Introduction: Anaplastic Lymphoma Kinase (ALK)-positive Anaplastic Large Cell Lymphoma (ALCL) is a subtype of T-cell lymphoma representing 10-15% of Non-Hodgkin Lymphoma in children and young adults. It is characterized by the presence of the oncogenic fusion protein nucleophosmin 1 (NPM1)-ALK. Treatment with standard chemotherapy regimens is highly effective, yet a significant number of patients relapse or experience treatment-related toxicities, resulting in a poor prognosis afterward. The ALK tyrosine kinase inhibitor (TKI) crizotinib is approved for use in children and young adults with relapsed or refractory disease. Despite improved patient survival, ALK TKIs do not achieve complete cure, with development of resistance or relapses in almost half of the patients, underscoring the need for more effective therapeutic strategies.

In this work, we aimed to identify T cell receptor (TCR) clonotypes targeting a human ALK immunogenic peptide presented by HLA-B*07:02 (B7) that we previously identified in ALK-positive cell lines and a biopsy of a patient with Non-small cell lung cancer[Mota et al, Nat Cancer 2023, PMID: 37430060] and to develop TCR-T cell therapy for ALK-rearranged ALCL.

Methods: To identify ALK-specific TCRs, we immunized transgenic mice expressing the human HLA-B*07:02 with four subcutaneous doses of the human ALK peptide RPRPSQPSSL. Activated CD137(4-1BB)+ /CD8+ T cells were sorted and subjected to single-cell sequencing. The most expanded TCR clonotypes identified (VDJ count ≥4) were cloned into a retroviral vector that was used to transduce human CD3+ T cells to produce ALK TCR-T cells. TCRs were characterized by ALK-specific dextramer and by pulsing ALK-, B7+ cells with different concentrations of peptide to measure the activation of T cells by CD137 expression and INF-γ release. In vitro killing activity of the selected ALK TCR-T cells was assessed using ALK+, B7+ (Karpas299), ALK+, B7- (JB6 and TS), and ALK-, B7+ (MAC-2A) cell lines. JB6, TS, and MAC-2A were additionally transduced with either HLA-B*07:02 or NPM-ALK plasmids to generate double-positive cell lines. To test the combination with crizotinib, Karpas299 cells were treated with 50nM crizotinib and co-cultured with ALK-TCR T cells.

Results: We identified 353 TCR clonotypes from HLA-B*07:02 transgenic mice vaccinated with the RPRPSQPSSL peptide. Gene set enrichment analysis (GSEA) comparing expanded (frequency >4) versus non-expanded (frequency <4) TCR clonotypes revealed upregulation of pathways involved in adaptive immune response, including T cell activation and proliferation, IFN-γ signaling, and cytokine release (adjusted p < 0.05).

Among the ALK-specific TCRs identified, we selected the two TCRs with the highest avidity, namely ALK-TCR #1 and ALK-TCR #2, that showed ALK-dextramer binding >95%. Interestingly, ALK-TCR #2 was binding the ALK-dextramer also in CD4+ T cells, suggesting a co-receptor-independent pMHC recognition. Functional avidity of the two ALK TCR-T cells demonstrated an EC50 of ~0.3-0.5μM, in line with previously published data on non-affinity matured TCRs. Both clonotypes showed >95% killing only of the ALK+, B7+ Karpas299 cells in an E:T ratio of 1:1, while no killing was observed with ALK+, B7- or the ALK-, B7+ cells. When JB6 and TS cells were transduced with HLA-B*07:02, or MAC-2A was transduced with NPM-ALK, ALK-TCR-T cells showed >95% killing, confirming that both ALK TCR-T #1 and #2 recognize specifically the ALK peptide RPRPSQPSSL presented by the HLA-B*07:02 and engage a potent anti-tumor activity.

Finally, we evaluated the outcome of combining the ALK TKI crizotinib, with the ALK TCR-T cells. While 50nM crizotinib alone decreased cell viability by 40%, the combination of ALK TCR-T and crizotinib resulted in >95% killing with both ALK TCR-T #1 and #2 even at unfavorable E:T ratios (1:5 and 1:10), showing a synergy between crizotinib and ALK TCR-T cells.

Conclusions: We identified ALK-specific TCRs that recognize an ALK peptide displayed by HLA-B*07:02 and proved their specificity and potent in vitro anti-tumor activity. Ongoing studies are investigating the therapeutic potential of ALK TCR-T cells in vivo using relevant ALK-positive ALCL mouse models. Results on these in vivo studies will be reported at the meeting. These results lay the basis for a potent and specific immunotherapy for patients with ALK-positive ALCL.

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2025
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