Enhancing CAR- and TCR-mediated targeting of cancer via an immune synapse-stabilizing receptor

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: major_revision

Abstract
Heterogeneity in antigen expression on cancer cells limits clinical benefit of engineered T-cell therapies and underpins treatment resistance through antigen escape. Here, we present a strategy to improve recognition and lysis of tumors with suboptimal antigen expression through a separate engineered receptor that selectively boosts cytotoxic signaling and immune synapse formation. This synapse-stabilizing receptor (SSR) harbors a modified linker for activation of T cells (LAT) endodomain that amplifies CD3ζ signaling upon binding to a secondary tumor antigen resulting in an augmented Ca2+ flux, activation of MAPK and NF-kB signaling, maturation of immune synapse, and enhanced T-cell degranulation. Removing C-terminal amino acids 178-233 in the LAT endodomain (LAT177) was necessary to minimize SSR-mediated cytotoxicity. In models of acute myeloid leukemia, we show that a CD38-targeting SSR boosts cytolysis of antigen-low cancer cells via a C-type lectin-like molecule-1 (CLL1)-specific chimeric antigen receptor (CAR) and a survivin-specific T cell receptor (TCR). Unlike another CAR, SSR does not produce significant cytotoxicity against normal CD38+ tissues. Our study thus shows that SSR arming enhances targeting of antigenically heterogeneous cancers without compromising safety and selectivity of therapeutic T cells.

Introduction
Chimeric antigen receptor (CAR) T cells can effectively target cancers with high and uniform antigen expression and produce immense clinical benefit in patients with lymphoid malignancies. Other cancers, including acute myeloid leukemia (AML) and many solid tumors, remain resistant to CAR T-cell therapy, in part due to the lack of “safe” tumor-specific target antigens and high heterogeneity of their expression on cancer cells1–3. Unlike T-cell receptors (TCRs), which require only several peptide-MHC molecules to trigger T-cell activation, CARs must engage a large number of target antigen molecules on the cell surface to initiate sufficient signaling4. This reduced sensitivity of CARs can help minimize damage to normal tissues that express the target antigen at a low level; however, it also underpins a major mechanism of cancer evasion from the adoptive cell therapy where partial antigen downregulation on tumor cells is sufficient to confer resistance to CAR T cells5,6. Similarly, downregulation of surface HLA levels has been associated with tumor relapse following allogeneic stem cell transplants and TCR T-cell therapy7. Combinatorial antigen targeting using several cytotoxic receptors (or a single receptor with two or more antigen binding domains) has been devised to overcome single antigen escape8,9. However, this approach must be developed with caution as multi-antigen targeting can broaden off-tumor toxicities and thus require additional mitigation strategies to prevent unwanted long-term T-cell activity10.

TCR- and CAR-embedded CD3z chains elicit similar signaling pathways, where Lck-phosphorylated ITAMs on the CD3z molecule recruit ZAP-70 kinase which, upon activation, phosphorylates linker for activation of T cells (LAT)4. LAT serves as a main scaffold for the recruitment of phospholipase C gamma 1 (PLC-γ1) and other adapter molecules that propagate downstream signaling cascades and remodeling cytoskeleton to facilitate formation of a cytotoxic immune synapse11–13. Therefore, LAT serves as both a key intermediate and an amplifier of CD3ζ signaling thus directly regulating antigen receptor sensitivity.

Here, we hypothesize T-cell activation and cytolysis of antigen-low cancer cells can be augmented by recruiting LAT to CAR or TCR. We surmise this non-cytotoxic synapse-stabilizing receptor (SSR) does not trigger T-cell activation per se but instead amplify CD3ζ signaling from the main antigen receptor thus improving T-cell degranulation and target cell killing. To that end, we engineer LAT-based receptors harboring an extracellular domain targeting CD38 – a surface antigen abundantly expressed in leukemia as well as in critical normal blood cells and primitive progenitors. In this report, we use preclinical models of AML to show a CD38-specific SSR arming improves CAR- and TCR-mediated cytolysis of antigen-low leukemia without producing additional toxicity against healthy CD38+ tissues. Our results suggest this modification can be applied broadly to other antigens and disease settings to improve antigen sensitivity of therapeutic T cells.

Results
CD38.SSR harboring a LAT177 endodomain improved antigen-specific cytolysis of AML by C-type lectin-like molecule-1 (CLL1). CAR T cells

To test the concept of LAT-mediated potentiation of CAR signaling, we created a prototypic synapse-stabilizing receptor (SSR) using a CD38-specific binder connected via a CD8a spacer and transmembrane region to the endodomain of linker for activated T cells (LAT). We co-expressed the CD38.SSR with a second-generation, CD28-costimulated CAR targeting CLL-1 (Fig. 1A), a myeloid antigen commonly expressed in AML at various levels. As both the CAR and the SSR utilize a CD8a spacer, we introduced the Cys164Ser mutation in the SSR spacer, abrogating covalent heterodimerization of the two receptors via a cysteine bond14. Further, full-length LAT endodomain (LATFL) has been shown to spontaneously oligomerize with SLP-76 via GADS/Grb2 adapter molecules13,15,16, producing spontaneous T cell activation which may be exacerbated by the expression of the SSR ligand CD38 on activated T cells17. Therefore, we created an additional SSR variant with a truncated LAT endodomain (LAT177) that retains PLC-γ1 binding via Tyr132 but lacks Tyr191 and Tyr226 residues required for GADS/Grb2 docking and SLP-76 oligomerization (Fig. 1A). CD38.SSRs harboring either LATFL and LAT177 endodomain were expressed at high levels with CLL1.CAR on T cells via gammaretroviral co-transduction routinely resulting in co-expression on >85% of T cells (Fig. 1B). Co-expression of SSR-LATFL and CLL1.CAR significantly reduced the expansion of dual-transduced T cells (Supplementary Fig. 1A), possibly reflecting excessive tonic CAR signaling amplified by the full-length LAT as T cells expressing SSR-LATFL alone demonstrated normal expansion (Supplementary Fig. 1A). In contrast, LAT177 truncation attenuated this effect and improved the expansion of SSR-armed CLL1.CAR T cells (Supplementary Fig. 1A). Unlike LATFL, LAT177-harboring CD38. SSR had a minimal effect on the differentiation status and subset composition of expanded T cells (Supplementary Fig. 1B). Notably, T cells expressing SSR-LATFL alone produced significant cytotoxicity against CD38+ target cell lines Molm13 and CCRF-CEM indicating activation of cytotoxic signaling upon engaging the full-length LAT endodomain thus acting as a “weak CAR” (Fig. 1C). In contrast, T cells expressing SSR-LAT177 had minimal activity against the same target cells (Fig. 1C) suggesting LAT truncation is required to minimize background cytotoxicity of the SSR. Given the “leakiness” of the full LAT endodomain in the CD38.SSR and cellular toxicity induced by its expression in T cells, we opted to utilize the CD38.SSR construct with the truncated LAT177 endodomain for subsequent analyses.

Next, we evaluated the activity of CD38.SSR in CLL1.CAR T cells targeting cancer cells with low antigen expression and compared it with other CD38-specific chimeric costimulatory receptors and a full-length cytotoxic CD38.CAR. We generated T cells expressing CLL1.CAR alone or in combination with a truncated (ΔSSR) construct or an SSR harboring either a TNFR superfamily endodomain (4-1BB or CD27), or an immunoglobulin superfamily endodomain (CD28), or a LAT177 endodomain, via gammaretroviral co-transduction. We also created a cytotoxic second-generation CD38.CAR (where the LAT endodomain was replaced with CD28 and CD3ζ signaling domains) as a control (Supplementary Fig. 2A). The expansion of SSR.LAT177-expressing T cells was comparable with those expressing a control truncated SSR lacking signaling endodomain (ΔSSR) or with T cells expressing SSR with a CD28 endodomain (Supplementary Fig. 2B–D). In contrast, expansion of T cells expressing SSRs with CD27 or 4-1BB endodomain was significantly impaired (Supplementary Fig. 2B–E). We have previously shown constitutive (tonic) TRAF2-NFkB signaling from chimeric TNFRSF endodomains produced cellular toxicity in T cells transduced with a gammaretroviral vector which is consistent with this observation18,19. Expectedly, the expansion of CLL1.CAR T cells co-expressing a full length CD38.CAR was also reduced, reflecting a CD38-directed T-cell fratricide20.

CLL1 expression in primary AML is very heterogeneous, ranging from dim/negative to bright, often within the same patient21,22. An AML line THP-1 represents the latter scenario whereas Molm13 AML cells express low levels of CLL1 insufficient for effective recognition and cytolysis by CLL1.CAR T cells22,23 (Fig. 1D). Both lines expressed CD38 on the cell surface (Fig. 1D). As expected, CLL1.CAR T cells displayed minimal activity against Molm13 cells (Fig. 1E). The cytotoxicity of CLL1.CAR T cell against Molm13 was only marginally improved by co-expressing a truncated non-signaling (ΔSSR) or CD27-harboring SSR (Fig. 1E). In contrast, CLL1.CAR T cells armed with CD28- or LAT177-based SSRs or a full cytotoxic CD38.CAR produced significantly greater killing of Molm13 and expansion upon a 3-day co-culture (Fig. 1E). However, CAR T cells expressing a CD28-based SSR also killed CCRF-CEM cells which do not express CLL1, reflecting unwanted CD38-driven activation that is independent of the CLL1.CAR (Fig. 1E). Co-expression of ΔSSR or SSR-LAT177 resulted in a reduced but still detectable cytotoxicity against control CCRF-CEM cells (Fig. 1E). This low-level cytotoxicity was further attenuated by either knocking out the CD3ε gene on T cells or removing MHC I on CCRF-CEM using CRISPR/Cas9, suggesting CD38 binding by SSR amplified allogeneic killing of mismatched cancer cells (Supplementary Fig. 3A–C). In contrast, CLL1 CAR T cells co-expressing SSR.CD28 displayed significant cytotoxicity against CCRF-CEM even after the knock-out of CD3ε or β2m indicating TCR/MHC-independent killing. CD3 editing did not attenuate killing of Molm13 by CAR.SSR T cells indicating CAR-mediated cytotoxicity (Supplementary Fig. 3D). Further, SSR co-expression improved killing of THP-1 AML but not KG1a, which expresses lower levels of CLL1 and CD38 (Supplementary Fig. 4A–C). CLL1.CAR T cells armed with either SSR-LATFL or SSR-LAT177 produced comparable improvement in killing Molm13 and CAR T-cell expansion (Supplementary Fig. 4D).

To further validate the potency of SSR in CAR sensitivity, we co-cultured CLL1.CAR.SSR T cells with a CLL1 low derivative of THP1 cells obtained by sort-purifying THP1 cells expressing dim levels of CLL1 (Fig. 1F). Consistent with the results obtained with Molm13, expression of CD38.SSR significantly improved cytotoxicity of CLL1.CAR T cells against THP1 CLL1 low cells, whereas unarmed CAR T cells showed only limited activity (Fig. 1G). Engaging CD38 on target cells is critical for the CD38.SSR activity as genetic ablation of the CD38 gene on Molm13 cells reduced the benefit of the SSR on CAR T-cell cytotoxicity and expansion during co-culture, suggesting SSR.LAT177-mediated augmentation of CLL1.CAR-mediated cytotoxicity was largely dependent on CD38 expression (Fig. 1H, I). SSR.LAT177 co-expression enhanced cytotoxicity and expansion of T cells expressing other CLL1.CAR constructs, including a CD28-costimulated CAR with a CD28 hinge and a 4-1BB costimulated CAR (Supplementary Fig. 5), suggesting the SSR is not sensitive to a CAR configuration or a choice of costimulation. These results indicate CD38.SSR with a LAT177 endodomain produced the highest augmentation of CAR T cell-mediated killing of CLL1-low AML without compromising CAR specificity.

CD38.SSR enhanced effector function and immune synapse formation of CAR-T cells

LAT is a scaffolding molecule that orchestrates TCR signal transmission by the Lck-activated ZAP70 kinase to the key downstream kinase PLCγ1 which directly binds to Tyr132 that is retained in the LAT177 endodomain. PLCγ1 promotes cleavage of PIP2 to diacyl glycerol (DAG) and IP3 resulting in Ca++ influx and activation of MAPK and NF-κB pathways triggering T-cell effector program. The live cell imaging revealed SSR co-expression improved Ca++ influx in CAR T cells upon a 30-min co-culture with Molm13 target cells compared to control unarmed CAR T cells (Fig. 2A, B). Intriguingly, the increase in calcium levels was also observed in T-cells expressing ΔSSR or SSR alone (Supplementary Fig. 6A), suggesting this effect is secondary to the SSR-mediated engagement of CD38 which mediates intracellular Ca++ release via NAADP signaling24–26.

Next, we evaluated activation of PLCγ and its downstream signaling in SSR-armed CLL1 CAR T cells stimulated with plate-bound recombinant CLL1 and CD38. We observed higher levels of PLCγ1 phosphorylation and the downstream MAPK (ERK1/2) and NF-kB pathways in SSR-expressing CAR T cells compared to control CAR T cells (Fig. 2C, D). Engagement of the SSR alone did not activate the PLCγ1 pathways in the absence of CAR signaling (Supplementary Fig. 6B, C). Mutation of the PLCγ1 binding site Y132 in the LAT177 endodomain minimized SSR activity upon coculture with Molm13, highlight the critical role of PLCγ1 in SSR.LAT177 function (Fig. 2E, F).

Next, we assessed how SSR affects the formation of CAR-mediated cytotoxic immune synapse using super-resolution confocal imaging of CLL1.CAR T cells exposed to Molm13 cells for 1h. While observing limited enhancement of lytic granule migration by SSR (Supplementary Fig. 7A, B), co-expression of SSR in CLL1.CAR T cells significantly improved polarization of filamentous actin (F-Actin) and the microtubule organizing center (MTOC) to the immune synapse, reflecting cytoskeleton reorganization and the formation of a more stable synapse (Fig. 3A). We also observed a trend towards enhanced recruitment of phospho-SLP76, a critical signaling partner of LAT (Fig. 3A, B). Expression of the SSR.LAT177 alone did not significantly affect polarization of the synaptic machinery (Fig. 3A, B), suggesting increased Ca++ influx was insufficient to trigger effector function of SSR-LAT177 expressing T cells in the absence of CAR signaling, consistent with prior findings.

In line with the augmentation of signaling and immune synapse formation, we found SSR co-expression on CLL1.CAR T cells increased intracellular production of IFN-γ in both CD4+ and CD8+ T cells and augmented IL-2 production by CD4+ T cells upon a 4-h co-culture with CLL1 low Molm13 cells (Fig. 3C and Supplementary Fig. 7C). We also observed higher production of granzyme B and degranulation, as measured by surface CD107a, in both CD4+ and CD8+ T cells suggesting SSR activity amplified the cytolytic program in CD4+ T cells (Fig. 3D). Of note, co-expression of a truncated SSR lacking the LAT177 endodomain (ΔSSR) had minimal effect on cytokine production and degranulation highlighting the active role of the LAT endodomain in potentiating CAR-mediated cytotoxicity.

To evaluate whether the SSR.LAT177-mediated enhancement of CAR cytotoxicity is a general phenomenon, we designed an SSR targeting GRP78, a protein upregulated on the cell surface of several tumors, including gliomas27. We co-expressed GRP78.SSR with a CAR targeting IL13Ra228, an antigen expressed in diffuse intrinsic pontine gliomas (DIPG) at varying levels (Supplementary Fig. 8A-C). GRP78.SSR improved the killing of two tumor cell lines derived from patients with DIPG and enhanced intracellular Ca++ flux in IL13Ra2 CAR T cells (Supplementary Fig. 8D, E). In contrast to the CD38.SSR, GRP78.SSR did not produce spontaneous Ca++ flux in CAR-negative T cells. Collectively, these results indicate SSR expression promotes the formation of a mature cytotoxic immune synapse between a CAR T cell and a target cell with low antigen expression, thus potentiating intracellular signaling and promoting cytokine production and degranulation.

SSR co-expression boosts anti-leukemic activity of CAR T cells in vivo

To evaluate the effect of SSR on CAR-T cell mediated tumor control in vivo, we utilized two mouse xenograft models of human AML. In the first model, we engrafted Molm13 cells into NSG mice lacking murine MHC Class I and Class II genes to reduce non-specific xenoreactivity by human T cells (NSG-MHCKO). Three days following AML injection, we injected a single dose of control non-transduced T cells or T cells expressing CLL1.CAR alone or in combination with CD38.SSR (Fig. 4A). Bioluminescence imaging of AML burden showed only minimal disease control by control unarmed CLL1.CAR T cells, whereas SSR.LAT177-coexpressing CAR T cells suppressed systemic expansion of leukemia and produced better expansion in peripheral blood in the first 10 days post-infusion, significantly extending the survival of animals (Fig. 4B–E). Of note, co-expression of a non-signaling ΔSSR did not enhance anti-tumor activity and expansion of CLL1 CAR T cells in vivo, suggesting LAT177 signaling is critical for the optimal SSR function in vivo (Fig. 4B–D). SSR-armed CLL1.CAR T cells had a higher frequency of CD62L+CD45RA+ naïve-like T cells 18 days post-infusion compared to unarmed CLL1.CAR T cells (Supplementary Fig. 9).

To validate these findings in another model of AML with suboptimal target antigen expression, we modified CLL1 low THP-1 cells to express firefly luciferase and engrafted into NSG-MHCKO mice followed by a single injection of CAR T cells (Fig. 4F). Again, CLL1.CAR T cells demonstrated poor activity against the antigen-low leukemia, and non-transduced or SSR-only T cells had minimal, if any, effect on tumor growth (Fig. 4G, H). By contrast, SSR-armed CLL1.CAR T cells controlled systemic leukemia in most animals, resulting in their long-term survival (Fig. 4I). SSR-armed CLL1.CAR T cells had reduced frequency of PD1+ LAG3+ cells compared to unarmed CAR T cells on day 61 post-infusion (Supplementary Fig. 10) possibly reflecting reduced systemic tumor burden. These results indicate SSR expression significantly boosts anti-leukemic activity of CLL1.CAR T cells against antigen-low AML in vivo.

Co-targeting CD38 through SSR enhances CAR-T targeting of primary AML but not healthy cells

For optimal activity, SSR must selectively improve CAR T-cell cytotoxicity against cancerous cells that express both CAR and SSR antigens without producing additional cytotoxicity against normal tissues that express the SSR target antigen. Among mature circulating cells, CD38 was expressed at the highest levels on CLL1+ monocytes and CLL1- NK cells and varying levels on T- and B-cells (Fig. 5A and Supplementary Fig. 11A-B). Co-expression of the CD38.SSR did not enhance cytotoxicity of CLL1.CAR T cells against autologous monocytes and produced no activity against CLL1-negative autologous T cells and B-cells (Fig. 5B). A weak activity of SSR-armed CLL1.CAR T cells was detected against CD38 bright NK cells, but it did not reach statistical significance, unlike in control CD38.CAR T cells (Fig. 5B, C and Supplementary Fig. 11C). T cells expressing SSR alone had no measurable activity against autologous lymphocytes but produced weak cytotoxicity against HLA-mismatched CD38-high T-ALL cell lines P12-ICHIKAWA and CCRF-CEM (Supplementary Fig. 11D, E).

In addition to mature blood cells, CD38 is expressed in primitive hematopoietic progenitors where its expression decreases at later stages of myeloid differentiation as CLL1 expression increases inversely3,29,30. To assess the activity of CD38.SSR-armed CLL1.CAR T cells against hematopoietic progenitors, we purified CD34+ cells from cord blood (Fig. 5C) and co-cultured them with control non-transduced or armed and unarmed CLL1.CAR T cells for 5h at an effector-to-target ratio of 10:1 followed by a 12-day culture on a semi-solid methylcellulose medium supportive of myeloid differentiation. We saw no activity of CLL1.CAR T cells with or without SSR against CLL1-negative erythroid progenitors (BFU-E, Fig. 5C). While granulocytic/monocytic progenitors (CLL1+) were expectedly targeted by CLL1.CAR T cells, addition of the SSR did not increase that cytotoxicity (CFU-GM, Fig. 5C). By contrast, co-expression of a cytotoxic CD38.CAR produced ablation of both types of CD38+ progenitors (Fig. 5C). Therefore, CD38.SSR does not exacerbate toxicity of CAR T cells against normal CD38+ tissues.

To evaluate the benefit of SSR in targeting primary AML, we co-cultured T cells with leukemic blasts obtained from two patients with treatment-refractory AML expressing CD38 and varying levels of CLL1 (Fig. 5D). SSR-armed CLL1 CAR T cells produced a significantly better killing of AML blasts from both patients, comparable with dual-CAR T cells (Fig. 5E). Therefore, SSR enhances killing of malignant blasts but not healthy CD38+ tissue.

CD38 SSR improved anti-tumor function of TCR-expressing T cells

Antigen heterogeneity does not affect only CAR T cells as tumor cells also commonly downregulate surface HLA molecules to evade TCR-mediated recognition. To assess the utility of SSR in potentiating TCR-mediated lysis of leukemia with low HLA expression, we tested its activity in T cells expressing a TCR targeting intracellular protein survivin/BIRC5 presented on HLA-A2 (Fig. 6A)31. SSR was co-expressed in Survivin TCR (Sur-TCR) T cells at high levels without affecting the expansion and subset composition of transgenic T cells derived from both HLA-A2- and HLA-A2+ donors indicating SSR expression does not enhance fratricide of Sur-TCR T cells which upregulate survivin during activation31 (Fig. 6B, C and Supplementary Fig. 12A–C). We postulated this is because SSR masks CD38 in T cells in cis thus preventing its recognition by the surrounding SSR T cells. Indeed, we observed complete loss of detectable surface CD38 in SSR-expressing T cells by flow cytometry utilizing an antibody binding to an epitope that overlaps with the SSR CD38 binder, supporting cis-masking (Supplementary Fig. 13A, B). To further test this hypothesis, we incubated control and SSR-armed CAR T cells with a cytotoxic CD38-specific monoclonal antibody daratumumab in the presence of purified NK cells. Expression of CD38.SSR prevented daratumumab-induced NK cell-mediated cell cytotoxicity to T cells illustrating protective masking of surface CD38 by the SSR (Supplementary Fig. 13C, D).

Next, we utilized three HLA-A2+ acute leukemia lines with a similar expression level of survivin but varying levels of surface HLA-A2, ranging from high in BV173 to intermediate in THP1 to low in TALL1 (Fig. 6D). While Sur-TCR T cells produced cytotoxicity against HLA-high BV173 cells, SSR co-expression further improved T-cell expansion in a 3 day co-culture (Fig. 6E and Supplementary Fig. 12D). We observed no cytotoxicity of unarmed Sur-TCR T cells against THP1 cells that express intermediate levels of HLA on the cell surface. However, SSR co-expression produced significant cytotoxicity against THP1 cells but did not improve T-cell expansion (Fig. 6E and Supplementary Fig. 12D). Finally, Sur-TCR T cells produced no killing of HLA-low cell line TALL1, regardless of the SSR co-expression, indicating the benefit of the SSR is limited to a dynamic range of main antigen expression. SSR-armed Sur-TCR T cells produced higher degranulation upon a short co-culture with BV173 and THP1 cells, and improved cytokine production upon co-culture with THP1 (Fig. 6F, G), consistent with observations in CAR T cells.

Next, we assessed whether SSR can improve the anti-tumor activity of Sur-TCR T cells in vivo using THP-1 cells resistant to Sur-TCR cytotoxicity. We established systemic THP1 leukemia via intravenous injection in NSG-MHCKO mice for 7 days and administered three doses of Sur-TCR T cells in the next week. Mice received IP injections of 2000 IU of exogenous rhIL2 twice a week for three weeks (Supplementary Fig. 14A). While unarmed Sur-TCR T cells had no significant activity against systemic leukemia compared to control (tumor only) condition, SSR-armed Sur-TCR T cells delayed leukemia progression and reduced levels of circulating AML blasts in peripheral blood (Supplementary Fig. 14B, C) significantly extending overall animal survival (Supplementary Fig. 14D). The activity of Sur-TCR T cells was transient and the SSR benefit limited (Supplementary Fig. 14D), likely due to the lack of critical Signals 2 and 3 that are required to maintain functional persistence of therapeutic T cells in a mouse. To further improve the in vivo efficacy, we modified Sur-TCR T cells to express membrane-bound IL15 thus providing continuous Signal 332–34 (Supplementary Fig. 15A–D). Mice were engrafted with THP-1 cells followed by a single injection of Sur-TCR T cells armed with ΔSSR or SSR (Fig. 6H). The cytokine modification slightly increased their persistence and anti-tumor activity of sur-TCR T cells (Supplementary Fig. 15E), and SSR co-expression enabled stronger anti-tumor control significantly prolonging animal survival (Fig. 6I–K). Consistent to CAR.SSR T cells, SSR-armed survivin T cells displayed a higher frequency of CD62L+CD45RA+ naïve-like T cells 27 days post-infusion compared to TCR.ΔSSR and unarmed TCR T cells (Supplementary Fig. 15F).

Taken together, these results demonstrate specific and potent augmentation of CAR- and TCR-mediated cytolysis of tumor cells expressing suboptimal levels of target antigens by a synapse-stabilizing receptor harboring LAT177 endodomain.

Methods
Cell lines
All leukemia cell lines are human-derived. THP1 (Male, TIB-202), HL60 (Female, CCL-240), KG1a (Male, CCL-246.1), and CCRF-CEM (Female, CCL-119) are from American Type Culture Collection (ATCC, Manassas, VA). Molm-13 (Male, ACC554) and P12-ICHIKAWA (Male, ACC34) are from Leibniz Institute DSMZ. Molm13, HL60 and KG1a were maintained in IMDM (Gibco™, Thermo Fisher Scientific) with 20% fetal bovine serum (FBS, Gibco™, Thermo Fisher Scientific). THP1, CCRF-CEM, P12-ICHIKAWA, T-ALL1 and BV173 were maintained in RPMI1640 (HyClone™, Cytovia) with 10-20% FBS. All media contained and 2 mM L-GlutaMAX (Gibco™, Thermo Fisher Scientific). THP1-CLLlow was generated through sorting natural CLL1 low cells from parental THP1 using SH800S Cell Sorter (Sony Biotechnology). Molm13-CD38KO was engineered by genetic knock-out of hCD38 using a CRISPR/cas9 system described below. All cell lines were subjected to Mycoplasma testing routinely, and have been authenticated with STR analysis in the Cytogenetics and Cell Authentication Core of MD Anderson.

Gene Knock-outs
sgRNA of hCD38 (Target sequence: CGGTCTCGGGAAAGCGCT), hCD3ε (Target sequence: CACTCACTGGAGAGTTCT) and hTRAC (AGTCTCTCAGCTGGTACA) were design using CRISPRscan and COSMID algorithms, and generated by an in vitro transcription system established previously47,48. For knock-out of hCD38 in AML cell lines, briefly, 1µg of CD38 sgRNA and 1µg Cas9 protein (Integrated DNA Technologies) were electroporated into 0.25×106 Molm13 resuspended in 10µl buffer R with one 1650-V, 20-ms pulse following the manufacturer’s instruction of Neon Transfection System (Thermo Fisher Scientific). Residual CD38-positive Molm13 cells were further depleted using Magnetic MicroBeads (Miltenyi Biotech). For knock-out of hCD3 and hTRAC in T cells, 1µg of CD3ε sgRNA and 1µg Cas9 protein were electroporated into 0.25×106 activated T cells at day 2 post-stimulation resuspended in 10µl buffer T with three 1600-V, 10-ms pulses, and maintained in 2ml CTL medium containing 20% FBS and 10ng/ml IL7/IL15 overnight followed by retroviral transduction.

Constructs of SSRs and retrovirus production
CD38.SSR were generated by Infusion Cloning (NEBuilder HiFi DNA Assembly, NEB) of synthesized dsDNA of CD38-specific single chain variable fragments (scFv) derived from anti-human CD38 mAb (Clone HB7, Patent No. 770718) with the PCR fragments of CD8α-hinge and transmembrane region including point mutation at C164S and the endo-domains of LAT (28–233 a.a or 177 a.a) amplified from a human complementary DNA library derived from T cells using CloneAmp HiFi PCR Premix (Takara Bio), followed by Sanger sequencing to confirm identity (Supplementary Fig. 16). CD38 CAR was generated by replacing the endodomain of SSRs with CD28 and CD3ζ domains from a cytotoxic CAR49. To clone GRP78.SSR, the extracellular domain of CD38.SSR was replaced with the PCR fragment of a GRP78-specific peptide CTVALPGGYVRVC fused with a mutant IgG4 hinge and a CD28 transmembrane domain amplified from GRP78.CAR27. Gammaretroviral vector (SFG) supernatant was made from 293T cells as previously described50. Briefly, 0.6×106 293T cells were seeded in a well of 6-well plates overnight, followed by transfection with 1.4µg SFG vector, 1.4µg Peg-Pam-e (MoMLV gag-pol proteins) and 1.1µg RD114 (envelope protein) using a polyetherimide (PEI) transfection system under manufacturer’s protocol (PEIpro®, Polyplus). The whole virus supernatant was collected at 48h or 72h post transfection and preserved at −80°C for upcoming transduction.

T-cell activation and transduction
Activated T cells were generated by activating PBMC on 24-well plate pre-coated with 500µl of 1µg/ml OKT3 (Ortho Biotech, Bridgewater) and anti-CD28 antibodies (BD Biosciences) for 2 days in 2ml CTL medium (45% Click’s medium, 45% RPMI 1640, 10% FBS and 2 mM L-GlutaMAX). Co-transduction was performed by pre-coating viral supernatant with the CLL1.CAR or Sur-TCR:CD38.SSR ratio 1:1 (1ml) in RetroNectin (Takara Bio)-coated 24-well plate at 4122g for 1h at 32°C, followed by removal of virus supernatant and spinoculation of 1×105 activated T cells in 1ml CTL medium at 1000g for 10 mins. The medium was replenished in the next day, and transduced T- cells were expanded and maintained in CTL medium containing 10ng/ml recombinant human IL7 and IL15. T-cell growth and viability were assessed using Trypan Blue and a hemocytometer. T-cell phenotype was monitored by flow cytometry after T-cell transduction. CAR T cells were collected for functional analysis 7 days post transduction.

For generation of GRP78.SSR armed IL13Ra2.CAR T cells, sequential transduction was performed by transduction of 2.5×105 T cells with 500µl IL13Ra2.CAR viral supernatant on day 2 and 500µl GRP78.SSR viral supernatant on day 3 post T-cell activation, respectively. CAR expression and T cell phenotype were determined at 5 to 6 days post-transduction.

Co-culture and cytotoxicity assays
To examine the cytotoxicity of CAR and TCR T cells against leukemia cell lines, 20,000 target cells expressing green fluorescent protein and firefly luciferase (GFP.FFLuc) were co-cultured with 5,000 effector cells at 1:4 E:T ratio without cytokines in each well of a flat-bottom 96-well plate. Residual tumor cells and effector cells were distinguished by GFP expression and quantified on day 0, day 1 and day 3 by flow cytometry using CountBright Counting Beads (Thermo Fisher Scientific), including 7-AAD (BD) to exclude dead cells. To measure cytotoxicity against CD38-postive primary cells, 0.2×106 autologous PBMCs isolated from health donors and labeled with 1μM eFluor 670 (Invitrogen) were co-cultured with 50000 effector cells at 1:4 E:T ratio in 400µl CTL medium (IL15/1L7 free) in each well of a flat-bottom 48-well plate. Purified NK cells were isolated using EasySep Human NK Cell Isolation Kit (STEMCELL). Residual efluor+ monocytes (CD14+), T cells (CD3+), NK cells (CD3-CD56+) and B-cells (CD19+) were quantified 24h later by flow cytometry as described above.

To examine the cytotoxicity of IL13Ra2.CAR T cells against solid tumor cells in luciferase-based assays, 30,000 Firefly luciferase (ffLuc)-expressing tumor cells were seeded onto 96-well/white clear bottom plate (Thermo Fisher Scientific) and incubated with varying amounts of CAR T cells to assess cytotoxicity at a range of E:T ratios. Twenty-four hours later, the remaining tumor cells were quantified by luminescence when adding D-luciferin (Perkin Elmer) at 0.3mg/mL in tumor media. Media only and tumor only served as controls to assess percent cytotoxicity. Luminescence was measured by using an Infinite 200 Pro MPlex plate reader (Tecan, Männedorf, Switzerland).

Primary AML
For co-culture with primary AML blasts, 50,000 PBMCs isolated from patients with AML (recruited to the CARMEN study NCT04219163) were labeled with 1μM eFluor 670 (Invitrogen), and co-cultured with 50,000 effector cells at 1:1 E:T ratio in 200µl CTL medium (without cytokines) in each well of a flat-bottom 96-well plate. Residual efluor+ AML blasts (CD33+CD34+) were quantified by flow cytometry. The study was conducted under an IND approved by the IRB and the FDA according to the principles of the Declaration of Helsinki. All participants gave informed consent prior to enrollment and any study procedures.

Colony formation assay
CD34+ cord blood (CB) cells sorted by Magnetic MicroBeads (Miltenyi Biotech) was a gift from the laboratory of Dr. Masataka Suzuki. Two thousand CD34+ CB cells were co-cultured with 20,000 effector cells in a flat-bottom 96 well plate for 5h, followed by resuspended in 1ml semi-solid methylcellulose gels containing recombinant cytokines required for colony formation of hematopoietic cells (H4434 Classic, MethoCult, STEMCELL) for 12 days. Colony formation of myeloid cells derived from CD34+ CB cells, including burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte, macrophage (CFU-GM), were identified and quantified under manufacturer’s instruction.

Flow cytometry
In general, cells were stained with fluorochrome-conjugated antibodies in PBS (1% FBS) for 30 min at 4°C, and after wash steps, all of samples were acquired by the Northern Lights™ flow cytometer (Cytek Biosciences). Ultimately, the cell populations were gated and analyzed using FlowJo (BD Biosciences). Human BD Fc Block (BD Bioscience) was included for characterization of AML cell and PBMCs to reduce background of myeloid cells. The antibodies used in the studies are further categorized and listed below.

T-cell phenotyping: PE-NGFR (1:200) (clone C40-1457, BD, 557196), AF647-CLL1 CAR idiotype (1:200) (Clone MM162, Monoclonal Antibody Core, University of Texas M.D. Anderson Cancer Center), APC-mTCR beta (1:200) (clone H57-597, eBioscience, 17-5961-82), PerCPCy5.5-CD38 (1:100) (clone HIT2, BD, 551400), BV421-CD3 (1:200) (clone UCHT1, BioLegend, 300434), BV421-HLA-A2 (1:200) (clone BB7.2, BioLegend, 343326), PE-Cy7-CD4 (1:400) (clone L200, BD, 560644), FITC-CD8 (1:100) (Clone SK1, BD, 347313), V450-CCR7 (1:100) (clone 150503, BD, 560863), ECD-CD62L (1:200) (clone DREG56, IOTest, IM2713U), APC-AF750-CD45RA (1:200) (clone 2H4, IOTest, A86050), Alexa Fluor 647-AffiniPure Goat Anti-Mouse IgG, F(ab’)₂ fragment specific (1:200) (Jackson ImmunoResearch, 115-606-006) and Alexa Fluor 647-G4S Linker (1:200) (clone E7O2V, Cell Signaling Technology, 69782).

Characterization of AML cell and PBMCs: BV421-CLL1 (1:200) (clone 50C1, BD, 74929), PerCP-Cy5.5-CD38 (1:100) (clone HIT2, BD, 551400), PE-HLA-A2 (1:100) (clone BB7.2, BioLegend, 343306), BV510-hCD45 (1:200) (clone HI30, BD, 563204), APC-CD3 (1:200) (Clone UCHT1, IOTest, IM2467U), PE-CD14 (1:100) (Clone MφP9, BD, 347497), FITC-CD56 (1:100) (Clone NCAM16.2, BD, 340723) and PE-Cy-CD19 (1:200) (clone SJ25C1, BD, 557835). The nuclear protein Survivin was stained with AF750-survivin (1:40) (clone 91630, R&D, IC886S) using True-Nuclear™ Transcription Factor Buffer set (BioLegend, 424401).

Intracellular staining: APC-IFNγ (1:100) (clone B27, BD, 554702), FITC-TNFα (1:100) (clone Mab11, BioLegend, 502906), BV-421-IL12 (1:100) (clone 5344.111, BD, 562914), APC-CD107a (1:100) (clone H4A3, BD, 641581) and V450-GzB (1:100) (clone GB11, BD, 561151).

Detection of cytokine production and degranulation of lytic granules
Molm13 target cells were co-cultured with 0.1×106 CAR T cells at 1:1 E:T ratio for 4h at present of monensin (GolgiStop™ protein transport inhibitor BD Biosciences), followed by staining CD4 and CD8 surface markers for 30min. Subsequently, the stained cells were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) under manufacturer’s instruction, and subjected to staining and analysis of IFNγ, TNFα, and IL2 and lytic granules (Granzyme B) by flow cytometry. For degranulation of lytic granules, the CD107a antibody was added into the T-cell medium during the 4-h co-culture for detection of the release of granules.

Confocal microscopy
The protocol for microscopic immune synapse characterization was established as previously described51. Poly-Lysine (PLL (Sigma-Aldrich, P4707))-coated coverslips were prepared using N1 coverslips (Thermo Fisher Scientific, 12-545-80P) overnight at 4°C. Then, they were washed with PBS and filled with media until Molm13 cells were seeded. Next, 100,000 Molm3 cells were plated onto the pre-coated coverslips and cultured for 30 mins at 37°C/5% CO2. Then CAR T cells were added on top and co-cultured for 1h at 37°C/5% CO2. After co-culture, cells were washed with cold PBS and fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences, 15710) for 10 min at room temperature. Fixed cells were washed twice with PBS, and the remaining PFA was inactivated with blocking buffer (PBS-2% BSA (Sigma-Aldrich, A9418) and 1.5M glycine (Sigma-Aldrich, G8898)) for 10min at room temperature. Cells were permeabilized by adding permeabilization buffer (PBS, 0.2% BSA and 0.05% saponin; Sigma-Aldrich, 47036) for 20min at room temperature. Cells were washed twice with permeabilization buffer before primary antibody incubation and diluted in permeabilization buffer, following the manufacturerʼs instructions. All the primary antibodies were incubated at 4°C overnight. Cells were washed with permeabilization buffer and incubated with secondary antibodies for 2h at room temperature. Finally, cells were washed with permeabilization buffer and PBS before letting them dry for 1h at room temperature. Then, coverslips were mounted onto slides using Fluoromount (Thermo Fisher Scientific, 00-4958-02).

Primary antibodies and probes with their dilutions are as follows: anti-human Lamp1 (1:50) (Abcam, ab25630); phalloidin-Alexa Fluor 647 (1:200) (Thermo Fisher Scientific, A22287); anti-pSLP76 (1:100) (Abcam, AB75829); anti-a-Tubulin (1:1000) (Abcam, ab6160); anti-GzB (1:100) (Cell signaling, 17215). Secondary antibodies with their dilutions are as follows: anti-rabbit Alexa Fluor 488 (1:200) (Thermo Fisher Scientific, A32731), anti-rat Alexa fluor plus 405 (Thermo Fisher Scientific, Cat# A48261), and anti-mouse Alexa Fluor 568 (1:200) (Thermo Fisher Scientific, A-11004).

Images were acquired in a Airyscan confocal microscope (Zeiss LSM980), and the processing and analysis were performed with Fiji (ImageJ) software (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012)). Single-cell images shown in the figures were cropped from a larger field. Image brightness and contrast were manually adjusted. To analyze lysosome and pSLP76 distribution in T cells, T cells were segmented by using celltrace violet signal, and the immune synapse was manually identified as the point of interaction between CAR T cells and tumor cells. To identify the polarization/recruitment of different labels, the center of mass of each fluorescence was identified and their relative distance from the IS was automatically calculated.

Live cell imaging of calcium influx
Tumor cells were labeled with CellTracker Red CMTPX (Invitrogen, Cat: C34552) or CellTrace Far Red (Invitrogen, Cat: C34564) (1:500) for 30 mins and then washed and maintained in culture media until image acquisition. 1×105 Molm13 cells were seeded onto PLL-coated μ-slide 8 well chambers (Ibidi, Cat: 80807) and 1×105 DIPG007 and DIPG7c cells were seeded onto the same chambers coated with GelTrex (Thermo Fisher Scientific, Cat: A1413302). Tumor cells were then incubated for 30min at 37°C and 5% CO2. 1×106 CAR T cells were resuspended in 1mL of PBS and labeled with CellTrace Violet (1:1000) (Thermo Fisher Scientific, Cat: C34557) and CAL590 (ATT bioquest, Cat: 20510) or CAL520 (ATT bioquest, Cat: 21130) (1:500) for 1h and then washed and maintained in T cell media until image acquisition. At a time of image acquisition, 1×105 CAR T cells were added to each well preloaded with tumor cells, and the image acquisition was initiated once T cells were detected in the visual field. Images were acquired in a spinning disc confocal microscope (Zeiss Axio Observer with CSU-X spinning disc), using a 63× objective. The acquisition parameters were a 4D image (60min of acquisition with 1min of frame, and 20µm of height with a Z-step of 2µm).

The processing and analysis were performed with FIJI (ImageJ) software48. Cell tracking49 and Ca2+ influx were performed using Trackmate plugin. All tumor and CAR T cell interactions were recorded over time, and Ca2+ influx was measured as the maximum fluorescence emitted by CAL520/590 signal which was normalized by its value before the first peak of calcium influx upon tumor interaction. Total Ca2+ influx was quantified as the area under curve. (AUC) of Ca2+ influx registry until 20 mins of interaction.

Western blotting
To investigate LAT downstream signaling, 1×106 CAR.SSR T cells were loaded into a well of a 24-well plate pre-coated with 0.3μg anti-CLL1 CAR antibody and 1μg recombinant human CD38 (Sino Biological, Cat: 10818) for 30min on ice, followed by stimulation at 37°C in a water bath for indicated time-points. Immediately, the stimulated T cells were harvested and lyzed with 100µl RIPA buffer (Sigma-Aldrich, Cat: R0278) containing the cocktail of proteinase/phosphatase inhibitors (Thermo Fisher Scientific, Cat: 178440) for 30min, and the protein lysates were collected after centrifugation at 8000g for 10mins. The protein samples were further prepared with 4x Laemelli buffer (Bio-Rad) with β-mercaptoethanol, and 20μl of each sample was loaded into a well of 7.5% or 10% sodium dodecyl sulfate polyacrylamide gels for electrophoresis (SDS-PAGE, Bio-Rad) in parallel. Subsequently, the proteins were transferred into nitrocellulose (NC) membranes (Bio-Rad) and blocked in PBS with 0.2% Tween 20 and 5% skimmed milk for 1h before antibody incubation. The NC membranes were incubated with primary antibodies at 4°C overnight, followed by the incubation of secondary antibodies goat anti-mouse IRDye 680RD (1:5000) (LI-COR Biosciences, Cat: 925-68070) or goat anti-rabbit IRDye 800CW (1:10000) (LI-COR Biosciences, Cat: 925-32211) for 1h at room temperature. Finally, the blots were developed using the LI-COR Odyssey CLx (LI-COR Biosciences) and quantified by ImageJ. The primary antibodies used are listed as follows: anti-phospho-LAT (Y132) (1:500) (Invitrogen, Cat: 44-224), anti-phospho-PLCγ1 (Y783) (1:500) (Cell Signaling Technology, Cat: 14008), anti-phospho-ERK1/2 (T202/Y204) (1:1000) (Cell Signaling Technology, Cat: 9101), anti-phospho-NFκB (S536) (1:500) (Cell Signaling Technology, Cat: 3033), and anti-GAPDH (1:3000) (Santa Cruz, Cat: sc-47724).

AML mouse xenograft models
Mouse work was performed using NSG-MHC class I/II DKO mice (NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl/SzJ, stock no. 025216) and NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, stock no. 005557), purchased from The Jackson Laboratory and bred separately in a pathogen free mouse room of Baylor College of Medicine animal facility. Mice were maintained under standard housing conditions.

To evaluate the efficacy of CAR.SSR T cells on antigen-low AML models, 0.5×106 Molm13-GFP/FFLuc cells or 1×106 THP1-CLL1low-GFP/FFLuc cells were intravenously (i.v.) injected into each NSG-MHC class I/II DKO mouse aged 6-10 weeks (including both male and female), followed by infusion of CAR.SSR T cells expanded and collected at day 7 post retroviral transduction 3 days after tumor engraftment. To evaluate the efficacy of Sur-TCR.SSR T cells on AML mice, 1×106, THP1-GFP/FFLuc cells were i.v. injected into each NSG-MHC class I/II DKO mouse, followed by infusion of 3 doses of 4×106 TCR.SSR T cells 7 days after tumor engraftment. 2000 IU recombinant human IL2 (NIH) were intraperitoneally administered twice a week for the following three weeks after T-cell infusion. In experiments with Sur-TCR.SSR.mbIL15 T cells, 1×106 THP1-GFP/FFLuc cells were injected per mouse i.v., followed by a single dose of 10×106 TCR.SSR.mbIL5 T cells i.v. 7 days after tumor injection. The details of mouse age and sex in each experiment have been disclosed in the Source Data file.

Tracking total tumor burdens, each mouse was intraperitoneally administered with 100µl D-luciferin (30 mg/mL; XenoLight, Revvity), and the bioluminescence imaging was detected after 10 mins using an IVIS Lumina II imaging system (Caliper Life Sciences), followed by quantification of radiance in Living Image software (Caliper Life Sciences). To track circulated tumor cells and T cells, 50µl peripheral blood was obtained from the tail vein of mice, and red blood cells were removed by 1ml RBC Lysis Buffer (BD Biosciences), followed by quantification of tumor counts (hCD45+GFP+) and CAR (hCD45+CD3+CAR+) or TCR (hCD45+CD3+TCR+) T-cell counts and phenotyping by flow cytometry using CountBright Absolute Counting Beads and 7-AAD. Mice showing signs of distress, immobility, disease progression, such as paralysis and distended abdomen, and/or weight loss exceeded 20% of baseline were euthanized in compliance with the Baylor College of Medicine IACUC (protocol AN-4758). CO2 euthanasia was conducted according to the IACUC-approved Euthanasia in Rodents Policy by use of automated CO2 euthanasia chambers with a SMARTBOX auto CO2 system (Euthanex, E-Z Systems).

Daratumumab-induced cytotoxicity
Twenty thousand CAR T cells were labeled by Cell Proliferation Dye eFluor 670 and co-culture with 60,000 NK cells purified by EasySep Human NK Cell Isolation Kit (STEMCELL) in presence of 10μg/ml daratumumab (Selleckchem) or IgG1 isotype (BioXCell) control in 200μl CTL medium for 4h, followed by staining apoptotic cells with PB-annexin V (BioLegend) in Annexin V Binding Buffer (BioLegend), and analysis by flow cytometry.

Statistical analysis
Statistical analysis was performed and plotted using GraphPad Prism 10 software (GraphPad Software), and details are described in figure legends. In co-culture assays, data were pooled and analyzed with replica including T cells derived from different donors from independent experiments. The statistical significance between two groups was determined by two-tailed unpaired Student’s t test in mouse experiments. The statistical analysis including multiple comparisons was performed using analysis of variance (ANOVA), and the P value was adjusted by Tukey’s correction or Dunnett’s correction. The difference between the two groups in a time course were calculated and compared by mean area under curve (AUC). The significance in Kaplan–Meier survival curves was determined by Mantel–Cox log-rank test.

Software
Data analysis was performed using indicated licensed software. Image in Fig. 2A was created in BioRender licensed under CC BY 4.0

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The original data plotting the graphs and uncropped WB blots have been uploaded as a Source Data file, and supplementary data are provided in Supplementary information. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes the development of a synapse-stabilizing receptor (SSR) designed to enhance the cytotoxic activity of CAR and TCR redirected T cells in preclinical models of acute myeloid leukemia (AML). The SSR comprises a truncated endodomain of the proximal T cell signaling molecule LAT which is joined to a CD38-scFV via a CD8 hinge/transmembrane domain. A point mutation in the hinge domain of the CD8 (C164S) is required to limit SSR to CAR heterodimerization and reduce killing through the SSR. In the presence of the SSR, the authors show enhanced in vitro killing and improved in vivo tumor control by CLL-1 CAR T cells of AML cell lines expressing low levels of CLL-1. The observed enhancement is linked through imaging experiments to improved immune synapse formation that is dependent on CD38 expression. Modest improvements in in vitro killing and in vivo tumor control have also been detected when the SSR was expressed together with a survivin-specific TCR.\nOverall, this work describes a novel and interesting platform that can exploit T cell signaling networks to enhance the efficacy of adoptive cell therapies. This will be well received by the field and stands a good chance of being adopted by other researchers. The work is well suited for this journal. However, there are multiple concerns that should be addressed experimentally prior to publication.\nMajor comments\nImaging to determine mechanism of SSR mediated enhancement on the synapse: The authors show that the expression of the SSR results in increased calcium flux following antigen challenge, suggesting enhanced PLC-gamma activity. However, this happens even in the absence of CAR, raising the question of whether the assay was correctly performed or whether the overexpressed SSR drives baseline phosphorylation. Can the calcium flux be repeated in a flow based assay?\nIf this Ca flux without CAR signaling is real, would a mutation at Y132 abrogate the enhanced calcium flux? In addition, the authors should image PLC-gamma or a further downstream interacting molecule at the SSR/synapse to show the mechanism is indeed PLCgamma related as it seems unlikely that PLCgamma is being recruited and activated in the absence of CAR signal.\nAlso, in 2A/B, the conditions include NT, CAR, CAR + SSR, and SSR alone. However, in the quantification it is listed as CAR + ΔSSR (figure 2C). The latter would be the appropriate control as, hypothetically, CD38 engagement by the ΔSSR could lead to improved assembly of the immune synapse after CLL-1 binding. This should be clarified and repeats of the experiment should be performed with the ΔSSR.\nDoes the immune synapse of CAR+SSR at low CLL-1 densities looks compared to the immune synapse of CAR only at high CLL-1 densities? In other words, is LAT overexpression overall restoring the quality of the immune synapse to that which is seen with high antigen density? Additionally, an important technical question is does MOLM-13 include CLL-1 negative cells? If so, could this affect the results as authors may have imaged a CLL-1 negative cell?\nSpecificity of the SSR approach: The concept of the SSR is to enhance killing only in the presence of the target of the cytotoxic CAR. However, the authors see killing on CLL1 negative cells in Figure 1E (brown vs gold). This is obfuscated a bit by using a log axis. This should be changed to linear and discussed head on. Quite annoyingly, the authors switch from log in Fig 1 to linear in supplemental figure 3e which is inconsistent and highlights the data the authors wish to stand out. This should be consistent. Additionally, there is no statistical difference between the CD28- and LAT- SSR killing of CCRF-CEM in figure 1E so the SSR should not be deemed cleaner than the CD28 approach.\nThe authors then show that TCR KO rescues this unwanted activity which is reassuring. However, it is unclear whether this has to do with the mere presence of TCR vs allo-reactivity which the authors would like to ‘blame.’ This is extremely important to clarify and to answer this question, the authors could KO B2M/CIITA in the tumor cell lines and repeat the experiment. It also has not been evaluated if TCR KO could also abrogate the killing of CCRF-CEM (CLL1-) by the CD28-SSR which should be approached since the SSR is deemed by the authors as more specific.\nAvidity: The experiment using acoustic force seems not to have worked correctly. The NT T cells (no CAR) have a higher avidity than the CAR or CAR+ SSR which doesn’t make sense as the CAR (and CAR+SSR) has a binder for the tumor cells. This should be removed or controls showing the assay worked correctly included (eg add a CD38 CAR and/or show these conditions against a CLL1 high cell line).\nMechanism of in vivo efficacy: Importantly, the in vivo experiments, it would be appropriate to include a CAR + ΔSSR control to rule out that the enhanced activity derives from the LAT signaling (even better would be a full SSR bearing a mutation at Y132).\nGeneralizability: Translation of this approach is the most important potential output of this work, but only a single CAR design has been tested in vivo. Would the SSR work to enhance a CAR with a CD28 hinge domain (which may not dimerize with the SSR)? What about a CAR that utilizes a 4-1BB signaling? This should be answered in vivo.\nTCR: The enhancement of survivin-specific T cells is modest – is this a difference between CAR and TCR (eg TCR better at utilizing LAT so this is less relevant) – authors should discuss. The control group in the in vivo experiment figure are listed as “tumor only” (and not “control non-transduced cells” as stated in the text). This experiment should indeed include “control non-transduced cells” as well as SSR T cells +/- ΔSSR, to rule out any survivin independent reactivity that could be exacerbated by the SSR, the 3 T cell injections and the IL-2 administration.\nAntigen downregulation: In a manuscript about overcoming low antigen density, it would be good to understand what antigen looks like in tumor cells at relapse in vivo – this would help define the limits of the novel system and drive future work.\nOther comments:\n1) Statistics in the confocal imaging- There seem to be no statistical differences of CAR + SSR compared to the CAR alone condition, all stats shown appear to be NT to CAR + SSR and should be re-done with the relevant comparisons to CAR alone – otherwise it appears disingenuous.\n2) Figure 1I, is there statistical difference in the killing of the CD38 KO cells – why? Please discuss\n3) Figure 2 C-D- the color of the CAR + ΔSSR is different between graphs and legend.\n4) In figure 3B-3E the number of mice and timepoints is not consistent in assessments of AML, CAR T cells, and survival. Eg – one mouse in CAR.SSR has tumor burden on D5 but then goes away – where is that mouse on survival curve? Overall, there appear to be more mice in D than B.\n5) line 140: reference to supplementary figure 4 seems incomplete as the sentence mentions expansion and that is not shown.\n6) The killing of normal tumor blood cells in figure 4B-C killing data are from over 24h, while for tumor cells throughout they do 3 days. The authors should repeat with longer time points and a higher E:T ratio to truly assess the risk of on-target toxicity through CD38. For NK cells in 4B, there appears to be some (non statistically significant killing). Is the CD38 high population being eliminated (can look by flow?)?\n7) The label of the y axis in figure 4b needs to be adjusted\n8) 5G - In the text the authors mention enhanced cytokine production against BV173 but the figure only shows data for THP-1.\n9) The masking data appears highly out of place and is difficult to follow.\n10) All BLI should be shown as log values which is standard\n11) line 303: the statement in the discussion that the LAT SSR enhances CAR signaling is not supported by data. This could be true if the experiments described in the major critique section are performed to demonstrate PLC-gamma recruitment etc.", "reviewer_2": "Tsai and colleagues have submitted a manuscript, where they describe a novel fusion receptor, that ameliorates synapse formation based on engineered signaling, conditionally to boost CAR T cell efficacy. Given the interest in cellular therapeutics the strategy is of potential value to the field and the specific approach employed novel. The manuscript is well written and easy to follow but I have the following concerns that requires attention before further consideration:\n1) The impact in mice models is a bit underwhelming. Approximately an increase in median survival of 10-15 days in the MOLM-Model and the TCR-model and two animals clearing disease in the THP-1 model. While this may be sufficient to generate proof-of-concept, it is unlikely to be sufficient to trigger translational efforts around this. Additional evidence is required (see also point 2). This could for example include primary AML samples demonstrating similar enhancement\n2) In the in vitro experiments provided it looks that the delta SSR has activity (see for instance calcium flux, cytokine production and perhaps even degranulation). This means that delta SSR is an essential control to distinguish the effect of additional binding and of LAT signaling. This is an essential control (CAR+delta SSR) missing in the in vivo experiments.\n3) The legends are sometimes hard to follow, as the authors change the order of conditions across some graphs (for example 2C-D and E-F) and even misallocate some labels (see same figure 2). This needs correction.\n4) It is difficult to follow what the nature of the data is that is shown. Are the n numbers donors? Replicates? Independent – dependent? This needs to be made very clear for each figure and subfigure\n5) How was cell line identity ascertained", "reviewer_3": "The present manuscript intends to improve the efficacy of CAR T cells against tumors with suboptimal antigen expression such as antigen-low AML. It relies on the co-expression of a CAR targeting CLL-1 and containing a CD28 costimulatory domain and of a separate CAR called synapse-stabilizing receptor (SSR) that harbors a modified LAT cytoplasmic domain and targets CD38, a second tumor antigen expressed on AML. Providing additional exogenous LAT scaffolds and co-localizing them with the regular CD3-zeta containing CAR is expected to boost CAR T cell activation and it constitutes the rationale beyond the approach. A truncated LAT cytoplasmic domain (LAT177) that retains the PLC-g1 binding tyrosine residue (Tyr) but lacks Tyr191 and Tyr226 residues which are required for SLP-76 docking was found to reduce tonic signalling and spontaneous cytotoxicity and in turn used in most of the study. In contrast to THP-1 cells, Molm-13 AML cells express low levels of CLL1 insufficient for effective recognition and cytolysis by regular CLL1.CAR T. In contrast, CLL1.CAR T-cells armed with LAT177-based SSR killed Molm-13 AML. The use of CD3-edited CAR.SSR T-cells further demonstrating the specificity CAR-mediated cytotoxicity. Consistent with the results of an ultrasonic assay showing that the expression of SSR-LAT177 did not increase the avidity of CLL1.CAR T-cells binding to Molm-13, a series of experiments involving super-resolution confocal imaging showed that coexpression of the CLL1 CAR and of SSR boosted signaling and immune synapse formation. Two mouse xenograft model of human AML were used to evaluate the in vivo efficacy of SSR-coexpressing CLL1 CAR T cells and corresponding to NSG mice lacking murine MHC Class I and Class II genes and engrafted with Molm3 or CLL1low THP-1 cells. Both models showed that SSR expression significantly boosts anti-leukemic activity of CLL1.CAR T cells against antigen-low AML in vivo. Importantly, simultaneous targeting CD38 and CLL1 SSR did not exacerbated off-tumor toxicity against normal cells/tissues expressing CD38. Finally, coexpression of CD38 SSR also improved the anti-tumor efficacy of a survivin specific TCR. Therefore, this thorough and solid study validate the translational potential of synapse-stabilizing receptor.\nSpecific comments\n1/ The method of gammaretroviral co-transduction leading to high levels with of both CAR needs to be more precisely described.\n2/ The sentence ‘expansion of T-cells expressing SSRs with CD27 or 4-1BB endodomains was significantly impaired likely due to the toxicity from tonic TNFR signaling commonly observed with gammaretroviral vectors’ is a bit cryptic and might need to be further developed."}, "author_response": "We would like to thank the Reviewers for their constructive comments that allowed us to improve the manuscript. We have addressed all these queries in our point-by-point response below. As a result, we have updated all Figures, expanded their number from 5 to 6, and updated Supplementary Figure 3 with new data. We have also added 6 new Supplementary Figures (5, 6, 8, 11, 14 and 15). The revised manuscript file with new and modified Figures and with the amended text highlighted in green is included in this re-submission. We hope the modified version is now acceptable for publication\nReviewer #1 (Remarks to the Author):\nMajor comments\nImaging to determine mechanism of SSR mediated enhancement on the synapse: The authors show that the expression of the SSR results in increased calcium flux following antigen challenge, suggesting enhanced PLC-gamma activity. However, this happens even in the absence of CAR, raising the question of whether the assay was correctly performed or whether the overexpressed SSR drives baseline phosphorylation. Can the calcium flux be repeated in a flow based assay?\nIf this Ca flux without CAR signaling is real, would a mutation at Y132 abrogate the enhanced calcium flux? In addition, the authors should image PLC-gamma or a further downstream interacting molecule at the SSR/synapse to show the mechanism is indeed PLCgamma related as it seems unlikely that PLCgamma is being recruited and activated in the absence of CAR signal.\nResponse:\nWe appreciate the on-point comments from the Reviewer. Calcium influx requires continuous receptor engagement by target cells or antibodies. For flow-based Ca++ assays, antibody stimulation is most commonly used because cell-cell interactions are disrupted during flow cytometry for the accurate measurement in single cells. In contrast, live microscopy can distinguish CAR/SSR T cells from target cells and thus quantify calcium flux only in T cells engaging AML. Indeed, we did not detect any calcium influx in CAR T cells co-cultured with Molm-13 by flow cytometry (Figure 1A). We attempted to overcome this limitation by stimulating instead with anti-CLL1 CAR antibody and recombinant CD38. Unlike the anti-CLL1 antibody, recombinant CD38 failed to induce Ca++ signaling in SSR-expressing T cells, likely highlighting the need for immobilization-mediated crosslinking (Figure 1B). However, in both cases we did not observe increased baseline Ca++ flux in SSR-expressing T cells suggesting CD38 engagement by the SSR in cis does not trigger Ca++ signaling.\nFigure 1. Flow cytometry-based calcium influx assays. Calcium influx in CAR T cells upon co-culture with Molm-13 at 1:1 E:T ratio (A) or stimulated with 0.3 µg anti-CLL1specific antibodies and 1µg recombinant CD38 in a flow tube. Calcium signal was tracked by CAL520-AM.\nTo further evaluate the role of PLCγ1 signaling in SSR, we co-transduced CLL1 CAR with SSR with a LAT177 containing a Y132F mutation to prevent PLCγ1 binding (Figure 2A) and examined the cytotoxicity of CAR.SSRY132F T cells against Molm13 compared with control CAR.SSR T cells. The Y132F mutation abrogated SSR activity resulting in no benefit in cytotoxicity or CAR-T expansion (Figure 2B). These data highlight the critical role of PLCγ1 in SSR.LAT177 function. These data are added to the main manuscript as Supplementary Figure 6\nFigure 2. Mutation of PLCγ1 binding site in SSR reduced the cytotoxicity of CLL1 CAR T cells. (A) Schematic of SSR construct containing a point mutation Y132F in the LAT endo-domain. (B) Flow plots showing the co-expression of CAR and SSR or SSR with Y132F mutation in LAT endo-domain on day 6 post transduction. (C) Residual tumor counts (left) and the fold T-cell expansion normalized to day0 (right) in a 3 day co-culture with Molm13. The subsets including 2 independent donors from one experiment.\nNext, we evaluated PLCγ1 signaling in SSR-armed CLL1 CAR T cells via the Western blot. We observed higher levels of PLCγ1 phosphorylation and the downstream ERK1/2 and NF-kB pathways in SSR-expressing CAR T cells after short stimulation with anti-CLL1 CAR ideotype antibodies and recombinant CD38-coated plate compared to control CAR T cells (Figure 3A, B). Collectively, these results demonstrate SSR activates PLCγ1 mediated signaling in CAR-T cells. These data have been added to the manuscript as Figure 2D, E\nFigure 3. SSR improved the activation of PLCγ1-mediated pathways in CLL1 CAR T cells. (A) Western blots showing phospho-proteins and GAPDH loading control in CAR or CAR.SSR T-cells stimulated with plate-bound anti-CLL1 CAR antibody and recombinant CD38. (B) Quantification of fold change of pPLCγ1, pERK1/2 and pNF-KB normalized to GAPDH by densitometry.\nAlso, in 2A/B, the conditions include NT, CAR, CAR + SSR, and SSR alone. However, in the quantification it is listed as CAR + ΔSSR (figure 2C). The latter would be the appropriate control as, hypothetically, CD38 engagement by the ΔSSR could lead to improved assembly of the immune synapse after CLL-1 binding. This should be clarified and repeats of the experiment should be performed with the ΔSSR.\nResponse:\nWe thank the Reviewer for the important comment. Indeed, the SSR-alone condition in Figure 2C was mislabelled and is now corrected in the modified Figure 2 in the main manuscript (Figure 4). We have also repeated the experiments including ΔSSR with T cells derived from additional donors for immune synapse characterization. We observed co-expression of ΔSSR had only slight effects on actin polymerization and MTOC polarization (Figure 5).\nFigure 4. CD38.SSR enhanced effector function and immune synapse polarization of CAR T cells upon co-culture with antigen-low AML. (A) Calcium flux in T cells interacting with Molm13 over the indicated timepoints, and the mean area under the curve (AUC) from 0 to 20 mins at post contact. Each dots represent single cells. Data combined from 2 independent donors and experiments. (B) Representative fluorescence confocal microscopy of F-actin, SLP76, WASP and microtubule organizing center (MTOC) as well as lytic granule markers Lamp1 and GzB after 1 hour co-culture of CLL1.CAR T cells with Molm13. White bars denote 10 µm. Quantitative single-cell analysis of the distance from (C) F-actin, MTOC, (D) LAMP1 and granzyme B (GzB) to the immune synapse. Each dots represent single cells. Percentages of (E) IFNγ/IL-2 positive and (F) GzB/CD107a double positive population CD4 and CD8 subsets in CLL1.CAR T cells after a 4-hour co-culture with Molm13 at a 1:1 E:T ratio. Subsets include 3 or 4 donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, or Kruskal-Wallis test with Dunn’s post-test for single cell quantification. Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ***P<0.001. ns, non-significant).\nFigure 5. The effect of SSR and ΔSSR on the maturation of immune synapse in CLL1 CAR T cells. Measurement of the distance of filamentous actin (F-actin) and MTOC (A) and pSLP76 (B) to the immune synapse in individual T cells. Individual dots represent single cells derived from 4 (A) or 3 (B) independent donors compared to CAR. P values were determined using one way ANOVA with Dunnet’s correction for multiple comparisons. Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ns, non-significant).\nDoes the immune synapse of CAR+SSR at low CLL-1 densities looks compared to the immune synapse of CAR only at high CLL-1 densities? In other words, is LAT overexpression overall restoring the quality of the immune synapse to that which is seen with high antigen density? Additionally, an important technical question is does MOLM-13 include CLL-1 negative cells? If so, could this affect the results as authors may have imaged a CLL-1 negative cell?\nResponse: These are all great points. To address them, we generated CLL1-high Molm13 using ectopic expression of human CLL1 ORF via gammaretroviral transduction, resulting in a high surface CLL1 level (Figure 6A). I contrast to the parental Molm-13, the CLL1 high Molm-13 cells were highly susceptible to CLL1 CART killing, with very little additional benefit provided by the SSR (Figure 6B, C). Unfortunately, CLL1 overexpression changed the morphology of AML cells precluding effective attachment to a microscopy slide and making impossible imaging studies. Regarding the CLL1-negative Molm-13 cels, it is highly unlikely they are present at meaningful frequencies as SSR-armed CLL1 CART cells (but not control T cells or those expressing SSR alone) eliminate >99.9% of Molm-13 cells in vitro and in vivo suggesting CLL-1 is expressed in vast majority of AML blasts at targetable levels. Further, these antigen-negative cells would be present at the same frequency in co-cultures with both SSR-armed and unarmed conditions thus equalizing the chance of their imaging in both instances.\nFigure 6. Overexpression of CLL1 re-sensitized Molm13 to CLL1 CAR T cells. (A) Flow histogram showing the CLL1 levels in parental Molm13 and CLL1-high Molm13, and (B) the flow plots and bar charts showing the residual tumor counts (GFP+) after a 3 day co-culture with Molm13 at a E:T ratio 1:4. Data represent two independent donors.\nSpecificity of the SSR approach: The concept of the SSR is to enhance killing only in the presence of the target of the cytotoxic CAR. However, the authors see killing on CLL1 negative cells in Figure 1E (brown vs gold). This is obfuscated a bit by using a log axis. This should be changed to linear and discussed head on. Quite annoyingly, the authors switch from log in Fig 1 to linear in supplemental figure 3e which is inconsistent and highlights the data the authors wish to stand out. This should be consistent. Additionally, there is no statistical difference between the CD28- and LAT- SSR killing of CCRF-CEM in figure 1E so the SSR should not be deemed cleaner than the CD28 approach.\nThe authors then show that TCR KO rescues this unwanted activity which is reassuring. However, it is unclear whether this has to do with the mere presence of TCR vs allo-reactivity which the authors would like to ‘blame.’ This is extremely important to clarify and to answer this question, the authors could KO B2M/CIITA in the tumor cell lines and repeat the experiment. It also has not been evaluated if TCR KO could also abrogate the killing of CCRF-CEM (CLL1-) by the CD28-SSR which should be approached since the SSR is deemed by the authors as more specific.\nResponse: We understand the frustration although it wasn’t our intent to deceive the reader. To address the important points raised by the Reviewer, we conducted a series of additional experiments.\nFirst, we repeated the experiments comparing the activity of CLL1 CAR T cells armed with SSR-CD28 with SSR-LAT177and pooled results from 6 independent donors. While SSR-LAT177 and SSR-CD28 showed comparable activity against Molm13 (CLL1-low cells, Figure 7A), we observed statistically higher non-specific killing of CLL1-negative CCRF-CEM by SSR-CD28 armed CLL1 CART (on par with a control dual-CAR condition) which translated to a higher T-cell expansion indicative of their activation (Figure 7B). These data have been integrated into the manuscript as Figure 1E.\nFigure 7. SSR-LAT177 arming selectively increases T-cell cytotoxicity against CLL1-low AML. Residual tumor counts (left) and the fold T-cell expansion normalized to day 0 (right) after a 3 day co-culture with Molm13 (A) and CCRF-CEM (B) at a E:T ratio1:4. Data represent 6 donors from 3 independent experiments. for each. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ***P<0.001. ns, non-significant).\nThen, we aimed to further dissect the mechanism of CLL1-independent killing of CCRF-CEM; all results are shown in a linear scale to avoid confusion. We generated CD3epsilon-knockout T cells and transduced them with CLL1 CAR or SSR or both. Like in the previous experiment, CLL1 CAR T cells armed with SSR-CD28 produced non-specific killing by both WT and CD3KO T cells resulting in their expansion while the activity of SSR-LAT177 was substantially attenuated by CD3 removal (Figure 8A). Further, to address the Reviewer’s query on the role of TCR expression in SSR-mediated non CAR-specific killing we deleted b2M in CCRF-CEM using CRISPR/Cas9. CCRF-CEM do not express HLA class II obviating the need for CIITA knockout (Figure 8B).\nNotably, deletion of b2M did not significantly change cytotoxicity of SSR-armed CLL1 CAR T cells (Figure 8C). However, the cytotoxicity of SSR-LAT177 armed CAR T cells was similar to that armed with a truncated non-signaling SSR suggesting CCRF killing was mediated by CD38 engagement rather than LAT signaling (Figure 8C).\nTo better understand the activity of SSR-armed T cells against normal and malignant CD38+ cells, we performed co-cultures with T-ALL lines P12 ICHIKAWA and CCRF-CEM and normal autologous T, B, and NK cells. All these cell types had different expression of CD38 (Figure 8D). To minimize alloreactivity against T-ALL cell lines, we knocked out TRAC in T cells and modified them to express either truncated SSR or SSR-LAT177. Both SSR-armed T cells produced minimal cytotoxicity against normal and malignant cell lines after a 3-day co-culture, with no difference observed between ΔSSR and SSR-LAT177 armed T cells (Figures 8E, F). These results strongly indicate the lack of non-specific killing of CD38+ cells through the SSR and have been included in the manuscript as a Supplementary Figure 11.\nFigure 8. The effect of TCR expression and activity on SSR-aided cytotoxicity against the CLL1-negative cell line CCRF-CEM. (A) Residual tumor counts normalized to NT (left) and the fold T-cell expansion (right) in a 3 day co-culture of WT or CD3εKO CAR T cells with CCRF-CEM at 1:4 E:T ratio. Data include 5 donors from 3 independent experiments. (B) Histograms show the expression of DR/DP/DQ (MHCII) and β2M (MHCI) on CCRF-CEM. (C) Normalized residual tumor counts of WT or β2M KO CCRF-CEM in a 3 day co-culture with CAR T cells. Data include 3 donors from 2 independent experiments. (D) CD38 expression in T-ALL lines and PBMCs. Residual counts of T-ALL cells (E) or PBMCs (F) after a 3 day co-culture with SSR T cells at E:T 1:4. P values were determined using two way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M.\nAvidity: The experiment using acoustic force seems not to have worked correctly. The NT T cells (no CAR) have a higher avidity than the CAR or CAR+ SSR which doesn’t make sense as the CAR (and CAR+SSR) has a binder for the tumor cells. This should be removed or controls showing the assay worked correctly included (eg add a CD38 CAR and/or show these conditions against a CLL1 high cell line).\nResponse: The data have been removed from the Figures.\nMechanism of in vivo efficacy: Importantly, the in vivo experiments, it would be appropriate to include a CAR + ΔSSR control to rule out that the enhanced activity derives from the LAT signaling (even better would be a full SSR bearing a mutation at Y132).\nResponse: We repeated in vivo experiment including the CAR+ ΔSSR and SSR alone (Figure 9A). Consistent with previous results, CAR.SSR T cells displayed potent anti-leukemic activity improving T-cell expansion and significantly prolonging animal survival (Figure 9B-E). In contrast, co-expression of ΔSSR did not enhance CLL1 CAR T-cell anti-tumor activity in vivo suggesting LAT signalling is critical to mediate optimal SSR function in vivo (Figure 9B-E). These data have been added to the manuscript in Figure 4.\nFigure 9. Anti-tumor activity of SSR-armed CLL1 CAR T cells against Molm-13. (A) Schematic timeline of the experiment. (B) Leukemia progression monitored by bioluminescence and (C) CAR T-cell counts (hCD45+hCD3+CAR+) in peripheral blood were tracked at indicated days post CAR T-cell infusion. (D) AUC of CAR-T expansion between day 0 and day 14. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ***P<0.001. ns, non-significant). (E) Overall mouse survival in the Molm13 AML model with 5 mice for each group. Statistical significance for survival analysis was determined by a log-rank test.\nGeneralizability: Translation of this approach is the most important potential output of this work, but only a single CAR design has been tested in vivo. Would the SSR work to enhance a CAR with a CD28 hinge domain (which may not dimerize with the SSR)? What about a CAR that utilizes a 4-1BB signaling? This should be answered in vivo.\nResponse: To address this question, we generated two variants of the original CLL1 CAR (CD8H-CD28z): one with a CD28hinge (CD28H-CD28z) and another where the CD28 costimulation was replaced with 41BB (CD8H-41BBz) (Figure 10A). While all CARs were expressed alone or in combination with the SSR in T cells (Figure 10B), expression of the 41BB costimulated CAR (CD8H-41BBz) impaired T-cell expansion after transduction (Figure 10C). This phenomenon is commonly observed with CARs expressed from gammaretroviral vectors (Gomes-Silva et al., Cell Reports 2017) and, unfortunately, did not allow generating enough CAR T cells to perform a meaningful in vivo experiment. The same outcome was observed in our previous study with CLL1 CAR T cells (Atilla et al., JITC 2021). However, CAR-T cell numbers were sufficient for in vitro assays. In those, we observed an SSR-mediated enhancement of CLL1 CAR T-cell cytotoxicity and T-cell expansion upon co-culture with CLL1-low AML regardless of the CAR structure (Figure 10D, E). These data have been added to the manuscript (Supplementary Figure 5).\nFigure10. SSR-mediated enhancement of CLL1 CARs with different configurations. (A) The schematic of variant CLL1 CAR with CD8 hinge (CD8H-28z), CD28 hinge (CD28H-28z) or 41BB intracellular domain (CD8H-41BB), and (B) co-expression of CAR and SSR at 7 days post transduction. (C) Expansion of T cells expressing different CAR constructs. Residual tumor counts (D) and T-cell expansion normalized to day 0 (E) in a 5 day co-culture with Molm13 at a E:T ratio1:4. The subsets includes 3 donors from on experiments. P values were determined using two way ANOVA with Tukey’s correction for multiple comparisons, The XY graphs show mean+S.E.M.\nIn addition, we showed another SSR targeting GRP78 improves cytotoxicity, expansion and Ca++ flux in IL13Ra2-targeting CAR T cells upon co-culture with DIPG cell lines (See Figure 13 below).\nTCR: The enhancement of survivin-specific T cells is modest – is this a difference between CAR and TCR (eg TCR better at utilizing LAT so this is less relevant) – authors should discuss. The control group in the in vivo experiment figure are listed as “tumor only” (and not “control non-transduced cells” as stated in the text). This experiment should indeed include “control non-transduced cells” as well as SSR T cells +/- ΔSSR, to rule out any survivin independent reactivity that could be exacerbated by the SSR, the 3 T cell injections and the IL-2 administration.\nResponse: The main factor limiting TCR T-cell activity is their short functional persistence in vivo owning to the lack of Signals 2 and 3. The SSR does not provide those signals but instead amplifies Signal 1 from the CD3 zeta chain via LAT-mediated signaling. This increases cytotoxicity but does not prolong T-cell persistence which has to be boosted through additional engineering, such as ectopic expression of costimulatory receptors or cytokines.\nWith that in mind, we modified Survivin TCR T cells to express membrane-bound IL-15 to provide Signal 3 (Figure 11A, B, C) and incorporated the TCR+ΔSSR control, as requested by the Reviewer. Arming SurTCR T cells with IL15 only slightly increased their persistence and anti-tumor activity, likely reflecting the lack of a critical Signal 2 that is normally embedded into a CAR. Co-expression of a ΔSSR did not alter anti-leukemic activity of T cells while SSR-LAT177 co-expression mediated stronger anti-tumor control significantly prolonging animal survival (Figures 11D, E, F). These results reiterate the important of LAT signaling in the SSR and were added to the manuscript in Figure 6\nFigure 11. Anti-AML activity of SurTCR.SSR T cells armed with IL-15 (A) Schematic of a membrane-bound (mb)IL-15, containing IL15 fused with CD80 transmembrane region. A surrogate marker containing flag epitope on a CD8a stalk is integrated into the construct (top). Schematic timeline for sequential transduction is shown on the bottom. (B) Representative flow plots showing co-expression of TCR, SSR and mbIL15 on day 7 post transduction. (C) Schematic timeline of the mouse experiment. (D) Leukemia progression was monitored by bioluminescence and tumor burden AUC from day 0 to day 26. (E) Absolute TCR T-cell counts (hCD45+hCD3+TCR+CD8+) in peripheral blood were detected at indicated days post TCR T-cell infusion. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M. (*P<0.05). (F) Overall mouse survival in the THP1 model, and the cohort include 19 mice (NT, n=5, TCR, n=5, TCR ΔSSR, n=5, TCR.SSR, n=4). Statistical significance for the survival curve was determined by a log rank test.\nAntigen downregulation: In a manuscript about overcoming low antigen density, it would be good to understand what antigen looks like in tumor cells at relapse in vivo – this would help define the limits of the novel system and drive future work.\nResponse: We agree on the importance of this analysis and have attempted it previously. However, AML relapses in CAR.SSR T-cell treated mice were confined to the jaw/skull area resulting in their deformation and requiring mouse euthanasia (Figure 12). We were unsuccessful in retrieving these AML cells for analysis.\nFigure 12. AML relapses in the Molm-13 model. (A) Flow plots show circulating Molm13 (GFP+) and T cells in peripheral blood at indicated days post the infusion of CAR.SSR T cells, and (B) bioluminescent imaging reveals the location of relapsed AML cells at day 31 post infusion.\nOther comments:\n1) Statistics in the confocal imaging- There seem to be no statistical differences of CAR + SSR compared to the CAR alone condition, all stats shown appear to be NT to CAR + SSR and should be re-done with the relevant comparisons to CAR alone – otherwise it appears disingenuous.\nResponse: We have updated the graphs and compared CAR+SSR to CAR alone as shown above.\n2) Figure 1I, is there statistical difference in the killing of the CD38 KO cells – why? Please discuss\nResponse: Although the effects of CAR.SSR T cells to Molm13 were largely reduced after CD38 KO, minimal residual toxicity could be due to alloreactivity of T cells amplified by the SSR-LAT177 expression. We have added a brief discussion to the text (Line#139-147). More detailed interrogation of SSR-mediated toxicities is provided above.\n3) Figure 2 C-D- the color of the CAR + ΔSSR is different between graphs and legend.\nResponse: We thank the reviewer for pointing out the mistake, and have corrected the mislabelling in the Figure 2 C-D.\n4) In figure 3B-3E the number of mice and timepoints is not consistent in assessments of AML, CAR T cells, and survival. Eg – one mouse in CAR.SSR has tumor burden on D5 but then goes away – where is that mouse on survival curve? Overall, there appear to be more mice in D than B.\nResponse: The discrepancy is due to limitations of data presentation as 0 tumor count detected in the PB could not be shown on a log scale. We have now added those time points with a value of “10” to reflect them in the graph (Figure 13).\nFigure 13 CLL1.CAR T cells armed with CD38.SSR suppressed progression of systematic leukemia and prolonged animal survival. (A) Schematic timeline of the experiment. (B) Leukemia progression was monitored in peripheral blood by flow cytometry (hCD45+GFP+). (C) Overall mouse survival in the Molm13 AML model, NT, n=6, CAR, n=6 and CAR.SSR, n=5. Statistical significance was determined by a Log-Rank test.\n5) line 140: reference to supplementary figure 4 seems incomplete as the sentence mentions expansion and that is not shown.\nResponse: We thank the Reviewer for the note and have added T-cell expansion data into the Supplementary Figure 4D.\n6) The killing of normal tumor blood cells in figure 4B-C killing data are from over 24h, while for tumor cells throughout they do 3 days. The authors should repeat with longer time points and a higher E:T ratio to truly assess the risk of on-target toxicity through CD38. For NK cells in 4B, there appears to be some (non statistically significant killing). Is the CD38 high population being eliminated (can look by flow?)?\nResponse: We understand a consistent time point would be better. However, monocyte survival from primary PBMCs is very brief, forcing shorter time course studies. We repeated the co-culture with autologous lymphocytes for 72h as shown in Figure8 above. No NK-cell killing was observed via the SSR. For the last question, CD38 detection is obfuscated by the scFv of CD38 SSR that recognizes the same epitope as the detection antibody.\n7) The label of the y axis in figure 4b needs to be adjusted\nResponse: We thank the Reviewer for the note. The label has been adjusted and updated in Figure 5B.\n8) 5G - In the text the authors mention enhanced cytokine production against BV173 but the figure only shows data for THP-1.\nResponse: We thank the Reviewer for pointing this out. We corrected the description in the text (Line# 291-294).\n9) The masking data appears highly out of place and is difficult to follow.\nResponse: We thank the Reviewer for pointing this out. CD38 masking data addresses an important question why there was no cell fratricide in SSR-armed sur-TCR T cells derived from HLA-A2 positive donors, as these cells should be Sur+ and CD38+. Furthermore, masking can potentially enable a combinatory therapy of CAR T cells and daratumumab in the future. We have clarified this in the text (Line#274-282).\n10) All BLI should be shown as log values which is standard\nResponse: We have changed all in vivo BLI data to a Log scale.\n11) line 303: the statement in the discussion that the LAT SSR enhances CAR signaling is not supported by data. This could be true if the experiments described in the major critique section are performed to demonstrate PLC-gamma recruitment etc.\nResponse: To address this point, we performed WB to show the activation of PLC-gamma-mediated pathway in CAR.SSR T cells, and the effect of disrupting PLC-gamma signaling in the SSR as shown above. These data have been added to the revised manuscript and discussed.\nReviewer #2 (Remarks to the Author):\nTsai and colleagues have submitted a manuscript, where they describe a novel fusion receptor, that ameliorates synapse formation based on engineered signaling, conditionally to boost CAR T cell efficacy. Given the interest in cellular therapeutics the strategy is of potential value to the field and the specific approach employed novel. The manuscript is well written and easy to follow but I have the following concerns that requires attention before further consideration:\n1) The impact in mice models is a bit underwhelming. Approximately an increase in median survival of 10-15 days in the MOLM-Model and the TCR-model and two animals clearing disease in the THP-1 model. While this may be sufficient to generate proof-of-concept, it is unlikely to be sufficient to trigger translational efforts around this. Additional evidence is required (see also point 2). This could for example include primary AML samples demonstrating similar enhancement\nResponse: We thank the Reviewer for bringing this up. Indeed, in vivo results could be “improved” by using less challenging models, such as increasing T-cell dose or decreasing tumor cell number or engraftment time. However, we did not optimize our models for these parameters. However, demonstrating the activity of SSR-armed CAR-T against primary tumor cells with “physiologic” levels of CLL-1 and CD38 is very important. We co-cultured CAR.SSR T cells with AML blasts isolated from peripheral blood of two patients. These blasts had a different pattern of CLL1 expression and retained CD38 expression (Figure 14A). CLL1 CAR T cells produced measurable killing of AML compared to control non-transduced T cells which was only minimally aided by a truncated SSR (Figure 14B). In contrast, co-expression of SSR-LAT177 significantly improved cytotoxicity of CLL1 CAR T cells on par with CLL1/CD38 dual CAR T cells (Figure 14B). We have added these data to the revised manuscript (Figure 5D, E)\nFigure 14. Co-culture of CAR.SSR T cells with primary blasts from AML patients. (A) Flow histograms show surface CLL1 and CD38 levels on AML blasts (CD33+/CD34+) from the PBMCs collected from 2 patients (Pt1 and Pt2). The residual AML blasts were detected at 24hrs or 72 hrs after co-culture with CLL1 CAR T cells in 1:1 E:T ratio. The subsets include 3 independent donors from one experiment. P values were determined using a one way ANOVA with Tukey’s correction for multiple comparisons, or paired Student’s t test for CAR vs CAR+SSR in residual AML blasts from Pt2 at72hrs. Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ***P<0.001. ns, non-significant).\nTo further evaluate the application of SSRs beyond AML, in collaboration with Dr Giedre Krenciute’s group at St Jude Children’s Research Hospital we developed an SSR targeting GRP78, an antigen commonly expressed in diffuse intrinsic pontine glioma (DIPG) and other cancers (Figure 14A, B). Co-expression of the GRP78 SSR improved the cytotoxicity of IL13Ra2 CAR T cells on against IL13Ra2-high and low patient-derived DIPG (Figure 14C, D), and enhanced calcium influx upon contact with DIPG cells (Figure 14E), supporting the broad role of SSR in improving CAR-T effector function. We have added these data to the revised manuscript (Supplementary Figure 8)\nFigure 14. GRP78 SSR enhances the activity of IL13Ra2 CAR T cells against patient-derived DIPG. (A) Schematic diagram showing the structure of IL13Ra2 CAR and GRP78 SSR fused with IgG4 hinge-CD28TM and LAT177 endo-domains, followed by truncated CD19 as a surrogate marker. (B) The co-expression of CAR and SSR shown at day 6 post transduction. (C) Histogram showing the IL13Ra2 and GRP78 levels on DIPG (Diffuse Intrinsic Pontine Glioma) patient-derived xenograft (PDX) cells. (D) The cytotoxicity assay was performed by co-culture of CAR.SSR T cells against FFluc+ DIPG cells at E:T ratios spanning from 2:1 to 1:16. (E) Calcium influx was tracked in CAR.SSR T cells generated from 3 donors (DN1-DN3) co-cultured with indicated DIPG cells at a E:T ratio 1:1, and (F) the mean area under the curve (AUC) from 0 to 30 mins at post-contact. XY and bart graphs show mean+S.E.M. P values were determined using one way ANOVA with Dunnett’s T3 correction for multiple comparison.\n2) In the in vitro experiments provided it looks that the delta SSR has activity (see for instance calcium flux, cytokine production and perhaps even degranulation). This means that delta SSR is an essential control to distinguish the effect of additional binding and of LAT signaling. This is an essential control (CAR+delta SSR) missing in the in vivo experiments.\nResponse: We repeated in vivo experiment including the CAR+ ΔSSR and SSR alone (Figure 15A). Consistent with previous results, CAR.SSR T cells displayed potent anti-leukemic activity improving T-cell expansion and significantly prolonging animal survival (Figure 15B-E). In contrast, co-expression of ΔSSR did not enhance CLL1 CAR T-cell anti-tumor activity in vivo suggesting LAT signalling is critical to mediate optimal SSR function in vivo (Figure 15B-E).\nFigure 15. Anti-tumor activity of SSR-armed CLL1 CAR T cells against Molm-13. (A) Schematic timeline of the experiment. (B) Leukemia progression monitored by bioluminescence and (C) CAR T-cell counts (hCD45+hCD3+CAR+) in peripheral blood were tracked at indicated days post CAR T-cell infusion. (D) AUC of CAR-T expansion between day 0 and day 14. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ***P<0.001. ns, non-significant). (E) Overall mouse survival in the Molm13 AML model with 5 mice for each group. Statistical significance was determined by a Log-Rank test for the survival curve.\n3) The legends are sometimes hard to follow, as the authors change the order of conditions across some graphs (for example 2C-D and E-F) and even misallocate some labels (see same figure 2). This needs correction.\nResponse: We thank the Reviewer for this comment and have corrected the figures\n4) It is difficult to follow what the nature of the data is that is shown. Are the n numbers donors? Replicates? Independent – dependent? This needs to be made very clear for each figure and subfigure\nResponse: The n numbers represent independent donors from 2 to 3 independent experiments. We have clarified this in figure legends.\n5) How was cell line identity ascertained\nReply: All cell lines (Molm13, THP1, CCRF-CEM, BV173, TALL1) used in the study have been authenticated with STR analysis in the Cytogenetics and Cell Authentication Core (UT MD Anderson Cancer Center). The latest analysis was performed on March 04 2025\nReviewer #3 (Remarks to the Author):\nSpecific comments\n1/ The method of gammaretroviral co-transduction leading to high levels with of both CAR needs to be more precisely described.\nResponse: We have amended the Methods section to include more details (Line #439-453).\n2/ The sentence ‘expansion of T-cells expressing SSRs with CD27 or 4-1BB endodomains was significantly impaired likely due to the toxicity from tonic TNFR signaling commonly observed with gammaretroviral vectors’ is a bit cryptic and might need to be further developed.\nReply: We thank the Reviewer for pointing this out and have explained this observation in more details (Line #123-125).\nReviewer #1 (Remarks to the Author):\nThe authors have addressed many of the prior concerns with thoughtful revisions, resulting in an improved manuscript. However, there are still some major issues that need to be addressed prior to publication – these are largely around tempering claims that are clearly not supported by the data. Also, we have major concerns with the dependability/reproducibility of the data for the calcium influx and this should likely be removed from the manuscript unless the authors can provide additional experimental data.\nCalcium influx results: The data presented are not sufficiently robust to support the conclusion that the enhanced calcium influx is dependent on the signaling contribution of the SSR. In fact, this reviewer has many questions about the dependability of this data. In Figure 2B, SSR expression appears to increase calcium influx even in the absence of the CAR. This is very surprising and unexpected as calcium influx has many downstream effects in T cells that do not aippear to occur in the SSR only control condition throughout the paper. In addition, in Supplementary Figure 8, this does not occur with the GPR78 SSR. Thus, it seems possible that there is a technical problem with the assay as performed in Figure 2B. Furthermore, in the flow-based assay shown in the response to reviewers, while the recombinant antibody triggered some calcium influx in the CAR-only condition, this response was not observed when the SSR was present. Based on these observations, one could speculate that the control CAR+ΔSSR would yield a similar result, thereby excluding a specific contribution from the LAT endodomain. In the absence of appropriate controls and explanations for the difference with the CD38 and GPR78 SSRs, the calcium experiments do not convincingly support the authors’ interpretation and should be removed from the manuscript.\nResponse:\nThe calcium influx assay in CD38 SSR alone was consistently observed in three independent experiments, making a technical error unlikely. We also observed background calcium changes in T cells expressing ΔSSR only (Figure 1A-C below), which were not significantly higher than the full-length SSR containing the LAT endodomain. Therefore, the spike in Ca++ was likely driven by the CD38 engagement on T cells or target cells by the SSR rather than by the SSR signaling endodomains. This increase could be attributed to the known enzymatic function of CD38 regulating calcium stores1, elevating intracellular NAADP levels and triggering calcium release from lysosomes2, 3. In contrast, GRP78 does not have a known Ca++ release function which explains the discrepancy between these SSRs. Nonetheless, co-engagement of the ΔSSR and the CAR only slightly increased calcium levels, while simultaneous engagement of both full-length SSR with the modified LAT and the CAR significantly enhanced calcium influx illustrating strong agonism by the LAT endodomain (Figure 1D). Increased calcium influx was further confirmed in GRP78-targeting SSR, which showed no background calcium spikes (Supplementary Fig. 8).\nFigure 1. Expression CD38.SSR enhanced calcium influx of CAR T cells in response to CLL1-low AML. (A) Calcium flux in SSR T cells interacting with Molm13 over the indicated timepoints in individual experiments, and (B) the mean area under the curve (AUC) from 0 to 30 mins post-contact. The calcium flux in (C) SSR T cells or (D) CAR.SSR T cells combined from 3 independent experiments, and (right) the mean area under the curve (AUC) from 0 to 30 mins post-contact. Each dot represents a single cell. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ns, non-significant).\nMechanistically, PLCγ1-mediated ERK/MAPK pathways were also enhanced in CAR.SSR T cells (main Fig. 2C and 2D), a response not observed in SSR T cells alone (Figure 2). Disruption of the PLCγ1-binding site Y132 on LAT abolished the SSR-mediated enhancement of CAR-T activity and expansion during co-culture with antigen-low AML cells (Figure 3), highlighting the critical role of LAT signaling in SSR function. Therefore, signaling pathways from CAR and SSR converged to activate T cells, whereas SSR alone, despite increasing calcium levels, was insufficient to trigger robust T-cell cytotoxicity and activation during co-culture. Overall, Ca++ flux data supports the intended function of the SSR. We have updated the main Figure 2B and discussed CD38-mediated calcium changes in the manuscript (Lines 168–173).\nFigure 2. The PLCγ1 mediated pathways in SSR T cells. (A) Western blots showing phosphoproteins and GAPDH loading control in NT control or SSR-only expressing T cells stimulated with plate-bound anti-CLL1 CAR antibody and recombinant CD38 at indicated doses for 2 mins. (B) Densitometry analysis of pPLCγ1, pERK1/2 and pNFKB normalized to GAPDH.\nFigure 3. Mutation of PLCγ1 binding site in SSR reduced the cytotoxicity of CLL1 CAR T cells against Molm13. (A) Schematic of SSR construct containing a point mutation Y132F in the LAT endo-domain, and representative flow plots (bottom) showing the co-expression of CAR and SSR or SSR with Y132F mutation in LAT endo-domain on day6 post transduction. (B) Residual tumor counts (left) and the fold T-cell expansion normalized to day0 (right) in a 3 day co-culture with Molm13. The subsets including 4 donors from two independent experiments. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean+S.E.M. (*P<0.05, ns, non-significant).\nWestern Blot signaling results: The authors should clarify how many independent experiments were performed and specify the number of biological replicates used. To ensure the robustness and reproducibility of the findings, the experiment should be repeated using cells from at least two to three independent donors in different experiments.\nResponse:\nWestern blot experiments were repeated in three independent experiments using three donors. The Western blotting results showed induction of the ERK/MAPK pathways in CAR.SSR T cells stimulated with low densities of anti-CLL1 CAR idiotype antibodies, as presented in one of the independent experiments (Figure 4).\nFigure 4. CD38.SSR improved the activation of PLCγ1 pathway in CLL1 CAR T-cells in response to low antigen density. (A) Western blots showing phospho-proteins and GAPDH loading control in CAR or CAR.SSR T-cess cells stimulated with plate-bound anti-CLL1 CAR antibody and recombinant CD38 at indicated doses for 2 mins. (B) Densitometry analysis of of pLAT, pPLCγ1and pERK1/2 normalized to GAPDH.\nContribution of LAT to the activity of the SSR vs avidity effects: While the in vivo contribution of the LAT component of the SSR is very clear, some of the enhancements observed in short term in vitro assays (e.g., calcium flux, cytotoxicity) are partially attributed to enhanced avidity resulting from CD38-scFv engagement alone. Examples include Figure 1E, 1G, and Supplementary 3C. The authors should address this and discuss the potential impact of increased avidity from the CD38-scFv on their findings. This concern is particularly relevant to the data presented in Supplementary Figures 5 and 8, where the absence of a ΔSSR control makes it difficult to isolate the specific contribution of the SSR. Furthermore, the new experiments shown in Supplementary Figure 11 with normal cells offer nearly no insight, as they should have been conducted using T cells co-expressing the SSR and a CAR to exclude the possibility that the SSR could exploit the CD3ζ provided in trans by the CAR.\nResponse:\nWe do not deny that expression of a ΔSSR slightly increases T-cell cytotoxicity – as the Reviewer has noted, likely due to the increased passive binding to the target cell and not due to the baseline changes in Ca signaling since this mild effect was lost when targeting a CD38KO target (main Fig. 1I). However, this effect was only observed in vitro, as CAR T cells armed with a ΔSSR did not have any difference in leukemia control in vivo or animal survival (main Fig. 4A-E) whereas CAR T cells co-expressing a full length SSR had a significantly better tumor control and prolongation of survival. Therefore, we concluded the modest effect of a ΔSSR, while consistently observed in vitro, has limited real-world implications, at least in the models we have tested. We have added a discussion on this point into the manuscript (Lines 348–354).\nThe aim of the new experiments in the Supplementary Figure 11 (D-E) was to provide additional data on the toxicity of SSR alone against CD38-high leukemia cells, compared with primary PBMCs, which express low to intermediate levels of CD38. The experiment was not designed to answer the question the Reviewer raised in the second round of reviews. We added a clarification in the manuscript (Lines 247–250).\nSpecificity of the SSR: Instead of recognizing that the CD38-SSR may have some off cancer effects, the authors appear to use statistical and graphical ‘slight of hand’ to minimize this data – this is incredibly frustrating to the reviewer. Instead of discussing that there appears to be some effect in vitro, the authors minimize these findings and make broad statements throughout the manuscript that are not supported by the data. This includes:\n1) “Line 137: CAR T cells co-expressing LAT177-based or CD27-based SSR produced only minimal cytotoxicity against CCRF139 CEM...”\n-this is not minimal – this is a log-based scale\n-should use terminology such as ‘reduced but not absent cytotoxicity’ instead of minimal\n-overall this section of the manuscript seems to be written in a manner in which the authors try not to acknowledge some background killing through the CD38-SSR- e.g -supplemental figure 3- For TCR KO and B2M-KO, this does not completely ablate the background killing – should make that clear (for CCRF-CEM – CD3KO maybe eliminates the stats, but the majority of donors still kill; and for B2M-KO, CAR vs CAR+SSR-LAT is very different – doesn’t matter if the deltaSSR is the same – even if this is from adhesion, the non-specific killing through CD38 is potentially real). Please improve the language to more accurately reflect the data.\n-Also the ICHIKAWA line doesn’t show what is claimed since the experiments were performed without a CD3z containing CAR. Please remove or add those controls.\nResponse:\nThere is no “sleight of hand” or other magic here. All cytotoxicity graphs are shown in a log scale, including CCRF-CEM. Yes, there is background cytotoxicity of CAR T cells co-expressing truncated or full length SSR against these cells, which is negated when targeting a CD38KO line (Figure 5). Coupled with the data on TCR KO (Supplementary Fig. 3), these results suggest that the background allogeneic killing of CCRF-CEM is enhanced through CD38 binding by an SSR.\nThe word “minimal” is subjective and in this case meant to imply this activity is unlikely to have clinically meaningful consequences. But we get the Reviewer’s point and have revised the language to indicate the activity is low but detectable.\nFigure 5. The toxicity of CD38 SSR-arming CLL1 CAR T-cells in CD38-high T-ALL cells. (A) Residual tumor counts after a 3 day co-culture with CCRF-CEM at a E:T ratio1:4. Data represent 6 donors from 3 independent experiments. (B) residual tumor counts of WT or CD38KO CCRF-CEM in a 3 day co-culture with CAR T cells. Data include 2 donors from one independent experiment. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M. (*P<0.05, **P<0.01, ***P<0.001. ns, non-significant).\n2) Line 157 – “Engaging CD38 on target cells is critical for the CD38.SSR activity as genetic ablation of the CD38 gene on Molm13 minimized the benefit of the SSR on CAR T-cell cytotoxicity and expansion during co-culture, suggesting SSR.LAT177- mediated augmentation of CLL1.CAR-mediated cytotoxicity was fully dependent on CD38 expression”\n-this is not fully dependent, you have nearly half a log difference in the CD38KO line from CAR+SSR-LAT vs CAR and this is statistically significant\n-temper language from “fully dependent” to ?perhaps mainly or largely dependent\nResponse:\nWe understand the frustration when data shown does not match the description but the reason for the choice of words is that we surmise these effects are mostly artefacts of an in vitro culture. However, there is no in vivo experiment to test this assumption so we changed “fully dependant” to “largely dependent” (Line#156).\n3) Line 243: “Co-expression of the CD38.SSR did not enhance cytotoxicity of CLL1.CAR T cells against autologous monocytes and produced no activity against CLL1-negative autologous T cells, B-cells, and NK-cells”\n-again problematic – fig 5B is 3 donors, the results are not significant likely because there is variability in the NK counts in the control condition, but if you look at % of cells remaining compared to the control for each donor – it is likely reduced (eg two highest points in CAR-SSR are lower than the two highest points in the NT control or the CAR only)\n-need to temper that language as some killing is likely happening and should use a different shape for each donor so the reader can see\n-also please show the flow plots of the CD38 on the remaining NK cells so can see if the CD38-high population was eliminated\nResponse:\nWe understand the point, which is similar to other points raised in this round. The lack of statistical significance precludes us from saying “there is an activity” so stating that would be incorrect (Figure 6A-B). We amended the description to reflect that there was a minor decrease that did not reach statistical significance, unlike with the CD38 CAR (Lines 245-247).\nCD38 undetectable on NK cells after co-culture with ΔSSR- or SSR-armed CAR T cells, likely reflecting trogocytosis or trans-masking of CD38 on target cells by the SSR (Figure 6C). This phenomenon was also observed in SSR T cells, where CD38 levels were reduced even in SSR-negative populations (Figure 6D).\nFigure 6. The toxicity of CD38 SSR-arming CLL1 CAR T-cells on primary hematopoietic cells. (A) CD38 expression in CCRF-CEM compared to PBMCs. (B) Residual counts of autologous NK after 24 hour co-culture with CAR T cells at E:T 1:4. The data includes 4 donors shown in different shapes. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, Bar graphs show mean+S.E.M. (C) A histogram showing detection of CD38 on residual NK cells after 24 hour co-culture. (D) Representative flow plots showing detection of CD38 on SSR T cells on day7 post transduction.\nStatistics on immune synapse imaging: Throughout this figure, the control for the CAR+SSR should be the CAR+ deltaSSR since the authors are saying LAT is responsible for enhancing the synapse and these specific conditions are clearly not numerically different but the stats are purposely not shown – need to show those statistics and comment in the manuscript – not compare them independently to CAR only\nResponse:\nWe do not make the conclusion that LAT is responsible for enhancing the synapse based on these data but rather describe the effect of the full length SSR. The description of the results is provided below.\n“Next, we assessed how SSR affects the formation of CAR-mediated cytotoxic immune synapse using super-resolution confocal imaging of CLL1.CAR T cells exposed to Molm13 cells for one hour. While observing limited enhancement of lytic granule migration by SSR (Supplementary Fig. 7A, B), co-expression of SSR in CLL1.CAR T cells significantly improved polarization of filamentous actin (F-Actin) and the microtubule organizing center (MTOC) to the immune synapse, reflecting cytoskeleton reorganization and the formation of a more stable synapse (Fig. 3A). We also observed a trend towards enhanced recruitment of phospho-SLP76, a critical signaling partner of LAT (Fig. 3A, B). Expression of the SSR.LAT177 alone did not significantly affect polarization of the synaptic machinery (Fig 3A, B), suggesting increased Ca++ influx was insufficient to trigger effector function of SSR-LAT177 expressing T cells in the absence of CAR signaling, consistent with prior findings. “\nTerminology of AND-gate: Authors refer to their approach as an “AND” gate in both the abstract and the discussion – this is not accurate as the authors do not claim both targets are necessary for activation (this manuscript is more in line with recent publication on an “IF-BETTER” approach in AML). This is not an AND-gate because the authors do not expect or even aim not to have cytotoxicity on CLL1 high tumor cells. In their own words, “We surmised this non-cytotoxic synapse-stabilizing receptor (SSR) will not trigger T-cell activation per se but instead amplify CD3z signaling from the main antigen receptor thus improving T-cell degranulation and target cell killing.” The terminology should be changed in the abstract and the discussion.\nResponse:\nWhile we do observe modest activity from the CAR alone in some in vitro models, the in vivo results (Fig 4A-E) demonstrate a clean “AND” gate as neither CLL1.CAR T cells alone or SSR T cells alone demonstrate any discernible activity whereas combinatorial expression produces the desired effect. The same is observed with the Survivin TCR against THP-1 (Fig. 6). We agree in other models the effect resembles the “IF-BETTER” gate, as illuminated in the discussion. We have removed the “AND” gate language from the abstract but still maintain the validity of the “AND” gate in Discussion.\nOther comments:\n-virtually all of the supplementary figure call outs are to the whole figure and not specific panels which really hurts the readability and made reviewing the manuscript much more difficult – this should be corrected\nResponse:\nThat is a good point. We have modified references to Supplementary Figures to specify panels.\n-In Figure 6E-F for the BV173 cell line, statistical comparisons should be performed against the CAR-only and CAR+ΔSSR control conditions and not against untransduced cells.\nResponse:\nWe agree and have updated the statistics here (Figure 8) and in the manuscript.\nFigure 8. The cytotoxicity of SSR.TCR T-cells on HLA-A2-high leukemia BV173. The residual tumor counts and TCR T cell expansion after 3-day co-culture with indicated cell lines at a E:T ratio 1:4. The subsets include 6 donors from 2 independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparisons. Bar graphs show mean+S.E.M.(*P<0.05).\n-Line 287 of the manuscript refers to Supplementary Figure 12E, which does not appear to exist.\nResponse:\nIndeed, it should read Supplementary Fig 12D. Fixed.\n-Supplementary Figure 5 doesn’t say what cell line was used\nResponse:\nWe have provided a clarification (Molm13) in the legend of Supplementary Figure 5.\n- Supplementary Figure 9D, top, rightmost flowplot – this seems somewhat impossibly negative for CD62L – was this an FMO tube or was antibody accidently not added? Something doesn’t look 100% right\nResponse:\nThere is no mistake. CLL1 CAR with the CD28-CD3ζ intracellular domain produces tonic signaling resulting in gradual depletion of CD62L+ cells.\nReference\n1. Lee HC, Deng QW, Zhao YJ. The calcium signaling enzyme CD38 - a paradigm for membrane topology defining distinct protein functions. Cell calcium 2022, 101: 102514.\n2. Churchill GC, Okada Y, Thomas JM, Genazzani AA, Patel S, Galione A. NAADP mobilizes Ca(2+) from reserve granules, lysosome-related organelles, in sea urchin eggs. Cell 2002, 111(5): 703-708.\n3. Fang C, Li T, Li Y, Xu GJ, Deng QW, Chen YJ, et al. CD38 produces nicotinic acid adenosine dinucleotide phosphate in the lysosome. The Journal of biological chemistry 2018, 293(21): 8151-8160.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed most concerns and tempered their claims to match their data.\nIn keeping with the response letter, I ask that they update the description of the western blot in the legend to indicate the number of times the experiment was performed.\nResponse:\nWe have added the description in the legend of Figure 2C (line 903)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most concerns and tempered their claims to match their data.\nIn keeping with the response letter, I ask that they update the description of the western blot in the legend to indicate the number of times the experiment was performed.", "reviewer_2": "For the avoidance of doubts, I will exclusively on the previous reviewer #2. From my perspective, the authors have done a very good job at adressing all my comments and concerns.", "reviewer_3": "The Authors have thoroughly addressed my comments as well as the substantial and appropriate comments made by the two other Reviewers."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed most concerns and tempered their claims to match their data.\nIn keeping with the response letter, I ask that they update the description of the western blot in the legend to indicate the number of times the experiment was performed."}, "author_response": "Response:\nWe have added the description in the legend of Figure 2C (line 903)."}]

Decision letter