A CD8αβ co-receptor modified to contain an intracellular CD28 signaling tail enhances TCR-engineered T cell function independent of solid-tumor-associated co-stimulatory ligands

A CD8αβ co-receptor modified to contain an intracellular CD28 signaling tail enhances TCR-engineered T cell function independent of solid-tumor-associated co-stimulatory ligands | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A CD8αβ co-receptor modified to contain an intracellular CD28 signaling tail enhances TCR-engineered T cell function independent of solid-tumor-associated co-stimulatory ligands Aude Chapuis, Shihong Zhang, Tzu-Hao Tang, Sinéad Kinsella, Francesco Mazziotta, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5939098/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Adoptive therapies using T cells genetically modified with T cell receptors (TCR)s have shown limited efficacy in the solid tumor setting. Although functional CD4 + and CD8 + T cells transduced with a TCR specific for HLA-A2-restricted melanoma-associated antigen A1 (MAGE-A1, T TCR−MA1−CD8αβ ) could be detected post-transfer and were safe in one patient who subsequently progressed, T TCR−MA1−CD8αβ were insufficient to sustain antitumor activity in “stress” mouse tumor models. Leveraging the obligate co-expression of CD8αβ required for engagement of CD4 + T cells expressing the TCR, we screened positive co-stimulatory signals tethered to the intracellular tail of CD8β and identified that CD28 reduced exhaustion, enhanced tumor infiltration and improved murine tumor control. Further modifications of the CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models. Thus, integrating co-stimulatory signals downstream of the TCR signaling complex can enhance TCR-engineered T cell function, independent of tumor-associated co-stimulatory ligand expression. Biological sciences/Immunology/Immunotherapy Health sciences/Diseases/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Translational immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations Yes there is potential Competing Interest. A.G.C. has received support from Elevate Bio, Juno Therapeutics, Lonza, and Affini-T. P.D.G. is on the Scientific Advisory Board of Celsius, Earli, Elpiscience, Immunoscape, Rapt, and Nextech, was a scientific founder of Juno Therapeutics, and receives research support from Lonza. A.G.C., T.M.S., and P.D.G. are co-founders of, have equity in, and receive research support from Affini-T. A.G.C, T.M.S. and M.M are listed as inventors on patents related to MA1 TCR and CD8/28 constructs. Supplementary Files ExtendedDataFig.1.pdf Extended Data 1 ExtendedDataFig.2.pdf Extended Data 2 ExtendedDataFig.3.pdf Extended Data 3 ExtendedDataFig.4.pdf Extended Data 4 ExtendedDataFig.5.pdf Extended Data 5 ExtendedDataFig.6.pdf Extended Data 6 ExtendedDataFig.7.pdf Extended Data 7 ExtendedDataFig.8.pdf Extended Data 8 ExtendedDataFig9.pdf Extended Data 9 SupplementaryTables18.pdf Table 1-8 Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5939098","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":412782556,"identity":"52ad3dbe-becb-427c-9659-4a6019b851c9","order_by":0,"name":"Aude 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CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e TTCR-MA1 cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eare safe when infused into one patient.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; Schematic of the multi-cistronic vector design used in pre-clinical studies.\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; Dose response curves (left panels) of IFNγ, TNFα, and IL-2 expression in response to decreasing peptide concentrations by CD8+ TTCR-MA1-CD8αβ (red), CD4 TTCR-MA1 (blue, open circles) and CD4 TTCR-MA1-CD8αβ (blue, full circles). Mean ± standard deviations (SD) from biological duplicates are shown. Mean EC50 (right panels) of IFNγ, TNFα, and IL-2 cytokine expression for the same previous 3 cell populations.\u003c/p\u003e\n\u003cp\u003e(C)\u0026nbsp; Growth kinetics (left panel) for the ME275 cell line in a live tumor visualization assay in the absence (gray line) or presence of CD4+ TTCR-MA1-CD8αβ (blue line), CD8+ TTCR-MA1-CD8αβ (red line), or CD4+ CD8 TTCR-MA1-CD8αβ (purple line). The E:T ratio was 5:1, and arrows indicate the addition of tumor cells to the culture. Final tumor integrated intensity (right panel) for the same experiment 6 days after start (144 hours). The mean and standard deviation (SD) of triplicate wells is shown. Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e\n\u003cp\u003e(D)\u0026nbsp; Proliferation of T cells was measured at 6 days post tumor simulation. Then change in absolute numbers of CD4 TTCR-MA1-CD8αβ (left panel) and CD8 TTCR-MA1-CD8αβ (right panel) in either alone (CD4 - blue bars, CD8 – red bars) or combined (purple bars) conditions. The mean ± SD from triplicated wells is shown (n=3). Unpaired two-tailed t-test.\u003c/p\u003e\n\u003cp\u003e(E)\u0026nbsp;\u0026nbsp; Schematic (left) and growth kinetics (right) of ME275 tumors engrafted subcutaneously into NSG mice with subsequent transfer of irrelevant T cells \u003csub\u003e(\u003c/sub\u003e\u003csub\u003e\u003cstrong\u003eTIrr.-CD8αβ\u003c/strong\u003e\u003c/sub\u003e\u003csub\u003e)\u003c/sub\u003e , CD4 \u003csub\u003e\u003cstrong\u003eTTCR-MA1-CD8αβ\u003c/strong\u003e\u003c/sub\u003e alone, CD8 \u003cstrong\u003eTTCR-MA1-CD8αβ \u003c/strong\u003ealone, or a 1:1 ratio of combined CD4+ and CD8+ \u003cstrong\u003eTTCR-MA1-CD8αβ, \u003c/strong\u003e22 days after implantation. The tumors were allowed to reach allowed to reach ~60mm\u003csup\u003e3\u003c/sup\u003e in size. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003e(F)\u0026nbsp;\u0026nbsp; Schematic (left) and growth kinetics (right) of A375F tumors engrafted into NSG mice with subsequent transfer of 1x 10^7 total T cells (TIrr.-CD8αβ, CD4 TTCR-MA1-CD8αβ alone, CD8 TTCR-MA1-CD8αβ alone, or a 1:1 ratio of combined CD4+ and CD8+ TTCR-MA1-CD8αβ) 12 days after implantation. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003e(G)\u0026nbsp; A375F tumor weight (top) and volume (bottom) from \u003cstrong\u003e(B) \u003c/strong\u003eassessed at sacrifice 21 days after T cell infusions for each indicated condition. One-way ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/caac8b2468c0a51ca767e988.png"},{"id":81028205,"identity":"8c1ca015-9ba9-4963-ba98-58e21ab52f33","added_by":"auto","created_at":"2025-04-21 10:56:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":225774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional CD4+ and CD8+ TTCR-MA1-CD8αβ\u0026nbsp;enhance intratumor proliferation but are insufficient to suppress tumor growth in a stress murine model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp;\u003c/strong\u003eContour flow plot of the patient’s CD4 TTCR-MA1-CD8αβ (top) and CD8 TTCR-MA1-CD8αβ (bottom) infusion product binding to CD28 (x-axis), CD62L (y-axis left panels) and CD127 (x-axis right panels).\u003c/p\u003e\n\u003cp\u003eB. IFNγ (x-axis) and TNFα (y axis) expression by the patient’s CD4 TTCR-MA1-CD8αβ (top) and CD8 TTCR-MA1-CD8αβ (bottom) infusion product after an 18-hour exposure to 10μM (left panels), 1 μM (middle panels), and no peptide stimulation (right panels).\u003c/p\u003e\n\u003cp\u003eC. Circulating\u0026nbsp;\u003csub\u003eTTCR-MA1-CD8αβ\u003c/sub\u003e\u0026nbsp;in PBMCs identified with p/HLA multimer at indicated timepoints (x axis) for the infused patient.\u003c/p\u003e\n\u003cp\u003eD. Schematic (left) and growth kinetics (right) of A375F tumors engrafted into NSG mice with subsequent transfer of 5x 10^6 total T cells (TIrr.-CD8αβ, CD4 TTCR-MA1-CD8αβ alone, CD8 TTCR-MA1-CD8αβ alone, or a 1:1 ratio of combined CD4+ and CD8+ TTCR-MA1-CD8αβ), 12 days after implantation. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003eE. A375F tumor volume (bottom) from\u0026nbsp;\u003cstrong\u003e(D)\u0026nbsp;\u003c/strong\u003eassessed at sacrifice 21 days after T cell infusions for each indicated condition. One-way ANOVA with Tukey’s multiple comparison test. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003eF. Expression of PD-1 (x-axis) and Tim 3 (y-axis) in CD4+ (left 3 panels) and CD8+ (right 3 panels) T cells identified in spleen (top panels) or tumor (lower panels) for the indicated conditions. Single cell suspensions were obtained at sacrifice, 21 days after T cell infusion from\u0026nbsp;\u003cstrong\u003e(D).\u0026nbsp;\u003c/strong\u003en=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003eG. Quantification of Tim3+ PD-1+ populations across the experimental groups from\u0026nbsp;\u003cstrong\u003e(D)\u0026nbsp;\u003c/strong\u003ein CD4+ (left panel) and CD8+ (right panel) for the indicated conditions. The mean ± SD of 6 tumors per group is shown. Unpaired two-tailed t-test. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H \u0026amp; I)\u0026nbsp;\u003c/strong\u003eHeatmap showing the expression of curated co-stimulatory (green box) and inhibitory (red box) genes for lung cancer\u0026nbsp;\u003cstrong\u003e(H)\u0026nbsp;\u003c/strong\u003eand melanoma\u0026nbsp;\u003cstrong\u003e(I)\u0026nbsp;\u003c/strong\u003epatients across annotated cell subsets. Higher expression values are depicted in red, while lower expression values are shown in blue.\u0026nbsp;\u003cstrong\u003e(J\u0026amp;K)\u0026nbsp;\u003c/strong\u003eUMAP plot showing the two-dimensional distribution of a costimulatory gene score (\u003cem\u003eCD80\u003c/em\u003e,\u0026nbsp;\u003cem\u003eCD86\u003c/em\u003e,\u0026nbsp;\u003cem\u003eCD40\u003c/em\u003e,\u0026nbsp;\u003cem\u003eTNFRSF4\u003c/em\u003e,\u0026nbsp;\u003cem\u003eTNFS9 and ICOSLG\u003c/em\u003e) in scRNAseq samples from lung cancer\u0026nbsp;\u003cstrong\u003e(J)\u0026nbsp;\u003c/strong\u003eand melanoma\u0026nbsp;\u003cstrong\u003e(K)\u0026nbsp;\u003c/strong\u003epatients.\u0026nbsp;\u003cem\u003eICOSLG\u0026nbsp;\u003c/em\u003eexpression was present in the melanoma dataset but not in the lung cancer dataset. Dark red indicates higher expression, while light grey marks regions with low score expression.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/f8a3ab6027eccffb9f4186a7.png"},{"id":81028203,"identity":"220b38ed-19a3-418e-a44f-a24f2a9f4481","added_by":"auto","created_at":"2025-04-21 10:56:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of chimeric CD8β chains functionally integrated into the immune synapse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schema of the multi-cistronic vector designs used to integrate CCRs downstream of CD8β.\u003c/p\u003e\n\u003cp\u003eB. MFI of CD8α (left), CD8β (middle) and MAGE-A1 p/HLA multimer (right) binding of CD4 transduced to express TTCR-MA1\u0026nbsp;with the indicated CCRs.\u003c/p\u003e\n\u003cp\u003eC. (left) Fit curves of IFNγ expression in response to decreasing peptide concentrations by CD4+ T cells transduced with TTCR-MA1 and the indicated CCRs as well as CD8α (negative control) measured 18 h post peptide stimulation. (right) Mean half maximal effective concentration (EC50) of IFNγ expression for CD4+ T cells transduced with the same constructs.\u003c/p\u003e\n\u003cp\u003eD. Fold change of IFNγ levels in TTCR-MA1\u0026nbsp;cells transduced to express the different CCR constructs and incubated for 18-hour with 1µM cognate peptide alone (left panel) or incubated with 1µM cognate peptide following 1 week of priming by co-culture with irradiated ME275 (right panel).\u003c/p\u003e\n\u003cp\u003eE. (left) Growth kinetics of the ME275 cell line in the absence (black lines) or presence of the indicated CCR constructs. An E:T ratio of 5:1 was used, and arrows indicate addition of tumor cells to the culture. (middle) Final tumor integrated intensity for the same experiment 16 days after start (384 hours). (right) Final lymphocyte counts were obtained after 16 days. The indicated p values were calculated by one-way ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/a743535a7c9475c01f1159d6.png"},{"id":81029779,"identity":"342147d6-3093-4fdb-a520-cd6e96206fa1","added_by":"auto","created_at":"2025-04-21 11:12:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1890668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCR enhances tumor control and T cell infiltration\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA. NSG mice were engrafted with 5*10^3 cells A375F and subsequently infused with CD4+ and CD8+ TTCR-MA1-CD8αβ CCR cells (TTCR-Irr.-CD8/CD28, TTCR-MA1-CD8αβ, TTCR-MA1-CD8/CD28) at a ratio of 1:1 12 days post engraftment. Survival analysis was carried out when tumor burden reached 1000mm\u003csup\u003e3\u003c/sup\u003e, and assessed tumor progression occurring 57 days vs. 36 days vs. 27 days post T cell transfer, respectively; Kaplan Meier test for analysis of survival applied. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003eB. Tumor burden measurements for NSG mice engrafted with 5*10^3 cells A375F and subsequently infused with CD4+ and CD8+ TTCR-MA1-CD8αβ\u0026nbsp;CCR cells (TTCR-Irr.-CD8/CD28,\u0026nbsp;TTCR-MA1-CD8αβ,\u0026nbsp;TTCR-MA1-CD8/CD28) at a ratio of 1:1 12 days post engraftment. One-way ANOVA with Tukey’s multiple comparison test. n=6 mice/group.\u003c/p\u003e\n\u003cp\u003eC. Representative IHC images of A375F tumors resected from NSG mice (same experiment as\u0026nbsp;\u003cstrong\u003eB\u003c/strong\u003e) showing DAPI (blue), CD4+ (red) and CD8+ (green) T cells of 4 groups: No T cells, irrelevant TCR (TTCR-Irr.-CD8/CD28) , CD8αβ (TTCR-MA1-CD8αβ), and CD8/CD28 CCR (TTCR-MA1-CD8/CD28). 2x and 10X magnification. n=6 mice/group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-G)\u0026nbsp;\u003c/strong\u003eTumor-infiltrating T cells to tumor cells ration\u0026nbsp;\u003cstrong\u003e(D)\u0026nbsp;\u003c/strong\u003efrom resected A375F tumors form NSG mice infused with TTCR-Irr.-CD8/CD28, TTCR-MA1-CD8αβ\u0026nbsp;\u003csup\u003eor\u003c/sup\u003e\u003csub\u003e\u0026nbsp;TTCR-MA1-CD8/CD28.\u003c/sub\u003e\u0026nbsp;The indicated p value was determined by Kruskal-Wallis test with Dunn’s multiple comparison test. CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;TCR-T cells and tumor cells ratio\u0026nbsp;\u003cstrong\u003e(E)\u003c/strong\u003e, p value determined by Kruskal-Wallis test. CD8\u003csup\u003e+\u003c/sup\u003eT cells and tumor cells ratio\u0026nbsp;\u003cstrong\u003e(F)\u003c/strong\u003e. CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cell ratio\u003c/p\u003e\n\u003cp\u003eG. showing CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cell proliferation, p value determined by Mann-Whitney unpaired two-tailed t-test. Analysis performed using HALO Link software on IHC slides. n=6 mice/group.\u003c/p\u003e\n\u003cp\u003eH. Flow plot representing Tim-3 and PD-1 double positive population of CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;TILs between TTCR-MA1-CD8αβ and TTCR-MA1-CD8/CD28 from tumor single cell suspension.\u003c/p\u003e\n\u003cp\u003eI. Bar graph showing PD-1+Tim3+ frequency of CD4+ and CD8+ TILs between TTCR-MA1-CD8αβ and TTCR-MA1-CD8/CD28. p value calculated Mann-Whitney unpaired two-tailed t-test. n=5-6/group.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/80e50b4a61d3433d278f09a0.png"},{"id":81030961,"identity":"361d73ef-7504-47ac-9b62-c6641f8067bd","added_by":"auto","created_at":"2025-04-21 11:20:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe chimeric CD8/CD28 co-receptor TCR-T cells have a reduced exhaustion(-like) phenotype\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA. Heatmap showing the top 20 differentially expressed genes identified within each cluster. The “top 20” refers to the 20 genes with the most significant differential expression across the identified clusters. The dendrogram on the left displays the similarity between the clusters, based on unsupervised clustering of gene co-expression patterns. The top dendrogram shows the relationships between the genes based on their expression patterns.\u003c/p\u003e\n\u003cp\u003eB. UMAP plot displaying the two-dimensional distribution of annotated T-cell transcriptional states, colored by subset. Subsets were annotated according to gene co-expression patterns in (A).\u003c/p\u003e\n\u003cp\u003eC. UMAP plot of T cells colored by density and split by group (from left to right: CD8-alone group, CD8 WT and CD8 CCR from the combination group). The plot shows the two-dimensional distribution of T cells, with color intensity representing cell density, where dark red indicates higher density.\u003c/p\u003e\n\u003cp\u003eD. Bar graph colored by the annotated T cells indicating the absolute (y-axis) abundance across the annotated subsets (x-axis) for the three groups.\u003c/p\u003e\n\u003cp\u003eE. Violin plots displaying the expression levels (y-axis) of stem-like (\u003cem\u003eTCF7, SELL, IL7R, LTB, LEF1, NELL2\u003c/em\u003e), proliferation (\u003cem\u003eMKI67\u003c/em\u003e,\u0026nbsp;\u003cem\u003eTOP2A\u003c/em\u003e,\u0026nbsp;\u003cem\u003ePCNA\u003c/em\u003e), and cytotoxicity (\u003cem\u003eKLRG1, KLRD1, NKG7, GZMB, GNLY, PRF1, GZMA, GZMM, CSTA, CSTW\u003c/em\u003e) markers across CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T-cell transcriptional states (x-axis). Statistical significance was assessed using the Wilcoxon rank sum test. Asterisks indicate the following thresholds of significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003eF. Tumor burden measurements for NSG mice engrafted with 5*10^3 cells A375F and subsequently infused with CD8+ only TTCR-MA1-CD8αβ , CD4+ and CD8+ (1:1 ratio combo) TTCR-MA1-CD8αβ , and CD4+ and CD8+ (1:1 ratio combo) TTCR-MA1-CD8/CD28 12 days post engraftment. n=6 mice/group.\u003c/p\u003e","description":"","filename":"Binder15.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/e12737c06034bed6675cc9f8.png"},{"id":81028593,"identity":"90fc7066-b419-45cf-a5d7-daf69dc641cf","added_by":"auto","created_at":"2025-04-21 11:04:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":107369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutations in the CD8-CD28 CCR enhances anti-tumor activity in MAGE and PRAME tumor models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schema showing the four motif mutations on the CD28 intracellular domain.\u003c/p\u003e\n\u003cp\u003eB. Dose response curve showing IFNγ and TNFα levels in CD4+ TTCR-MA1\u0026nbsp;CCR cells incubated with decreasing concentrations of cognate peptide overnight.\u003c/p\u003e\n\u003cp\u003eC. NSG mice were engrafted with treated with 5*10^3 A375F and subsequently infused with CD4+ and CD8+ T cells (TTCR-MA1-CD8αβ, TTCR-MA1-CD8/CD28, and TTCR-MA1-CD8/CD28mut) at a ratio of 1:1 on day 12 post engraftment. n=5-6 mice/group.\u003c/p\u003e\n\u003cp\u003eD. (left) Growth kinetics of the SKMEL-37 cell line in the absence (black lines) or presence of the indicated CCR constructs (TTCR-Irr.-CD8/CD28, TTCR-MA1-CD8αβ, TTCR-MA1-CD8/CD28, and TTCR-MA1-CD8/CD28mut) at a ratio of E:T 5:1. (right) Final lymphocyte counts were obtained after 16 days. The indicated p values were calculated by one-way ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e\n\u003cp\u003eE. Dose response curves showing IFNγ and TNFα levels in TTCR-PRAME-CD8αα,\u0026nbsp;TTCR-PRAME-CD8αβ,\u0026nbsp;TTCR-PRAME-CD8/CD28,\u0026nbsp;and TTCR-PRAME-CD8/CD28mut\u0026nbsp;cells incubated with decreasing concentrations of cognate peptide overnight.\u003c/p\u003e\n\u003cp\u003eF. (left) Growth kinetics of the PANC1 cell line in the absence (black lines) or presence of the indicated CCR constructs (TTCR-PRAME-CD8αα, TTCR-PRAME-CD8αβ, TTCR-PRAME-CD8/CD28,\u0026nbsp;\u003csup\u003eand\u003c/sup\u003e\u003csub\u003e\u0026nbsp;TTCR-PRAME-CD8/CD28mut)\u003c/sub\u003e\u0026nbsp;at a ratio of E:T 5:1. (right) Final tumor integrated intensity of Incucyte killing assay was analyzed. The indicated p values were calculated by one-way ANOVA with Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Binder16.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/50c68ee54fe50904279301b0.png"},{"id":81028215,"identity":"2fad5224-dc2a-4a32-b6aa-281d23313d47","added_by":"auto","created_at":"2025-04-21 10:56:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":177674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe gene expression profile of TTCR-MA1CD8/CD28mut\u0026nbsp;identified a balance between functional, exhaustion and persistence.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. GSEA pathway analysis of TTCR-MA1-CD8αα, TTCR-MA1-CD8/28, TTCR-MA1-CD8/28mut normalized by TTCR-MA1-CD8αβ. TTCR-MA1-CD8αβ, TTCR-MA1-CD8/CD28, and TTCR-MA1-CD8/CD28mut were stimulated with 1µM cognate peptide for 0 and 4 hours and firther analysed by bulk RNA sequencing.\u003c/p\u003e\n\u003cp\u003eB. Schematic of experimental layout showing TTCR-MA1-CD8/28\u0026nbsp;or TTCR-MA1-CD8/28mut\u0026nbsp;incubated with irradiated ME275 for 21days with tumor rechallenge every 7 days and further analyzed by flow cytometry. The cells were we challenged CCR-expressing cells with irradiated tumor cells every 7 days for 21 days and further stimulated with 1µM cognate antigen peptide for 18 hours.\u003c/p\u003e\n\u003cp\u003eC. PCA plot of bulk RNA-seq data, showing the first two principal components (PC1 and PC2). Data points represent individual samples, color-coded by condition to visualize any separation based on gene expression profiles. Ellipses, drawn at a 90% confidence level, indicate the range of variation within each condition, highlighting how closely related or distinct the three groups are from each other.\u003c/p\u003e\n\u003cp\u003eD. Heatmap showing the expression levels of differentially expressed genes across constructs (TTCR-MA1-CD8/CD28, TTCR-MA1-CD8/CD28mut) and stimulations (unstimulated vs. stimulated) in the rlog-transformed data. Rows represent genes, and columns represent individual samples (3 biological replicates per condition). Expression values are color-coded from low (blue) to high (red) based on z-scores. The dendrograms on the axes indicate hierarchical clustering of genes and samples.\u003c/p\u003e\n\u003cp\u003eE. Boxplots showing the z-scores for the expression of manually curated gene signatures of proliferation unstimulated and stimulated conditions. The z-scores were calculated from the averaged normalized counts of significant genes (padj \u0026lt; 0.05, log2 fold-change \u0026gt; 0.5). Each box represents the distribution of z-scores for a specific gene signature in the unstimulated and stimulated groups for TTCR-MA1-CD8/CD28 and TTCR-MA1-CD8/CD28mut, with the median indicated by the horizontal line within each box.\u003c/p\u003e\n\u003cp\u003eF. Boxplots showing the z-scores for the expression of manually curated gene signatures of exhaustion across unstimulated and stimulated conditions. The z-scores were calculated from the averaged normalized counts of significant genes (padj \u0026lt; 0.05, log2 fold-change \u0026gt; 0.5). Each box represents the distribution of z-scores for a specific gene signature in the unstimulated and stimulated groups for TTCR-MA1-CD8/CD28\u0026nbsp;and TTCR-MA1-CD8/CD28mut, with the median indicated by the horizontal line within each box.\u003c/p\u003e\n\u003cp\u003eG. Dot plot summarizing GSEA results for the 'Apoptosis' pathway across multiple comparisons. The x-axis represents the pathway of interest, while the y-axis denotes the pairwise comparisons between experimental conditions. Dot size corresponds to the -log10-transformed p-value, indicating the significance of enrichment, and dot color represents the normalized enrichment score (NES), with red indicating positive enrichment and blue indicating negative enrichment.\u003c/p\u003e\n\u003cp\u003eH. Flow cytometry quantification of Tim3+ PD-1+ double positive populations from TTCR-MA1-CD8/28\u0026nbsp;and TTCR-MA1-CD8/28mut\u0026nbsp;groups was measured at the end of the 21 days stimulation with irradiated ME275 incubation.\u003c/p\u003e\n\u003cp\u003eI. Flow cytometry quantification of IFNγ and TNFα expression in TTCR-MA1-CD8/28 and TTCR-MA1-CD8/28mut group was measured at the end of the 7 days stimulation with irradiated ME275 incubation.\u003c/p\u003e","description":"","filename":"Binder17.png","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/55c279a5242c5538dd817c3c.png"},{"id":100765827,"identity":"6f9eefcb-2ee0-44bd-aaab-69473eea1ad0","added_by":"auto","created_at":"2026-01-21 08:56:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4294833,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Zhangetalmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1_covered_4d7bbbfd-f323-4d7e-9888-c280bb788791.pdf"},{"id":81028588,"identity":"8dbb3ac9-eef0-4c62-9360-48336c8ccfae","added_by":"auto","created_at":"2025-04-21 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10:56:50","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":641007,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data 5\u003c/p\u003e","description":"","filename":"ExtendedDataFig.5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/02585256d6ca8bab0357801f.pdf"},{"id":81029782,"identity":"c39766fe-fbb6-4d7a-b10a-ce504d8c01c3","added_by":"auto","created_at":"2025-04-21 11:12:50","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1462547,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data 6\u003c/p\u003e","description":"","filename":"ExtendedDataFig.6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/05de9d09996acec96c9afacb.pdf"},{"id":81028223,"identity":"8a0d7636-8c3a-4b5b-a0b4-da8af9d82113","added_by":"auto","created_at":"2025-04-21 10:56:50","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":714008,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data 7\u003c/p\u003e","description":"","filename":"ExtendedDataFig.7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/f0ec094a7771c73056ab486c.pdf"},{"id":81028225,"identity":"0dd01e39-790b-4721-8a42-fb3592b8f1b1","added_by":"auto","created_at":"2025-04-21 10:56:50","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":730393,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data 8\u003c/p\u003e","description":"","filename":"ExtendedDataFig.8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/91e43f69f0bc6ce19963dae8.pdf"},{"id":81028217,"identity":"bfcc6057-5989-48b5-99fc-38af1524852f","added_by":"auto","created_at":"2025-04-21 10:56:50","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":849175,"visible":true,"origin":"","legend":"Extended Data 9","description":"","filename":"ExtendedDataFig9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/36d1a205d4d5cf0213ebf974.pdf"},{"id":81028597,"identity":"51fd745d-a3f4-48b2-ad35-5ee3af5da8fd","added_by":"auto","created_at":"2025-04-21 11:04:50","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":219891,"visible":true,"origin":"","legend":"Table 1-8","description":"","filename":"SupplementaryTables18.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5939098/v1/04e38fd23b1a3142082e155c.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nA.G.C. has received support from Elevate Bio, Juno Therapeutics, Lonza, and Affini-T. P.D.G. is on the Scientific Advisory Board of Celsius, Earli, Elpiscience, Immunoscape, Rapt, and Nextech, was a scientific founder of Juno Therapeutics, and receives research support from Lonza. A.G.C., T.M.S., and P.D.G. are co-founders of, have equity in, and receive research support from Affini-T. A.G.C, T.M.S. and M.M are listed as inventors on patents related to MA1 TCR and CD8/28 constructs.","formattedTitle":"A CD8αβ co-receptor modified to contain an intracellular CD28 signaling tail enhances TCR-engineered T cell function independent of solid-tumor-associated co-stimulatory ligands","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5939098/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5939098/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdoptive therapies using T cells genetically modified with T cell receptors (TCR)s have shown limited efficacy in the solid tumor setting. Although functional CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells transduced with a TCR specific for HLA-A2-restricted melanoma-associated antigen A1 (MAGE-A1, T\u003csub\u003eTCR\u0026minus;MA1\u0026minus;CD8αβ\u003c/sub\u003e) could be detected post-transfer and were safe in one patient who subsequently progressed, T\u003csub\u003eTCR\u0026minus;MA1\u0026minus;CD8αβ\u003c/sub\u003e were insufficient to sustain antitumor activity in \u0026ldquo;stress\u0026rdquo; mouse tumor models. Leveraging the obligate co-expression of CD8αβ required for engagement of CD4\u003csup\u003e+\u003c/sup\u003e T cells expressing the TCR, we screened positive co-stimulatory signals tethered to the intracellular tail of CD8β and identified that CD28 reduced exhaustion, enhanced tumor infiltration and improved murine tumor control. Further modifications of the CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models. Thus, integrating co-stimulatory signals downstream of the TCR signaling complex can enhance TCR-engineered T cell function, independent of tumor-associated co-stimulatory ligand expression.\u003c/p\u003e","manuscriptTitle":"A CD8αβ co-receptor modified to contain an intracellular CD28 signaling tail enhances TCR-engineered T cell function independent of solid-tumor-associated co-stimulatory ligands","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 10:56:45","doi":"10.21203/rs.3.rs-5939098/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"53aa0308-ecf8-48a5-be5b-a2d6be361a40","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44022277,"name":"Biological sciences/Immunology/Immunotherapy"},{"id":44022278,"name":"Health sciences/Diseases/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":44022279,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":44022280,"name":"Biological sciences/Immunology/Translational immunology"}],"tags":[],"updatedAt":"2026-01-21T08:37:35+00:00","versionOfRecord":{"articleIdentity":"rs-5939098","link":"https://doi.org/10.1038/s41467-025-67446-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-01-03 05:00:00","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-04-21 10:56:45","video":"","vorDoi":"10.1038/s41467-025-67446-5","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67446-5","workflowStages":[]},"version":"v1","identity":"rs-5939098","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5939098","identity":"rs-5939098","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}