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HLA-independent T cell receptors for targeting tumors with low antigen density

Abstract

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor–CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

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Fig. 1: TRAC locus editing to create an HLA-independent TCR (HIT receptor) directs TCR–CD3 complex-mediated antigen recognition.
Fig. 2: TRAC-HIT T cells exhibit increased sensitivity for tumor cells expressing low antigen levels relative to TRAC-CAR T cells.
Fig. 3: TRAC-HIT T cells exhibit increased degranulation and rapid killing activity upon exposure to low antigen levels.
Fig. 4: TRAC-HIT T cells outperform TRAC-CAR T cells in controlling low antigen tumor cells in vivo.

Data availability

All requests for raw and analyzed preclinical data and materials will be promptly reviewed by the corresponding authors (M.S. and J.M.S.) to determine if they are subject to intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a material transfer agreement (requested to Michel Sadelain). Sequences for the TRAC-HIT receptors have been submitted under patent no. WO2019157454A1 (19HIT). The TRAC-HIT sequences can be found in the Supplementary Information file.

Code availability

We have generated scripts for the automated analyses of the single-cell CTL assays as well as the actin, CAR, HIT and LAMP-1 signals in the confocal images. Request for these scripts will be promptly reviewed by the corresponding authors (M.S. and J.M.S.) to determine if they are subject to intellectual property or confidential obligations. Any script that can be shared will be released via a material transfer agreement (requested to Michel Sadelain).

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Acknowledgements

We thank Gertrude Gunset for logistical and technical assistance. We also thank the SKI Cell Therapy and Cell Engineering (CTCEF), Molecular Cytology, Flow Cytometry, Integrated Genomics Operation, Microchemistry and Proteomics, Antitumor Assessment and Animal Core Facilities for their expert assistance. This work was supported by the Lake Road Foundation, the Lymphoma and Leukemia Society, the Pasteur-Weizmann/Servier award, the Leopold Griffuel award and the NCI Cancer Center Support Grant no. P30 CA008748. SKI cores were in part supported by the Tow Foundation, Cycle for Survival, the Marie-Josée and Henry R. Kravis Center for Molecular Oncology and NCI grant P30 CA08748. A.D., M.S. and T.G. were supported by fellowships from the Canadian Institutes of Health Research, the Fogarty Foundation and the Alexander S. Onassis Public Benefit Foundation, respectively.

Author information

Authors and Affiliations

Authors

Contributions

J.M.-S. and J.E. designed the study, performed experiments, analyzed and interpreted data and wrote the manuscript. S.H. designed and performed experiments, and analyzed data. M.H., J.F., N.P., A.E.Z., Z.L., M.S., P.L.L., M. Saetersmoen, A.D. and M.M. performed experiments and analyzed data. A.I. performed statistical analysis. A.G.A., M.M.M., Z.Z., T.G., S.J.C.v.d.S. and F.T. performed experiments. M. Huse designed experiments. I.R., R.C.H. and C.H. designed experiments and interpreted data. M.S. designed the study, analyzed and interpreted data, and wrote the manuscript.

Corresponding authors

Correspondence to Jorge Mansilla-Soto or Michel Sadelain.

Ethics declarations

Competing interests

Memorial Sloan Kettering has submitted a patent application based in part on results presented in this manuscript (WO2019157454A1, J.M.-S., J.E. and M.S. are listed among the inventors). R.C.H. reports stock ownership in Merck. M.S. reports research funding from Juno Therapeutics, Fate Therapeutics, Takeda Pharmaceuticals and Atara Biotherapeutics, unrelated to the present research. M.S., I.R. and J.E. are scientific cofounders of Mnemo Therapeutics. M.S. serves on the scientific advisory board of St. Jude Children Research Hospital. All other authors declare no competing interests.

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Nature Medicine thanks Cliona Rooney and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 HIT receptor expression driven by the TCRα promoter rescues CD3 expression and directs lysis of CD19 + target cells.

a. Representative flow cytometry analysis showing HIT, CAR, and CD3 expression. TRAC-HIT and TRAC-CAR T cells were generated as in Fig. 1b. CD3 surface expression is only observed in TRAC-HIT T cells due to the presence of Cα and Cβ in the HIT receptor. b. HIT/CAR mean fluorescence intensity (MFI) measured by FACS using AF647-GAM. (left) HIT/CAR histograms (representative experiment) and (right) HIT/CAR MFI; n = 6 independent experiments, 3 donors. c. Representative cytotoxic activity using an 18 h bioluminescence assay, using firefly luciferase (FFL)-expressing NALM6 as targets cells (n = 2 independent experiments on 3 healthy donors). CD19-specific TRAC-HIT and TRAC-CAR T cells were generated using two different CD19-specific binding domains, SJ25C1 and FMC63. All data are mean ± s.e.m.

Extended Data Fig. 2 HIT receptors provide antigen-specific T cell-mediated cytotoxicity.

Cytotoxic activity using an 18 h bioluminescence assay, using firefly luciferase (FFL)-expressing targets cells (n = 2 independent experiments on 2 healthy donors). CD19-, BCMA-, and CD70-specific TRAC-HIT T cells, and untransduced (UT) T cell controls were incubated with either NALM6 (CD19 + ), MOLM13/CD19 (CD70 + , CD19 + ), MM1S (BCMA + , CD70 + , CD19 + ), SK-MEL-37/CD19 (CD19 + , CD70 + ), and knock-out (KO) control cell lines. CD19, BCMA, and CD70 genes were CRISPR/Cas9-edited in NALM6, MM1S, and MOLM13 cell lines, respectively. Top 4 groups show data for donor 1 (left panel), donor 2 (middle panel), and both together (right panel). All data are mean ± s.e.m.

Extended Data Fig. 3 HIT receptor elicits cytokine response upon antigen stimulation; cell surface HIT receptor expression is modulated by exposure to antigen.

a. TRAC-Untransduced (UT), TRAC-HIT (HIT) and TRAC-CAR (CAR) T cells were stimulated on CD19 + target for 24 h before supernatant were collected and analysed by flow cytometry to quantify IFNγ, IL-2, TNFα, and granzyme B (n = 3 independent experiments on 3 donors). b. TRAC-HIT and TRAC-CAR T cells stimulated on CD19 + target cells 1, 2 or 4 times over a 48 h period were analysed by flow cytometry using the GAM, CD4, and CD8 antibodies. Plots indicate relative HIT or CAR MFI (1 = MFI at 0 h) of CD4 and CD8 TRAC-HIT or TRAC-CAR T cells, respectively. (n = 3 independent experiments on 3 donors). c. Untransduced (UT), HIT and CAR T cells stimulated on CD19 + target cells either 0, 1, 2 or 4 times over a 48 h period were analysed by flow cytometry. Plots indicate the percentage of the CAR positive T cells measured by flow cytometry analysis of CD4 and CD8 (n = 3 independent experiments on 3 donors). All data are mean ± s.e.m.

Extended Data Fig. 4 Antigen binding depends on HIT receptor affinity.

a. Representative flow cytometry analysis showing CD19 binding. TRAC-HIT and TRAC-CAR T cells were incubated with a recombinant human CD19-Fc fusion, which was then detected using an anti-hFc-PE antibody. b. HIT/CAR mean fluorescence intensity (MFI) measured by FACS using AF647-goat anti human (GAH) antibody (representative experiment). c. Plot of CD19 binding (adjusted gMFI) vs CD19 binder affinity (n = 2 independent experiments). Geometric MFI for CD19 biding (PE signal from a.) was adjusted to the gMFI of the HIT/CAR receptor (AF647 signal from b.) All data are mean ± s.e.m.

Extended Data Fig. 5 HIT receptors provide greater antigen sensitivity than CARs.

a. Representative histogram of the CD19 expression in NALM6/WT, NALM6/Medium21, NALM6/Low21, and CRISPR-edited NALM6/Very Low (this study). b. NALM6/Very Low cells were used to generate single cell clones by limited dilution. CD19 expression was evaluated for each clone (blue histogram) along with the initial NALM6/Very Low cell population (red histogram); clone number indicated above the histogram plot. c. Schematics of the SIN lentiviral vector used to express low levels of CD19 in NALM6/12 cells. PGK100: short PGK promoter, which is a weak promoter24. d. Panel of NALM6 cells expressing different CD19 levels, which is represented as a histogram. NALM6/12-4 and NALM6/12-39 are derivatives of NALM6/12, which was transduced with the lentiviral vector described in c. e. Total CD19 protein quantification using mass spectrometry. Protein levels are expressed in terms of peptide abundance (A.U.), which can be compared across all samples analysed at the same time. A-D represent 4 independent analyses. AVG, average of A-D values. f. Representative cytotoxic activity using an 18 h bioluminescence assay, using FFL-expressing NALM6 as targets cells (clone numbers as in b and d); n = 2. CD19-specific TRAC-HIT and TRAC-CAR T cells were generated using two different CD19-specific binding domains, SJ25C1 and FMC63. Anova test was used to compare the CTL curves of all T cells for NALM6/2 and NALM6/7 cells. g. Representative cytotoxic activity using an 18 h bioluminescence assay, using FFL-expressing NALM6 as targets cells (same as in f); n = 2. CD19-specific TRAC-1928z, TRAC-19BBz, and TRAC-19z1 T cells were prepared as described in Materials and methods. Anova test was used to compare the CTL curves of all T cells for NALM6/2 cells. All data are mean ± s.e.m.

Extended Data Fig. 6 CD19, CD22, and BCMA HIT receptors elicit T cell-mediated lysis of multiple myeloma cells.

a. Representative flow cytometry analysis showing CD19, CD22, and BCMA expression in MM1S and NALM6/WT cells. b. Total CD19, CD22, and BCMA protein quantification using mass spectrometry. Protein levels are expressed in terms of peptide abundance (A.U.), which can be compared across all samples analysed at the same time. A-D represent 4 independent analyses. AVG, average of A-D values. c. Representative cytotoxic activity using an 18-h bioluminescence assay, using FFL-expressing MM1S as targets cells, which were incubated at the indicated E/T ratios with CD19-, CD22-, or BCMA-specific TRAC-HIT and TRAC-CAR T cells; n = 3. All data are mean ± s.e.m. Additional specificity studies are shown in Extended Data Fig. 2.

Extended Data Fig. 7 HIT T cells show increased signalling response to low antigen levels.

a. Gating strategy used to quantify ITAM3, ZAP70, and ERK1/2 phosphorylation in TRAC-HIT/CAR T cells (histograms shown in b.). b. Representative flow cytometry analysis showing histograms for intracellular phospho-ITAM3, phospho-ZAP70, and phospho-ERK1/2 in TRAC-HIT (left) and TRAC-CAR (right) T cells when incubated with NAML6/WT, NALM6/12-4, or NALM6/7 target cells at 1:2 ratio for 15 min, or with no target (No stim.). FMO: fluorescence minus one control. Geometric MFIs were obtained for each curve, and used to generate the plots presented in Fig. 2g. All data are mean ± s.e.m.

Extended Data Fig. 8 HIT T cells display increased degranulation upon stimulation.

a. Confocal and bright field images of conjugates of T cells expressing the CAR or HIT receptor and interacting for 30 min with NALM6/WT cells labeled for LAMP-1 (red channel) and Alexa Fluor 546 phalloidin (F-actin staining showed in green). Quantification of the lysosome polarity assessed as the distance to the immune synapse and compared with the average lysosomal distance to the total cortex of the T cell. This distance was normalized with the maximum distance found in the T cell and expressed as a polarity index between 1 (lysosomes at the synapse) and 0 (lysosomes at the opposite of the synapse. Data from two independent experiments; n = 59. Variance p-values were obtained by using unpaired t-test analysis. All data are mean ± s.e.m. Scale bar = 5 µm. White arrows indicate LAMP-1 signal. b. FACS plot gates used to quantify T cell degranulation (CD107a + cells). b. Representative (n = 2 independent experiments) analyses of CD107a levels in TRAC-HIT (top) and TRAC-CAR (bottom) T cells when incubated for 1 h (left) or 4 h (right) without or with NALM6 targets of different CD19 levels.

Extended Data Fig. 9 Control of low-antigen tumours by HIT T cells is enhanced by extending T cell persistence by co-expression of CD80 and 4-1BBL.

a. NALM6/12-4-bearing mice were treated with 4 × 105 TRAC-HIT T cells co-expressing the annotated costimulatory ligand. Tumour burden was quantified weekly over a 35-day period, at week 1 or week 3. Each line represents 5 mice. b. NALM6/12-4-bearing mice were treated with 4 × 105 CAR T cells (n = 5 per group; dot = one mouse) and euthanized at days 10 and 17 after infusion; bone marrow TRAC-HIT or TRAC-CAR T cells and NALM6 cells were analysed and counted by FACS. c. Same as in b, except that bone marrow TRAC-HIT or TRAC-CAR CD4 and CD8 T cells counted by FACS. Two-tailed unpaired Student’s t-tests were used for statistical analyses. All data are mean ± s.e.m. Tumor burden of individual animals are shown in Supplementary Fig. 2.

Extended Data Fig. 10 Co-expression of costimulatory ligands CD80 and 4-1BBL enhances the therapeutic potential of HIT cells.

a. Tumour burden (average radiance) of NALM6/12-4-bearing mice treated with 4 × 105 TRAC-HIT or TRAC-CAR T cells (n = 5), analysed through a 35-day period. b. Tumour burden (average radiance) of MM1S-bearing mice treated with 2 × 105 TRAC-HIT or TRAC-CAR T cells (n = 5), analysed through a 35-day period. c. Kaplan-Meier analysis of survival of MM1S-bearing mice treated with 4 × 105 BCMA-specific TRAC-HIT or TRAC-HIT + 80/BBL T cells (n = 5). d. Representative FACS plots of surface CD70 expression in activated CAR-T cells 4 days after electroporation without (mock) or with CD70-specific CRISPR/Cas9 RNP. e. Tumour burden (average radiance) of MOLM13-bearing mice treated with 4 × 105 TRAC-HIT or RV-CAR T cells (n = 5), analysed through a 35-day period. All data are mean ± s.e.m. Tumor burden of individual animals are shown in Supplementary Figs. 2,3.

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Mansilla-Soto, J., Eyquem, J., Haubner, S. et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 28, 345–352 (2022). https://doi.org/10.1038/s41591-021-01621-1

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