not , and COLO320DM cell lines stably expressing full-length wild-type or an EGFR mutant   Fig. , top. Coing Fig. , right. ing Fig. , middle.IgG Fig. , bottom.40. The results showed that the cetuximab variants exhibited ADCC  and antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab variants according to previously described methodsDCC Fig.  and CDC DCC Fig.  activitiG465R or affect EGFRG465R signaling, therefore the exogenous introduction of EGFR G465R in SW48 lowered the potency of cetuximab on tumor growth inhibition in the G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote) model, in comparison to the SW48 (WT-EGFR) model. On the other hand, the Ctx-W52D, capable of binding with EGFRWT and EGFRG465R, showed similar in vivo efficacy in two xenograft models  cell line-derived xenograft (CDX) models to evaluate the in vivo antitumor activity of Ctx-VY, Ctx-Y104D and Ctx-W52D. In the SW48 (WT-EGFR) xenograft model Fig. , tumor gels Fig. . No bodyels Fig. . In addiels Fig. , with reels Fig. . The in els Fig. . No bodyels Fig. . The in els Fig. . No bodyels Fig. . Furtherels Fig. . Immunos41 and bispecific antibodies (bsAbs)42. In this study, we successfully applied a structure-guided and phage-assisted evolution (SGAPAE) approach to direct the evolution of cetuximab, successfully reversing the acquired drug resistance conferred by EGFR ECD mutations. According to structural analysis and computational prediction of EGFR ECD with the S492R mutation, we identified 6 critical residues in the CDRs of cetuximab and created a 6-residue randomized phage-display library. After panning the epitope-restricted cetuximab mutant library, the cetuximab variants Ctx-VY and Ctx-Y104D were identified to exhibit restored binding affinity for EGFRS492R while maintaining their binding affinity for EGFRWT. The SGAPAE approach also reversed EGFRG465R-driven drug resistance to cetuximab, demonstrating its more general application. Another cetuximab variant, Ctx-W52D, with a single mutation in the cetuximab CDR, was shown to exhibit restored binding affinity for EGFRG465R. Intriguingly, all cetuximab variants maintained the functions of their parental antibody cetuximab, including blockade of EGFR activation and downstream signaling, induction of EGFR degradation, inhibition of cell proliferation, and CDC and ADCC activity. Collectively, these results demonstrate that with the SGAPAE approach, we efficiently achieved epitope-restricted evolution of cetuximab and identified cetuximab variants with minimal point mutations (1 or 2) to reverse EGFRS492R- or EGFRG465R- mediated drug resistance.Owing to deleterious single point mutations in the ectodomain of EGFR, such as S492R and G465R, acquired resistance to anti-EGFR therapeutic antibodies emerges during clinical antibody therapy, hindering further applications of antibody derivatives, including antibody-drug conjugates (ADCs)43.The application of SGAPAE in cetuximab evolution, which involves the use of the Rosetta platform, relies heavily on the structural information of the EGFR/cetuximab complex. Developing a computational model consistent with the binding characteristics of an antibody/antigen complex is essential for the prediction of potential hot spot residues for evolution. The Rosetta platform offers several advantages for evaluating the interfacial free energy. The InterfaceAnalyzer module of Rosetta determines the energy difference between the bound and unbound states of the interface, which is beneficial for evaluating the effect of a single point mutation on the affinity of cetuximab based on the known structure of the EGFR/cetuximab Fab complex. The flexible setting of constraints  in the Rosetta platform facilitates fast and exhaustive searching surrounding the point mutation. Finally, with the restricted binding site of the cetuximab variant, Rosetta provides the most accurate local docking pose after simply orienting the Fab in the expected binding site, leading to the identification of key amino acid residues for randomization. Therefore, the Rosetta platform in the SGAPAE approach was ideally suited to address EGFR single mutation -driven acquired resistance to cetuximab and should be effective for addressing single mutations in other receptors 44 and cytokine engineering, e.g., IL-2 bias screening45. However, to the best of our knowledge, phage display has not been applied to overcome single mutation-mediated antibody-drug resistance. Because of the limited key residues for randomization, the size of the cetuximab mutant library was reduced significantly to 106–107 phages, which exhibited superiority over conventional phage-display libraries with a size of 109–1012 phages in biased screening processes 46. The diversity of the semirationally designed cetuximab mutant library is confined within the binding epitope of the parental cetuximab structure, providing an obvious evolutionary direction toward EGFR and thereby retaining the highly similar binding site in the selected cetuximab variants. In combination with the known structural information and computational predictions, our SGAPAE approach was able to improve the screening efficiency for effective cetuximab variants. The binding epitope of the parental antibody is usually correlated with antibody efficacy. With the epitope-restricted cetuximab mutant phage-display library, we found that minimal point mutations or even a single point mutation  in the cetuximab CDR could completely reverse EGFRS492R- or EGFRG465R- mediated cetuximab resistance without altering the binding epitope or attenuating antibody efficacy. In addition, the evolved cetuximab variants  with one or two amino acid substitutions in the CDR, are less likely than other variants to perturb the rest of the antibody domains, inheriting the optimized physical and chemical properties of cetuximab to the greatest possible extent.To date, phage and yeast display technologies have been widely used in antibody discoveryS492R- or EGFRG465R- mediated resistance to cetuximab. Acquired drug resistance during antibody therapy has greatly undermined the effectiveness of antibody treatment due to frequent mutations in the target receptors . The SGAPAE approach provides an efficient and viable strategy to overcome single mutation-driven drug resistance while maintaining the druggability of parental antibodies. The SGAPAE approach also constitutes a promising strategy for efficiently evolution of other proteins, such as recombinant enzymes and cytokines, whose structural information is usually abundant and rapidly expanding owing to the rapid development of structural biology.In summary, we successfully applied the SGAPAE approach to efficiently reverse EGFRExperiments were carried out according to the National Institutes of Health (United States) Guide for the Care and Use of Laboratory Animals. All animal work was performed in accordance with the protocol approved by the Committee on the Ethics of Animal Experiments of Zhejiang University .2. COLO320DM cells were cultured in RPMI-1640 medium  supplemented with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin/streptomycin  at 37 °C in a humidified atmosphere containing 5% CO2. HEK293F cells were cultured in SMM 293-TI medium  supplemented with 0.5% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin at 37 °C under 5% CO2 in a New Brunswick S41i shaking incubator .NIH3T3 mouse embryonic fibroblasts were obtained from the Cell Bank of the Chinese Academy of Sciences . The human colorectal cancer cell lines SW48 and COLO320DM were purchased from Cobioer Biosciences . HEK293T cells and HEK293F cells were kindly provided by Professor Dr. Linqi Zhang . NIH3T3 cells, HEK293T cells, and SW48 cells were cultured in DMEM  supplemented with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin/streptomycin  at 37 °C in a humidified atmosphere containing 5% COBALB/c nude mice  were purchased from Shanghai SLAC Laboratory Animal Co., Ltd.  and housed under specific pathogen-free conditions. Experiments were carried out according to the National Institutes of Health (United States) Guide for the Care and Use of Laboratory Animals. All animal work was performed in accordance with the protocol approved by the Committee on the Ethics of Animal Experiments of Zhejiang University .https://github.com/schrodinger/PyMOL -open-source).The online server SWISS-MODEL was used to predict the 3D protein structures of S492R- and G465R-mutated EGFR and the cetuximab variants using the crystal structure of the wild-type EGFR/Fab complex for cetuximab (PDB code: 1YY9) as a template. Essential amino acid substitutions that lead to loss of cetuximab or panitumumab binding to EGFR were identified according to the literature (Refs). All predicted structures were decorated and displayed with the open-source version of the PyMOL program . Waters and other HET residues  in the crystal structure were removed. Missing loops and hydrogen atoms were added to the cleaned structure of the antibody/antigen complex, which was then subjected to initial refinement using the Relax application in Rosetta. The relaxed structure with the lowest energy was selected for subsequent mutant modeling.∆∆G) caused by EGFR point mutation was used as a criterion for method suitability testing. One hundred structural models were generated for each of the EGFRS492R/cetuximab and EGFRG465R /cetuximab complexes, and their corresponding ∆∆G values . S492R-, I491M-, K489E-, K467T-, G465R-, G465E-, S464L-, I462R-, R451C-, S442R- and V441D-mutated EGFR constructs were generated by site-directed mutagenesis using the wild-type EGFR construct as a template. Primers used in this experiment were listed in Supplementary Table EGFR mutant constructs by a Lipofectamine 3000 transfection kit according to the manufacturer’s protocol  and were then treated with hygromycin B  for 96 h. After immunostaining with a mouse polyclonal anti-Flag tag antibody  at a dilution of 1:500 and a FITC-conjugated goat anti-mouse IgG (H + L) secondary antibody at a dilution of 1:500, stable cells expressing wild-type or mutant EGFR were sorted by fluorescence-activated cell sorting (FACS) in a MoFlo Astrios EQ flow cytometer . Single NIH3T3 cell clones that stably expressed wild-type or mutant EGFR  were identified with an ACEA NovoCyteTM flow cytometer . Stable HEK293T cells harboring C-terminal eGFP-tagged human wild-type, S492R-mutated or G465R-mutated EGFR were generated as described above. Stable SW48 cells overexpressing G465R-mutated EGFR and stable COLO320DM cells overexpressing wild-type, S492R-mutated or G465R-mutated EGFR were also generated as described above.To generate cell lines with stable expression of EGFR and its mutants, the sequence encoding N-terminal Flag-tagged full-length human wild-type The fusion gene fragment of the human wild-type EGFR ectodomain  and the human IgG1 Fc-fragment were cloned into the expression vector pMH3. The S492R-EGFR-ECD-Fc, G465E-EGFR-ECD-Fc and G465R-EGFR-ECD-Fc mutant constructs were generated by site-directed mutagenesis. Primers used in this experiment were listed in Supplementary Table Escherichia coli XL1 BLUE to generate construct bacterial libraries. Each library was pooled and grown overnight at 30 °C in 2XTY broth supplemented with M13KO7 helper phage, 100 μg/mL ampicillin, 50 μg/mL kanamycin and 50 μg/mL tetracycline. Phages were precipitated with 20% PEG-8000/2.5 M NaCl followed by centrifugation at 5,000×g. The precipitated phages were washed with cold PBS before suspension in PBS (pH 7.4)-buffered 2% (w/v) nonfat milk (blocking buffer). To select phages that display cetuximab-derived scFvs capable of binding to EGFRS492R or EGFRG465R, recombinant phages were first prepanned with parental NIH3T3 cells in blocking buffer to remove unbound phages, and subsequently panned with S492R-EGFR-NIH3T3 cells or G465R-EGFR-NIH3T3 cells after blocking. After 2 h of incubation, the cells were washed ten times with PBS. Bound phages were then eluted by incubation with glycine elution buffer (pH 2.2) for 15 min and neutralized by the addition of 2 M Tris-HCl buffer (pH 8.0). Phages were further screened in WT-EGFR-NIH3T3 cells to obtain WT and mutant EGFR dual binders. The eluted phages were amplified by reinfection of E. coli XL1 BLUE, followed by two additional rounds of selection. Eluted phages from the third round of panning were used to reinfect E. coli XL1 BLUE, and single clones were then expanded in 96-well microtiter plates and induced to express soluble scFv antibody with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Culture supernatants containing the C-terminal E tag-fused scFv antibody were analyzed by enzyme-linked immunosorbent assays (ELISAs). ELISA plates were precoated with recombinant S492R-EGFR-ECD-Fc or G465R-EGFR-ECD-Fc, blocked with 5% (w/v) milk/PBS (pH 7.4), and then washed with PBST . Bound scFv antibodies were detected using an HRP-conjugated anti-E tag antibody  at a dilution of 1:1000 in 5% (w/v) milk/PBS (pH 7.4), followed by incubation with 3′,3′,5,5′-tetramethylbenzidine (TMB) substrate. Positive phage clones were sequenced to obtain various scFv sequences.The single-chain variable fragment (scFv) of cetuximab was cloned into the SfiI-NotI restriction sites of the phagemid vector pCANTAB-5E. On the basis of computational prediction, the S492R-associated library was constructed with random mutations in CDR2 and CDR3 of the VH domain , and CDR1 and CDR3 of the VL domain . The G465R-associated library was generated with random mutations in CDR2 and CDR3 of the VH domain . These residues were mutated to 20 other amino acids by introducing NNS  codons in the oligonucleotide primer, and the corresponding scFv gene fragments were amplified by PCR. Primers used in this experiment were listed in Supplementary Table VH and VL domain fragments from selected anti-EGFR scFvs were subcloned into the expression vector pMH3 to generate the full-length human IgG1 (Cκ) format. The cetuximab variants Ctx-Y104X constructs were generated by site-directed mutagenesis. Primers used in this experiment were listed in Supplementary Table S492R, EGFRG465E or EGFRG465R-expressing NIH3T3 cells were treated with serial concentrations of cetuximab, panitumumab or the cetuximab variants, and were then fluorescently labeled with the FITC-conjugated goat anti-human IgG (H + L) secondary antibody  at a dilution of 1:500 before flow cytometric analysis. FACS sequential gating strategy is shown in Supplementary Fig. To determine the binding affinity of therapeutic antibodies in cell lines, wild-type or mutant EGFR -expressing NIH3T3 cells were harvested by trypsinization and washed twice with cold PBS (pH 7.4). Each cell line was stained with cetuximab or panitumumab  on ice, washed twice with ice-cold PBS (pH 7.4) and incubated with a FITC-conjugated goat anti-human IgG (H + L) secondary antibody  at a 1:500 dilution in PBS (pH 7.4) for 30 min on ice. Samples were washed and resuspended in ice-cold PBS (pH 7.4) before analysis via an ACEA NovoCyteTM flow cytometer . The percentages of monoclonal antibody-stained cells among total EGFR-positive cells were determined. The mean fluorescence intensity (MFI) of the immunostained cells was also determined by flow cytometry. FACS sequential gating strategy is shown in Supplementary Fig. −4 nM to 100 nM. The plate wells were washed five times with PBST. Antibodies were detected with an HRP-conjugated polyclonal goat anti-human kappa light chain antibody  at a dilution of 1:500 using TMB as the substrate.The binding of cetuximab, panitumumab, and the cetuximab variants to EGFR-ECD was analyzed by ELISA. Ninety-six-well microtiter plates were coated with WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc, G465E-EGFR-ECD-Fc, and G465R-EGFR-ECD-Fc at a concentration of 1 µg/mL overnight at 4 °C. Antibodies were diluted in PBS with 5% (w/v) nonfat milk and added to the coated plates at concentrations ranging from 1 × 10Antibody binding affinities were determined by a NeoSPR-M100 instrument . Flow cells composed of carboxyl chips  were coupled with recombinant WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc and G465R-EGFR-ECD-Fc through amine-coupling chemistry, and the coupling buffer PBS was used as a reference. Cetuximab or the cetuximab variants were injected over the immobilized WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc or G465R-EGFR-ECD-Fc (coupled at densities ranging from 96-293 relative units to flow cells composed of carboxyl chips) at a flow rate of 20 µL/min at 25 °C in PBS. Flow cells were regenerated following each injection using 30 mM NaOH buffer. Kinetic parameters were analyzed by TraceDrawer software.2. Then, the cells were treated with 100 nM cetuximab, panitumumab, Ctx-VY, Ctx-Y104D, Ctx-W52D or control IgG for 2 h and further stimulated for 15 min with 10 ng/mL EGF. Lysates were subjected to western blot analysis for detection of EGFR downstream signaling by various primary antibodies: rabbit anti-EGFR and rabbit anti-pEGFR (Tyr1068) antibodies , rabbit anti-Akt and rabbit anti-pAkt (Ser473) antibodies , and rabbit anti-Erk and rabbit anti-pErk (Thr202/Tyr204) antibodies . HRP-conjugated goat anti-rabbit IgG (H + L)  at a dilution of 1:2000 was used as the secondary antibody. Immunoreaction signals were visualized by a ChemiDoc Touch Imaging System  with ECL reagents.WT-EGFR-NIH3T3, S492R-EGFR-NIH3T3 and G465R-EGFR-NIH3T3 cells were plated in 6-well plates and serum starved overnight in a 37 °C incubator with 5% CO2, and were then treated with cetuximab, panitumumab, Ctx-VY, Ctx-Y104D, Ctx-W52D or control IgG for 48 hours. Mean fluorescence intensity (MFI) values were normalized to those in the untreated controls. FACS sequential gating strategy is shown in Supplementary Fig. Antibody-induced EGFR-eGFP degradation was assessed by flow cytometry. HEK293T cells overexpressing WT-EGFR-eGFP, S492R-EGFR-eGFP and G465R-EGFR-eGFP were seeded in 48-well plates overnight in a 37 °C incubator with 5% CO2 and treated with serial concentrations of therapeutic antibodies or cetuximab variants. Three days post treatment, cell viability was evaluated using a Cell Counting Kit-8  and measuring the absorbance at 450 nm in a Model 680 microplate reader . The cell proliferation inhibition rate is shown relative to cells treated with PBS only. Data were analyzed in GraphPad Prism software.SW48, G465R-EGFR-SW48 and COLO320DM cells stably overexpressing wild-type or mutant EGFR were seeded in 96-well plates overnight in a 37 °C incubator with 5% CO5 human peripheral blood mononuclear cells (PBMCs) for 10 hours, and cell viability was determined by a lactate dehydrogenase (LDH) detection kit . The percentage of specific lysis was determined as follows: % lysis = /(maximum LDH release − spontaneous LDH release) × 100. All assays were performed in triplicates. The results are presented as the mean ± SD values. For the complement-dependent cytotoxicity (CDC) assays, antibodies were incubated with 1 × 104 target cells/well and 10% human serum for 2 hours. Cell viability was determined by a Cell Counting Kit-8 . The percentage of specific lysis was determined as follows: % lysis =  × 100. Experiments were performed in triplicate. The results are presented as the mean ± SD values.For the antibody-dependent cellular cytotoxicity (ADCC) assay, ten thousand target cells were coincubated with 5×10WT- or EGFRMut- positive cells were generated by subcutaneous inoculation of 5 × 106 SW48 (WT-EGFR), G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote), WT-EGFR-COLO320DM, S492R-EGFR-COLO320DM or G465R-EGFR-COLO320DM cells into the right flanks of nude mice. When the mean tumor volume reached ~50 mm3, the mice were randomly divided into 20 groups . Cetuximab or the cetuximab variants were injected intravenously into xenografted mice via the tail vein at a dosage of 25 mg/kg, once every 3 days for four times (q3d×4). Mouse body weights were measured once every 2–3 days with an electronic balance, and the tumor size was measured every 2–3 days with an electronic caliper; the tumor volume was calculated using the formula (L × W × W)/2, in which L represents the larger diameter of the tumor and W represents the smaller diameter. Tumor tissues were collected for preparation of paraffin sections for the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining assay, immunohistochemical staining (EGFR and Ki-67), and hematoxylin and eosin (H&E) staining. Organs, including the heart, liver, spleen, lung, and kidney, were isolated for preparation of paraffin sections for H&E staining. Serum was collected for measurement of ALT, AST, BUN, and Cr to evaluate liver and kidney function. A Cobas c311 analyzer  was used to test ALT, AST, BUN and Cr.Mouse xenograft models bearing EGFRTumor tissues were fixed with 10% formaldehyde and embedded in paraffin. After dewaxing, rehydration, and antigen retrieval, sections were blocked with goat serum blocking solution and were then stained using rabbit polyclonal anti-EGFR  or rabbit polyclonal anti-Ki-67  antibodies overnight at 4 °C. After staining with the HRP-conjugated goat anti-rabbit IgG (H + L) antibody  at 37 °C for 30 min, tumor tissue sections were visualized by a DAB detection kit  and an Olympus BX61 light microscope . For detection of apoptosis, paraffin-embedded sections of tumor tissues were labeled by a POD-TUNEL Kit (Roche Inc.) according to the manufacturer’s instruction. All images were acquired by an Olympus BX61 light microscope. Quantification was performed at ×400 magnification (3 images per tumor) using ImageJ 1.52a software.The statistical analysis is described in the “Methods” section or figure legends. All the experiments were performed at least in triplicate and repeated.Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6


The HPLC-DAD-HESI-MS/MS analysis performed on the extract obtained under such conditions, named optimized Annurca apple leaves extract (OAALE), led to the identification of twenty-three phenolic molecules, with fifteen of them quantified. To explore the nutraceutical potential of OAALE, the in vitro antioxidant activity was evaluated by DPPH, ABTS, and FRAP assays, resulting in 21.17 ± 2.30, 38.85 ± 0.69, and 34.14 ± 3.8 μmol Trolox equivalent/g of extract, respectively. Moreover, the IC50 of 0.330 mg/mL obtained from the advanced glycation end-product inhibition assay, further supported the antidiabetic potential of OAALE.Phlorizin is a plant-derived molecule with relevant anti-diabetic activity, making this compound a potential functional component in nutraceutical formulations for the management of glycemia. It is noteworthy that promising sources for the extraction of phlorizin include apple tree leaves, a by-product of apple fruit production. The main aim of this study was to optimize the extraction process of phlorizin from Annurca apple tree leaves (AALs) using response surface methodology (RSM), and to determine the potential nutraceutical application of the obtained extract. The results of the RSM analysis indicate a maximum phlorizin yield of 126.89 ± 7.579 (mg/g DW) obtained under the following optimized conditions: MeOH/H O-β-D-glucopyranoside) is the glucoside of phloretin, a member of dihydrochalcones, which are a family of bicyclic flavonoids. It was first isolated by a French scientist from apple tree bark in 1835  × 100, where Ai is the absorbance of the sample at t = 0, Af is the absorbance of the sample after the reaction time and Ac was the absorbance of the control (1000 µL of a methanol solution of DPPH + 100 µL of methanol). The obtained results are expressed in µmol of Trolox  equivalent (TE). Moreover, the results were also reported as EC50, which is the amount of antioxidant compound necessary to inhibit the initial DPPH• concentration by 50%  × 100, (2), where Ai is the absorbance of the sample at t = 0, Af is the absorbance after 2.5 min, and Ac is the absorbance of the control at time zero. Trolox was used as a standard antioxidant. The results are expressed both as µmol of TE and EC50, which is the amount of antioxidant necessary to decrease the initial ABTS•+ concentration by 50%  × 100, (4) where Fs is the fluorescence intensity in the presence of the sample; Fsb is the fluorescence intensity in the absence of fructose and glucose; Fc is the fluorescence intensity in the absence of sample; and Fcb is the fluorescence intensity in the absence of sample, fructose, and glucose. Finally, the results are reported as EC50.The inhibition of AGE generation by OAALE extract and the standard phenolic rutin was performed according to the method reported by Schiano et al.  with sli50 values determination were calculated using GraphPad Prism 8 software. The RSM optimization was performed with Minitab software version 21.1.0.Unless otherwise stated, all experimental results are expressed as the mean ± standard deviation (SD) of three repetitions. Graphics and ICp < 0.05) during the extraction process at a temperature lower than 40 °C − ion at m/z 197. The base peak ion at m/z 182 [M-H-CH3]− and the fragment ions of its tandem mass spectrum at m/z 179 [M-H-H2O]−, m/z 153 [M-H-CO2]− and m/z 138 [M-H-CO2-CH3]−, suggested the presence of a carboxylic acid, a methoxy and a phenolic group. According to the mass fragmentation pattern, compound 1 was identified as syringic acid [2 and 4 displayed a [M-H]− ion at m/z 163 and a base peak ion at m/z 119 [M-H-CO2]−. The fragment ions at m/z 145 [M-H-H2O]− and at m/z 135 [M-H-CO]− indicated the presence of the hydroxycinnamic acid scaffold and a phenol group. In agreement with the literature data, compounds 2 and 4 were annotated as p-coumaric acid isomers [3 showed a [M-H]− ion at m/z 353 and was putatively identified as a caffeoylquinic acid. The base peak ion at m/z 191 [M-H-CA]− and the fragment ion at m/z 179 [M-H-QA]− were due to the loss of the caffeic acid and the quinic acid group, respectively. By comparison with an authentic analytical standard, compound 3 was identified as chlorogenic acid [5) displayed a [M-H]− ion at m/z 179. The base peak ion at m/z 135 [M-H-CO2]− and the fragment ions at m/z 161 [M-H-H2O]− and m/z 107 [M-H-CO-CO2]− highlighted the linkage of a carboxylic acid and a phenolic acid. One procyanidin dimer B-type linkage (6) showed a [M-H]− ion at m/z 577 and a base peak ion at m/z 425 [M-H-C8H8O3]−, due to the RDA fission. The fragment ions at m/z 451 [M-H-C6H6O3]−, at m/z 289 [M-H-C15H12O6]− and at m/z 287 [M-H-C15H14O6]− were produced by the HRF and the QM cleavage, respectively. By comparison with the authentic analytical standard, compound 6 was identified as procyanidin B2 [7 displayed a [M-H]− ion at m/z 289 and was putatively identified as epicatechin. The base peak ion at m/z 245 [M-H-C2H4O]− and the fragment ion at m/z 137 [M-H-C8H8O3]−, due to the RDA fragmentation, were in agreement with the literature data [O-coumaroylquinic acid isomers (compounds 8 and 9) were tentatively identified. They showed a [M-H]− ion at m/z 337 and three fragment ions at m/z 191 [QA-H]−, at m/z 173 [QA-H-H2O]− and at m/z 163 [M-H-QA]−, due to the fragmentation of the quinic acid moiety. However, the base peak ion at m/z 173 [QA-H-H2O]− indicated the linkage between quinic acid and coumaric acid moieties with the 4-OH group. Therefore, compounds 8 and 9 were putatively identified as 4-O-coumaroylquinic acid isomers [O-hexoside isomers  were putatively detected. They showed an [M-H]− ion at m/z 463 and a base peak ion at m/z 301 [M-H-Hex]− due to the fragmentation of the hexoside group. The fragments at m/z 445 [M-H-H2O]− and at m/z 179 [M-H-Hex-C7H6O2]−, due to the RDA fragmentation, confirmed the presence of the flavonol scaffold and agreed with literature data [O-rutinoside isomers (compounds 11 and 13) were tentatively identified and displayed a [M-H]− ion at m/z 609. The base peak ion at m/z 301 [M-H-Glu-Rha]−, due to the loss of the disaccharide group, and the fragment ions at m/z 463 [M-H-Rha]− and at m/z 179 [M-H-Glu-Rha-C7H6O2]−, which derived from the RDA fragmentation, were consistent with the literature data [13 was identified as rutin by comparison with the authentic analytical standard. Compound 12 displayed a [M-H]− ion at m/z 433. The base peak ion at m/z 271 [M-H-Hex]− and the fragment ions at m/z 313 [M-H-C4H8O4]− and at m/z 151 [M-H-Hex-C8H8O]−, due to the RDA fragmentation, allowed the identification of the flavanone scaffold. Therefore, compound 12 was annotated as naringenin O-hexoside [15 showed a [M-H]− ion at 431. The base peak ion at m/z 269 [M-H-Hex]− and the fragment ion at m/z 311 [M-H-C4H8O4]− are derived from the cleavage of the hexoside group and the RDA fragmentation, respectively. Based on the tandem mass spectrum and by comparison with an analytical standard, compound 15 was identified as apigenin 7-O-glucoside. Compound 16 displayed a [M-H]− ion at m/z 593 and was annotated as kaempferol O-rutinoside. The base peak ion at m/z 285 [M-H-Pent-Hex]− and the fragment ions at m/z 327 [M-H-Pent-C4H8O4]− and m/z 257 [M-H-Pent-Hex-CO]− confirmed the linkage of the disaccharide rutinose and the aglycone kaempferol [18 showed a [M-H]− ion at m/z 433. The base peak ion at m/z 301 [M-H-Pent]− and the fragment ion at m/z 179 [M-H-Pent-C7H6O2]− allowed us to identify the presence of the pentoside group and the flavanol scaffold. Based on the tandem mass spectrum, compound 18 was annotated as quercetin O-pentoside. Quercetin O-rhamnoside (19) displayed an [M-H]− ion at m/z 447 and a base peak ion at m/z 301 [M-H-Rha]− for the loss of the rhamnoside unit. The fragment ions at m/z 429 [M-H-H2O]−, m/z 179 [M-H-Rha-C7H6O2]− and m/z 151 [M-H-Rha-C8H6O3]− confirmed the presence of the flavanol scaffold and are consistent with the literature data. Compound 21 displayed a [M-H]− ion at m/z 435. The base peak ion at m/z 273 [M-H-Hex]− and the prominent fragment ion at m/z 167 [M-H-Hex-C7H6O]− indicated the presence of the chalcone scaffold and the linkage of the hexoside group. Based on these data and by comparison with an analytical standard, compound 21 was identified as phloridzin. Kaempferol 3-O-rhamnoside (22) showed a [M-H]− ion at m/z 431. Its tandem mass spectrum displayed a base peak ion at m/z 285 [M-H-Rha]− and two fragment ions at m/z 327 [M-H-C4H8O3]− and m/z 179 [M-H-Rha-C7H6O]−, due to the loss of the sugar moiety and RDA fragmentation [22 was confirmed by comparison with the analytical standard. Compound 23 displayed a [M-H]− ion at m/z 273. Its tandem mass spectrum is characterized by a base peak ion at m/z 167 [M-H-C7H6O]− and a fragment ion at m/z 125 [M-H-C9H8O2]−, which is linked to the presence of the chalcone moiety. Based on these data and by comparison with an analytical standard, compound 23 was identified as phloretin [OCE polyphenolic composition was characterized by HPLC-HESI-MS/MS, as reported in gic acid . Compoun isomers . Compounnic acid . Caffeicnidin B2 . Compounure data . Two 4-O isomers . Four quempferol . Compounentation . The idehloretin .The antiradical potential of apple leaves, as vegetal matrices , conside50, which is the quantity of antioxidants necessary to decrease the concentration of the initial solution by 50% [50 of 0.828 mg/mL for the DPPH assay and 0.542 mg/mL for the ABTS assay. Therefore, these results would support the relevant potential application of OAALE as a source of antiradical agents, with the indubitable benefit of re-evaluating food waste. It is noteworthy that increasing evidence from in vitro and clinical trials indicates that oxidative stress may play a relevant role in the pathogenesis of diabetes. High levels of free radicals, and the concomitant decrease in antioxidant defense mechanisms, may lead to the injury of biological structures, which is recognized as the main pathological origin for the generation and development of diabetes-related complications [Regarding the antiradical activity, OAALE has shown a higher relevant activity compared to AAF  . Moreoven by 50% . Figure ications .50 value was 0.330 mg/mL. In this regard, phlorizin and phloretin may be considered the main actors of OAALE potential valuable biological activity. In support of this hypothesis, these two molecules represent the main polyphenolic components of OAALE and, as reported in other studies, both of them demonstrated the inhibition of AGEs formation in a concentration-dependent manner, at a concentration range of 0.01–1.0 mM [Increasing evidence has identified the formation of advanced glycation end-products (AGEs) as a major pathogenic risk agent related to hyperglycemia and diabetes-related complications. It is also well known that the continuous AGEs accumulation in tissues and organs is directly linked to the development of chronic diabetic-related complications, such as retinopathy, nephropathy, neuropathy, and macrovascular disease ,42. AGEs1–1.0 mM .50 of 0.330 mg/mL would be attributed to the synergic action of dihydrochalcones and other polyphenols contained in OAALE.In this context, although dihydrochalcones were the most abundant molecules in OAALE, we also showed a valuable concentration of other classes of polyphenols . The extract obtained under optimized conditions (OAALE) was also chemically characterized and its in vitro potential biological activity was tested. The promising results about the antioxidant activity and the inhibition of AGEs formation may suggest that AALs are a powerful functional ingredient, useful for the formulation of nutraceutical products for the management of diabetes disease. Further investigations about the beneficial potential exerted by the formulation in a diabetes model are required to assess the effective application in the management of this pathological condition. In addition, future perspectives include the possibility of performing a toxicological analysis aiming to exclude the possible residues of the means used to treat apple trees.