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

Abstract

Adoptive transfer of T cells engineered with tumor-specific T cell receptors (TCRs) has shown limited efficacy in solid tumors, hindered by insufficient persistence, tumor trafficking, and dependence on tumor-associated co-stimulatory ligands. In a phase I trial (NCT04639245) for patients with metastatic MAGE-A1-expressing tumors and adequate organ function; one participant received treatment, which was well-tolerated. In this case and NSG murine models, infusion of CD4/CD8 T cells co-expressing a class-I MAGE-A1-specific TCR and CD8αβ, failed to control tumor progression. To enhance function downstream of TCR signaling, here we investigate the adaptability of TCR components to synthetic modification. Leveraging the obligate co-expression of CD8αβ required for class-I TCR function in CD4 T cells, we identify CD8β as a tractable site for engineering without loss of function. In vitro screening demonstrates incorporation of the CD28 intracellular tail, yielding a CD8/CD28 chimeric co-receptor, most effectively enhances cytokine production, T cell persistence, and tumor control in immunodeficient murine models while preserving stem-like transcriptional features compared to native CD8β. Further rational modification of the CD28 binding motifs improves tumor control in vivo with increased intratumoral accumulation and reduced exhaustion. This benefit also extends to PRAME and WT1-specific TCRs in vitro supporting generalizability.

Fig. 1. Functional CD4 T_TCR-MA1 cells enhance MAGE A1-specific CD8⁺ T_TCR-MA1 cells in vitro and in vivo.

A Schematic of the multi-cistronic vector design used in pre-clinical studies. B Dose response curves (left panels) of IFNγ, TNFα, and IL-2 expression in response to decreasing peptide concentrations by CD8 T_{TCR-MA1-CD8αβ} (red), CD4 T_TCR-MA1 (blue, open circles) and CD4 T_{TCR-MA1-CD8αβ} (blue, full circles). Mean EC₅₀ (right panels) of IFNγ, TNFα, and IL-2 cytokine expression for the same previous 3 cell populations. C Growth kinetics (left panel) for the ME275 cell line in a live tumor visualization assay (Incucyte S3) in the absence (gray line) or presence of CD4 T_{TCR-MA1-CD8αβ} (blue line), CD8 T_{TCR-MA1-CD8αβ} (red line), or CD4 + CD8 T_{TCR-MA1-CD8αβ} (purple line). The E:T ratio was 2:1, and arrows indicate the addition of tumor cells to the culture. Final tumor integrated intensity (right panel) for the same experiment at 144 and 192 h. The mean ± standard deviation (SD) of triplicate wells is shown. p value determined by a one-way ANOVA with Tukey’s multiple comparison test. (n = 3 wells/group). D Counts of survived T cells were measured at 144 h post tumor simulation from Incucyte S3 live tumor assay (C). Change in absolute numbers of CD4 T_{TCR-MA1-CD8αβ} (left panel) and CD8 T_{TCR-MA1-CD8αβ} (right panel) in either alone (CD4 - blue bars, CD8 - red bars) or combined (purple bars) conditions compared to seeded cells. The mean ± SD from triplicated wells is shown (n = 3 wells/group). p value determined by unpaired two-tailed t-test. E Schematic (left) and representative growth kinetics (right) of 1 × 10⁶ ME275 tumors engrafted subcutaneously into NSG mice with subsequent transfer of 1 × 10⁷ equal numbers of total irrelevant T cells (T_Irr.-CD8αβ), CD4 T_{TCR-MA1-CD8αβ} alone, CD8 T_{TCR-MA1-CD8αβ} alone, or a 1:1 ratio of combined CD4 and CD8 T_{TCR-MA1-CD8αβ}, 22 days after engraftment. The tumors were allowed to reach ~60mm³ in size before T cell infusion (n = 3 mice/group, 2 tumors/mouse). F Schematic (left) and representative growth kinetics (right) of 1 × 10⁵ A375F tumors engrafted into NSG mice with subsequent transfer of 1 × 10⁷ equal numbers of total T cells (T_Irr.-CD8αβ, CD4 T_{TCR-MA1-CD8αβ} alone, CD8 T_{TCR-MA1-CD8αβ} alone, or a 1:1 ratio of combined CD4 and CD8 T_{TCR-MA1-CD8αβ}) 12 days after engraftment. The tumors were allowed to reach ~60mm³ in size before T cell infusion (n = 3–4 mice/group, 2 tumors/mouse). G A375F tumor volume from (F) assessed at sacrifice 8 days after T cell infusions for each indicated condition. p value determined by one-way ANOVA with Tukey’s multiple comparison test. (n = 4 mice for T_Irr.-CD8αβ group and n = 3 mice for CD4 T_{TCR-MA1-CD8αβ} alone, CD8 T_{TCR-MA1-CD8αβ} alone, or a 1:1 ratio of combined CD4 and CD8 T_{TCR-MA1-CD8αβ} groups, 2 tumors/mouse averaged). All data are presented as mean ± SEM. Panels E, F created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f.

Fig. 2. Infusion of functional CD4 and CD8 T_{TCR-MA1-CD8αβ} are not associated with excessive toxicity in one patient and require enhancement to achieve tumor burden reduction.

A Contour flow plot of the patient’s CD4 T_{TCR-MA1-CD8αβ} (top) and CD8 T_{TCR-MA1-CD8αβ} (bottom) infusion product binding to CD28 (x-axis), CD62L (y-axis left panels) and CD127 (x-axis right panels). B IFNγ (x-axis) and TNFα (y axis) expression by the patient’s CD4 T_{TCR-MA1-CD8αβ} (top) and CD8 T_{TCR-MA1-CD8αβ} (bottom) infusion product after an 18-h exposure to 10 μM (left panels), 1 μM (middle panels), and no peptide stimulation (right panels). C Circulating T_{TCR-MA1-CD8αβ} in PBMCs identified with p/HLA multimer at indicated timepoints (x-axis) for the infused patient. D Schematic (left) and growth kinetics (right) of 1 ×10⁵ A375F tumors engrafted into NSG mice with subsequent transfer of 5 × 10⁶ total T cells (T_Irr.-CD8αβ, CD4⁺ T_{TCR-MA1-CD8αβ} alone, CD8⁺ T_{TCR-MA1-CD8αβ} alone, or a 1:1 ratio of combined CD4 and CD8 T_{TCR-MA1-CD8αβ}), 12 days after engraftment. (n = 3 mice/group, 2 tumors/mouse, data shown are representative of three independent experiments.) Image created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f. E A375F tumor volume from (D) assessed at sacrifice 8 days after T cell infusions for each indicated condition. Data shown are representative of three independent experiments. p value determined by Kruskal-Wallis with Dunn’s multiple comparison test. (n = 6 mice/group, 2 tumors/mouse averaged, from 2 separate experiments). All data are presented as mean ± SEM. F Quantification of Tim3⁺/PD-1⁺, Tim3⁺/CD39⁺ and 2B4⁺ binding across the experimental groups from (D) in CD4⁺ (top panel) and CD8+ (bottom panel) T_{TCR-MA1-CD8αβ} for the indicated conditions. Single cell suspensions were obtained at sacrifice, 8 days after T cell infusion. Graph represents mean ± SD. p values determined by unpaired two-tailed t-test. (n = 3 mice, 6 tumors/group, 2 tumors/mouse). G Quantification of IFNγ⁺ /TNFα⁺ binding after 1 μM cognate peptide stimulation for 18 h ex vivo across the same experimental groups from (D) in CD4 (left) and CD8 (right) T_{TCR-MA1-CD8αβ} for the indicated conditions. Single cell suspensions were obtained at sacrifice, 6 days after T cell infusion. Graph represents mean ± SD. p values determined by unpaired two-tailed t-test. (n = 3 mice/group, 2 tumors/mouse). H, I Heatmap showing the expression of curated co-stimulatory (green box) and inhibitory (red box) genes for lung cancer (H) and melanoma (I) patients across annotated cell subsets. Higher expression values are depicted in red, while lower expression values are shown in blue. J, K UMAP plot showing the two-dimensional distribution of a costimulatory gene score (CD80, CD86, CD40, TNFRSF4, TNFS9 and ICOSLG) in scRNAseq samples from lung cancer (J) and melanoma (K) patients. ICOSLG expression was present in the melanoma dataset but not in the lung cancer dataset. Dark red indicates higher expression, while light gray marks regions with low score expression.

Fig. 3. Generation of chimeric CD8β chains functionally integrated into the immune synapse.

A Schematic of the multi-cistronic vector designs used to integrate CCRs downstream of CD8β. Image created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f. B Histogram of CD8α (left), CD8β (middle), and MAGE-A1 p/HLA multimer (right) binding of CD4 transduced to express T_TCR-MA1 with the indicated CCRs after transduction for equal transduction copy numbers and post-REP (rapid expansion protocol) p/HLA multimer binding. C (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 CD8αα (negative control), measured 18h post peptide stimulation. (right) Mean half maximal effective concentration (EC₅₀) of IFNγ expression for CD4 T cells transduced with the same constructs. D Fold change of IFNγ levels in T_TCR-MA1 cells transduced to express the different CCR constructs and incubated for 18 h with 1 µM cognate peptide alone (left panel) or incubated with 1 µM cognate peptide following 1 week of priming by co-culture with irradiated ME275 (right panel). E (left) Growth kinetics of the ME275 cell line in the absence (black lines) or presence of CD4 with the indicated CCR constructs from a live tumor visualization assay (Incucyte S3). An E:T ratio of 10:1 was used, and tumors were added to the culture every 72 h. (middle) Final tumor integrated intensity for the same experiment 16 days after start (384 h). (right) Final lymphocyte counts were obtained after 16 days. p-values determined by one-way ANOVA with Tukey’s multiple comparison test. (n = 3 wells/group). All data are presented as mean ± (SD).

Fig. 4. CCR enhances tumor control and T cell survival in vivo.

A Representative of tumor burden measurements (left) for NSG mice engrafted with 1 × 10⁵ cells A375F and subsequently infused with a total of 5 × 10⁶ CD4 and CD8 T_{TCR-MA1-CD8αβ} CCR cells (T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28)} at a ratio of 1:1 12 days post engraftment. Final tumor volume of tumor-bearing mice (right panel) between days 6–8 after infusion (n=9 mice/group, 2 tumors/mouse averaged, from 3 separate experiments). p value determined by Kruskal-Wallis with Dunn’s multiple comparison test. All data are presented as mean ± SEM. B Representative IHC images of A375F tumors resected from NSG mice (same experiment design as A) showing DAPI (blue), CD4 (red), and CD8 (green) T cells of 4 groups: No T cells, irrelevant TCR (T_{TCR-Irr.-CD8/CD28}), CD8αβ (T_{TCR-MA1-CD8αβ}), and CD8/CD28 CCR (T_{TCR-MA1-CD8/CD28}). 2X and 10X magnification. Scale bars represent 100 µm and 500 µm. The white box represents the area enlarged in the 500 µm images underneath the top images. C–F Tumor-infiltrating T cells to tumor cells ratio (C) from resected A375F tumors from NSG mice infused with T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, or T_{TCR-MA1-CD8/CD28}. The indicated p-value was determined by a Mann–Whitney unpaired two-tailed t-test. CD4 TCR-T cells and tumor cells ratio (D), p value determined by Welch’s unpaired two-tailed t-test. CD8T cells and tumor cells ratio (E), p value determined by Mann-Whitney unpaired two-tailed t-test. CD4 and CD8 T cell ratio (F), p value determined by Welch’s unpaired two-tailed t-test. Analysis performed using HALO Link software on IHC slides. (n = 3 mice, 6 tumors/group, 2 tumors/mouse). All data are presented as mean ± SEM. G Bar graph showing PD-1⁺/Tim3⁺, CD39⁺/Tim3⁺, and PD-1⁺/TIGIT⁺ frequency of CD4 and CD8 TILs between T_{TCR-MA1-CD8αβ} and T_{TCR-MA1-CD8/CD28}. Single cell suspensions were obtained at sacrifice, 8 days after T cell infusion from (same experiment design as A). p value determined by unpaired two-tailed t-test. (n = 3 mice, 6 tumors/group, 2 tumors/mouse). The graph represents mean ± SD. H Quantification of IFNγ⁺/TNFα⁺ populations after 1 μM cognate peptide stimulation for 18 h ex vivo across the same experimental groups from (A) in CD4 (left panel) and CD8 (right panel) T_{TCR-MA1-CD8αβ} for the indicated conditions. Single cell suspensions were obtained at sacrifice, 6 days after T cell infusion. Graph represents mean ± SD. Unpaired two-tailed t-test. (n = 3 mice, 5-6 tumors/group, 2 tumors/mouse). I NSG mice were engrafted with 1 × 10⁵ cells A375F and subsequently infused with a total of 5 × 10⁶ CD4 and CD8 T_{TCR-MA1-CD8αβ} CCR cells (T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28}) at a ratio of 1:1 6 days post engraftment. Survival analysis was carried out when tumor burden reached 500 mm³ and assessed tumor progression occurring 44 days vs. 23 days vs. 16 days post T cell infusion, respectively; the Kaplan-Meier test for analysis of survival was applied. p values determined by a log-rank test. (n = 6 mice/group). Panels A & I were created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f.

Fig. 5. The chimeric CD8/CD28 co-receptor TCR-T cells have a reduced exhaustion(-like) phenotype in vivo.

A 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. B 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). C UMAP plot of T cells colored by density and split by group (from left to right: CD8-alone T_{TCR-MA1-CD8αβ} group, CD8 in T_{TCR-MA1-CD8αβ} and CD8 in T_{TCR-MA1-CD8/CD28} 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. D Violin plots displaying the expression levels (y-axis) of stem-like (TCF7, SELL, IL7R, LTB, LEF1, NELL2), proliferation (MKI67, TOP2A, PCNA), and cytotoxicity (KLRG1, KLRD1, NKG7, GZMB, GNLY, PRF1, GZMA, GZMM, CSTA, CSTW) markers across CD8 T-cell transcriptional states between CD8-alone and CD8 in T_{TCR-MA1-CD8αβ} combo (x-axis). Statistical significance was assessed using a two-sided Wilcoxon rank sum test. Boxplots within violins show the median, interquartile range (box; 25th–75th percentiles), and whiskers extending to 1.5× IQR. E Tumor burden measurements for NSG mice engrafted with 1 × 10⁵ cells A375F and subsequently infused with a total of 5 × 10⁶ T cells of CD8-only T_{TCR-MA1-CD8αβ} or CD4 and CD8 (1:1 ratio combo) T_{TCR-MA1-CD8αβ} 12 days post engraftment. (n = 3 mice, 2 tumors/group averaged). p value determined by unpaired two-tailed t-test. Graph represents mean ± SEM. F Violin plots displaying the expression levels (y-axis) of stem-like (TCF7, SELL, IL7R, LTB, LEF1, NELL2), proliferation (MKI67, TOP2A, PCNA), and cytotoxicity (KLRG1, KLRD1, NKG7, GZMB, GNLY, PRF1, GZMA, GZMM, CSTA, CSTW) markers across CD8 T-cell transcriptional states between T_{TCR-MA1-CD8αβ} and T_{TCR-MA1-CD8/28} (x-axis). Statistical significance was assessed using a two-sided Wilcoxon rank sum test. Boxplots within violins show the median, interquartile range (box; 25th–75th percentiles), and whiskers extending to 1.5× IQR. G Tumor burden measurements for NSG mice engrafted with 1 × 10⁵ cells A375F and subsequently infused with total 5 × 10⁶ T cells of CD4 and CD8 (1:1 ratio combo) T_{TCR-MA1-CD8αβ}, and CD4 and CD8 (1:1 ratio combo) T_{TCR-MA1-CD8/28} 12 days post engraftment. (n = 3 mice, 2 tumors/group averaged). p value determined by unpaired two-tailed t-test. The graph represents mean ± SEM.

Fig. 6. CD8-CD28 CCR tail mutations enhance anti-tumor activity in MAGE tumor models.

A Schematic of the four motif mutations on the CD28 intracellular domain. B Dose response curve showing IFNγ and TNFα levels in CD4 T_TCR-MA1 CCR cells incubated with decreasing concentrations of cognate peptide overnight. C NSG mice were engrafted with 1 × 10⁵ A375F and subsequently infused with total 5 × 10⁶ CD4 and CD8 T cells (T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut}) at a ratio of 1:1 on day 12 post engraftment. Graph represents mean ± SEM. (n = 3 mice/group, 2 tumors/mouse, data shown are representative of two independent experiments). D NSG mice were engrafted with 1 × 10⁵ A375F and subsequently infused with total 2.5 × 10⁶ CD4 and CD8 T cells (T_{TCR-Irr.-CD8/28}, T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut}) at a ratio of 1:1 on day 12 post engraftment. (n = 3mice/group, 2 tumors/mouse, data shown are representative of two independent experiments.) The graph represents mean ± SEM. E A375F tumor volume assessed at sacrifice 9 days after T cell infusions for each indicated condition same as (D). p value determined by one-way ANOVA with Tukey’s multiple comparison test. The graph represents mean ± SEM. (n = 4 mice for T_{TCR-Irr.-CD8/28} group and n = 5 mice for T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut} groups, data shown are representative of two independent experiments). F Growth kinetics of the SK-MEL-37 cell line (left) in the absence (black lines) or presence of CD4 T cells with the indicated CCR constructs (T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut}) at a ratio of E:T 5:1 in a live tumor visualization assay (Incucyte S3). Final lymphocyte counts (right) were obtained after 84 h. The graph represents mean ± SD. p-values determined by one-way ANOVA with Tukey’s multiple comparison test. (n = 3 wells/group). G Non-humanized MISTRG mice were engrafted with 1 × 10⁶ SK-MEL-37 (left) and subsequently infused with a total of 2 × 10⁷ CD4 and CD8 T cells (T_{TCR-Irr.-CD8/28}, T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut}) at a ratio of 1:1 on day 21 post engraftment (n = 4 mice, 2 tumors per mouse). SK-MEL-37 tumor volume (right) assessed at 62 days after T cell infusions for each indicated condition. The graph represents mean ± SEM. p-value determined by a two-tailed unpaired t-test. (n = 4 mice/group, 2 tumors/mouse averaged). Panels C,D, and G were created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f.

Fig. 7. The gene expression profile of T_{TCR-MA1CD8/CD28mut} identified a balance between functional, exhaustion and persistence.

A GSEA pathway analysis of cytokine pathways (Biocarta), cytokine activity (GOMF), cytokine and inflammatory response (WP), and the Th1/Th2 pathway (Biocarta) of CD4 T_{TCR-MA1-CD8/28}, T_{TCR-MA1-CD8/28mut} normalized by T_{TCR-MA1-CD8αβ}. T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut} were stimulated with 1 µM cognate peptide for 4 h and further analyzed by bulk RNA sequencing. p values were determined by two-sided Mann–Whitney U-test. B Schematic of experimental layout showing CD4 T_{TCR-MA1-CD8/28} or T_{TCR-MA1-CD8/28mut} incubated with irradiated ME275 for 21days, with tumor rechallenge every 7 days. Cells were stimulated with 1 µM cognate peptide for 18 h or without stimulation and further analyzed by flow cytometry and bulk RNA-seq downstream analysis. Image created in BioRender. Tang, A. (2026) https://BioRender.com/f64l203. C 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. D Heatmap showing the expression levels of differentially expressed genes across constructs (T_{TCR-MA1-CD8/CD28}, T_{TCR-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. E Boxplots showing the z-scores for the expression of manually curated gene signatures of proliferation in unstimulated and stimulated conditions. The z-scores were calculated from the averaged normalized counts of significant genes (p_adj <0.05, log₂ fold-change > 0.5). Each box represents the distribution of z-scores for a specific gene signature in the unstimulated and stimulated groups for T_{TCR-MA1-CD8/CD28} and T_{TCR-MA1-CD8/CD28mut}. Boxplots show the IQR; lines denote medians. The bounds of the box represent the 25th and 75th percentiles; whiskers span 1.5x IQR. F 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 <0.05, log2 fold-change > 0.5). Each box represents the distribution of z-scores for a specific gene signature in the unstimulated and stimulated groups for T_{TCR-MA1-CD8/CD28} and T_{TCR-MA1-CD8/CD28mut}. Boxplots show the IQR; lines denote medians. The bounds of the box represent the 25th and 75th percentiles; whiskers span 1.5x IQR. G 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. Enrichment p-values were calculated by two-sided permutation testing. H Flow cytometry quantification of Tim3⁺ PD-1⁺ double positive populations from T_{TCR-MA1-CD8/28} and T_{TCR-MA1-CD8/28mut} groups was measured at the end of the 21 days stimulation with irradiated ME275 incubation. The graph represents mean ± SD. p-value determined by a two-tailed unpaired t-test. (n = 3 wells/group). I Flow cytometry quantification of IFNγ⁺ and TNFα⁺ expression in T_{TCR-MA1-CD8/28} and T_{TCR-MA1-CD8/28mut} group was measured at the end of the 7 days stimulation with irradiated ME275 incubation. The graph represents mean ± SD. p value determined by one-way ANOVA with Tukey’s multiple comparison test. (n = 3 wells/group).