Introductory paragraph The adoptive transfer of anti-CD19 chimeric antigen receptor (CAR)-engineered T cells has shown impressive clinical responses in patients with refractory B-cell malignancies1–7. However, the therapeutic effects of CAR-T cells targeting other malignancies have not yet resulted in significant clinical benefit8–11. Although inefficient tumor trafficking and various immunosuppressive mechanisms can impede CAR-T cell effector responses, the signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals, including T cell receptor (TCR) engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3)12. However, CAR gene constructs currently being tested in the clinic contain a CD3z (TCR signaling) domain and a costimulatory domain(s) but not a domain transmitting signal 313–18. Here, we have developed a novel CAR construct capable of inducing cytokine signaling upon antigen stimulation. This new generation CD19 CAR encodes a truncated cytoplasmic domain of IL-2Rβ and a STAT3-binding YXXQ motif together with CD3z and CD28 domains (28-ΔIL2RB-z (YXXQ)). The 28-ΔIL2RB-z (YXXQ) CAR-T cells showed antigen-dependent JAK-STAT3/5 pathway activation, which promoted their proliferation and prevented terminal differentiation in vitro. The 28-ΔIL2RB-z (YXXQ) CAR-T cells demonstrated superior in vivo persistence and antitumor effects in both liquid and solid tumor models compared with CAR-T cells with a CD28 or 4-1BB domain alone. Taken together, these results suggest that our new generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicities in the clinic. Clinical translation of this novel CAR is warranted.

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Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T cell grafts from third-party donors will enable widespread use of this modality if allogeneic T cell responses are effectively controlled. Here, we generated HLA class I, class II, and T cell receptor triple knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA deficient T cells were targeted by natural killer cells, they persisted better than HLA sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice.When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMC compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease, but retained antitumor responses. These results demonstrated the benefit of HLA class I, class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy. SynopsisAntitumor T cell grafts concurrently ablated of HLA class I, class II, and TCR molecules evade allogeneic T cell responses and can be used as a universal T cell source for adoptive cancer immunotherapy.

T cell receptor (TCR)α and β chains cooperatively recognize peptide-MHC (pMHC) complexes. It has been shown that a ‘chain-centric’ TCR hemichain can, by itself, dictate MHC-restricted antigen specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α chain-centric HLA-A*02:01(A2)/MART127–35 TCRα, clone SIG35α, into A2-matched and unmatched post-thymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127–35. While the generated A2/MART127–35-specific T cells used various TRBV genes, TRBV27 predominated with >102 highly diverse and unique clonotypic CDR3β sequences. T cells individually reconstituted with various A2/MART127–35 TRBV27 TCRβ genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127–35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-antigens. Our results indicate that, although a chain-centric TCR hemichain determines antigen specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising antigen specificity. TCR chain centricity can be exploited to generate a thymically unselected antigen-specific T cell repertoire, which can be used to isolate high avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery due to tolerance.

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Classical antigen processing leads to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. Here we show that, unlike other class II molecules, prevalent HLA-DP molecules with β-chains encoding Gly84 (DP84Gly) constitutively present endogenous peptides. DP84Gly does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region, nor does it present CLIP. However, Ii does facilitate the transport of DP84Gly from the endoplasmic reticulum (ER) to the endosomal/lysosomal pathway by transiently binding DP84Gly via a non-CLIP region(s) in a pH-sensitive manner. Accordingly, like class I, DP84Gly constitutively presents endogenous peptides processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP). Therefore, DP84Gly, found only in common chimpanzees and humans, uniquely uses both class I and II antigen-processing pathways to present peptides derived from intracellular and extracellular sources.

Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A Ã 24:02/Wilms tumor 1 235-243 (A24/WT1 235 ) and cross-reactive with B Ã 57:01 (B57). The TAK1b, but not the TAK1a, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRa counterchains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1b-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1 235 and/or to B57. T cells reconstituted with these TCRa genes along with TAK1b possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/ peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs.