Rapid enrichment of progenitor exhausted neoantigen-specific CD8 T cells from peripheral blood

Abstract

Neoantigen-reactive peripheral blood lymphocytes (NeoPBL) are tumor-specific T cells found at ultra-low frequencies in the blood. Unlike tumor-infiltrating lymphocytes (TIL), NeoPBL exist in a favorable less dysfunctional phenotypic state in vivo, but their rarity has precluded their effective use as cell therapy. Leveraging a priori knowledge of bona fide neoantigens, we combined high-intensity neoantigen stimulation with bead extraction of neoantigen peptide-pulsed target cells to enable the enrichment of NeoPBL to frequencies comparable to ex vivo cultured TIL over a 28-day period. Throughout this process, NeoPBL demonstrate specific reactivity against autologous tumor organoids and maintain memory-like features, including elevated expression of CD28 and TCF7. We additionally demonstrate that NeoPBL reactivity is polyclonal, encompassing multiple clonotypes that are detectable within in vivo TIL populations, underscoring physiological specificity for the targeted neoantigens. This streamlined process yields clinically relevant cell doses and enables identification and expansion of blood-derived neoantigen-specific TCRs. By potentially avoiding additional surgical risks and protracted delays of TIL and individualized TCR-engineered methods, the NeoPBL platform may have clinical and practical advantages. Ultimately, NeoPBL combines intrinsic cell fitness, minimal invasiveness and rapidity to potentially facilitate personalized adoptive cell therapy for cancer.

Figure 1.. NeoExpand-REP enhances expansion, preserves diversity, and improves phenotype and function of TCR-T cells.

(A) TCR-T cells targeting TP53 R175H (HLA-A*02:01-restricted) were generated from patient PBL were plated alone (control) or at a 1:5 E:T ratio with neoantigen peptide-pulsed PBL (neoantigen stim). Flow cytometry plots show 4–1BB expression on CD8⁺mTCRβ⁺ gated cells after 16 hours, after which cells were transferred to REP conditions (with OKT3, allogeneic feeders and IL2). (B) Fold expansion of all lymphocytes (CD4⁺, CD8⁺, transduced or untransduced) from 4196 TCR-T cultures after 14 days in rapid expansion protocol (REP) or NeoExpand-REP. (C) Flow cytometry data showing CD8 and mTCRβ expression on lymphocytes and (D) fold expansion of CD8⁺mTCRβ⁺ T cells during REP or NeoExpand-REP. (E) Phenotypic characterization of CD8⁺mTCRβ⁺ T cells by flow cytometry showing expression of CD39 and CD69. (F) Functional cytokine responses (TNF-α and IFN-γ) in sorted CD8⁺mTCRβ⁺ T cells following 16-hour co-culture with HLA-A*02:01⁺ TYK-nu tumor cells; bar graphs show technical replicates. (G) Tumor cell killing by sorted CD8⁺mTCRβ⁺ T cells (equal cell numbers) measured by luminescence in an Incucyte assay. (H) Tumor growth in NSG mice bearing established TYK-nu xenografts treated with either 4.4×10⁵ or 1.3×10⁶ TCR-T cells expanded via REP or NeoExpand-REP. Mice received one intravenous and two intraperitoneal doses of IL-2. Tumors were measured weekly by a blinded investigator. (I) Schematic of scRNA-seq workflow: TCR-T cells (from patients 4141 and 4196, both targeting TP53 R175H, with low and high avidity respectively) were expanded for 14 days via REP or NeoExpand-REP, sorted for CD8⁺mTCRβ⁺ expression, and captured for sequencing. (J) TCR diversity shown by D50 index—the number of clonotypes contributing 50% of the repertoire. Values for 4141 and 4196 TCR-T cells before and after expansion are shown. (K) Volcano plot showing differentially expressed genes (DEGs) in the top 5 expanded clonotypes present in both REP and NeoExpand-REP using a combined data set with both 4141 and 4196 TCR-T cells.

Figure 2.. NeoSelect rapidly expands rare NeoPBL to clinically relevant frequencies.

(A) Schematic comparing a standard REP, NeoExpand-REP and NeoSelect. (B) Representative flow cytometry showing the vμ7.1⁺ fraction of CD8⁺ gated T cells (top panels, gated CD8⁺) and cytokine responses (bottom panels, gated on CD8⁺vβ7.1⁺ T cells) of TP53-targeted NeoSelect cultures on day 14 to peptide rechallenge. (C) 4–1BB and OX40 expression on day 28 CD8⁺ T cells following 16-hour rechallenge with neoantigen peptides or tumor organoids. Gated on vβ7.1⁺ (TP53) or vβ5.1⁺ (PSMB9). (D–E) Peptide titration curves using mutant and wild-type peptides for TP53 (D) and PSMB9 (E); gating as indicated. (F) TP53/B*07:02 tetramer staining and vβ7.1 expression in day 28 cultures, gated on CD8⁺ T cells. (G) CD39 and CD69 expression (left) or CD27 and CD28 expression (right) in tetramer^+/− β7.1^+/− quadrants from TP53-targeted day 28 NeoSelect cultures. (H) CD39/CD69 and CD27/CD28 expression in CD8⁺ T cells gated on vβ5.1⁺ (predominantly PSMB9-reactive) versus non-reactive vβ families. (I) Schema for manufacturing a NeoPBL treatment product. This process is modeled based on TIL workflows, which require 25–30 days of ex vivo culture and are later expanded for 14 days to create an infusion product. (J) Expression of CD8 and vμ7.1, and cytokine responses (TNF-α, IFN-γ) following neoantigen rechallenge, of day 42 NeoPBL infusion product.

Figure 3.. Reactivity-seq defines the identity, diversity, and transcriptional state of NeoPBL.

(A) Single-cell transcriptomic data from patient 4556 TIL, showing UMAP clustering; TIL Cluster 9 exhibits the strongest NeoTCR8 signature score and was used to predict the original neoTCRs. (B) Reactivity-seq analysis of day 28 NeoSelect cultures from patient 4556, sorted on CD8⁺ T cells with or without peptide stimulation. Characteristic genes of the reactivity cluster (PBL Cluster 9) are shown in the middle. UMAPs of day 28 NeoSelect cultures combined and separated by hashed sample identifiers are shown on the right. (C) Projection of reactive (confirmed and high-probability) clonotypes onto the 4556 TIL UMAP reveals 9 of 13 NeoPBL clonotypes were present in TIL. The ‘neoTCR8’ cluster is circled in red. Also projected are the bystander (abundant in both NeoSelect cultures, no compelling evidence of reactivity) and indeterminant clonotypes (enriched in one culture, no compelling evidence of reactivity). (D) Reactivity cluster (PBL C9) clonotypes highlighted in resting-state cells from day 28 NeoSelect cultures. (E) Single-cell UMAP of day 28 NeoPBL cultures with annotated clusters. (F) Differential expression analysis comparing upregulated genes in clusters 4 and 5, which were enriched for NeoPBL clonotypes.

Figure 4.. NeoSelect uncovers a broad and functionally poised repertoire of tumor-reactive clonotypes.

(A) Pie chart showing the distribution of clonotypes in TP53-targeted NeoSelect cultures categorized as confirmed (cloned and tested), high-probability (based on > 100-fold relative enrichment compared to PSMB9 cultures and evidence of reactivity from 4–1BB sorting or reactivity-seq), indeterminant (> 100-fold relative enrichment but no other supporting evidence of reactivity), or bystander (present in both NeoSelect cultures at similar frequencies). (B) Pie chart showing clonotype classification in PSMB9-targeted NeoSelect cultures using the same criteria as in (A). (C) Single-cell transcriptomic cluster assignment for each TCR identified in TP53-targeted (first column) or PSMB9-targeted (second column) NeoSelect cultures on day 28. Graphs show reactive (red, first row), indeterminant (grey, second row), bystander (blue, third row) and singleton/doublets (fourth row). Each dot shows the percentage of a given clonotype present in the indicated transcriptomic cluster shown in Fig. 3E. (D) Cluster identity and I2 cell type scores for TCR13 and TCR17 when found as reactive NeoPBL (in PSMB9-targeted cultures) or as bystanders (in TP53-targeted cultures). (E) Cluster identity and I2 scores for TCR1 when found as reactive NeoPBL (in TP53-targeted cultures) or as a bystander (in PSMB9-targeted cultures).