T cells engineered against Dickkopf-1-A2 complex can be used to treat HLA-A2^+ solid and hematologic cancers

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
Although chimeric antigen receptor (CAR)-T cells are promising effector cells to treat hematologic tumors, developing effective CAR-T cells for solid tumors remains challenging. Dickkopf-1 (DKK1) protein is widely expressed by human hematologic and solid tumors. Using the sequence of murine or humanized monoclonal antibody recognizing DKK1-A2 complexes (DKK1-P20 peptide in the context of HLA-A*0201) that are detected on all examined HLA-A2+ tumor samples but not normal tissues except tonsils, we generate DKK1-A2 CAR-T cells that specifically and effectively lyse HLA-A2- and DKK1-expressing tumor cells but not blood or bone marrow cells from HLA-A2+ healthy donors. In xenograft models of human myeloma, pancreatic, lung, and breast cancers and patient-derived xenograft of pancreatic cancer, DKK1-A2 but not CD19 CAR-T cells effectively control or eradicate established tumors without detectable toxicities in NSG or human DKK1 and HLA-A2-trangenic mice. This study indicates that DKK1-A2 CAR-T cells may be used to treat human cancers.

Introduction
Chimeric antigen receptor (CAR)-T cells have demonstrated remarkable clinical success in the treatment of hematologic tumors, especially those targeting antigens such as CD19 and BCMA1–6. These therapies have significantly advanced the management of blood cancers, including lymphomas, some forms of leukemia, and most recently multiple myeloma (MM)7. However, developing effective CAR-T cell therapies for solid tumors remains challenging8. Although some tumor antigens, such as Mucin-19, Glypican-310 and Claudin 18.211 have been explored in research, identifying safe and effective antigens for CAR-T cell therapies for both hematologic and solid tumors continues to be a major hurdle12.

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the Wnt/\beta-catenin signaling pathway13. DKK1 binds to LRP5/6 co-receptors on the cell surface, preventing them from interacting with Wnt proteins14,15. DKK1 overexpression has been observed in various cancers, including MM16, pancreatic cancer17, lung cancer18, breast cancer19, and hepatocellular cancer20. In MM, elevated DKK1 level leads to bone lesions and osteoporosis due to the imbalance in bone remodeling21. In certain tumors, DKK1 also promotes tumor progression by creating a microenvironment that supports cancer growth, affects immune response, and facilitates metastasis22,23. Conversely, DKK1 has limited expression in normal tissues, with the exception of the placenta and testis24,25. This reciprocal expression makes DKK1 an attractive target for cancer immunotherapy26, particularly in developing CAR-T cell therapies for both MM and solid tumors.

To target DKK1 for cancer immunotherapy, we identified a DKK1 peptide, P20 (ALGGHPLLGV), that showed an excellent binding affinity to HLA-A*0201 (for short, HLA-A2 or A2), which is a widely distributed HLA serotype found in 30–50% of the human population25,27. We developed a T cell receptor-like monoclonal antibody (C2) that recognizes DKK1-P20 peptide in the context of HLA-A2 molecules (thereafter, referred as DKK1-A2 complex)28. This C2 antibody can directly induce apoptosis in DKK1-A2 positive cancer cells and indirectly lyse DKK1-A2 positive cancer cells by mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Furthermore, C2 antibody was therapeutic against established cancers in human xenograft mouse models. Notably, C2 antibody did not bind to or kill HLA-A2 positive blood or bone marrow cells in vitro, nor caused tissue damage in tumor-free or tumor-bearing HLA-A2-transgenic mice. The exceptional specificity and safety profile make the C2 antibody a promising candidate for developing CAR-T cell therapy.

In this study, we developed a DKK1-A2 CAR, incorporating the single-chain variable fragment (scFv) of C2 antibody along with 4-1BB and CD3\zeta costimulatory domains to generate human DKK1-A2 CAR-T cells. We hypothesized that this CAR-T cell therapy could effectively target HLA-A2 positive MM and a broad range of solid tumors, including pancreatic cancer, lung cancer, and breast cancers that express DKK1. Our results clearly showed that DKK1-A2 CAR-T cells have a strong anti-tumor effect in vitro and in vivo against established hematologic and solid tumors with a favorable safety profile, and DKK1-A2 CAR-T cells may be a promising therapeutic tool to treat DKK1-A2 positive human cancers.

Results
DKK1 and DKK1-A2 expression profiles in different cancers and normal tissues
To evaluate DKK1 expression in hematologic tumors, three gene sequencing datasets from myeloma29, lymphoma30, and leukemia31 were analyzed. DKK1 mRNA was highly expressed in many hematologic tumors, particularly in MM and acute leukemia originated from B cells (Fig. 1a). MM exhibited the highest DKK1 expression among various hematologic tumors, reaching up to around 70-fold higher than plasma cells from healthy donors (Fig. 1a). To determine DKK1 expression in solid tumors, its gene expression profiles across all tumor samples from GEPIA2 database32 were utilized for analysis (Fig. 1b). Most TCGA solid tumor types highly expressed DKK1 mRNA compared to paired normal tissues, particularly in pancreatic adenocarcinoma (PAAD)33 that expressed more than 90-fold DKK1 than normal pancreatic tissues (Fig. 1b). Pancreatic ductal adenocarcinoma (PDAC) patients with a higher expression of DKK1 had inferior overall and disease-free survivals than patients with a lower expression of DKK1 (Fig. 1c, d). Similarly, poor prognosis was also found in other solid tumors with higher DKK1 expression, such as lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and head and neck squamous cell carcinoma (HNSC) (Supplementary Fig. 1a–d). Oncomine datasets were used to validate DKK1 expression in tumor types of interest. PDAC34, non-small cell lung cancer (NSCLC)35, and triple negative breast cancer (TNBC)36 exhibited higher DKK1 mRNA expression compared to normal tissues (Fig. 1e). Immunohistochemistry was utilized to determine the expression of DKK1 protein and DKKI-A2 complex by tumor tissues. Our results showed that DKK1 protein was not detected in normal pancreatic, lung, or breast tissues from HLA-A2 positive donors (Fig. 1f). However, all tumor tissues from 10 randomly selected PDAC (Fig. 1g), NSCLC (Fig. 1h), or TNBC (Fig. 1i) patients expressed high levels of DKK1 protein. Among these patients, 4/10, 6/10, and 6/10, respectively, are HLA-A2 positive, and tumor samples from all these HLA-A2 positive patients expressed DKK1-A2 complexes stained by C2 antibody (Fig. 1g–i). Similarly, tissue array analysis showed that all of 40 NSCLC tumor tissues but none of 10 normal lung tissues highly expressed DKK1 protein, and 8 out of the 40 NSCLC patients were HLA-A2 positive and stained positive with C2 antibody (Supplementary Fig. 1e). One DKK1 negative and HLA-A2 positive TNBC sample was included and stained negative for C2 antibody (Supplementary Fig. 1f). Immunochemistry staining of DKK1, HLA-A2 and DKK1-A2 complex was controlled by isotype controls (Supplementary Fig. 1g). Together, these data revealed that DKK1 protein is highly expressed by multiple tumors, its expression is associated with poor survival of some cancer patients, and all HLA-A2-positive tumors express high levels of DKK1-A2 complexes recognized by C2 antibody.

DKK1-A2 CAR-T cells display strong anti-tumor activity in vitro
As C2 antibody recognizes DKK1-A2 complex on tumor cells but not normal tissues, we generated DKK1-A2 CAR-T cells to target tumor cells that express DKK1-A2 complex (Fig. 2a). DKK1-A2 CAR consisted of the scFv derived from C2 antibody, 4-1BB, and CD3\zeta intracellular domains. Herein, both of murine C2 antibody (mC2) and humanized C2 antibody (hC2) were used to generate DKK1-A2 CAR-T cells. If not otherwise specified, DKK1-A2 CAR-T cells refer to mC2 CAR-T cells. CD19 CAR-T cells were generated and utilized as a control. Enhanced green fluorescent protein (eGFP) was co-expressed with CAR to track CAR-T cells (Fig. 2b). The average transduction efficiency of activated human T cells was around 70% (Fig. 2c). To evaluate the anti-tumor activity of DKK1-A2 CAR-T cells, MM, PDAC, NSCLC, and TNBC cell lines were used. As DKK1 is widely expressed by the tumor cell lines, C2 antibody could bind to HLA-A2 positive tumor cell lines, including JeKo-1, U266, KMS-26, CFPAC-1, PANC-1, NCI-H441, and MDA-MB-231 (Fig. 2d), but not HLA-A2 negative tumor cell lines (Supplementary Fig. 2a). CD19 expression on the tumor cells was also determined by flow cytometry. JeKo-1 and Daudi but not MM cell lines expressed CD19 (Supplementary Fig. 2b). The proliferation of CAR-T cells was corroborated by Celltrace dilution assay, as DKK1-A2 CAR-T cells exhibited a strong proliferation in response to HLA-A2 positive tumor cells compared to CD19 or control CAR-T cells (Fig. 2e). Cytolytic activity of DKK1-A2 CAR-T cells against tumor cells was determined by a firefly luciferase (Luc)-based cytotoxicity assay. DKK1-A2 CAR-T cells significantly lysed HLA-A2 positive tumor cells in a dose-dependent manner (Fig. 2f). We also assessed the anti-tumor activity of DKK1-A2 CAR-T cells against HLA-A2-transduced tumor cell lines (Supplementary Fig. 2c) and observed significant cytolytic activity and enhanced proliferation of DKK1-A2 CAR-T cells against MM.1S-A2, KMS-12-BM-A2, or PL45-A2 tumor cells (Supplementary Fig. 2d, e). DKK1-knockout (KO) JeKo-1 cells were generated by CRISPR-Cas9 (Supplementary Fig. 2f) and cytolytic activity and proliferation of DKK1-A2 CAR-T cells were not observed against JeKo-1-KO cells (Supplementary Fig. 2g, h). The anti-tumor activity of DKK1-A2 CAR-T cells was further supported by IFN-\gamma and TNF-\alpha production by both CD4+ and CD8+ DKK1-A2 CAR-T cells in their co-culture with tumor cell lines by flow cytometry (Fig. 2g, h) and ELISA (Supplementary Fig. 2i, j). Overall, DKK1-A2 CAR-T cells exhibit strong target-specific anti-tumor activity against HLA-A2 positive tumor cells in vitro.

Therapeutic efficacy of DKK1-A2 CAR-T cells in human MM xenograft models
To evaluate the in vivo anti-tumor effects of DKK1-A2 CAR-T cells, we first established and used MM xenograft tumor models. U266 MM cells were implanted intravenously into NSG mice. Four weeks later, mice were infused intravenously with either control, CD19, or DKK1-A2 CAR-T cells, and tumor burden, determined by measuring the amounts of human immunoglobulin lambda (Ig\lambda) chain secreted by MM cells, was monitored weekly by ELISA (Fig. 3a). DKK1-A2 CAR-T cells effectively controlled U266 cell growth, and all mice remained alive at 7 weeks post CAR-T cell infusion, while CD19 CAR-T cells failed to do so (Fig. 3b, c). The body weight of all treated mice was not significantly changed after CAR-T cell therapy (Fig. 3d). Similar data were obtained from DKK1-A2 positive and CD19 negative KMS-2637 MM model (Supplementary Fig. 3a–d).

Therapeutic efficacy of DKK1-A2 CAR-T cells in human solid tumor xenograft models
We then assessed the anti-tumor efficacy of DKK1-A2 CAR-T cells against human solid tumors in their xenograft mouse models. First, firefly luciferase-transduced CFPAC-1 PDAC tumor cells were implanted subcutaneously 1 week before CAR-T cells infusion, and tumor volume and bioluminescence intensity were monitored weekly (Supplementary Fig. 3e). DKK1-A2 CAR-T cells effectively controlled tumor growth, evidenced by bioluminescence and survival rate, and body weight was not significantly changed after CAR-T cell therapy (Supplementary Fig. 3f–i). More importantly, we developed and used a PDAC orthotopically xenografted model by implanting CFPAC-1-Luc tumor cells into mice pancreas38 (Fig. 3e). Again, DKK1-A2 CAR-T cells exhibited effective and safe anti-tumor activity, evidenced by bioluminescence, survival, and body weight monitoring (Fig. 3f–i). Second, we used an NSCLC metastatic model by injecting Luc-transduced NCI-H441 tumor cells intravenously into NSG mice (Fig. 3j). DKK1-A2 CAR-T cells significantly reduced metastatic tumor burden, and all DKK1-A2 CAR-T cell-treated mice achieved a superior survival rate. Body weight was decreased after control, and CD19 CAR-T cell therapy, mainly due to tumor progression, and body weight was not significantly changed after DKK1-A2 CAR-T cell therapy (Fig. 3k–n). Third, the anti-tumor efficacy of DKK1-A2 CAR-T cells was examined in orthotopic (Fig. 3o) and subcutaneous (Supplementary Fig. 3j) Luc-transduced MDA-MB-231 TNBC models. Again, DKK1-A2 CAR-T cell treatment effectively controlled MDA-MB-231 tumor growth and resulted in long-term survival in all mice in the orthotopic model. DKK1-A2 CAR-T cell therapy did not significantly affect the body weight of all mice (Fig. 3p–s). Similarly, DKK1-A2 CAR-T cells exhibited strong anti-tumor efficacy in the subcutaneous model (Supplementary Fig. 3k–n). Also, the TUNEL assay was performed in CFPAC-1 orthotopic, NCI-H441 metastatic, and MDA-MB-231 orthotopic models after CAR-T cell therapy. We observed CAR-T cell infiltration coupled with tumor apoptosis in the tumor tissues after DKK1-A2 CAR-T cell but not control or CD19 CAR-T cell treatment (Supplementary Fig. 4a–c). In most of these studies, CD19 CAR-T cells were used as a control, and no anti-tumor effect was observed.

Therapeutic efficacy of DKK1-A2 CAR-T cells in PDAC patient-derived xenograft model
Patient-derived xenograft (PDX) models effectively replicate the histology and genetic profile of the original patient tumors39. In collaboration with our surgical oncologists, we obtained fresh tumor samples from 7 PDAC patients. Primary PDAC tumors were expanded in the flanks of NSG mice for 1–3 passages, allowing for greater expansion of tumor tissues40 (Supplementary Fig. 5a, b). All primary PDAC tumor tissues expressed a high level of DKK1 protein, and DKK1-A2 complexes were detected in two HLA-A2 positive patients (PDAC #1 and #14; Fig. 4a). Proliferation of DKK1-A2 CAR-T cells was found in their co-culture with PDAC #1 and #14 tumor cells but not with HLA-A2 negative PDAC cells (Fig. 4b). Celltrace Violet-labeled primary PDAC cells were co-cultured with control, CD19, or DKK1-A2 CAR-T cells for 3 days, and DKK1-A2 but not CD19 CAR-T cells eliminated nearly all HLA-A2 positive PDAC #1 tumor cells but not HLA-A2 negative PDAC #2 cells (Fig. 4c, d). Activation of DKK1-A2 CAR-T cells was also supported by higher levels of IFN-\gamma, TNF-\alpha, and Granzyme B production by both CD4+ and CD8+ CAR-T cells in co-culture with PDAC #1 cells (Fig. 4e–g). To characterize primary and passage tumors of PDAC #1 PDX model, tumor tissues were digested and analyzed by flow cytometry (Supplementary Fig. 5c). Tumor cells were distinguished by carcinoembryonic antigen (CEA) staining. The primary and F3 passage tumors retained the same cellular composition, and nearly all the tumor cells were CEA, DKK1, and DKK1-A2 positive (Supplementary Fig. 5d). The results were also confirmed by immunochemistry staining with both low and high magnifications (Supplementary Fig. 5e, f). To evaluate the anti-tumor efficacy of DKK1-A2 CAR-T cells in the PDX model, expanded PDAC #1 tumor fragments were implanted subcutaneously into NSG mice, and CAR-T cells were infused intravenously 2 weeks post tumor inoculation (Fig. 4h). DKK1-A2 CAR-T cells effectively controlled tumor growth in vivo, and all mice achieved superior survival up to 8 weeks (Fig. 4i, j). Flow cytometry analysis showed that large numbers of circulating and resident (in spleen and bone marrow) DKK1-A2 but not control or CD19 CAR-T cells were detected at the end of the experiment (Fig. 4k). Collectively, these results showed that DKK1-A2 CAR-T cells are highly therapeutic against established hematologic and solid tumors, including primary tumors from HLA-A2 positive patients in vivo.

Generation of DKK1-A2 CAR-T cells using a humanized C2 antibody
For future clinical application of DKK1-A2 CAR-T cells, we generated CAR-T cells with the scFv of the hC2 antibody constructed from the mC2 antibody. To compare the cytolytic activity of mC2 and hC2 CAR-T cells, U266, CFPAC-1, or primary PDAC tumor cells were used as targets and co-cultured with the CAR-T cells at different E:T ratios in a luciferase-based cytotoxicity assay. Both mC2 and hC2 CAR-T cells showed strong cytolysis against U266 and CFPAC-1 tumor cells (Fig. 5a, b), and both mC2 and hC2 CAR-T cells exhibited similar cytolytic activity against primary PDAC #1 cells at 24- or 72-h co-culture (Fig. 5c, d). To evaluate the anti-tumor activity of the CAR-T cells in vivo, U266, KMS-12-BM-A2, or MM.1S-A2 MM cell lines were utilized to establish xenograft models by tail vein injection into NSG mice (Fig. 5e and Supplementary Fig. 6a, e). Although both mC2 and hC2 CAR-T cells effectively controlled tumor growth, hC2 CAR-T cells, compared to mC2 CAR-T cells, showed a superior anti-tumor ability in U266 MM model evidenced by relatively lower human Ig\lambda (tumor burden) (Fig. 5f) and better mouse survival (Fig. 5g). All mice treated with hC2 CAR-T cells displayed long-term survival and remained tumor free at the end of the experiment (week 12) (Fig. 5g). In MM.1S-A2 (Supplementary Fig. 6a–d) and KMS-12-BM-A2 (Supplementary Fig. 6e–h) mouse models, hC2 CAR-T cells also showed better anti-tumor activities evidenced by long-term survival rate up to 8 weeks post CAR-T cell infusion. Moreover, we evaluated the therapeutic efficacy of mC2 and hC2 CAR-T cells in PDAC mouse models (Fig. 5h and Supplementary Fig. 6i). mC2 and hC2 CAR-T cells showed comparable anti-tumor activities in CFPAC-1 xenograft model (Fig. 5i–k). However, in the primary PDAC #1 PDX model, hC2 CAR-T cells showed a better anti-tumor activity compared to mC2 CAR-T cells, and all hC2 CAR-T cell-treated mice achieved long-term survival over 10 weeks (Supplementary Fig. 6j, k). Hence, while both mC2 and hC2 CAR-T cells exhibit comparable cytolytic ability in vitro, hC2 CAR-T cells show a better anti-tumor activity in vivo.

hC2 CAR-T cells display central memory phenotype and exert better anti-tumor effects in vivo
Multiple mechanisms and clinical studies have shown that less differentiated memory T cells are required for a sustained CAR-T cell persistence in vivo41–43. To evaluate whether mC2 and hC2 CAR-T cells display distinct memory phenotypes, CAR-T cells generated from 4 healthy donors were analyzed for CCR7 and CD45RO expression by flow cytometry. hC2 CAR-T cells contained a higher percentage of cells (70%) that were CCR7+ and CD45RO+ (central memory phenotype), while mC2 CAR-T cells had a large population of CCR7– and CD45RO+ effector memory cells compared to their counterpart (Fig. 6a, b). In co-culture with DKK1-A2 positive JeKo-1 tumor cells, hC2 CAR-T cells rapidly differentiated into effector memory phenotype to execute cytolysis activity, while the memory phenotype of mC2 CAR-T cells had not significantly changed (Fig. 6c, d). mC2 and hC2 CAR-T cells were infused into JeKo-1 bearing mice for 7 days, and CCR7 expression on hC2 CAR-T cells was much higher than that of mC2 CAR-T cells, indicating hC2 CAR-T cells as less differentiated memory T cells in vivo (Supplementary Fig. 7a, b). Central memory-like hC2 CAR-T cells secreted lower amounts of IFN-\gamma and TNF-\alpha in both CD4 and CD8 positive subsets in response to JeKo-1, but Granzyme B secretion was similar to mC2 CAR-T cells (Fig. 6e–g). Notably, re-stimulation of the CAR-T cells by adding more tumor cells to the co-culture led to significantly higher IFN-\gamma, TNF-\alpha, and Granzyme B production by hC2 CAR-T cells compared to mC2 CAR-T cells (Fig. 6h–j). To further evaluate the long-term response of the CAR-T cells, we performed tumor rechallenge experiments. CAR-T cell-infused JeKo-1-bearing NSG mice were rechallenged with JeKo-1 tumor cells at 4 weeks post CAR-T cell infusion (Fig. 6k). In hC2 CAR-T cell-treated mice, tumor volumes were much lower, and more mice remained alive post tumor rechallenge (Fig. 6l, m). Improved hC2 CAR-T cell persistence was found in blood, lymph node, and spleen post tumor rechallenge (Fig. 6n). We also observed similar results in U266-rechallenge model that was controlled with naïve mice (Supplementary Fig. 7c–e). In CFPAC-1 rechallenge model, although all mice remained alive post-tumor rechallenge, tumor burdens in hC2 CAR-T cell-treated mice were significantly lower than those treated with mC2 CAR-T cells (Supplementary Fig. 7f–i). Together, these findings showed that hC2 CAR-T cells display a less-differentiated central memory phenotype that may have contributed to the long-term anti-tumor immunity against both MM and PDAC.

DKK1-A2 CAR-T cells do not cause a damage to normal tissues
We assessed the expression of DKK1-A2 complex on human normal tissues with commercially available tissue arrays. Thirty-five different types of normal tissues from three HLA-A2 positive donors were stained with DKK1, HLA-A2, and DKK1-A2 (C2) antibodies. Although some normal human tissues expressed DKK1 protein, all human normal tissues were stained negative for DKK1-A2 complex except the tonsil tissues (Fig. 7a and Supplementary Fig. 8a, b), indicating that the target for DKK1-A2 CAR-T cells is not present on most human tissues. To evaluate the potential hematological toxicity of DKK1-A2 CAR-T cells, peripheral blood mononuclear cell (PBMC) and bone marrow mononuclear cells (BMMC) from HLA-A2 positive donors were co-cultured with CAR-T cells and cell apoptosis was analyzed by flow cytometry (Supplementary Fig. 8c). CD19 CAR-T cells effectively lysed B cells in both PBMC and BMMC, while mC2 or hC2 CAR-T cells did not cause significant cell death, evidenced by Annexin V and Caspase 3 staining in cell populations of PBMC or BMMC (Supplementary Fig. 8d–g). We then established a tripartite co-culture model44 consisting of CAR-T cells, autologous monocytes and JeKo-1 cells for 24 h (Fig. 7b). In co-culture with hC2 CAR-T cells, significantly lower levels of human IFN-\gamma and IL-6 were observed in comparison to co-culture with mC2 CAR-T cells (Fig. 7c). To further assess the safety profile, we utilized a cytokine release syndrome model45 by infusing a large dose (50\times10^6 per mouse) of CAR-T cells intraperitoneally into JeKo-1 bearing mice (Fig. 7d). Similar to what was observed in vitro, CD19 and mC2 CAR-T cells expressed a comparable level of human IFN-\gamma and mouse IL-6, while hC2 CAR-T cells had attenuated cytokine release detected in peritoneal lavage fluid of treated mice (Fig. 7e). Body weight loss was also monitored as a readout. Both mC2 CAR-T cells and positive control CD19 CAR-T cells induced a more serious body weight loss compared to hC2 CAR-T cells (Fig. 7f).

Finally, we investigated the potential efficacy and toxicity of DKK1-A2 CAR-T cells in immunocompetent mice. We generated transgenic C57BL/6 mice expressing human DKK1 and HLA-A2 molecules (hDKK1-A2 mice) and developed murine CAR-T cells using mouse splenocytes targeting murine CD19 or human DKK1-A2 complex for in vivo tests. We first examined the potential hematological toxicity of the murine CAR-T cells. After intravenous infusion, murine CD19 CAR-T cells led to depletion of mouse B cells, while murine DKK1-A2 CAR-T cells did not affect cell populations in blood, spleen, or bone marrow (Fig. 7g–i). Moreover, we utilized the MM 5TGM1 and pancreatic cancer Pan02 syngeneic mouse models46,47 by transducing murine 5TGM1 and Pan02 cells with human DKK1 and HLA-A2 molecules. After the tumor developed, mice were infused with CD19 or DKK1-A2 murine CAR-T cells. The results showed that DKK1-A2 but not CD19 CAR-T cells effectively controlled tumor growth in murine 5TGM1- and Pan02-bearing mice without obvious body weight change (Supplementary Fig. 9a–f). Since platelets were reported to express both DKK1 and MHC molecule48, we evaluated platelet cell count in hDKK1-A2 mice post CAR-T cell treatment. No significant change in platelet count was observed 7 days after CAR-T cell infusion, suggesting that DKK1-A2 CAR-T cells do not cause substantial depletion of circulating platelets (Supplementary Fig. 9g). Overall, these results clearly showed that DKK1-A2 CAR-T cells display a favorable safety profiles and may be less or non-toxic in human patients.

Methods
Ethical statement
Our research complies with all relevant ethical regulations. The use of primary tumor tissues was approved by the Institutional Review Board at the Houston Methodist Research Institute. All animal studies were approved by the Institutional Animal Care and Use Committee of the Houston Methodist Research Institute.

Human and mouse cell lines
Human mantle cell lymphoma cell line JeKo-1, Burkitt’s lymphoma cell line Daudi, MM cell lines U266, KMS-26, ARP-1, KMS-11, KMS-12-BM, and MM.1S, PDAC cell lines CFPAC-1, PANC-1, MIAPaCa-2, and PL45, NSCLC cell lines NCI-H441 and NCI-H266, and TNBC cell lines MDA-MB-231 and MDA-MB-468 were purchased from American Type Culture Collection or obtained from Arkansas Cancer Research Center. KMS-12-BM-A2, KMS.1S-A2, and PL45-A2 were generated by transducing with the human gene HLA-A*0201. JeKo-1-KO was generated by CRISPR/Cas9 gene-editing technology. Firefly Luciferase stably transduced-JeKo-1, Daudi, U266, KMS-26, CFPAC-1, PANC-1, NCI-H441, MDA-MB-231, KMS-12-BM-A2, KMS.1S-A2, and PL45-A2 were generated by transducing with luciferase gene. Murine MM 5TGM1-DKK1-A2 and Pan02-DKK1-A2 cells were generated by transducing the cells with human HLA-A2 and DKK1 genes. All cell lines were cultured in complete RPMI 1640 medium or Dulbecco modified Eagle’s medium supplemented with 10% FBS and 1% penicillin and streptomycin.

Animals
NSG and SCID mice were purchased from the Jackson Laboratory. hDKK1-A2 mice were generated by crossing HLA-A2 transgenic mice with DKK1 transgenic mice by Cyagen Biosciences. Male and female 6-8-week-old mice were used in the studies. Mice were maintained in American Association of Laboratory Animal Care-accredited facilities, and all studies were approved by the Institutional Animal Care and Use Committee of Houston Methodist Research Institute. We have complied with all relevant ethical regulations. For MM mouse models, the maximal tumor burden permitted by our ethics committee is the point when they develop signs of hindlimb paralysis. For solid tumor mouse models, the maximal tumor burden permitted by our ethics committee is no more than 20 mm in diameter. In our study, the maximal tumor burden was not exceeded.

Human samples
Human whole peripheral blood was purchased from the Gulf Coast Regional Blood Center, Houston, Texas. Human PDAC, NSCLC, and TNBC tissues were obtained from surgical resections of patients at Houston Methodist Hospital. Human tumor tissues were used for establishment of primary patient-derived tumor cells and xenograft model, as well as immunohistochemistry and tissue microarray studies. Utility of human samples in this study was approved by the institutional review board at the Houston Methodist Research Institute.

Plasmid construction and retrovirus preparation
Plasmid construction and retrovirus preparation were described previously67. Gene sequence of light and heavy chain of murine or humanized C2 antibody were obtained from cDNA of C2 hybridomas amplified by using PCR. mC2, hC2, or FMC63 scFv-based chimeric antigen receptor were cloned into SFG retroviral vector, followed by CD8\alpha hinge and transmembrane, 4-1BB intracellular costimulatory domain, CD3\zeta intracellular signaling domain and eGFP. 293T cells were seeded in 100 mm cell culture dish and transfected with a plasmid mixture of chimeric antigen receptor retroviral vector, packaging plasmid encoding Gag/Pol, and envelope plasmid encoding RD114 by using Lipofectamine 3000 transfection reagent. Culture supernatant was collected 48 and 72 h after transduction, and retrovirus was precipitated by PEG-400.

Retrovirus transduction and CAR-T cell culture
Transduction of T cells was described previously68,69. Peripheral blood mononuclear cells were obtained by Ficoll density gradient centrifugation. CD3+ T cells were isolated by immunomagnetic negative selection kit from STEMCELL Technologies. T cells were activated with CD3/CD28 Dynabeads or 10 \mu g/mL anti-CD3/CD28 antibodies for 24 h and transduced with retrovirus on RetroNectin-coated plates. Three days later, T cells were expanded in complete RPMI 1640 medium supplemented with human IL-2. Two weeks after transduction, T cells were collected for in vitro or in vivo experiments. Transduction efficiency was determined by the percentage of eGFP expression analyzed by flow cytometry.

Flow cytometry
Tumor cell lines were harvested and resuspended in FACS buffer (PBS with 1% FBS and 1 mM EDTA). First, tumor cells were stained with FcR blocker and surface-stained with BUV395-conjugated HLA-A2 antibody (BD Bioscience, 1 \mu g/mL) and Alexa Fluor 647-conjugated mC2 antibody (1 \mu g/mL). After wash with FACS buffer, tumor cells were fixed and permeabilized by using BD Cytofix/Cytoperm Fixation/Permeabilization Kit. Tumor cells were intracellularly stained with DKK1 antibody (Santa Cruz Biotechnology, 1 \mu g/mL), and then Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Life Technologies, 1 \mu g/mL). For the PDX model, tumor tissues were digested with Collagenase and DNase I. Tumor cells were harvested and stained with purified CEA antibody (NeoBiotechnologies, 1 \mu g/mL), followed by staining with Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (Life Technologies, 1 \mu g/mL), along with anti-DKK1, HLA-A2, or mC2 antibodies as described above. All fluorescent antibodies were controlled by corresponding isotype control (1 \mu g/mL). After wash with permeabilization buffer, tumor cells were resuspended in FACS buffer and analyzed by flow cytometry.

Cytotoxicity assay
Luciferase reporter assay was used to evaluate the cytotoxicity of CAR-T cells. Firefly luciferase stably transduced tumor cells were seeded onto a 96-well plate overnight at 0.05 million per well. The next day, serial dilutions of CAR-T cells were prepared according to the effector to target (E:T) ratio and incubated with target cells for the indicated time. The supernatant was gently discarded, D-luciferin was added to each well for 5-min incubation, and data was measured by a microplate reader. The cytotoxicity of CAR-T cells was quantified by converting the obtained values to percentage of specific lysis by the following equation: [1-(S-E)/(T-B)]*100%, where S is the value of luminescence of the sample well, E is the value of luminescence of the effector cell well, T is the mean value of luminescence of target cell well, and M is the mean value of luminescence of blank well. Flow cytometry-based cytotoxicity assay was also used to evaluate lysis ability of CAR-T cells70. Briefly, target cells were stained with Celltrace Violet and co-cultured with effector cells at an E:T ratio of 1:1 for 24–72 h. After that, percentages of tumor cells and CAR-T cells were analyzed by flow cytometry.

Proliferation assay
CAR-T cells were stained with Celltrace Violet at the concentration of 1 \mu M and then co-cultured with tumor cells at a E:T ratio of 1:1 for 72 h. Proliferation of CAR-T cells was determined by dilution of Celltrace Violet in CAR-T cells. Data were obtained with flow cytometry and analyzed by FlowJo software.

Cytokine assay
Cytokine secretion analysis was performed using flow cytometry-based techniques71. Briefly, CAR-T cells were incubated with target tumor cells in 96-well plates overnight in the presence of phorbol myristate acetate, ionomycin, and brefeldin A. After that, cells were harvested then stained with FcR blocker and fluorescent antibodies specific to CD3, CD4, and CD8 on ice. After fixation and permeabilization, cells were intracellularly stained with fluorescent antibodies specific to IFN-\gamma, TNF-\alpha, or Granzyme B. Data were acquired with flow cytometry and analyzed with FlowJo software. Cytokine secretion was also analyzed by ELISA according to the manufacturer’s instructions.

Human xenograft mouse models
Luciferase stably transduced human myeloma U266, KMS-26, KMS-12-BM, and MM.1S, pancreatic cancer CFPAC-1 and PANC-1, lung cancer NCI-H441, and breast cancer MDA-MB-231 xenograft models were used to evaluate the therapeutic efficacy of CAR-T cells in vivo. For MM models, tumor cells were inoculated intravenously into NSG mice. For subcutaneous solid tumor models, tumor cells were inoculated subcutaneously into NSG mice. For metastatic tumor models, tumor cells were inoculated intravenously into NSG mice. For orthotropic PDAC or breast tumor models, tumor cells were implanted into the pancreas or mammary pad of NSG mice, respectively. Control, CD19, mC2, or hC2 CAR-T cells were intravenously transferred into tumor-bearing mice. For the JeKo-1 rechallenge experiment, JeKo-1 tumor cells were inoculated subcutaneously into the contralateral side of the mouse back relative to the primary tumor. For the U266 rechallenge experiment, U266 tumor cells were inoculated intravenously into the mice. For the CFPAC-1-Luc rechallenge experiment, CFPAC-1-Luc tumor cells were inoculated subcutaneously into the contralateral side of the mouse back relative to the primary tumor. The numbers of rechallenge tumor cells were the same as the primary tumor inoculation. Tumor burdens were evaluated weekly by bioluminescence on IVIS imaging system and images were processed by Living Image software72. For MM models, serum levels of human Ig\lambda secreted by MM cells were determined by ELISA73. Survival and body weight of tumor bearing mice were monitored. Tumor volume was calculated by the formula: tumor volume=0.5*length*width^2. Mice were sacrificed when undergoing cancer cachexia, or tumor diameter exceeded more than 20 mm, or tumor volume exceeded more than 2000 mm^3. Mice were sacrificed at the indicated time point for ex vivo analysis.

Patient-derived tumor cells and xenograft model
Human PDAC tumor tissues were obtained from surgical resections of PDAC patients at Houston Methodist Hospital. Necrotic tissue and non-tumor tissue were removed under sterile conditions, and tumor tissues were cut into small pieces (2–4 mm^3). Primary PDAC tumors were expanded in the flanks of NSG mice for 1–3 passages, allowing for greater expansion of tumor tissues40. For primary patient-derived tumor cells, tumor fragments from xenograft mice models were digested by DNase I and collagenase IV. Single tumor cell suspension was filtered for cytotoxicity, proliferation, and cytokine assay in vitro as described previously74. Tumor fragments were implanted into subcutaneous space of 6–8 weeks NSG mice. Fourteen days post-tumor implantation, mice were treated with CAR-T cells intravenously. Tumor volume and body weight were monitored weekly, and survival was monitored daily. Tumor volume was calculated by the formula: tumor volume=0.5*length*width^2. Mice were sacrificed when undergoing cancer cachexia, tumor diameter exceeded more than 20 mm, or tumor volume exceeded more than 2000 mm^3. Mice were sacrificed at the indicated time point for ex vivo analysis.

Immunohistochemistry, immunofluorescence, and tissue microarray
Human PDAC, NSCLC, and TNBC tumor and normal tissues were obtained from Houston Methodist Hospital. Immunohistochemistry was described before75. Formalin-fixed, paraffin-embedded tissue sections (4–5 \mu m) were deparaffinized in xylene and rehydrated through graded ethanol. Slides were treated with a heat-induced epitope retrieval solution for 10–20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, followed by incubation with a blocking solution to prevent non-specific binding. Sections were incubated with the primary antibody of DKK1 (Santa Cruz Biotechnology, 10 \mu g/mL), HLA-A2 (Santa Cruz Biotechnology, 10 \mu g/mL), DKK1-A2 (mC2/hC2, 10 \mu g/mL), or CEA (NeoBiotechnologies, 10 \mu g/mL) overnight at 4 °C. All samples were controlled with corresponding isotype control at the same concentrations. A secondary antibody conjugated to horseradish peroxidase was applied, and the signal was visualized using diaminobenzidine as the chromogen. Slides were analyzed under a microscope. Immunofluorescence was described before76. Mouse tumor tissues were collected, embedded into OCT compound, and rapidly frozen in dry ice. The frozen blocks were then mounted on a cryostat and sectioned. Sections were collected on glass slides, fixed, and then stained with the TUNEL assay kit (Thermo Fisher) and DAPI. Slides were analyzed under a microscope. Tissue microarray was purchased from Tissue Array company. Slides were stained with an antibody against DKK1, HLA-A2, and DKK1-A2 as described above and analyzed under a microscope. Quantitative data of the DKK1-A2 complex of tissue microarray were analyzed and obtained by ImageJ software.

Cytokine release syndrome model
For the cytokine release syndrome model in vitro, a tripartite coculture of CAR-T cells, autologous monocytes, and JeKo-1 tumor cells were performed for 24 h at a ratio of 1:1:144. Supernatants were collected, and human IFN-\gamma and IL-6 were measured by ELISA. For cytokine release syndrome model in vivo45, SCID mice were inoculated with JeKo-1 tumor cells intraperitoneally for 2 weeks followed by a large dose (50 \times 10^6 per mouse) of CAR-T cells infusion intraperitoneally. Peritoneal lavage was collected 3 days post CAR-T cells infusion, and human IFN-\gamma and murine IL-6 were measured by ELISA.

Hematologic toxicity assay
For in vitro hematologic toxicity assay, CAR-T cells were co-cultured with PBMC or BMMC for 4 h at a ratio of 1:1. Cultured cells were stained with anti-human CD11b, CD11c, CD3, CD4, CD8, CD19, NK1.1, CD34, and CD71 antibodies. Cell apoptosis was determined by staining the cells with anti-human Annexin V or Caspase 3 antibodies and analyzed by flow cytometry. For in vivo hematologic toxicity assay, hDKK1-A2 mice were infused with murine CAR-T cells intravenously. Blood, spleen, and bone marrow were collected on 7 days post CAR-T cell infusion. Single cell suspension was prepared and stained with anti-mouse CD11b, CD11c, CD3, CD4, CD8, CD19, and NK1.1 antibodies. Cell counts were normalized by counting beads and analyzed by flow cytometry. For the platelet cell count experiment, hDKK1-A2 mice were transferred with control, CD19, mC2, or hC2 CAR-T cells. One week after CAR-T cell transfer, whole blood was collected, and platelets were stained with CD41 and CD61 antibodies. Platelet cell count was analyzed by flow cytometry.

Statistical analysis
All graphs and statistical analysis were generated by GraphPad Prism software. Student’s t test or ANOVA analysis was performed for statistical analysis. Log-rank test was performed for survival analysis. Simple linear regression was performed for correlation analysis. A p value <0.05 was considered statistically significant. Results are presented as mean \pm SEM unless otherwise indicated.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review of the Manuscript: “T Cells Engineered against Dickkopf-1-A2 Complex Can Be Used to Treat HLA-A2⁺ Solid and Hematologic Cancers”\nIn this manuscript, the authors describe the generation and evaluation of CAR T cells targeting the DKK1-P20 peptide presented in the context of HLA-A2, using a previously described C2 monoclonal antibody. Both murine (mC2) and humanized (hC2) CAR constructs are developed and tested for antigen-specific cytotoxicity. The study includes in vitro assays, xenograft models using cell lines, and limited testing in patient-derived xenografts (PDXs). The authors also explore potential off-tumor toxicity and cytokine release in both in vitro and in vivo settings.\nWhile the targeting strategy leveraging HLA-A2-restricted DKK1 expressionrepresents a potentially valuable approach for tumor-specific CAR T cell therapy, the manuscript has limitations that affect the strength of the conclusions and the translational relevance of the study. Addressing these concerns would significantly strengthen the claims and translational relevance of the work. .\nMajor points:\n1. Novelty and redundancy in expression analysis (fig. 1): the expression data and staining presented in Figure1 are very similar to those shown in a previous publication from the same group (Fig 1 of Qian, J., et al. (2024).). As such, the novelty of these data is limited.\n2. Additionally, while relative expression to normal tissues is shown, the authors do not provide absolute expression levels or the number of normal tissues tested, although more data is provided in fig 7. This is critical, as low expression in normal tissues does not equate to absence of expression. Of samples analyzed showing DKK1 expression, is there also correlation or data available to show HLA2 positivity of the DKK1 pos samples?.\n3. To strengthen the specificity claims, the authors could knock out DKK1 in HLA-A2+ tumor cell lines used in functional assays. Currently, no HLA-A2+ DKK1neg cell line is used as a negative control although irrelevant CAR is used in some assays. I would suggest including this critical control. The inclusion of DKK1-KO cells would be essential to validate antigen specificity.\n4. Authors generate a manufactured product that is 100% CAR+. This transduction efficiency does not reflect typical clinical manufacturing outcomes. This raises concerns about the physiological relevance of the results. The authors should consider lower MOIs or provide a rationale for the current transduction strategy. High transduction efficiency likely results in multiple viral integrations per cell and unnaturally high CAR expression levels. The authors should either reduce MOI or provide justification for the approach.\n5. The PDX models (Fig 4) are inadequately characterized. There is no validation that primary tumors and PDX passages (especially F3) retain the same cellular composition/tumor microenvironment or DKK1-HLA-A2 expression as primary tumors. The authors should provide side-by-side staining (e.g., mC2/hC2 vs. isotype) on primary tumors and F3 PDXs, along with gating strategies and markers to distinguish tumor cells from TME components. Are all the cells of PDAC TME retained in PDAC #1 and are the different cells composing the TME in these pdac samples expressing DKK1-A2?\n6. Tumor clearance in PDX models suggests either uniform target expression or enrichment for DKK1-A2⁺ cells during passaging. However, Fig 4 shows DKK1-A2 complexes expression and looks that positive tumors (#1 and #14) have negative cells. Please explain if all PDAC cells of F3 PDAC#1 used for in vivo express DKK1-A2\n7. Some of the cells look negative in the flow histograms included in the figure. However, tumors are completely cleared. Additionally, although the manuscript generalizes findings to “primary tumors,” only one primary PDAC sample underwent three passages and was used for efficacy testing in vivo. This limitation requires tempering or moderating the conclusions\n8. Authors inject high CAR T cell doses (up to 10⁷ cells/mouse) in the in vivo studies, despite 100% CAR⁺ cell products. This raises concerns about strength of the product. Moreover, this also raises concerns about xenogeneic GvHD. The authors should include toxicity metrics such as weight loss. Dosing inconsistencies (2×10⁶, 4×10⁶, 1×10⁷) also require clarification. Additionally, authors should explain dosing inconsistencies for u266 model since in fig 5 authors inject 2e6 cart/mouse and fig3 and in 6 use 4e6 car t per mouse. The rationale for this variability should be explained.\n9. Rechallenge studies lack naïve mouse controls, which are standard in tumor rejection models. It is unclear whether these experiments were repeated independently and how representative the data are. Clarify whether naïve controls were used in other experiments and provide full experimental details.\n10. In Fig. 6, phenotypic differences between mC2 and hC2 CAR T cells are presented without matched-donor statistics. The authors claim functional advantages for hC2 CARs but provide no direct functional comparison in the Jeko model. Without supporting in vitro or in vivo efficacy data, this remains speculative\n11. Fig. 7a and 7b should include quantitative data, not just representative images of TMAs. Panel b should be redesigned for clarity and visual consistency, suitable for a high-impact journal.\n12. Authors investigate potential toxicity of DKK10A2 CAR T cells in syngeneic models. Is the expression levels of HLA2 and DKK1 in these mice comparable to human samples? How is the expression regulated? Authors should show the expression levels in the potential target cells if aim to prove lack of toxicity.\n13. They use a CRS in vivo model to assess toxicity, and refer the Nature paper of Giavridis et al in 2018. In fact in that 2018 paper authors prove the role of macrophages and show weigh loss as one of the readouts. If there is crs in this model, authors should show weight loss, and actually a pos control of CRS since the levels of mIL-6 and other values look not too high compared to the manuscript referenced and actually macrophages and not monocytes trigger such phenotype.\nMinor points:\nIn Figure 2, the inclusion of untransduced (UTD) T cell controls alongside CAR19 T cells would strengthen the claims of antigen-specific cytotoxicity.\nThe authors demonstrate effective killing by mC2 CAR T cells compared to CAR19 T cells in Figure 2. However, incorporating UTD T cells as an additional control would further support the specificity of the observed effect.\nThe nomenclature for mC2 is inconsistent throughout the manuscript. The construct is initially referred to as DKK1A2 CAR and later as mC2. Please ensure consistent terminology throughout.\nColor coding for CAR types is also inconsistent across figures (e.g., CAR19 is shown in red in early figures, but hC2 is shown in red in later figures), making visual interpretation difficult. To avoid confusion, avoid using the same color for different groups across figures, even if they do not appear in the same panel.\nThe authors state that CAR T cells do not elicit significant cytotoxicity against PBMCs/BMMCs, but only Annexin V staining is shown. Additional assays (e.g., viability dyes, caspase activation, or cytotoxicity assays) would strengthen this conclusion. Furthermore, DKK1 and HLA-A2 expression in these samples should be assessed by flow cytometry using the C2 antibody.\nThe section title “DKK1-A2 CAR-T cells generated from humanized C2 antibody” is awkward. A clearer title might be “Generation of DKK1-A2 CAR T cells using a humanized C2 antibody.”\nDosing units should be presented consistently throughout the manuscript (e.g., use either 1×10⁷ or 10×10⁶ uniformly).\nCell line nomenclature is inconsistent across figures (e.g., CFPAC-1 vs. CFPAC-1-luc). The authors should standardize this terminology and clarify whether the same cell lines are used across in vitro and in vivo assays. Additionally, bioluminescence data appear to be missing for CFPAC-1-luc in the in vivo experiments.\nIn figures measuring immunoglobulin lambda (Igλ) chain levels, some panels show individual tumor values while others show only averages. Please harmonize data presentation across figures for clarity and consistency.", "reviewer_2": "This manuscript demonstrates that CAR-T cell therapy targeting Dickkopf-1 (DKK1) is effective against HLA-A2-positive solid and hematologic cancers. DKK1 plays a role as an antagonist of the Wnt/β-catenin pathway and its overexpression in a variety of tumors has been observed. In particular, high expression of DKK1 is associated with malignant tumor progression. In certain tumors, DKK1 also promotes tumor progression by creating a microenvironment that affects immune response.\nThe authors have previously reported that the P20 (ALGGHPLLGV) peptide of DKK1 protein shows excellent binding affinity to HLA-A0201. The authors also revealed that DKK1 P20 peptide bound to HLA-A2 were presented as natural antigen on the surface of multiple myeloma cell that simultaneously expressed DKK1 and HLA-A2. The authors then developed a murine monoclonal antibody (C2 antibody) that specifically recognizes the DKK1 P20-HLA-A2 complex expressing on the tumor cell surface and reported that this antibody alone exerts antitumor effects against various human cancer cells. Based on the previous research works, the authors generated novel DKK1-A2 CAR-T cells which specifically recognize and destroy tumor cells expressing DKK1-A2 complex in this manuscript.\nDKK1-A2 CAR-T cells expressing demonstrated prominent anti-tumor effects against various tumor cell lines both in vitro and in vivo. DKK1-A2 CAR-T cells showed long-term antitumor effect via induction of memory cell population without severe damages to normal tissues or obvious hematologic toxicity. Since DKK1 is expressed in various tumors but not in normal organs, DKK1-A2 CAR-T therapy developed by the authors may be a promising new therapeutic modality.\nThe data presented are clear and convincing, while the reviewer needs to point out that this manuscript, as a preclinical model study for CAR-T cell based therapeutics, is still lacking in some respects. Given that one of the major challenges in applying CAR-T cell therapy to solid tumors is overcoming the immunosuppressive tumor microenvironment and poor trafficking of T cells into tumor tissues, the efficacy observed in the NSG xenograft model may not fully predict clinical success. The absence of myeloid cells, stromal components, and suppressive immune population in NSG mice raises questions about the translationability of the findings. There are several critiques to which the authors should address.\nMajor points\n1. If the efficacy of DKK1-A2 CAR-T in solid tumors is emphasized, validation in a more physiologically relevant model, such as a humanized mouse model with reconstituted human immune components or syngeneic models with murine CAR constructs, would strengthen the translational potential of the study.\n2. Lines 93 to 97. It is surprising that all tumors from 30 different cancer patients express DKK1 because several studies have shown that DKK1 expression levels vary by cancer types. DKK1-negative samples should be included in Fig. 1g-i as negative controls to ensure the reliability of these assay. In addition, immune histochemical study using isotype control Abs should be performed.\n3. Fig. 2f. It seems that CD19 CAR-T cells shows the cytotoxicity against H441 cells. The authors should comment on the results.\n4. The experiments with CD19 CAR-T cells are required in Fig. 3e as a control.\n5. Fig. 3g, k, and o demonstrated the trend of tumor size or IVIS signal intensity affected by CAR-T cells. The author should show the histological images of tumor specimen resected at the time point before tumor disappears and prove the DKK1-A2 CAR-T cell infiltration in the tumor tissues in couple with tumor apoptosis in xenograft tumor models.\n6. Fig. 4 and Extended Fig. 4 are important experiments. The authors should include DKK1-low expressing PDXs in the assay design. Histology of PDX and an IHC study of DKK1 should be shown in both low and high magnification\n7. The results Fig. 7a and b are hard to see. They are important data to consider the presence or absence of adverse effects. The authors mention that DKK1-A2 is highly expressed in the tonsil, but it is difficult to read from the figure. In addition, it would be better to show a magnified image of 7b in the extended data.\n8. Platelets express both high levels of DKK1 and MHC molecule [https://doi.org/10.1161/ATVBAHA.109.189761]. Does DKK1 expressed on the cell surface of platelet? Was there no thrombocytopenia induced by DKK1-A2 CAR-T cells? Simple blood cell count test to evaluate the platelet cell count using hDKK1-A2 mice should be performed in Fig. 7.\n9. This study is a preclinical model of novel CAR-T therapy. The authors should describe the limitation of this model more explicitly and mention the reasons why DKK1-A2 CAR-T is promising for broad range of solid tumors where CAR-T has previously been ineffective in Discussion. In addition, the possible immunosuppressive mechanism by DKK1, including the signal transduction system, should be discussed.\nMinor points\n1. Fig. 1f-i. Appropriate scale bars should be included in the panels.\n2. For all IHC analysis, the antibodies used and their concentrations should be precisely described in the “Methods” section to ensure reproducibility.\n3. The details of FACS analysis in Fig. 2d should be described in “Methods” section. They need to include the antibodies used, their concentration, protocols for membrane permeabilization and fixation, and conjugation of fluorescence dye to antibodies.\n4. The explanation for the “percentage (%)” in y-axis of each panel should be described in the legend of Fig. 4k.\n5. The explanation for “Tn/Tscm/Tcm/Tem/Temra” in the panel should be described in the legend of Fig. 6c and d. It should state what the “percentage (%)” to what is shown on the y-axis.", "reviewer_3": "This manuscript reports the development of DKK1-A2 CAR-T cells that selectively target the DKK1-P20 peptide in complex with HLA-A2, a combination highly expressed in hematologic and solid tumours, but largely absent from normal tissues. The authors demonstrate that these engineered T cells exhibit potent and specific antitumor activity in vitro and xenograft models, including patient-derived xenografts (PDX), while minimizing off-tumor toxicity. The study also compares murine and humanized scFvs, showing improved persistence and a central memory phenotype with the humanized version. Their results suggest the applicability of DKK1-A2 CAR-T cells in the treatment of human cancer. The manuscript is timely, well-structured, and of high translational relevance.\nGiven that DKK1 is a secreted protein, the potential for soluble DKK1 to interfere with CAR binding or trigger tonic signaling is not explored. Could the authors clarify whether this was evaluated in vitro, particularly with regard to background activation or impaired target recognition?\nThe authors provide subset-resolved intracellular cytokine data (% IFN-γ⁺ and TNF-α⁺) for both CD4⁺ and CD8⁺ CAR-T cells across multiple tumor targets, which is appreciated. If feasible, reporting absolute cytokine concentrations (e.g., pg/mL) from bulk culture supernatants via ELISA or multiplex assay would complement these findings and support a functional comparison of the two subsets.\nOff-target effects due to cross-reactive peptide–HLA complexes are a known concern in TCR-mimic CAR-T approaches. While the authors report no reactivity against healthy HLA-A2⁺ PBMCs and BMMCs, has the CAR been tested against a broader panel of primary human cells or iPSC-derived tissues (e.g., cardiomyocytes, hepatocytes)? Alternatively, the authors may consider discussing limitations of the current off-target screening strategy.\nThe authors note that the hC2 antibody has lower binding affinity to the DKK1-A2 complex than the original murine C2 antibody, which they propose as a possible explanation for the improved persistence and central memory phenotype of hC2 CAR-T cells. While this is a reasonable hypothesis, it would strengthen the mechanistic interpretation if quantitative binding data (e.g., KD values from SPR, BLI, or ELISA) were provided to directly compare the affinities of hC2 and mC2 antibodies.\nThe current CAR is restricted to HLA-A2, which, while prevalent (~30–50% worldwide), does not provide universal coverage. Have the authors considered whether other DKK1-derived peptides might be presented by additional HLA alleles? Addressing this in the Discussion as a future direction would strengthen the translational scope.\nThe expression of the DKK1-A2 complex in tonsils is briefly mentioned but not discussed in terms of clinical safety. As tonsils are lymphoid tissues that may be exposed to circulating T cells, it would be helpful to clarify whether this represents a potential site of on-target off-tumor toxicity.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers’ Comments\nWe would like to thank the reviewers for careful and thorough reading of this manuscript and for\nthe thoughtful comments and constructive suggestions, which have helped to improve the\nquality of this manuscript. The corresponding changes and refinements made in the revised\npaper are summarized in our response below. Please note that the specified changes or\nmodifications in the revised version of the manuscript are highlighted by red color.\nReviewer #1 (Remarks to the Author):\nMajor points:\n1. Novelty and redundancy in expression analysis (fig. 1): the expression data and staining\npresented in Figure1 are very similar to those shown in a previous publication from the same\ngroup (Fig 1 of Qian, J., et al. (2024).). As such, the novelty of these data is limited.\nThe expression profiles of DKK1 and DKK1-A2 complex are essential for these studies, and we\nmake sure that no similar data are presented in these two manuscripts. The expression data in\nFig. 1 highlighting the expressions of DKK1 and DKK1-A2 complex in solid tumors are different\nfrom previous publication (Qian, J., et al. 2024). We employed IHC staining of DKK1 and DKK1-\nA2 complex on pancreatic cancer, lung cancer and triple negative breast cancer, and also used\ncommercial tissue array consisting randomly selected 40 lung cancers and 10 normal lung\ntissues to confirm the expression profile. In addition, we analyzed overall and disease-free\nsurvival correlation of some selected solid tumors. These data indicate the high expression of\nDKK1 and DKK1-A2 complex on solid tumors, which were not included in our previous\npublication. We believe that these data, together with the expression data from our previous\npublication, collectively present a comprehensive expression profile of DKK1 and DKK1-A2 in\nboth hematologic and solid tumors.\n2. Additionally, while relative expression to normal tissues is shown, the authors do not provide\nabsolute expression levels or the number of normal tissues tested, although more data is\nprovided in fig 7. This is critical, as low expression in normal tissues does not equate to absence\nof expression. Of samples analyzed showing DKK1 expression, is there also correlation or data\navailable to show HLA2 positivity of the DKK1 pos samples?\nWe appreciate the comments. Data showing relative expressions of DKK1 were generated from\nmRNA sequencing data of public databases, which makes it impossible to obtain absolute\nexpression levels. In addition, HLA typing is usually done using whole-genome sequencing and\nwhole-exome sequencing, so there is no available data to analyze the correlation between HLA-\nA2 and DKK1 expression based on these datasets. According to our previous publications\n(Qian, J., et al. Blood. 2007) and normal tissue array in Fig. 7, DKK1 is not expressed in most\nnormal tissues except tonsils. Based on the suggestion, we have updated the number of tissues\ntested in Fig. 1 legend.\n3. To strengthen the specificity claims, the authors could knock out DKK1 in HLA-A2+ tumor cell\nlines used in functional assays. Currently, no HLA-A2+ DKK1neg cell line is used as a negative\ncontrol although irrelevant CAR is used in some assays. I would suggest including this critical\ncontrol. The inclusion of DKK1-KO cells would be essential to validate antigen specificity.\nWe appreciate the comments. We have now generated DKK1-knockout JeKo-1 tumor cells\nusing CRISPR/Cas9 gene editing. As expected, DKK1-A2 CAR-T cells showed markedly\nreduced cytotoxicity and proliferation against DKK1-knockout tumor cells compared to the wild-\ntype tumor cells. These new data are now shown as Supplementary Fig. 2f-h.\n4. Authors generate a manufactured product that is 100% CAR+. This transduction efficiency\ndoes not reflect typical clinical manufacturing outcomes. This raises concerns about the\nphysiological relevance of the results. The authors should consider lower MOIs or provide a\nrationale for the current transduction strategy. High transduction efficiency likely results in\nmultiple viral integrations per cell and unnaturally high CAR expression levels. The authors\nshould either reduce MOI or provide justification for the approach.\nCAR-T cells in this study were generated from health donors, and T cell transduction efficiency\nin health donor T cells are often higher than that from tumor patients, as tumor patients often\npresent impaired T cell function and quality. In fact, T cell transduction efficiency in this study\nranged from 50% to 90% depending on different donors, and the best transduction efficiency\ndata was shown in Fig. 2. We have now revised Fig. 2c to show a more representative T cell\ntransduction efficiency data (around 70%) and the Results section to clarify potentially\nmisleading information about T cell transduction efficiency.\n5. The PDX models (Fig 4) are inadequately characterized. There is no validation that primary\ntumors and PDX passages (especially F3) retain the same cellular composition/tumor\nmicroenvironment or DKK1-HLA-A2 expression as primary tumors. The authors should provide\nside-by-side staining (e.g., mC2/hC2 vs. isotype) on primary tumors and F3 PDXs, along with\ngating strategies and markers to distinguish tumor cells from TME components. Are all the cells\nof PDAC TME retained in PDAC #1 and are the different cells composing the TME in these\npdac samples expressing DKK1-A2?\nWe appreciate the comments. To characterize the PDX model more adequately, we analyzed\ntumor microenvironment of PDAC #1 primary and F3 tumors by flow cytometry. The results\nshowed that F3 tumor retained the same tumor composition as primary tumor, and both of\nprimary and F3 tumor highly expressed CEA, DKK1, HLA-A2 and DKK1-A2 complex. The\nresults were also confirmed by immunochemistry staining of primary and F3 tumors in both low\nand high magnification. The new data with flow along with gating strategies and IHC images are\nnow shown as Supplementary Fig. 5c-f.\n6. Tumor clearance in PDX models suggests either uniform target expression or enrichment for\nDKK1-A2⁺ cells during passaging. However, Fig 4 shows DKK1-A2 complexes expression and\nlooks that positive tumors (#1 and #14) have negative cells. Please explain if all PDAC cells of\nF3 PDAC#1 used for in vivo express DKK1-A2.\nWe appreciate the comments. We re-analyzed flow data of Fig. 4a to exclude debris and dead\ncells, and the data showed that all PDAC cells of PDAC#1 and #14 expressed DKK1-A2\ncomplex. The new results are shown as Fig. 4a.\n7. Some of the cells look negative in the flow histograms included in the figure. However, tumors\nare completely cleared. Additionally, although the manuscript generalizes findings to “primary\ntumors,” only one primary PDAC sample underwent three passages and was used for efficacy\ntesting in vivo. This limitation requires tempering or moderating the conclusions\nWe appreciate the comments. PDX tumors were completely cleared post CAR-T cells infusion\nas all PDAC cells expressed DKK1-A2 complex according to re-analyzed flow data of Fig. 4a.\nIndeed, only one primary PDAC model was used for in vivo efficacy experiment, mainly due to\nthe difficulties and long time to establish stable PDX models. We have revised the related\nDiscussion to moderate the conclusions and state this limitation.\n8. Authors inject high CAR T cell doses (up to 10⁷ cells/mouse) in the in vivo studies, despite\n100% CAR⁺ cell products. This raises concerns about strength of the product. Moreover, this\nalso raises concerns about xenogeneic GvHD. The authors should include toxicity metrics such\nas weight loss. Dosing inconsistencies (2×10⁶, 4×10⁶, 1×10⁷) also require clarification.\nAdditionally, authors should explain dosing inconsistencies for u266 model since in fig 5 authors\ninject 2e6 cart/mouse and fig3 and in 6 use 4e6 car t per mouse. The rationale for this variability\nshould be explained.\nWe appreciate the comments. In this study, we typically used a dose of 10×106 for solid tumor\ntreatment and a moderate dose of 4×106 for myeloma treatment, as solid tumors often present\nphysical, metabolic and immunologic barriers and show inferior efficacy compared to\nhematologic tumors. We believe that our CAR-T dosing strategies are similar to previous\npublications, eg. Du, H., Cancer Cell. 2019; Liu, L., Nature Commun. 2020. A preclinical study\n(Milone, M,. Molecular Therapy. 2009) of CTL019 (later KYMRIAH), the first FDA-approved\nCAR-T cell therapy, showed most leukemia clearance post 5×106 CAR-T cell injection. Since\nDKK1-A2 complexes have limited expression on normal tissues, and moderate doses of CAR-T\ncells were used, symptoms related to GvHD were not observed during our animal study.\nNevertheless, safety is still a major concern in this study, and we have included weight loss data\nin Fig. 3.\nWe fully understand the concern about dosing inconsistencies for U266 models. Generally,\n4×106 CAR-T cells were used in U266 models as shown in Fig. 3 and Fig. 6, and we did not\nobserve any tumor relapse over more than 3 months. To compare in vivo efficacy and tumor\nrelapse between hC2 and mC2 CAR-T cells within a short period of time (less than 3 months), a\nlower dose of 2×106 CAR-T cells were used in Fig. 5 U266 experiment.\n9. Rechallenge studies lack naïve mouse controls, which are standard in tumor rejection\nmodels. It is unclear whether these experiments were repeated independently and how\nrepresentative the data are. Clarify whether naïve controls were used in other experiments and\nprovide full experimental details.\nWe appreciate your comments. In the U266 rechallenge studies, naïve mouse controls were\nincluded in another experiment. The new data are now shown as Supplementary Fig. 7c-e. In\naddition, we have provided experimental details in the Methods section.\n10. In Fig. 6, phenotypic differences between mC2 and hC2 CAR T cells are presented without\nmatched-donor statistics. The authors claim functional advantages for hC2 CARs but provide no\ndirect functional comparison in the Jeko model. Without supporting in vitro or in vivo efficacy\ndata, this remains speculative.\nWe appreciate the comments. We have re-analyzed the data using matched-donor and paired\nstatistical tests and updated the new data in Fig. 6b,d. This matched-donor analysis confirms\nthe significance of the phenotypic differences observed. In addition, we performed in vivo\ntherapeutic experiment in Jeko-1 bearing mice. The data showed a long-term persistence and\nlower relapse after hC2 CAR-T cell therapy compared to mC2 CAR-T cells. These data further\nsupport the functional advantages of hC2 CARs. We have updated the new data in Fig. 6k-n.\n11. Fig. 7a and 7b should include quantitative data, not just representative images of TMAs.\nPanel b should be redesigned for clarity and visual consistency, suitable for a high-impact\njournal.\nWe appreciate the comments. We have included quantitative data of normal tissue array in\nSupplementary Fig. 8b. For clarity and visual consistency, previous Fig. 7b has been\nredesigned and moved to Supplementary Fig. 8a with magnified image.\n12. Authors investigate potential toxicity of DKK10A2 CAR T cells in syngeneic models. Is the\nexpression levels of HLA2 and DKK1 in these mice comparable to human samples? How is the\nexpression regulated? Authors should show the expression levels in the potential target cells if\naim to prove lack of toxicity.\nWe appreciate the comments. DKK1 expression and regulation in mice are very similar with that\nin human (Lieven, O., et al. Developmental Biology. 2010). Briefly, DKK1 is highly expressed\nduring embryonic development and then silenced in almost all adult tissues. In pathological\nconditions, DKK1 is frequently upregulated in various tumors, osteolytic lesion and some\ninflammatory diseases. In order to obtain DKK1-A2 transgenic mice, firstly we knocked in\nhuman DKK1 gene into the mouse DKK1 allele to obtain DKK1 transgenic mice. Then DKK1\ntransgenic mice were crossing with HLA-A2 transgenic mice from the Jackson Lab. The\nexpressions of DKK1 and HLA-A2 in DKK1-A2 transgenic mice are shown below. Very similar to\nnormal human tissues, DKK1 expression is absent from all selected tissues but not nasal-\nassociated lymphoid tissue (NALT).\n13. They use a CRS in vivo model to assess toxicity, and refer the Nature paper of Giavridis et\nal in 2018. In fact in that 2018 paper authors prove the role of macrophages and show weigh\nloss as one of the readouts. If there is crs in this model, authors should show weight loss, and\nactually a pos control of CRS since the levels of mIL-6 and other values look not too high\ncompared to the manuscript referenced and actually macrophages and not monocytes trigger\nsuch phenotype.\nWe appreciate the comments. To further strengthen our model, we have now included a positive\ncontrol group treated with CD19 CAR-T cells as described in Giavridis et al. Nature Medicine\n2018, and also, we showed weigh loss as readout in this CRS model. We have updated the\ndata in Fig. 7f.\nAnother concern regards monocytes used in in vitro CRS models. We co-cultured CAR-T cells\nwith monocytes and tumor cells, and IFN- and IL-6 were used as readouts. This model was\nadapted from Arcangeli et al. JCI. 2022 studies. The authors showed that CRS is associated\nwith monocyte activation. In addition, the Nature paper of Giavridis also showed that monocytes\ntogether with macrophages and dendritic cells were the main source of IL-6 that trigger CRS.\nMinor points:\n1. In Figure 2, the inclusion of untransduced (UTD) T cell controls alongside CAR19 T cells\nwould strengthen the claims of antigen-specific cytotoxicity.\nWe appreciate the comments. We have included non-transduced T cell control in Fig. 2f\ncytotoxicity assays.\n2. The authors demonstrate effective killing by mC2 CAR T cells compared to CAR19 T cells in\nFigure 2. However, incorporating UTD T cells as an additional control would further support the\nspecificity of the observed effect.\nWe appreciate the comments. Non-transduced T cell control in the cytotoxicity assays was\nupdated in Fig. 2f.\n3. The nomenclature for mC2 is inconsistent throughout the manuscript. The construct is initially\nreferred to as DKK1A2 CAR and later as mC2. Please ensure consistent terminology throughout.\nWe appreciate the comments. To avoid confusion, we have revised the manuscript to ensure\nconsistent use of terminology. We now refer to the construct uniformly as mC2 or hC2 CAR, and\nDKK1-A2 CAR as mC2 and hC2 CAR.\n4. Color coding for CAR types is also inconsistent across figures (e.g., CAR19 is shown in red in\nearly figures, but hC2 is shown in red in later figures), making visual interpretation difficult. To\navoid confusion, avoid using the same color for different groups across figures, even if they do\nnot appear in the same panel.\nWe appreciate the comments. We have revised all figures to ensure that each CAR type is\nrepresented by a unique color. Non-transduced T cell is shown in black, CD19 CAR in green,\nmC2 CAR in red, and hC2 CAR in blue.\n5. The authors state that CAR T cells do not elicit significant cytotoxicity against\nPBMCs/BMMCs, but only Annexin V staining is shown. Additional assays (e.g., viability dyes,\ncaspase activation, or cytotoxicity assays) would strengthen this conclusion. Furthermore, DKK1\nand HLA-A2 expression in these samples should be assessed by flow cytometry using the C2\nantibody.\nWe appreciate the comments. We have now performed the caspase activation assays by flow.\nThe results showed minimal caspase levels against PBMC and BMMC. In addition, DKK1 and\nHLA-A2 expression on PBMC and BMMC were also assessed by flow. These new data are\nupdated to Supplementary Fig. 8c-g.\n6. The section title “DKK1-A2 CAR-T cells generated from humanized C2 antibody” is awkward.\nA clearer title might be “Generation of DKK1-A2 CAR T cells using a humanized C2 antibody.”\nWe appreciate the comments. The section title is revised to “Generation of DKK1-A2 CAR T\ncells using a humanized C2 antibody.”\n7. Dosing units should be presented consistently throughout the manuscript (e.g., use either\n1×10⁷ or 10×10⁶ uniformly).\nWe appreciate the comment. We have presented the dosing units consistently as ×10⁶.\n8. Cell line nomenclature is inconsistent across figures (e.g., CFPAC-1 vs. CFPAC-1-luc). The\nauthors should standardize this terminology and clarify whether the same cell lines are used\nacross in vitro and in vivo assays. Additionally, bioluminescence data appear to be missing for\nCFPAC-1-luc in the in vivo experiments.\nWe appreciate the comments. We have reviewed and standardized the cell line nomenclature\nthroughout the manuscript. Specifically, CFPAC-1-Luc refers to the luciferase-expressing\nvariant used both in vitro and in vivo. Bioluminescence data for CFPAC-1-luc are included in\nFig. 3f and Supplementary Fig. 3f.\n9. In figures measuring immunoglobulin lambda (Igλ) chain levels, some panels show individual\ntumor values while others show only averages. Please harmonize data presentation across\nfigures for clarity and consistency.\nWe appreciate the comments. To improve clarity and consistency, we now display averages of\nimmunoglobulin lambda (Igλ) chain levels across all relevant figures.\nReviewer #2 (Remarks to the Author):\nMajor points\n1. If the efficacy of DKK1-A2 CAR-T in solid tumors is emphasized, validation in a more\nphysiologically relevant model, such as a humanized mouse model with reconstituted human\nimmune components or syngeneic models with murine CAR constructs, would strengthen the\ntranslational potential of the study.\nWe appreciate the comments. We agree that a more physiologically relevant solid tumor model\nwould strengthen the translational potential. To validate this, we established syngeneic models\nof DKK1-A2 transgenic mice bearing murine pancreatic cancer cell line Pan02 expressing\nhuman DKK1 and HLA-A2. Tumor-bearing mice were treated with DKK1-A2 or mCD19 murine\nCAR-T cells, tumor volume and body weight were monitored. The results showed that DKK1-A2\nbut not CD19 murine CAR-T cells effectively controlled tumor growth in murine Pan02-bearing\nmice, and CAR-T cell infusion did not cause weight loss in all mice groups. The new data are\nshown as Supplementary Fig. 9d-f.\n2. Lines 93 to 97. It is surprising that all tumors from 30 different cancer patients express DKK1\nbecause several studies have shown that DKK1 expression levels vary by cancer types. DKK1-\nnegative samples should be included in Fig. 1g-i as negative controls to ensure the reliability of\nthese assay. In addition, immune histochemical study using isotype control Abs should be\nperformed.\nWe appreciate the comments. We agree that DKK1 expression varies among different cancer\ntypes as shown by DKK1 mRNA sequencing data in Fig. 1a-b. Among all the cancer types,\npancreatic, lung and triple negative breast cancer express the highest levels of DKK1. The\ncommercial lung cancer tissue array in Supplementary Fig. 1e showed that all 40 randomly\nselected lung cancers express DKK1, but none of 10 normal lung tissues does. To ensure the\nreliability, isotype control and DKK1-negative sample were performed and the data are updated\nin Supplementary Fig. 1f-g.\n3. Fig. 2f. It seems that CD19 CAR-T cells shows the cytotoxicity against H441 cells. The\nauthors should comment on the results.\nWe appreciate the comments. CD19 CAR-T cells should not have shown cytotoxicity against\nH441 lung cancer, as H441 cells do not express CD19. The confusing results might be caused\nby experiment error. We have repeated this cytotoxicity assay and no obvious cytotoxicity\nagainst H441 cells was observed. The new data are shown in Fig. 2f.\n4. The experiments with CD19 CAR-T cells are required in Fig. 3e as a control.\nWe appreciate the comments. We used CFPAC-1 PDAC orthotopically xenografted models in\nFig. 3e. It was really hard to establish this model as CFPAC-1 tumor cells were implanted into\nthe pancreas of the mice. Due to the limited number of model mice, CD19 CAR-T cell control\nwas not included in this experiment. While in other pancreatic cancer mice models\n(Supplementary Fig. 3e and Fig. 4h), experiments were strictly conducted using CD19 CAR-T\ncell control.\n5. Fig. 3g, k, and o demonstrated the trend of tumor size or IVIS signal intensity affected by\nCAR-T cells. The author should show the histological images of tumor specimen resected at the\ntime point before tumor disappears and prove the DKK1-A2 CAR-T cell infiltration in the tumor\ntissues in couple with tumor apoptosis in xenograft tumor models.\nWe appreciate the comments. To validate CAR-T cell infiltration and tumor cell apoptosis, we\ncollected tumor tissues at the end point. Tumor tissues were freshly frozen and stained with\nTUNEL Alexa Fluor 594. The results revealed that tumors treated with mC2 CAR-T cells\nshowed CAR-T cell infiltration coupled with tumor cell apoptosis. The new data are updated in\nSupplementary Fig. 4a-c.\n6. Fig. 4 and Extended Fig. 4 are important experiments. The authors should include DKK1-low\nexpressing PDXs in the assay design. Histology of PDX and an IHC study of DKK1 should be\nshown in both low and high magnification.\nWe appreciate the comments. We agree that DKK1-low PDX model should be designed as a\ncontrol in this experiment. However, as pancreatic cancer patients express highest level of\nDKK1, we have not yet identified DKK1-low pancreatic cancer samples in all collected\npancreatic cancer samples. Nevertheless, in response to Reviewer #1 third comment, we have\ngenerated DKK1-knockout JeKo-1 tumor cells using CRISPR/Cas9 gene editing. As expected,\nDKK1-A2 CAR-T cells showed markedly reduced cytotoxicity and proliferation against DKK1-\nknockout tumor cells compared to the wild-type tumor cells. These new data are now shown as\nSupplementary Fig. 2f-h. Although these are different tumors, the new data do indicate that\ntumor-expressing DKK1 protein is crucial for CAR-T cell-mediated cytotoxicity against the tumor\ncells.\nBased on the suggestion, histology and IHC of PDX in both low and high magnification are\nupdated in Supplementary Fig. 5e,f.\n7. The results Fig. 7a and b are hard to see. They are important data to consider the presence\nor absence of adverse effects. The authors mention that DKK1-A2 is highly expressed in the\ntonsil, but it is difficult to read from the figure. In addition, it would be better to show a magnified\nimage of 7b in the extended data.\nWe appreciate the comments. We provide clearer images of normal tissue array in Fig. 7a, and\nformer Fig. 7b is moved to Supplementary Fig. 8a with magnified image. In addition,\nquantitative data of DKK1-A2 expression on normal tissue array is updated in Supplementary\nFig. 8b to present data more clearly.\n8. Platelets express both high levels of DKK1 and MHC molecule\n[https://doi.org/10.1161/ATVBAHA.109.189761]. Does DKK1 expressed on the cell surface of\nplatelet? Was there no thrombocytopenia induced by DKK1-A2 CAR-T cells? Simple blood cell\ncount test to evaluate the platelet cell count using hDKK1-A2 mice should be performed in Fig.\n7.\nWe appreciate the comments. Platelets have been reported to express DKK1, as well as MHC\nmolecules. To address the potential on-target, off-tumor effect of DKK1-A2 CAR-T cells on\nplatelets, we monitored peripheral platelet cell count in hDKK1-A2 transgenic mice following\nCAR-T cell infusion. As shown in Supplementary Fig. 9g, no significant low platelet count was\nobserved, suggesting that DKK1-A2 CAR-T cells do not cause substantial depletion of\ncirculating platelets.\n9. This study is a preclinical model of novel CAR-T therapy. The authors should describe the\nlimitation of this model more explicitly and mention the reasons why DKK1-A2 CAR-T is\npromising for broad range of solid tumors where CAR-T has previously been ineffective in\nDiscussion. In addition, the possible immunosuppressive mechanism by DKK1, including the\nsignal transduction system, should be discussed.\nWe appreciate the comments. We have described the limitations of models and therapeutic\npotentials in solid tumors in Discussion section.\nMinor points\n1. Fig. 1f-i. Appropriate scale bars should be included in the panels.\nWe appreciate the comments. Scale bars are included in Fig. 1f-i.\n2. For all IHC analysis, the antibodies used and their concentrations should be precisely\ndescribed in the “Methods” section to ensure reproducibility.\nWe appreciate the comments. Antibodies and concentrations for IHC are now described in\nMethods section.\n3. The details of FACS analysis in Fig. 2d should be described in “Methods” section. They need\nto include the antibodies used, their concentration, protocols for membrane permeabilization\nand fixation, and conjugation of fluorescence dye to antibodies.\nWe appreciate the comments. Antibodies, concentrations and protocols for Fig. 2d are now\ndescribed in Methods section.\n4. The explanation for the “percentage (%)” in y-axis of each panel should be described in the\nlegend of Fig. 4k.\nWe appreciate the comments. The percentage (%) means the percent eGFP-expressing CAR-T\ncells in blood cells or splenocytes or bone marrow cells. We have now included these\ninformation in Legend of Fig. 4k.\n5. The explanation for “Tn/Tscm/Tcm/Tem/Temra” in the panel should be described in the\nlegend of Fig. 6c and d. It should state what the “percentage (%)” to what is shown on the y-\naxis.\nWe appreciate the comments. We have now described the memory phenotype and meaning of\npercentage (%) in the Legend of Fig. 6a-d.\nReviewer #3 (Remarks to the Author):\n1. Given that DKK1 is a secreted protein, the potential for soluble DKK1 to interfere with CAR\nbinding or trigger tonic signaling is not explored. Could the authors clarify whether this was\nevaluated in vitro, particularly with regard to background activation or impaired target\nrecognition?\nWe appreciate the comments. DKK1-A2 CAR-T cells were generated from C2 antibody. As we\nshowed previously (Qian, J., et al. 2024), C2 antibody can only recognize DKK1-P20\n(ALGGHPLLGV) and HLA-A2 complex presented on the cell surface and should not bind with\nsoluble DKK1 or HLA-A2 or DKK1-P20 peptide. Therefore, soluble DKK1 would not trigger tonic\nsignaling or background activation on CAR-T cells. Nevertheless, based on the comment, we\nevaluated CAR-T cell activation in the content of different concentrations of soluble DKK1\novernight, and PMA/Iono were used as a positive control. IFN- and TNF- levels were\nanalyzed by flow, and background activation or impaired target recognition were not detected.\n2. The authors provide subset-resolved intracellular cytokine data (% IFN-γ⁺ and TNF-α⁺) for\nboth CD4⁺ and CD8⁺ CAR-T cells across multiple tumor targets, which is appreciated. If\nfeasible, reporting absolute cytokine concentrations (e.g., pg/mL) from bulk culture supernatants\nvia ELISA or multiplex assay would complement these findings and support a functional\ncomparison of the two subsets.\nWe appreciate the comments. To functionally compare these two subsets, CD4+ and CD8+\nCAR-T cells were separated by flow sorting, then co-cultured with multiple tumor cells, and\ncytokine secretion was analyzed by ELISA. The data showed specific IFN- and TNF-\nsecretion which suggested similar results compared to flow data. The new data are updated in\nSupplementary Fig. 2i,j.\n3. Off-target effects due to cross-reactive peptide–HLA complexes are a known concern in TCR-\nmimic CAR-T approaches. While the authors report no reactivity against healthy HLA-A2⁺\nPBMCs and BMMCs, has the CAR been tested against a broader panel of primary human cells\nor iPSC-derived tissues (e.g., cardiomyocytes, hepatocytes)? Alternatively, the authors\nmay consider discussing limitations of the current off-target screening strategy.\nWe appreciate the comments. Our current study demonstrated no reactivity against a limited\npanel of PBMC and BMMC. Actually, we are planning into future studies to better characterize\npotential off-target risks by using primary human cells. Based on the suggestion, we have added\na discussion section in the revised manuscript outlining these limitations of the current off-target\nscreening strategy and emphasizing the need for comprehensive evaluation.\n4. The authors note that the hC2 antibody has lower binding affinity to the DKK1-A2 complex\nthan the original murine C2 antibody, which they propose as a possible explanation for the\nimproved persistence and central memory phenotype of hC2 CAR-T cells. While this is a\nreasonable hypothesis, it would strengthen the mechanistic interpretation if quantitative binding\ndata (e.g., KD values from SPR, BLI, or ELISA) were provided to directly compare the affinities\nof hC2 and mC2 antibodies.\nWe appreciate the comments. We have performed quantitative binding assessments using\nSPR. The KD value (nM) of hC2 antibody was approximately tenfold larger than that of mC2\nantibody, indicating that hC2 exhibited lower binding affinity compared with mC2. We have\nupdated KD valve in the Discussion section.\nLigand Analyte ka (1/Ms) kd (1/s) KD (nM) Rmax (RU) Chi2 (RU2)\nmC2 DKK1-A2 2.35E+05 2.84E-04 1.82 41.54 0.0197\nhC2 DKK1-A2 3.42E+05 6.51E-04 19 66.6 0.619\n5. The current CAR is restricted to HLA-A2, which, while prevalent (~30–50% worldwide), does\nnot provide universal coverage. Have the authors considered whether other DKK1-derived\npeptides might be presented by additional HLA alleles? Addressing this in the Discussion as a\nfuture direction would strengthen the translational scope.\nWe appreciate the comments. Yes, we are working on screening new antibodies that recognize\nother HLA alleles presenting DKK1 peptides. We have addressed this future direction in the\nDiscussion section.\n6. The expression of the DKK1-A2 complex in tonsils is briefly mentioned but not discussed in\nterms of clinical safety. As tonsils are lymphoid tissues that may be exposed to circulating T cells,\nit would be helpful to clarify whether this represents a potential site of on-target off-tumor\ntoxicity.\nWe appreciate the comments. We observed that human tonsils express DKK1-A2 complex and\nwe agree that target expression in tonsils may present possible safety concern. We think that\nthe possible reason would be tonsil-related inflammation. We have clarified the potential site of\non-target off-tumor toxicity in the Discussion section.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my previous concerns, often with substantial new experimental data, re-analysis, and figure revisions. Importantly, the major issues that required additional evidence, such as the inclusion of DKK1-KO control, improved PDX model characterization, matched-donor statistical analyses, and refinements to the CRS model, were resolved.\nSeveral sections that previously required clarification were meaningfully improved. In particular, the expanded heterologous tumor analyses and survival correlations in Figure 1 provide better context for the clinical relevance of DKK1 expression, and the authors’ moderation of claims in the Discussion enhances the overall balance of the work.\nOverall, I believe the manuscript has been substantially improved and meets the standards for publication.", "reviewer_2": "I confirmed that all my comments and critiques have been thoroughly addressed and am satisfied with this revised version. I have no further comments.", "reviewer_3": "The authors have fully addressed all my concerns. The newly added experimental data and thoughtful clarifications significantly strengthen the manuscript. I support its publication in its current form.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter