Engineering programmable CAR and antigen pairing via drug-gated light activation

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Chimeric antigen receptor (CAR) T-cell immunotherapy has achieved clinical success, but remains limited in solid
tumors by antigen escape and tumor heterogeneity. Here, we engineer a high-affinity R-phycoerythrin (PE)-binding
monobody to create a PE-programmable CAR toward user-defined antigens sequentially or simultaneously. To
reduce off-tumor toxicity, we implement a drug-gated light-activation approach (DGLA) strategy to spatially
confine CAR function. We further couple DGLA-controlled tumor antigen presentation with synNotch-mediated
programmable antigen-targeting CAR (sPAT) to establish programmable CAR-antigen pairing (DGLA-sPAT).
This system induces clinically validated antigens on tumor cells as local ‘training centers’ that recruit and activate
sPAT CAR T cells, enabling elimination of entire tumor populations through broadly expressed tumor antigens
using PE-conjugated antibodies. In vivo, DGLA-sPAT manifests local T cell activation and potent tumor
suppression with minimal off-tumor toxicity. Thus DGLA-sPAT provides a modular and spatially controlled
framework to overcome antigen escape and heterogeneity while improving safety in solid-tumor CAR-T therapy.

Introduction
Chimeric antigen receptor (CAR) T cell-based therapies have achieved tremendous success in treating blood
tumors1-4. While these therapies have shown promising outcomes for solid tumors in some cases, there are still
challenges that must be overcome before their broad application5-11. One major obstacle is antigen escape, where
tumor cells down-regulate the expression of antigens targeted by CAR T cells, resulting in tumor relapse4, 12, 13. In
addition, tumor heterogeneity is challenging for current CAR T therapies, most of which are single-antigen targeted4.
CAR T cells that can simultaneously target multiple antigens have been developed and have demonstrated
successful outcomes14-17; however, broadening the spectrum of target antigens also increases the risk of off-tumor
toxicity, especially when the therapeutic cells are delivered systemically18-20. Constitutively active CAR T cells are
difficult to control once infused into patients and hence not well versed in addressing these adversities21-23.

Programmable antigen targeting (PAT) CAR, which binds to an adapter molecule that can be conjugated to various
antibodies against different tumor antigens8, 24-26, have been developed to address the antigen escape and tumor
heterogeneity19, 27. The PAT CAR designs offer several potential advantages, including: (1) flexibility in antigen
choice, which can be challenging to predict due to the heterogeneous complexity of physiology in different patients;
(2) versatility in applications that allow the same CAR T cell product to be used for different antigen-targeting
demands. However, immunogenicity and off-tumor toxicity still present potential safety concerns for the PAT CAR
designs28-30.

In this study, we aim to develop potent CAR T cells with programmable antigen targeting to address antigen escape
and tumor heterogeneity and yet achieve safe CAR T therapies31-34 (Fig. 1a). We developed a new PAT CAR based
on PEbody, which is a monobody derived from human fibronectin type III via directed evolution by our group to
be a specific binder of phycoerythrin (PE)35. This PEbody PAT CAR can be programmed to target multiple user-
defined antigens via PE-conjugated antibodies sequentially or simultaneously. PE is considered biocompatible and
safe and has been widely used in food, cosmetics, pharmaceutical, and biomedical industries36-38. As such,
compared with other PAT CARs that may cause immunogenicity28-30, PAT CARs with the fully human-derived
PEbody and its target PE can have better biocompatible immunogenicity35, 39, 40.

To minimize the off-tumor side effects, especially when the PEbody PAT CAR T cells are empowered with
broadened multi-antigen targeting capability, we have implemented the tamoxifen-gated photo-activatable Cre
(TamPA-Cre) approach to confine the PEbody PAT CAR T cell activity in the local tumor regions41. This drug-
gated light-activation (DGLA) mechanism regulated by the FDA-approved drug tamoxifen can define a time
window for intended light activation, thus minimizing Cre activity leakage and basal noise originating either from
spontaneous pMag-nMag dimerization or unintended activation by ambient light sources41, 42. This DGLA-
controlled PAT CAR T approach (DGLA-PAT) should (1) allow targeting of user-defined tumor-associated
antigens to overcome challenges in solid tumor therapies, such as antigen escape and tumor heterogeneity6, 10, 12, 43;
(2) provide the controllability of the therapeutic T cells’ cytotoxicity in space and time thus minimizing the off-
tumor toxicity22, 31, 33. We further extended the DGLA-PAT approach to combine DGLA-controllable tumor antigen
presentation and synNotch-mediated PAT CAR (sPAT CAR) for programmable CAR-antigen pairing (DGLA-
sPAT), which can induce clinically validated antigen presentation on the surface of cancer cells to serve as ‘training
centers’ for the recruitment and activation of sPAT CAR T cells. These trained or activated sPAT CAR T cells can
then attack the entire cancer cell population at tumor sites, guided by PE-conjugated antibodies targeting broadly
expressed tumor antigens. As these ‘training centers’ can be precisely confined at local tumor regions by light, this
DGLA-sPAT approach is not limited by targeting antigens that are exclusively expressed on tumor sites, which can
be challenging to identify, while ensuring safe and efficient tumor clearance. Consistently, our in vivo tests in mice
showed a local sPAT CAR T cell activation and effective tumor suppression with minimal off-tumor toxicity. As
such, we have developed DGLA cells with programmable CAR and antigen pairing to target solid tumors with high
flexibility, killing efficiency, and safety.

In summary, we establish a programmable CAR platform based on a PEbody that enables flexible multi-antigen
targeting and combine it with a drug-gated light activation system to spatially restrict CAR activity. We further
integrate inducible tumor priming with synNotch-regulated CAR expression to generate localized ‘training centers’
that activate therapeutic T cells only within illuminated tumor regions. This strategy achieves effective tumor
control in vivo while maintaining minimal off-tumor activity. Beyond this specific implementation, the framework
provides a generalizable approach for decoupling antigen choice from CAR engineering and for enforcing spatial
control over cell therapies. Such programmable and locally confined immunotherapies offer a path toward safer
targeting of heterogeneous solid tumors and expand the design space for next-generation cellular therapies.

Results
A PEbody-based PAT CAR. We developed a PAT CAR using PEbody35 and the third generation CAR design
containing the human T cell co-stimulatory domains CD28 and 4-1BB, and the intracellular signaling domain of
CD3ζ44 (referred to as PEbody PAT CAR). As such, PEbody PAT CAR can be programmed to target the PE tag
on different antibodies recognizing their corresponding tumor-associated antigens (Fig. 1b, Supplementary Fig.
1a).

We firstly verified the programmable antigen-targeting capability of PEbody PAT CAR T cells using different PE-
conjugated antibodies that target CD19, CD20, and CD38 on different tumor cell lines (Fig. 1c, Supplementary
Fig. 1b). The orthogonality of PEbody PAT CAR was also confirmed (Supplementary Fig. 1c-d), demonstrating
its ability to target various tumors and antigens with high specificity. Consistent with the PAT CAR design, the
cytotoxicity of PEbody PAT CAR showed a dose-dependent pattern on antibody concentration (Fig. 1d). Increasing
the effector-to-target (E/T) ratio also led to more effective killing of the target cells without significantly affecting
the basal cytotoxicity (Fig. 1e, Supplementary Fig. 1e). To further demonstrate the broad applicability of PEbody
PAT CAR, we tested its cytotoxicity against several solid tumor cell lines, including breast cancer cell line MCF-
7 (targeting MUC1 or HER2), the aggressive triple-negative breast cancer cell line MDA-MB-231 (referred to as
MDA, targeting MUC1 or PD-L1), as well as the prostate cancer cell line PC-3 (targeting PSMA), all demonstrating
efficient killing performance with the corresponding target antibodies (Fig. 1f). Additionally, the PEbody PAT
CAR T cells demonstrated robust upregulation of T cell activation markers CD25 and CD69 upon target
engagement (Supplementary Fig. 1f), accompanied by cytokine secretion (Supplementary Fig. 1g). These results
established that PEbody PAT CAR can be programmed by different antibodies to target corresponding tumor
antigens with high specificity.

Using PEbody PAT CAR T cells to mitigate antigen escape and heterogeneity of tumors. Antigen escape has
become one of the major causes of relapse after CAR T therapies45-47. We tested the programmable feature of
PEbody PAT CAR in an antigen-escape model of CD19, one of the prevailing causes of relapse after CD19CAR T
treatment45-47. We reasoned that the same PEbody PAT CAR T cells can not only target CD19+ tumor cells, but
also be redirected to target the CD19-negative cells in the event of antigen escape, by supplementing with other
PE-conjugated antibodies such as those against CD20, another antigen on the target tumor cells (Fig. 2a). To
mimic antigen escape, we generated CD19-negative Nalm6 cells via CRISPR-Cas9 mediated gene knockout (Fig. 2b,
Supplementary Fig. 2a). We observed a decreased killing capability of PEbody PAT CAR T cells (targeting CD19)
as the percentage of CD19-negative cells in the tumor population increased (antigen escape), which can be fully
restored by switching to the PE-conjugated CD20 antibody while using the same CAR T cells (Fig. 2c).

As PEbody PAT CAR can also be programmed to target multiple antigens simultaneously, we further tested its
cytotoxicity against heterogeneous tumors. With a mixed population of CD19− and CD19+ Nalm6 cells (1:1), the
PEbody PAT CAR T cells can kill ~50% of the total tumor cells when targeting CD19 only (using PE-conjugated
CD19 antibody), while a significant killing (>80%) can be achieved with both CD19 and CD20 antibodies present
in the co-culture (Fig. 2d). Consistently, using engineered PC-3 prostate cancer cell lines (either CD19+/PSMA−
or CD19−/PSMA+, Supplementary Fig. 2b-c), the PEbody PAT CAR T cells also demonstrated excellent
cytotoxicity against the heterogeneous population (CD19+/PSMA−: CD19−/PSMA+ = 1:1) only when both CD19
and PSMA antibodies were present (Fig. 2e), with each antibody contributing to the killing of its corresponding
subpopulation (Fig. 2f).

DGLA-PAT CAR T cells. The PEbody PAT CAR framework enables targeting of multiple antigens, a property
useful for combating antigen heterogeneity and escape in solid tumors, but broad targetability can raise the risk of
on-target/off-tumor effects14-17. To enhance safety while preserving flexibility, we integrated our drug (tamoxifen)-
gated light-inducible (DGLA) gene expression system, TamPA-Cre41, to develop DGLA-PAT CAR so that the
action of PEbody PAT CAR T cells can be confined at tumor sites to reduce the off tumor cytotoxicity (Fig. 3a).

We firstly demonstrated that bioluminescence reporter gene (Fluc) can be controlled by DGLA (Fig. 3b), and
verified the inducible expression of CD19CAR in primary T cells (Fig. 3c), which can be translated into cytotoxicity
against CD19+ target cells (Fig. 3d). Consistently, DGLA also allowed precise regulation of the PEbody PAT CAR
expression in T cells by light (Fig. 3e), which can then be programmed to attack the target tumor cells, leading to
controllable T cell activation (Fig. 3f) and cytotoxicity against target tumor cells (Fig. 3g). We further applied this
DGLA-PAT CAR approach to address the antigen escape with the CD19 model introduced in Fig. 2a. Indeed, the
“escaped” (CD19-negative) tumor cells can be contained by switching targeted antigen from the initial CD19 to
CD20 (Fig. 3h), which can be precisely controlled by drug-gated light stimulation (induced vs. non-induced) to
reduce off-tumor toxicity and achieve multi-antigen targeting, thereby addressing antigen escape and tumor
heterogeneity in a single approach.

DGLA tumor priming and PAT CAR killing in vitro. We further extended the potential of DGLA-PAT CAR
for tumor priming and immunotherapy, using DGLA for local priming of tumor cells to guide the PAT CAR T cells
for tumor killing. This delivery of DGLA and PAT CAR gene cassettes into separate cell types should reduce the
genetic load for each cell type and thereby enhance the delivery efficacy (Fig. 4a). In this design, the tumor cells
will be firstly vaccinated by a clinically validated non-pathogenic viral vector, adeno-associated virus (AAV) 48, to
express DGLA and its inducible reporter for user-defined antigen presentation (e.g., clinically validated antigen,
the truncated CD19 or tCD19 in this case). As tCD19 may likely be induced only in a subpopulation of the tumor
cells due to limitation on efficiencies of gene delivery and DGLA activation, a direct targeting of the induced tCD19
could result in incomplete eradication of the entire tumor cell population. Instead, the subpopulation of tumor cells
with induced tCD19 could be designed to serve as ‘training centers’ for systemically delivered T cells engineered
with anti-CD19 synthetic notch receptor (synNotch), and transactivate the expression of PEbody PAT CAR
(synNotch-mediated PAT CAR, or sPAT CAR, Fig. 4b and Supplementary Fig. 3a). As such, the light-activated
and primed subpopulation of tumor cells can “train” and activate the sPAT CAR T cells, which can be programmed
to target antigen(s) of the whole cancer cell population at the local tumor site6. As the CAR expression will gradually
decay when the sPAT CAR T cells leave the “vaccinated” and light-primed local tumor region, this will allow
potent but less-ideal antigens to be used as targets for systemically introduced sPAT CAR T cells to ensure effective
tumor killing with minimal off-tumor toxicity. We named this specific strategy DGLA-sPAT CAR, with
programmable CAR killing guidable by DGLA local priming of vaccinated tumors.

To test the DGLA-sPAT CAR system, HEK 293T cells were engineered to universally express PD-L1 to mimic
the tumor antigen, as well as DGLA and its reporter, which can be induced to express tCD19 (Supplementary Fig.
3b). The sPAT CAR T cells were then introduced for co-culture, followed by the addition of PE-conjugated PD-
L1 antibody to target the PD-L1+ cells (Supplementary Fig. 3c, upper panel). We observed over 90% tumor killing
within 24 hr in the group where target cells had been induced to express tCD19 (Supplementary Fig. 3c, lower
panel), whereas the killing activity was minimized in the absence of antibody or DGLA induction. Similar killing
effect by the sPAT CAR T was also observed when tCD19 was induced in the breast cancer cell line MDA-MB-
231 expressing the endogenous PD-L1 (Fig. 4c).

We further tested the specificity of the DGLA-sPAT CAR approach using the engineered PC-3 cell lines (CD19+
or PSMA+ PC-3, Supplementary Fig. 3d-e). While sPAT CAR T cells can efficiently kill CD19+ PC-3 cells (with
antibody targeting CD19), they did not have significant cytotoxicity against CD19−/PSMA+ PC-3 cells even in the
presence of the PE-conjugated PSMA antibody, as PEbody PAT CAR cannot be induced. In the case of partial
CD19 induction in a heterogeneous tumor population, as mimicked here by the mixture of CD19+/PSMA− and
CD19−/PSMA+ PC-3 cells (Supplementary Fig. 3f), the sPAT CAR T cells can still be sufficiently activated to
target the heterogeneous population, with antibodies guiding the target antigens.

Due to the ‘training center’ effect, we reasoned that the percentage and level of induced tCD19 in primed tumor
cells and hence the light stimulation time can be less stringent while maintaining efficient killing. To test this,
different lengths of light stimulation were applied to the tumor cells41, and the expression of tCD19 was measured
at the 24, 48, and 72 hr time points after onset of light stimulation (Fig. 4d). We observed that the tCD19 expression
was mainly affected by the illumination time duration, with moderate and gradual increase after illumination
stopped (i.e., 48 and 72 hr time points). Specifically, with 24 hr illumination, over 85% of the tumor cells can be
induced to express tCD19, in contrast to less than 1% tCD19 expressing cells in the group without light stimulation
(Fig. 4e). Thus, antigen induction can be tightly controlled by light. Interestingly, we observed that over 50% of
the tumor cells can be induced to express tCD19 with 5 hr of illumination, which can be readily translated into
effective killing through the sPAT CAR mechanism6. Indeed, we observed strong killing of target tumor cells under
this shortened light stimulation, which is similar to that of the 24 hr illumination group (Fig. 4f). Even with 1 hr
illumination, ~50% of killing can be obtained. These results suggest that DGLA-sPAT CAR can achieve efficient
tumor killing with flexible illumination conditions.

DGLA-guided tumor priming in vivo. We then tested whether DGLA can guide gene expression in mice. Nalm6
cells engineered with DGLA and the inducible firefly luciferase (Fluc) reporter were subcutaneously inoculated in
the left and right flanks of the same mice (Fig. 5a). For DGLA, we administered tamoxifen to the mice one day
before light stimulation, as well as 5 hr before light stimulation to ensure ERT2-regulated nuclear translocation of
molecular regulators in tumor cells (Fig. 3a). We then applied pulsatile blue light (5 sec per min) to one side of the
mice for 12 hr, while keeping the other side in the dark as control. Bioluminescence imaging was used here to
monitor Fluc gene expression before and after light stimulation, with constitutively expressed Renilla luciferase
(Rluc) to serve as normalization reference. Our results showed that blue light led to a ~35-fold increase of gene
expression on day 3 when compared with the dark control (Fig. 5b). Moreover, the tamoxifen drug gating
mechanism worked robustly in vivo, as no gene activation was observed with or without light stimulation in the
absence of tamoxifen. Overall, our results demonstrated that DGLA can be used to achieve drug-gated light-
controllable gene expression in vivo.

Tumor homing and local activation of sPAT CAR T cells in vivo. In parallel, we tested whether the systemically
injected sPAT CAR T cells can home to and be activated locally at the tumor region in vivo that expresses the
designed antigen (tCD19 in this case). To this end, one flank of the mice was inoculated with MDA-MB-231 cells
expressing tCD19 (‘tCD19+ MDA’), while the other flank inoculated with control MDA cells without tCD19
(‘tCD19− MDA’) (Fig. 5c). The expression of tCD19 did not affect the behavior of MDA tumor cells
(Supplementary Fig. 4a). The CD19-targeting sPAT CAR T cells were then intravenously introduced into the
mice. To visualize the homing and activation of sPAT CAR T cells, we also cloned the firefly luciferase gene into
the synNotch reporter cassette downstream of the PEbody PAT CAR (Gal4UAS-Pmin-PEbody PAT CAR-P2A-
Fluc, Fig. 5c enlarged panel from sPAT CAR T), so that activation of the sPAT CAR T cells can be visualized by
bioluminescence in vivo49. Fluc bioluminescence was only observed at the side with ‘tCD19+ MDA’ tumor cells
following T cell injection, but not at the ‘tCD19− MDA’ tumors (Fig. 5d-e, Supplementary Fig. 4b). The early
rise in Fluc signal in Fig. 5e reflects synNotch activation of homed T cells rather than proliferation, as expansion-
driven Fluc expression also requires subsequent PEbody CAR induction. These results suggest that the homing and
local activation of sPAT CAR T cells only happened at the designated target tumor site (tCD19+), even though the
control side mimicking the normal tissues (tCD19−) has similar antigen profiles except tCD19.

To assess whether the sPAT CAR T cells could suppress the target tumor (‘tCD19+ MDA’), we also intravenously
delivered PE-conjugated PD-L1 antibody one day after T cell injection, which can trigger the killing activity of the
trained sPAT CAR T cells against MDA cells. Although MDA cells endogenously express PD-L150, tumor
suppression was only observed at the ‘tCD19+ MDA’ tumor, with minimal effect observed at the ‘tCD19−’ tumor
(mimicking normal tissue expressing the same PD-L1 antigen as the tumor) (Fig. 5f, Supplementary Fig. 4c). The
cytotoxicity of sPAT CAR T cells can hence be restricted to the local region of tumors expressing the clinically
validated antigen, allowing the discrimination between tumors and normal tissues via the tCD19 antigen, thereby
mitigating off-tumor toxicity after infusion.

DGLA-sPAT CAR T immunotherapy in vivo. We further examined the integrated DGLA-sPAT CAR T approach
with MDA cells pre-engineered with the DGLA-inducible tCD19 (Fig. 6a), instead of constitutively expressing
tCD19 as shown in Fig. 5c. After tumors were established, mice were primed with tamoxifen, and one side of the
mice was subject to blue light stimulation as described earlier in Fig. 5b (referred to as ‘light side’), while the other
side was kept in dark (referred to as ‘dark side’). Two days after light stimulation, sPAT CAR T cells were
intravenously introduced, followed by injection of PE-conjugated PD-L1 antibody every two days to trigger tumor
killing (Fig. 6b). Our results demonstrated that tumor growth on the light side was effectively suppressed by the
sPAT CAR T cells, while the dark side remained unaffected (Fig. 6c-d). Similar results were observed in an
additional study using the prostate cancer cell line PC-3 targeting the PSMA antigen (Supplementary Fig. 5),
confirming the broad applicability of this approach. Furthermore, the DGLA-sPAT CAR T cells were detected in
the peripheral blood and spleen after treatment, remaining non-activated (Supplementary Fig. 6a). In contrast, the
tumor-infiltrating DGLA-sPAT CAR T cells were clearly activated with significant PAT CAR expressions
(Supplementary Fig. 6b), consistent with the localized activation observed in tumors. More importantly,
hematoxylin and eosin (H&E) staining of major organs alongside kidney and liver functional tests revealed no
treatment-related lesions or toxicities (Supplementary Fig. 7), indicating the DGLA-sPAT CAR system's potential
as a safe and effective treatment for solid tumors.

In addition to using pre-engineered tumor cells, we further assessed the system’s applicability to tumors that are
vaccinated by DGLA gene cassettes in situ, which better mimics clinical conditions. We generated tumors on both
flanks of mice using wild type MDA-MB-231 cells, with one side serving as the target tumor and the other side
mimicking a normal organ/tissue with a similar antigen expression profile (Fig. 6e). To achieve DGLA-guided
tCD19 expression in the tumor cells, the adeno-associated viruses (AAVs) encoding the DGLA and the inducible
tCD19 reporter were delivered intratumorally (Supplementary Fig. 8a-b). Two days later, mice were primed with
tamoxifen and blue light was applied to the target tumor side to induce tCD19 antigen presentation (Fig. 6f). The
sPAT CAR T cells were then intravenously injected and subsequently “trained” and activated by the induced tCD19
in the tumor (Supplementary Fig. 8c) to express PEbody CAR. Subsequent injection of PE-conjugated PD-L1
antibody guided the activated sPAT CAR T cells to recognize and target PD-L1 on all MDA cells. Although this
treatment was initiated at a later stage using more established tumors (day 15 after tumor inoculation), we still
observed significant tumor growth slowdown and eventual reduction in size following the injection of sPAT CAR
T cells and targeting antibody (Fig. 6g-h). In contrast, the control tumors mimicking normal tissues continued to
grow exponentially, indicating minimal off-tumor toxicity of the DGLA-sPAT CAR approach. Another control
experiment further confirmed that the suppression effect was specifically mediated by the designed sPAT CAR T
cells (Supplementary Fig. 9).

To compare the safety and efficacy of the DGLA-sPAT approach with conventional CAR T therapy in vivo,
particularly to evaluate mitigation of on-target off-tumor toxicity (OTOT), we generated a PSMA⁺ tumor model
(Supplementary Fig. 10a), in which human PSMA was also expressed in the mouse liver via lentiviral
transduction20, 51 (Supplementary Fig. 10b), followed by treatment with either DGLA-sPAT CAR T cells or
conventional anti-human PSMA CAR T cells52. Successful hepatic expression of human PSMA was validated by
in vivo and ex vivo bioluminescence imaging (Supplementary Fig. 10c-d). Although both conventional PSMA-
CAR T and DGLA-sPAT CAR T effectively controlled tumor growth (Supplementary Fig. 10e-g), mice receiving
conventional CAR T displayed apparent weight loss (Supplementary Fig. 10h), indicative of a heightened
systemic toxicity burden53. Systemic cytokine profiling further revealed elevated cytokine levels in the conventional
CAR T group (Supplementary Fig. 10i-k), consistent with an exaggerated inflammatory response54. Along with
increased serum alanine aminotransferase (Supplementary Fig. 10l) and histological evidence of dense CAR-T
infiltration and hepatocyte damage (Supplementary Fig. 10m), these findings indicate liver OTOT caused by
conventional PSMA-CAR T treatment. In contrast, no significant OTOT-associated immune infiltration or tissue
damage was observed in the examined organs from the DGLA-sPAT-treated mice, confirming its substantially
improved safety profile. Collectively, these results demonstrate that DGLA-sPAT CAR T therapy achieves potent
antitumor activity while spatially restricting CAR T cytotoxicity, enabling a generalizable and antigen-
programmable platform for safe and effective treatment of solid tumors.

Methods
Ethics statement. Animal studies were conducted in compliance with all relevant ethical regulations and were
approved by the Institutional Animal Care and Use Committee (IACUC) of University of Southern California
(Protocol No. #21479). The maximal permitted tumor size/burden approved is 1.5 cm in diameter. Tumor growth
was monitored 3 times per week, and this limit was not exceeded in all experiments. Animals were euthanized in
accordance with the approved protocols if humane endpoints were reached. Sex was not considered in experiment
design and analysis as outcomes were not expected to yield meaningful sex-specific differences, and the objectives
of the study were not centered on sex-dependent effects.

Cell culture. Human cell lines HEK 293T (ATCC CRL-3216), MCF-7 (ATCC HTB-22), MDA-MB-231 (ATCC
HTB-26), and PC-3 (ATCC CRL-1435), all obtained from ATCC, were cultured in Dulbecco's Modified Eagle
Medium (DMEM, Gibco, 11995073) supplemented with 100 U/mL penicillin-streptomycin (Gibco, 15140122), 2
mM L-glutamine (Gibco, 25030149), 1 mM sodium pyruvate (Gibco, 11360070), and 10% Fetal Bovine Serum
(FBS, Gibco, 10438026). Human cell lines Jurkat (ATCC TIB-152) and Nalm6 (gift from Michel Sadelain lab,
ATCC CRL-3273) were cultured in RPMI 1640 (Gibco, 22400105), supplemented with 100 U/mL penicillin-
streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, and 10% FBS. Human peripheral blood mononuclear
cells (PBMCs) were isolated from buffy coats obtained from the San Diego Blood Bank using lymphocyte
separation medium (Corning, 25072CV) according to the manufacturer’s instructions. The PBMCs were provided
as fully de-identified, unidentifiable research-grade products. The study protocol and material usage were reviewed
by the University of Southern California Institutional Review Board (IRB), which determined that the project does
not meet the definition of human subjects research under 45 CFR 46.102(e)(1). Human T cells were subsequently
isolated from purified PBMCs using a pan-T cell isolation kit (Miltenyi, 130-096-535). Human T cells were
cultured in X-VIVO 15 medium (Lonza, 04-418Q) supplemented with 5% FSBS, 10 μM 2-mercaptoethanol (Gibco,
31350010), and 100 IU/mL recombinant human IL-2 (PeproTech, 200-02), with Dynabeads Human T-Activator
CD3/CD28 (Gibco, 11131D) following the manufacturer’s protocol. All the cell cultures were maintained at 37 C
with 5% CO2.

Cloning. Primers for PCR amplification were synthesized by Integrated DNA Technologies (IDT Custom DNA
Oligo service; sequences listed in Supplementary Table 1). PEbodyCAR construct (PEbody-CD28-4-1BB-CD3z):
PEbody fragment was PCR-amplified from DNA template reported35; CAR fragment CD28-4-1BB-CD3z was
amplified from a third generation anti-CD19 CAR (CD19 scFv-CD28-4-1BB-CD3z). TamPA-Cre (DGLA)
constructs including Cre-loxP based CD19CAR reporter were PCR-amplified from constructs reported41 (Addgene
#192636 and #176311). The truncated CD19 (tCD19) reporter was constructed by replacing CD19CAR with tCD19
(amino acid 1-313) as amplified from Addgene #174610 with codon optimization for human cell line expression
(Integrated DNA Technologies, CA). For lentivirus production, the lentiviral vector backbone from Addgene
#79125 was used. For adeno-associated virus (AAV) production, the AAV2 vector backbone from Addgene #59462
was used. AAVs were produced by GT3 Core Facility of the Salk Institute. PCR amplifications were performed
using Q5 high fidelity DNA polymerase (NEB, M0491S). DNA fragments were purified by agarose gel
electrophoresis followed by column-based purification (Zymo Research, D4008). All the Constructs were generated
by Gibson assembly (NEB, E2611L). Bacterial transformations were performed using NEB Stable Competent E.
coli following the manufacturer’s instructions (NEB, C3040H).

Lentivirus production. HEK 293T cells were seeded one day before transfection in a 10-cm tissue culture dish
and were transfected with 5 μg of psPAX2 (Addgene #12260), 5 μg of pMD2.G (Addgene #12259), and 10 μg of
the corresponding transfer plasmid, using the ProFection Mammalian Transfection System (Promega, E1200).
The medium was replaced with fresh medium at 6 hours post-transfection. Lentivirus-containing supernatants were
harvested at 48 hours post-transfection, filtered through a 0.45 μm filter (Nalgene, 725-2545), and concentrated
100x in PBS with Lenti-X Concentrator (Takara, 631232). To determine the virus titer, HEK 293T cells were
seeded at a density of 0.1 million cells per well in a 24-well plate and infected with serial dilutions of the
concentrated virus. The titer was calculated as infectious units per milliliter (IFU/mL).

Lentivirus infection. For infection of human cell lines, lentivirus was directly added to the culture medium with a
multiplicity of infection (MOI) of 1-3, and the infection efficiency was checked at day 3 post infection. For the
infection of primary human T cells, the naive CD3+ T cells were activated by CD3/CD28 Dynabeads (Gibco,
11141D) at 1:1 ratio for 48-72 hr, and were infected in RetroNectin coated wells (Takara, T100B) following a
spinoculation protocol (1500 g, 1 hr, 32 ℃) with an MOI of 5-10. Infection efficiency was checked at day 3 post
infection.

Live-cell imaging. Primary human T cells transduced to express PEbody CAR-GGSGGT-eGFP (day 5-7 post-
transduction) were washed and resuspended in phenol-red-free RPMI supplemented with 10 mM HEPES and 2%
FBS. Live cells were imaged on a Nikon ECLIPSE Ti inverted microscope equipped with a CCD camera, using a
100× objective.

Activation markers and cytokine measurements. A total of 0.1 million constitutive or inducible Jurkat T cells
or primary T cells were co-cultured with 0.1 million Nalm6 cells with 3 nM PE-conjugated anti-human CD19
antibody (clone HIB90, mouse IgG1, κ, BioLegend 302208) in a 96-well plate for 24 hr. Gating strategies were
exemplified in Supplementary Figure 11. Supernatants were collected for secreted IFN‑γ, TNF, and IL‑2
measurement via ELISA (Invitrogen 88-7316-22, 88-7346-22, 88-7025-22) following manufacturer’s instructions.
Cells were washed once with PBS, and resuspended in 100 uL PBS, and stained with APC-conjugated anti-human
CD69 antibody (clone FN50, mouse IgG1, κ, BioLegend 310910) or APC-conjugated anti-human CD25 antibody
(clone BC96, mouse IgG1, κ, BioLegend 302609) following the manufacturer’s instructions (Supplementary Table
2). The stained cells were analyzed on a BD Accuri C6 flow cytometer.

In vitro killing assays. Killing efficiencies of T cells were determined based on the firefly luciferase activity of
the remaining target cells after co-culture using Bright-Glo Luciferase Assay System (Promega, E2610) following
the manufacturer’s instructions. PEbody PAT CAR T killing assay: 0.1 million T cells (engineered or non-
engineered) were co-cultured with 0.1 million target cells expressing firefly luciferase with the corresponding PE-
conjugated antibody in a 96-well plate for 24 hr. To target different antigens, the following PE-conjugated
antibodies were used: CD19 (clone HIB19, mouse IgG1, κ, BioLegend 302208), CD20 (clone 2H7, mouse IgG2b,
κ, BioLegend 302306), CD38 (clone HB-7, mouse IgG1, κ, BioLegend 356604), MUC1 (clone 16A, mouse IgG1,
λ, BioLegend 355604), HER2 (clone 24D2, mouse IgG1, κ, BioLegend 324406), PD-L1 (clone 29E.2A3, mouse
IgG2b, κ, BioLegend 329706), PSMA (LNI-17, mouse IgG1, κ, BioLegend 342504). Data were normalized to
tumor-only control wells where no T cells were added.

CRISPR-Cas9 knockout of CD19 gene. Nalm6 cells were knockout by transfection with Cas9 protein-sgRNA
(5’-CUAGGUCCGAAACAUUCCAC-3’, Invitrogen) RNP. Specifically, the SF Cell Line 4D-Nucleofector X Kit
(Lonza) was used following the manufacturer’s instructions. RNP was prepared by incubating mixture of 2 uL Cas9
protein (30 pmol/uL) and 3 uL sgRNA (20 pmol/uL) for 15 min at 37 C. A total of 0.2 million of Nalm6 cells were
transfected with the prepared RNP using program CV-104 in 20 uL Nucleocuvette Strips (Lonza, V4SC-2096).
CD19 knockout on genetic level was verified by ICE analysis (Inference of Crispr Edits, Synthego). CD19 knockout
on protein level was further verified by flow cytometry after immunostaining (clone HIB19, mouse IgG1, κ,
BioLegend 302222) with isotype control (mouse IgG1, κ, BioLegend 400130), and CD19-negative clones were
isolated by FACS sorting (Sony Biotechnology, SH800S) and amplified for later experiments.

Antigen escape model. CD19-knockout Nalm-6 cell line was generated by CRISPR-Cas9, followed by verification
of CD19 expression via flow cytometry. The 25%, 50%, 75%, and 100% CD19 loss cells were generated by mixing
CD19-knockout Nalm-6 cells (0% CD19+) with wild-type CD19⁺ Nalm-6 cells (100% CD19+) at the defined ratios
to simulate varying levels of antigen escape.

DGLA CAR T killing assay. T cells expressing DGLA and CAR reporter were treated with 500 nM 4-OHT
(Sigma-Aldrich, H7904) for 3 hr, followed by blue light illumination (5 mW/cm2, 5 sec per min, for 12 hr). A total
of 0.1 million T cells were co-cultured with 0.1 million target cells expressing firefly luciferase in a 96-well plate
for 24 hr. Luciferase activities were measured as described above.

DGLA tCD19 based killing assay. Tumor cells expressing DGLA and tCD19 reporter were treated with 4-OHT
for 3 hr, followed by blue light illumination (5 mW/cm2, 5 sec per min, for 12 hr). A total of 0.1 million anti-CD19
synNotch PEbody PAT CAR T cells were co-cultured with 0.1 million target cells (treated as described above or
non-treated as control) expressing firefly luciferase in 96-well plate for 6 hr, followed by addition of the
corresponding PE-conjugated antibody and co-culture for another 24 hr. Luciferase activities were measured as
described above.

Migration and invasion assays. MDA-MB-231 cell lines were starved for 12 h in 0.5% FBS DMEM, trypsinized
and resuspended in 1% FBS DMEM. For migration, 5 × 10⁴ cells in 0.25 mL were seeded into 24-well 8-µm PET
inserts (Corning 353097); the lower wells contained 0.75 mL 10% FBS DMEM. For invasion, inserts were first
coated on ice with 50 µL Matrigel Matrix (Corning 354248) diluted 1:3 in cold serum-free DMEM, polymerized
30-40 min at 37 C, and hydrated 30 min in serum-free DMEM before seeding. All wells were incubated for 16 h
at 37 C, 5 % CO₂. Percentages of migration/invasion were calculated using the counted cell number on the
underside of inserts compared with the seeding cell number per insert.

Serum cytokine, urea, and alanine transaminase (ALT) assays. Whole blood was collected from mice at
indicated time points, allowed to clot for 30 min at 25 C, and centrifuged at 2,000 × g for 15 min at 4 C to obtain
serum. Serum cytokine concentrations were quantified using ELISA kits following the manufacturer’s instructions
(Invitrogen IFN-γ: 88-7316-22; IL-2: 88-7025-22; TNF: 88-7346-22). Serum urea and alanine transaminase (ALT)
levels were measured using the Urea Fluorometric Assay Kit (Cayman, #700620) and the Alanine Transaminase
Colorimetric Activity Assay Kit (Cayman, #700260), respectively, according to the manufacturers’ protocols.

In vivo gene activation. Mice were housed in an SPF barrier facility under a 12-hour light/12-hour dark cycle at
an ambient temperature of 20-26 °C and relative humidity of 40-60%. Mice were commercially purchased (no
breeding) and experimental/control mice were housed in the same room under identical conditions but maintained
in separate cages. Eight-week-old NOD/SCID/IL2rγnull (NSG, Jackson Laboratory, strain 005557) male mice were
subcutaneously injected with 1 million Nalm6 cells engineered to express DGLA, its reporter loxP-ZsG-loxP-Fluc,
and Rluc on the left and right flanks. For tamoxifen priming, tamoxifen (Sigma-Aldrich, 10540‐29‐1) was dissolved
in corn oil (Sigma-Aldrich, C8267) at a concentration of 20 mg/ml by rotation at 37°C until completely dissolved.
A dose of 100 uL tamoxifen solution was administered through intraperitoneal injection at 24 hr and 5 hr before
light stimulation. Blue light stimulation (460 nm, 5 W/m2, 5 sec/min) was applied for 12 hr on the ‘light’ side; and
the ‘dark’ side was protected from light. Bioluminescence imaging (BLI) was performed using an In vivo Imaging
System Lumina LT Series III (PerkinElmer). Images were analyzed with Living Image software (PerkinElmer).
The luciferase activity of Fluc and Rluc was measured using their respective substrates, D-luciferin (GoldBio,
LUCK-100) and coelenterazine (GoldBio, CZ10), following the manufacturers’ protocols. To prevent interference
between the two bioluminescence signals, the BLI of Fluc and Rluc in the same mouse was typically performed 4
hr apart. Imaging settings were kept constant throughout the entire experiment. The maximum tumor size permitted
is 1.5 cm in diameter, and all experiments were conducted in strict adherence to this limit. Tumor size was measured
using digital calipers, and animals were monitored three times per week throughout the study. Criteria for early
termination include significant body-weight loss (~15-20%), poor body condition or dehydration, inability to reach
food or water, severe lethargy or unresponsiveness, persistent pain or distress, abnormal respiration or neurologic
signs, large or ulcerated tumors exceeding approved size limits, serious infection or non-healing wounds, and any
condition judged by veterinary staff to compromise animal welfare. Mice were euthanized by cervical dislocation
by trained and experienced staff at experiment end points.

In vivo T cell homing and activation. Eight-week-old NSG (Jackson Laboratory, strain 005557) female mice
were subcutaneously injected with 1 million MDA-MB-231 cells expressing tCD19 (tCD19+) on one flank, and
wild type (tCD19−) on the other flank, both expressing Rluc for tumor growth monitoring. For in vivo tCD19
induction experiments, 1 million of engineered DGLA-tCD19 MDA-MB-231 cells or DGLA-tCD19 PC-3 cells
were injected on each flank of mice; all the tumor cells express Fluc for tumor growth monitoring. For in vivo gene
delivery and tCD19 induction experiments, 1 million of wild type MDA-MB-231 cells were injected on each flank
of mice; a total volume of 50 uL of purified AAV viral vector mixtures encoding DGLA and its reporter loxP-
ZsGreen-loxP-tCD19 was injected intratumorally per tumor. All the tumor cells express Fluc for tumor growth
monitoring. For DGLA activation, the tamoxifen priming and light stimulation were performed as described in the
above section. When tumors are palpable, a total of 5 million human T cells transduced to express anti-CD19
synNotch and its reporter Gal4UAS-PEbodyCAR-P2A-Fluc (for synNotch activation monitoring and tumor killing)
or Gal4UAS-PEbodyCAR (for tumor killing only) were injected through tail vein. BLI for Fluc and Rluc was
performed as described above. For tumor killing, a dosage of 8 ug of anti-human PD-L1 antibody (for MDA tumor,
clone 29E.2A3, mouse IgG2b, κ, BioLegend 329706) or anti-human PSMA antibody (for PC-3 tumor, clone LNI-
17, mouse IgG1, κ, BioLegend 342504) was injected through tail vein every two days. The tumor size was
monitored by Rluc or Fluc signals as indicated.

Ex vivo flow cytometry and frozen-section histology/IHC. At endpoint, spleen, peripheral blood, and tumors
were harvested. Single-cell suspensions were prepared as following procedures. Spleen: mechanical dissociation
through a 70-µm strainer with ACK RBC lysis (Gibco A1049201, 2-5 min, RT); peripheral blood: EDTA-
anticoagulated blood with ACK lysis (5 min, RT); tumor: mincing and digestion in DMEM containing collagenase
D (Roche 11088858001, 1 mg/mL) and DNase I (Roche 04716728001, 0.1 mg/mL) for 30 min at 37 °C, quenched
with 10% FBS and filtered (70 µm). Cells were washed in FACS buffer (PBS, 2% FBS, 2 mM EDTA). For staining,
1-2 × 10⁶ cells were incubated 20 min at 4 °C with anti-human CD3-APC (clone OKT3; mouse IgG2a, κ; BioLegend
317317) and R-PE (Cayman 16637) to detect PEbody PAT CAR, washed, and resuspended in FACS buffer. Data
were acquired on a BD Accuri C6 and analyzed in FlowJo v10. For histology, heart, liver, lung, spleen, kidney, and
brain were embedded in OCT, snap-frozen on dry ice, stored at -80 °C, and cryosectioned at 10 µm onto Superfrost
Plus slides. Sections were fixed in 4% paraformaldehyde (10 min, RT), rinsed in PBS, air-dried, and stained with
hematoxylin and eosin (H&E). For immunohistochemistry of tCD19 on frozen tumor sections, tumors were
harvested on day 8 after AAV delivery and light stimulation, embedded in OCT, snap-frozen, and sectioned at 10
µm. Sections were fixed in 4% paraformaldehyde (10 min, RT), air-dried, quenched with 0.3% H₂O₂ in methanol
(10 min), rinsed in PBS, and blocked in 5% BSA/PBS (30 min, RT). Slides were incubated with rat anti-CD19
(clone 6OMP31; Thermo Fisher 53-0194-82; 1:500 in blocking buffer) overnight at 4 °C, washed in PBS-T (0.05%
Tween-20), incubated with HRP-conjugated goat anti-rat IgG (H+L) (Thermo Fisher 31470; 1:500) for 1 h at RT,
developed with Pierce™ DAB Substrate (Thermo Fisher 34002) for ~2 min, rinsed, counterstained with
hematoxylin, blued, dehydrated, cleared in xylene, and coated on coverslips. Imaging was performed on an EVOS
M7000 (Thermo Fisher Scientific).

In vivo comparison of DGLA-sPAT CAR-T and conventional CAR-T therapy. Human PSMA was ectopically
expressed in the livers of 8-week-old NSG male mice (Jackson Laboratory, strain 005557) by tail-vein injection of
a lentiviral vector encoding PGK-nLuc-P2A-hPSMA (1×10⁸ TU/µL, 40 µL, diluted in 120 µL sterile DPBS).
Hepatic nLuc expression was verified by luminescence imaging using the Nano-Glo fluorofurimazine in vivo
substrate (Promega, N4100) according to the manufacturer’s instructions. Seven days after hepatic transduction,
mice were inoculated subcutaneously with 0.5×10⁶ PSMA⁺ PC-3 tumor cells expressing the DGLA-inducible
tCD19 cassette and constitutive firefly luciferase. For the DGLA-sPAT group, tamoxifen (20 mg/mL in sterile corn
oil; 75 mg/kg body weight) was administered intraperitoneally on days 11 and 12 after tumor implantation, followed
by localized blue-light stimulation at the tumor site 4 h after tamoxifen on day 12 (460 nm, 5 mW/cm², 5 s/min,
total 12 h). On day 14, all mice received a single intravenous infusion of 5×10⁶ untransduced T cells (UTD),
DGLA-sPAT CAR-T cells, or conventional PSMA CAR-T cells. Tumor burden was monitored by IVIS bioluminescence
imaging following D-luciferin administration (GoldBio, LUCK-1G). Peripheral blood was collected at three time
points, before infusion (pre-infusion), after DGLA induction (post-DGLA), and after CAR-T infusion (post-
infusion), for quantification of systemic cytokines (human IFN-γ, IL-2, TNF; Invitrogen kits 88-7316-22, 88-7025-
22, 88-7346-22). Serum ALT was measured post-infusion using the Cayman ALT Activity Assay (700260). Mice
were euthanized when tumor diameter exceeded 1.5 cm or when ulceration or necrosis was observed. At study
endpoints, major organs (heart, liver, lung, spleen, kidney) were harvested and processed for H&E staining to assess
tissue pathology. For ex vivo liver bioluminescence imaging, mice were injected with fluorofurimazine substrate
immediately before euthanasia, and freshly dissected livers were imaged using the IVIS system.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors developed a novel CAR that can be programmed with PE-conjugated antibodies to target multiple user-defined antigens, which allows for overcoming antigen escape and tumor heterogeneity. In this CAR-T system, by integrating PAT CAR and DGLA gene expression，DGLA-PAT CAR can confine the CAR T activation in the local tumor tissue and reduce off-target toxicity. this is a meaningful study，However, there are some experiments and controls that detract from the significance of this work. The following suggestions also should be taken into account.\n1.The authors emphasize the local infiltration and activation of CAR-T and speculate that the expression of CAR-T diminishes after it migrates out of the tumor tissue. In animal models, it is recommended to supplement data related to the detection of CAR-T in peripheral blood, spleen, and draining lymph nodes after CAR-T treatment. In addition, it is recommended to add safety-related studies, such as liver and kidney function tests, as well as pathologic evaluation of important organs.\n2.In Figure 2b, Isotype as a control should be added to verify the knockdown effect of CD19 in Nalm-6 cells.\n3.CD19 is a lineage marker for B cells, and the authors applied CD19+/PSMA-, as well as CD19-/PSMA+ prostate cancer PC-3 cells in their experiments, which required the detection of CD19 as well as PSMA expression in PC-3 cells.\n4.In Figure 3, the authors constructed Tam-light induced PAT CAR-T and verified the ability to target kill target cells. However, the result does not seem to be sufficiently confirmed to attenuate the off-tumor toxic response to CAR-T therapy.\n5.Figure S3b, Lack of statistical analysis results for left and right side tumors before treatment. From the results of Day15, before treatment, the left side tCD19+ MDA tumors were smaller than the right side tCD19- MDA tumors, and the results of pre-treatment statistical analysis were required to ensure the reliability of the results. In addition, considering the possible effects of gene editing on MDA-MD-231 cells, the effects of expressing tCD19 on the proliferation, invasion and migration of MDA-MD-231 need to be verified.\n6.In figure 3h, the results showed a difference in cytotoxicity between the non-induced and induced groups in CD19- Nalm-6 cells (100% loss of CD19 antigen). However, the non-induced group were normal activated T cells and the induced group were CAR-T cells that could target CD19. It is theoretically implausible that there is a difference in cytotoxicity between these two groups in CD19- cells.\n7.The activation of CAR-T cells requires more detailed characterization, such as activation markers and cytokines.", "reviewer_2": "This manuscript from Ziliang Huang and colleagues described a strategy of T cell engineering for cancer therapy, which combines the applications of a PE-binding protein domain, the synNotch-regulated gene expression system, the tamoxifen-induced protein translocation system, and the light-responsive protein dimerization system. The authors aim to use the presented system to solve the current problems in T cell-mediated cancer therapy including the antigen loss, tumor heterogenicity, off-tumor toxicity, etc. They clearly showed that their DGLA-sPAT CAR system could work in experimental models based on CD19, PD-L1 as model antigens. Below are concerns that if addressed could expand the significance of the studies and offer clarification on certain points.\n1. PEbody is generated by directed evolution. It’s not clear whether PEbody can also bind cell/tissue in host, which means using it in a CAR might be risky for an on-target/off-PE effect. If this is the first case of using it in a CAR, there could great value in investigating this attribute in the present study.\n2. In the DGLA-PAT CAR design, tumor antigen is induced by light and Tamoxifen, PAT CAR expression is induced by synNotch recognition of induced tumor antigen, PAT CAR-T killing is induced by PE-antibodies. The concern is that due to multiple gatekeeping events, the eventual percentage of cancer-fighting CAR-T cells seems to be on the lower side of transferred CAR-T cells. According to Fig3c, e, f, the CAR induction is around 15-20%, activation is around 20%. Can the authors articulate or speculate as to why only a subset of these CARs are activated?\n3. For the concept of “training center” tumor, the initial step is T cells expressing synNotch recognize either endogenous or DGLA-induced antigen, which means CAR-T cell activity is still dependent on one antigen, and antigen loss or downregulation will likely make such CAR-T therapy nonresponsive. Can the authors please address (defend or refute this assertion) this important issue either experimentally or in text.\n4. In Fig 6, it seems that a clear interpretation of these data would require evaluating the effects of blue light treatment and AAV infection in one tumor side, control groups without sPAT CAT T cells.\n5. Overall, the in vivo CAR T immunotherapy studies need to be strengthened. Although experiments described in Fig6 showed that DGLA-PAT CAR strategy could work to initiate tumor control based on inducible tCD19 expression and anti-PD-L1 treatment, there is no evidence that DGLA-PAT CAR is better than traditional CAR-T strategy in tumor control. And although the concept is novel, there is concern that given that the DGLA-PAT CAR strategy is more complicated in cell engineering, stricter in tumor killing initiation, possibly resulting in less tumor-fighting CAR T cells, a parallel comparison between DGLA-PAT CAR and traditional CAR is recommended. Can the authors please address (defend or refute this assertion) this important issue either experimentally or in text.\n6. Only one tumor type, the MDA cell line, was used for in vivo tumor therapy study. To support the concept of “versatile” therapy strategy, multiple tumor types, antigens and antibodies need to be tested in more than one tumor model in vivo.\n7. The description of the results is not clear. In the spirit of rigor and transparency, it is critical that experimental details be provided.\nMinor concerns,\n1. The surface expression of PEbody PAT CAR in T cells is not confirmed. This is important to know as this will impact signaling and ultimately efficacy.\n2. In Fig1f, it seems that MCF-7 is not sensitive to killing by anti-HER2 and anti-MUC1. Please elaborate in the discussion.\n3. Information of the antibodies used are not provided.\n4. In Fig2c, details regarding the generation of 25%,50%,100% CD19 loss cell lines are not required.\n5. In Fig3 and the verbiage in line 127-144 appears to be irrelevant to the topic of this manuscript. Also, although interesting, it is not clear how plausible it is for a CAR T therapy to engineer T cells with four transgenes for inducible CAR expression by Tam and light.\n6. In Fig6, direct evidence for in situ AAV-injection and light-induced tCD19 expression in MDA tumor is not provided (although the concept is interesting). The PEbody CAR-T cell infiltration in tumor has not been thoroughly characterized nor has their function.", "reviewer_3": "This is a timely article reporting the development of a monobody based PE targeting CAR (PAT) that allows for T cell reactivity to be directed to tumor antigens via PE-conjugated antibodies. The authors demonstrate the ability of PAT T cells to kill tumor cells in a specific manner based on the presence of PE-conjugated antibodies. They further improve the CAR safety profile by engineering the PAT under a light/tamoxifen controlled Cre recombinase (DGLA-PAT CAR), which restricts CAR activity to local illuminated areas and requires the presence of tamoxifen. The authors also engineered a synNotch regulated PAT (sPAT), whereby the PAT expression is induced by priming of the SynNotch antigen. Most notably, the authors devised a CAR-antigen pairing system, whereby tumor antigen expression is controlled by the DGLA system and T cell reactivity is controlled by the sPAT modules (DGLA-sPAT). In this strategy, tumor cells can be vaccinated with an inducible antigen that can then “train” T cells to react specifically at the tumor site through the synNotch module. In murine flank models of human breast cancer, the authors show MDA cells engineered or vaccinated with the DGLA inducible CD19 cassette, express the target antigen only under illumination and with tamoxifen treatment. Administration of sPAT T cells and PE-conjugated PD-L1 antibody resulted in tumor regression only in tumors that were illuminated with light. These efforts are framed in the context of antigen escape, tumor heterogeneity, and safety concerns arising from clinical experience of standard CAR-T therapy, particularly for solid tumors. The impact and novelty of the manuscript is well suited for Nature Communications. The following revision experiments/edits would strengthen the manuscript:\n1. In cytotoxicity assays (Figure 1 and Figure 3), the authors should include a conventional CAR control as a benchmark. It is unclear whether the inducible CAR formats reported in this study are of similar potency as standard CARs used in the field. The authors do a good job of motivating the clinical relevance of the study (i.e. addresses antigen heterogeneity, safety, and the biocompatibility of PE), but having a benchmark for potency is also needed to assess the clinical relevance. In addition to tumor killing, measuring the levels of cytokine secretion (i.e. IFNy, IL-2) from tumor cocultures is appropriate for assessing T cell functionality.\n2. Similar to comment 1, Mock (untransduced) T cells are the appropriate negative controls in cytotoxicity assays that allow the reader to assess the leaky or non-specific activity of the system and should be included.\n3. In Figure 3C, are the cells gated on transgene+? It is unclear if the relatively low induction of CD19 CAR-T (~15%) is because of inefficiencies of the inducible system or if it is due to low transduction efficiencies. The authors should clarify these details.\n4. The cytotoxicity data presented in this study are normalized. The authors should indicate what the plots were normalized to.\n5. In Figure 5E, it is unclear if the increase in fluc signal is driven by T cell expansion or synNotch activation since there is no constitutive and orthogonal luciferase that was coexpressed in the T cells. Did the authors administer PE conjugated antibody in this experiment? Adequate clarification or additional experiments should be provided to help interpret the source of the increasing fluc signal.\n6. The authors should add a reference for the following study that demonstrated an inducible CAR regulated by tamoxifen activated recombinase.\nChakravarti, Deboki, et al. \"Inducible gene switches with memory in human T cells for cellular immunotherapy.\" ACS synthetic biology 8.8 (2019): 1744-1754."}, "author_response": "We thank the reviewers for their thorough evaluation of our manuscript and their constructive feedback. We have conducted additional experiments extensively and addressed all comments and suggestions, which have helped us improve the clarity and quality of our work. Below, we provide detailed responses to each reviewer's comments and describe the revisions made to address their concerns. We believe these changes have significantly strengthened the manuscript.\nReviewer #1 (Remarks to the Author)\nIn this study, the authors developed a novel CAR that can be programmed with PE-conjugated antibodies to target multiple user-defined antigens, which allows for overcoming antigen escape and tumor heterogeneity. In this CAR-T system, by integrating PAT CAR and DGLA gene expression, DGLA-PAT CAR can confine the CAR T activation in the local tumor tissue and reduce off-target toxicity. this is a meaningful study. However, there are some experiments and controls that detract from the significance of this work. The following suggestions also should be taken into account.\n1.The authors emphasize the local infiltration and activation of CAR-T and speculate that the expression of CAR-T diminishes after it migrates out of the tumor tissue. In animal models, it is recommended to supplement data related to the detection of CAR-T in peripheral blood, spleen, and draining lymph nodes after CAR-T treatment. In addition, it is recommended to add safety-related studies, such as liver and kidney function tests, as well as pathologic evaluation of important organs.\nResponse: We appreciate this constructive suggestion and have added new data on CAR-T cell detection and safety-related studies. Briefly, we performed detection of DGLA-PAT CAR-T cells in peripheral blood and spleen after CAR-T treatment (Fig. S6a). The draining lymph nodes cannot be located due to their under development in NSG mice (Chappaz et al., J Immunol, 2010). The non-activated T cells, via anti-human CD3 antibody staining, are readily detectable in these locations, but none of them were activated as shown by the negative signals in PAT-CAR staining. Consistent with our deign, the DGLA-PAT CAR-T cells were significantly activated in tumor region evidenced by the clear PAT CAR staining (Fig. S6b), confirming the intended tumor-specific activation.\nHistopathology study, via H&E staining, of heart, lung, spleen, kidney, liver, and brain after CAR-T treatment (Fig. S7a), as well as liver and kidney functional tests were further performed (Fig. S7b). No treatment-related lesions or toxicities are observed. These results verified the safety of our approach. The Materials and Methods (line 538-558) and Results (line 249-254) sections have been updated accordingly.\n2.In Figure 2b, Isotype as a control should be added to verify the knockdown effect of CD19 in Nalm-6 cells.\nResponse: Thank you for pointing this out. We have now included the isotype control staining result to verify the effect of CD19-knockout in the Nalm-6 CD19-KO cell line (Fig. S2a, line 112-113). The isotype control profile has also been included in the revised Fig. 2B (CD19 staining histogram), and noted in the figure legend.\n3.CD19 is a lineage marker for B cells, and the authors applied CD19+/PSMA-, as well as CD19-/PSMA+ prostate cancer PC-3 cells in their experiments, which required the detection of CD19 as well as PSMA expression in PC-3 cells\nResponse: We have now validated the CD19 and PSMA antigen profiles of the engineered PC-3 cell lines and included the data in the revised manuscript (Fig. S2b-c, line 122-123).\n4.In Figure 3, the authors constructed Tam-light induced PAT CAR-T and verified the ability to target kill target cells. However, the result does not seem to be sufficiently confirmed to attenuate the off-tumor toxic response to CAR-T therapy.\nResponse: We appreciate your insightful feedback regarding the evaluation of off-tumor toxicity attenuation in our study. In response, we would like to clarify two important points:\nFirst, we have demonstrated in Fig. 3g-h that the cytotoxicity of DGLA PAT CAR-T cells can be effectively regulated through external stimuli such as light. These results indicate that the cytotoxicity of the engineered DGLA CAR-T cells can be precisely confined at the tumor site, supporting the potential for reducing off-tumor toxicity through on-demand activation such as light stimulation on tumor regions as shown in Fig. 5c-f and Fig. 6.\nSecond, due to the low efficiency of delivering all the three gene cassettes into primary T cells, it would be difficult to perform a direct in vivo validation with this initial design. Recognizing these limitations, we subsequently transitioned to an alternative design that is more suitable for in vivo evaluation (Fig. 4a-b), as detailed in the later sections of our manuscript, which demonstrated attenuated off-tumor toxic response to CAR-T therapy (Fig. 5c-f and Fig. 6).\n5.Figure S3b, Lack of statistical analysis results for left and right side tumors before treatment. From the results of Day15, before treatment, the left side tCD19+ MDA tumors were smaller than the right side tCD19- MDA tumors, and the results of pre-treatment statistical analysis were required to ensure the reliability of the results. In addition, considering the possible effects of gene editing on MDA-MD-231 cells, the effects of expressing tCD19 on the proliferation, invasion and migration of MDA-MD-231 need to be verified.\nResponse: We appreciate the reviewer’s concern regarding potential baseline differences in tumor size prior to CAR-T-cell treatment. We have performed the statistical analysis on tumor growth comparing the left and right sides, each normalized to their respective Day 7 measurements, to evaluate any pre-treatment imbalance. As shown in Fig. 5f (associated with IVIS images of Fig. S4c in the revised manuscript), there was no statistically significant difference between the left side (tCD19+) and right side (tCD19-) MDA-MB-231 tumors prior to CAR-T-cell injection (noted as “ns” in Fig. 5f, p > 0.05). A significant divergence emerged only after CAR-T treatment, confirming that the therapeutic effect, rather than an initial size bias, accounts for the observed difference on tumor growth.\nRegarding the potential effects of gene editing and tCD19 expression on MDA-MB-231 cell behavior, we respectfully note that tCD19 is a truncated, non-signaling surface marker that has been extensively validated as a clinically safe selection marker (Di Stasi et al., N Engl J Med, 2011; Terakura et al., Blood, 2012; Lee et al., J Immunother Cancer, 2023). It lacks intracellular signaling domains and has no known impact on cell proliferation, migration, or invasion. This inert nature has been demonstrated in multiple cell types, including T cells and hematopoietic stem cells, without affecting their biological behavior (Park et al., Sci Transl Med, 2020). We have also performed a comparison of proliferation, migration, and invasion between tCD19+ and tCD19- MDA-MB-231 lines, and observed no significant difference (Fig. S4a, line 217-218). Therefore, the use of tCD19 in our study is unlikely to alter the intrinsic properties of MDA-MB-231 cells.\n6.In figure 3h, the results showed a difference in cytotoxicity between the non-induced and induced groups in CD19- Nalm-6 cells (100% loss of CD19 antigen). However, the non-induced group were normal activated T cells and the induced group were CAR-T cells that could target CD19. It is theoretically implausible that there is a difference in cytotoxicity between these two groups in CD19- cells.\nResponse: We thank you for this critical note. As correctly noted, CD19- Nalm-6 cells (with complete loss of CD19 expression) should not exhibit differential sensitivity to non-induced T cells versus induced PEbody CAR-T cells, as the antigen is absent. The small but statistically significant difference observed in the initial experiment is possibly attributable to experimental variation rather than true antigen-specific cytotoxicity. To clarify this point, we have re-conducted this assay with more biological repeats (n = 4). The updated results, now included in the revised Fig. 3h, show no significant difference between these two groups, consistent with the PEbody CAR design.\n7.The activation of CAR-T cells requires more detailed characterization, such as activation markers and cytokines.\nResponse: We thank you for this important suggestion. To provide a more comprehensive characterization of CAR-T cell activation, we have performed additional experiments to characterize surface activation markers and cytokine release. Specifically, we analyzed the expression of CD69 and CD25 by flow cytometry (Fig. S1f), and measured the secretion of key cytokines including IFN-γ, TNF-α, and IL-2 via ELISA (Fig. S1g). These results further confirm the antigen-specific activation of CAR-T cells and are now included in the revised manuscript (line 101-103).\nReviewer #2 (Remarks to the Author)\nThis manuscript from Ziliang Huang and colleagues described a strategy of T cell engineering for cancer therapy, which combines the applications of a PE-binding protein domain, the synNotch-regulated gene expression system, the tamoxifen-induced protein translocation system, and the light-responsive protein dimerization system. The authors aim to use the presented system to solve the current problems in T cell-mediated cancer therapy including the antigen loss, tumor heterogenicity, off-tumor toxicity, etc. They clearly showed that their DGLA-sPAT CAR system could work in experimental models based on CD19, PD-L1 as model antigens.\nBelow are concerns that if addressed could expand the significance of the studies and offer clarification on certain points.\n1. PEbody is generated by directed evolution. It’s not clear whether PEbody can also bind cell/tissue in host, which means using it in a CAR might be risky for an on-target/off-PE effect. If this is the first case of using it in a CAR, there could great value in investigating this attribute in the present study.\nResponse: We thank you for highlighting this important point. In the current work, we have assessed the binding specificity of PEbody when used as the antigen binding motif in CAR against several human cell lines originated from different tissues including blood (Nalm-6, Fig. 1c), breast (MCF-7, MDA-MB-231, Fig. 1f), and prostate (PC-3, Fig. 1f). Our results showed that when PE-conjugated antibody is absent, there is no binding/killing against the target cells, indicating the binding of PEbody towards host cells/tissues is minimal. To further address the potential concern of “on-target/off-PE” effects in vivo, we performed histological studies of important organs, including heart, lung, spleen, kidney, liver, and brain (Fig. S7a), as well as liver and kidney functional assays (Fig. S7b), after PEbody PAT CAR T treatment. Our results revealed no signs of tissue damage or inflammation, supporting the conclusion that PEbody CAR-T cells do not exhibit detectable off-PE toxicity in host tissues. These new results have been included in the revised manuscript (line 252-254).\n2. In the DGLA-PAT CAR design, tumor antigen is induced by light and Tamoxifen, PAT CAR expression is induced by synNotch recognition of induced tumor antigen, PAT CAR-T killing is induced by PE-antibodies. The concern is that due to multiple gatekeeping events, the eventual percentage of cancer-fighting CAR-T cells seems to be on the lower side of transferred CAR-T cells. According to Fig3c, e, f, the CAR induction is around 15-20%, activation is around 20%. Can the authors articulate or speculate as to why only a subset of these CARs are activated?\nResponse: We appreciate your thoughtful comment regarding the proportion of activated CAR-T cells in the DGLA-PAT CAR design. As noted, Fig. 3c, e, f reflect results from the DGLA-PAT CAR T system, in which PAT CAR expression is directly induced in T cells via light- and tamoxifen-dependent transcription, without involvement of the synNotch mechanism. The observed CAR expression (~15-20%) and subsequent activation (~20%) are influenced by several factors. First, the efficiencies of tamoxifen-induced nuclear translocation and light-induced pMag-nMag dimerization are inherently variable in primary human T cells, and may constrain the overall induction rate (Allen et al., ACS Synth Biol, 2019). Second, the induction of large genetic cargos, such as the CAR construct, tends to be less efficient compared to smaller reporters like GFP, as we have observed in previous studies (Wu et al., Nat Biomed Eng, 2021). Consistently, the induction efficiency of the PEbody CAR (~1200 bp) is higher than that of CD19CAR (~1600 bp) (Fig. 3e vs 3c). Third, expression levels of exogenous optogenetic components (e.g., pMag and nMag) are tightly regulated in T cells, further limiting the maximal response to light stimulation (Huang et al., Sci Adv, 2020).\nTo address these limitations, we later implemented the DGLA-sPAT CAR T design, in which tamoxifen and light are used to induce antigen expression in tumor cells rather than directly driving CAR expression (Fig. 4a,b). This approach achieves a much higher antigen induction efficiency (~86%, partly attributed to the small tCD19 size, as shown in Fig. 4d,e), which then activates PEbody PAT CAR expression in T cells via a synNotch mechanism with substantially improved efficiency (Fig. 4f). The modularity and scalability of the DGLA-sPAT design allow for better control and higher activation efficiency in engineered CAR-T cells.\n3. For the concept of “training center” tumor, the initial step is T cells expressing synNotch recognize either endogenous or DGLA-induced antigen, which means CAR-T cell activity is still dependent on one antigen, and antigen loss or downregulation will likely make such CAR-T therapy nonresponsive. Can the authors please address (defend or refute this assertion) this important issue either experimentally or in text.\nResponse: We thank you for raising this important point regarding potential antigen loss and its impact on CAR-T responsiveness in the “training center” tumor strategy. In the DGLA-sPAT CAR T design, the expression of the tumor antigen that serves as the input for synNotch activation is not reliant on endogenous antigen expression, regulation, or evasion, but rather induced synthetically via the DGLA system, which is activated by tamoxifen and light with high efficiency (as shown in Fig. 4d,e). Once triggered, this induction results in stable and constitutive expression of the synthetic antigen on the tumor cell surface.\nFurthermore, the DGLA gene cassette is stably integrated in the genome of tumor cells at a much higher copy number than that of typical endogenous antigen genes. This makes antigen loss or downregulation much more unlikely, thereby mitigating a major cause of resistance commonly seen in conventional CAR-T therapies. Since this is a synthetic and controllable gene cassette, we can also integrate genomic insulators flanking the inducible gene in the future for the stable maintenance of synthetic gene expression (Cabrera et al., Cell Syst, 2022).\nTaken together, the DGLA-sPAT CAR T system is designed to be resilient against antigen escape, as the “training center” antigen is not subjected to the selective pressures that typically drive immune evasion through loss of endogenous targets. We have added this part in Discussion of the revised manuscript (line 341-347).\n4. In Fig 6, it seems that a clear interpretation of these data would require evaluating the effects of blue light treatment and AAV infection in one tumor side, control groups without sPAT CAT T cells.\nResponse: We thank you for raising this important point. To directly assess the potential effects of blue light exposure and AAV delivery independent of sPAT CAR T cell activity, we performed an additional in vivo control study using a bilateral tumor model (Fig. S9). In this study, one tumor received both blue light illumination and AAV administration, while the contralateral tumor received no treatment (Fig. S9a,b). No sPAT CAR T cells were administered in this experiment. The results showed no significant difference in tumor growth between the treated and untreated sides (Fig. S9c,d), indicating that blue light and AAV delivery alone do not induce tumor regression. These findings, included in line 270-272 of the revised manuscript, support the conclusion that the therapeutic effects observed in Fig. 6 are specifically attributable to sPAT CAR T cell activity, rather than nonspecific effects of the light or vector delivery.\n5. Overall, the in vivo CAR T immunotherapy studies need to be strengthened. Although experiments described in Fig6 showed that DGLA-PAT CAR strategy could work to initiate tumor control based on inducible tCD19 expression and anti-PD-L1 treatment, there is no evidence that DGLA-PAT CAR is better than traditional CAR-T strategy in tumor control. And although the concept is novel, there is concern that given that the DGLA-PAT CAR strategy is more complicated in cell engineering, stricter in tumor killing initiation, possibly resulting in less tumor-fighting CAR T cells, a parallel comparison between DGLA-PAT CAR and traditional CAR is recommended. Can the authors please address (defend or refute this assertion) this important issue either experimentally or in text.\nResponse: We appreciate your comment regarding the need to evaluate the DGLA-PAT CAR strategy in comparison to traditional CAR-T therapies. Compared to traditional CAR-T approaches, the DGLA-sPAT CAR design offers two major advantages.\n(1) Spatially confined CAR-T activation. Traditional CAR-T therapies often suffer from on-target/off-tumor toxicity, especially in solid tumors where target antigens may also be expressed at low levels on healthy tissues (Morgan et al., Mol Ther, 2010; Flugel et al., Nat Rev Clin Oncol, 2023). Particularly, in a bilateral model, one side representing the target tumor and the other mimicking normal tissue with a similar antigen profile, traditional CAR-T cells injected at the target tumor side still trafficked to and killed the distal tumor mimicking normal tissue expressing similar antigens, modeling potential off-tumor effects (Wu et al., Nat Biomed Eng, 2021). In contrast, the DGLA-sPAT CAR system enables precise spatial control of cytotoxicity via light-directed local induction of synthetic tumor antigens (Fig. 5c-e), allowing T cell activation confinement strictly within illuminated tumor regions (Fig. 5f, Fig. 6). This spatial gating offers a path to improved safety of DGLA-sPAT CAR over the traditional CAR T therapy.\n(2) Antigen flexibility to overcome tumor heterogeneity and escape. Traditional CARs are hardwired to recognize a single predefined antigen, which can lead to tumor relapse due to antigen heterogeneity or antigen loss. Our strategy enables dynamic antigen control, allowing CAR-T cells to be reprogrammed to respond to different or multiple antigens either simultaneously or sequentially introducing corresponding antibodies. This capability, as demonstrated in Fig. 1c,f (antigen switching), Fig. 2d,e (tumor heterogeneity), and Fig.2c and Fig. 3h (antigen escape), provides a modular platform to adapt CAR-T function in response to evolving tumor phenotypes.\nIn summary, compared with traditional CAR-T approaches, DGLA-sPAT CAR offers two key advantages: light-gated spatial confinement of cytotoxicity to reduce on-target/off-tumor toxicity and programmable multi-antigen control to address heterogeneity and antigen escape. We have emphasized this point in Discussion in the revised manuscript (line 318-321).\n6. Only one tumor type, the MDA cell line, was used for in vivo tumor therapy study. To support the concept of “versatile” therapy strategy, multiple tumor types, antigens and antibodies need to be tested in more than one tumor model in vivo.\nResponse: We appreciate your suggestion regarding the need to demonstrate the versatility of our therapeutic strategy across multiple tumor models. To this end, we have conducted an additional in vivo experiment using a different tumor type: the prostate cancer cell line PC-3 with prostate-specific membrane antigen PSMA (Fig. S5). In this model, NSG mice were inoculated bilaterally with the engineered PC-3 cells with DGLA-inducible tCD19 (Fig. S5a). After systemic administration of sPAT CAR T cells via tail vein injection, we delivered an anti-PSMA-PE antibody to guide CAR-T killing (Fig. S5b). Consistent with our previous findings, the DGLA-sPAT CAR T system effectively controlled tumor growth, demonstrating high antigen specificity and minimal off-target effects (Fig. S5c,d). Importantly, the contralateral tumors, representing tissues with a similar antigen expression profile but without DGLA-induced target antigen presentation, showed no significant regression. These results support the generalizability and adaptability of our approach to different tumor types, antigens, and antibody-PE conjugates, further reinforcing the versatility of the DGLA-sPAT CAR T platform. We have included this result (line 247-249) and updated the Materials and Methods (line 520-536) in the revised manuscript.\n7. The description of the results is not clear. In the spirit of rigor and transparency, it is critical that experimental details be provided.\nResponse: We thank you for emphasizing the importance of clarity and transparency in data presentation. In response, we have revised the manuscript to include more and substantial experimental details in Results and Materials and Methods sections. These updates clarify key aspects of the study design, experimental procedures, and data interpretation to ensure rigor and reproducibility. We believe these additions will help readers more clearly understand and evaluate the findings presented. Particularly, we have provided more details for the experiments below:\nActivation markers and cytokine measurements (line 441-449)\nIn vitro killing assays (line 451-461)\nCRISPR-Cas9 knockout of CD19 gene (line 463-472)\nAntigen escape model experiments (line 474-477)\nTumor migration and infiltration assays (line 491-498)\nLiver and kidney functional tests (line 500-504)\nIn vivo T cell homing and activation (line 520-536)\nEx vivo flow cytometry and frozen-section histology/IHC (line 538-558)\nMinor concerns,\n1. The surface expression of PEbody PAT CAR in T cells is not confirmed. This is important to know as this will impact signaling and ultimately efficacy.\nResponse: We thank you for pointing out this important consideration. To confirm surface expression of the PEbody PAT CAR in T cells, we have performed live cell imaging of transduced T cells using EGFP as marker to verify the surface expression of PEbody CAR (Fig. S1a, line 88 in the revised manuscript). The results clearly demonstrate that the PEbody PAT CAR is expressed on the T cell surface, supporting its proper localization and potential for antigen recognition and signaling.\n2. In Fig1f, it seems that MCF-7 is not sensitive to killing by anti-HER2 and anti-MUC1. Please elaborate in the discussion.\nResponse: We thank you for the opportunity to further clarify our findings regarding MCF‐7 cell sensitivity to PEbody CAR‐mediated killing. In our system, the PEbody CAR T cells utilize antibodies to bridge between the CAR and tumor antigens such as HER‐2 and MUC1 on the target cells. The efficacy of this approach is highly dependent on the expression level and conformation of these antigens on tumor cells. MCF‐7 cells are well documented to express low levels of HER‐2 (Slamon et al., Science, 1987; Holliday et al., Breast Cancer Res, 2011). Consequently, the low HER‐2 density on MCF‐7 cells results in fewer available binding sites for the anti‐HER2 adapter, thereby reducing the effective engagement of the PEbody CAR T cells. Similarly, although MCF‐7 cells do express MUC1, the antibody adapter’s ability to mediate effective CAR T cell activation could be influenced by the antigen’s conformation and glycosylation status. Gendler provided an in‐depth analysis of MUC1 structure and highlighted how its glycosylation can modulate antibody binding (Gendler, J Mammary Gland Biol Neoplasia, 2001), while epitope accessibility and post‐translational modifications of MUC1 have been reported to affect the efficacy of antibody‐mediated targeting (Movahedin et al., Glycobiology, 2017). Thus, the suboptimal presentation of MUC1 on MCF‐7 cells likely contributes to the reduced cytotoxic response observed with anti‐MUC1 adapter‐mediated targeting.\nIn summary, the adapter-based mechanism of our PEbody CAR T cells is critically dependent on both the density and conformational state of the target antigens. The inherently low HER‐2 expression and the less favorable presentation of MUC1 on MCF‐7 cells explain the limited cytotoxicity observed in our experiments. We have incorporated these points in Discussion (line 349-362) to provide a clearer rationale for the observed differential sensitivity of MCF‐7 cells.\n3. Information of the antibodies used are not provided.\nResponse: We have now included detailed information for the antibodies used in Materials and Methods section, including clone names, sources, and catalog numbers to ensure clarity and reproducibility.\nLine 442-443:\nPE-conjugated anti-human CD19 antibody (clone HIB90, mouse IgG1, κ, BioLegend 302208)\nLine 446-448:\nAPC-conjugated anti-human CD69 antibody (clone FN50, mouse IgG1, κ, Biolegend 310910)\nAPC-conjugated anti-human CD25 antibody (clone BC96, mouse IgG1, κ, Biolegend 302609)\nLine 455-460:\nCD19 (clone HIB19, mouse IgG1, κ, BioLegend 302208)\nCD20 (clone 2H7, mouse IgG2b, κ, BioLegend 302306)\nCD38 (clone HB-7, mouse IgG1, κ, BioLegend 356604)\nMUC1 (clone 16A, mouse IgG1, λ, BioLegend 355604)\nHER2 (clone 24D2, mouse IgG1, κ, BioLegend 324406)\nPD-L1 (clone 29E.2A3, mouse IgG2b, κ, BioLegend 329706)\nPSMA (LNI-17, mouse IgG1, κ, BioLegend 342504)\nLine 468:\nCD19 antibody for flow cytometry (clone HIB19, mouse IgG1, κ, Biolegend 302222)\nLine 470:\nIsotype control (mouse IgG1, κ, BioLegend 400130)\nLine 532-535:\nAnti-human PD-L1 antibody (clone 29E.2A3, mouse IgG2b, κ, BioLegend 329706)\nAnti-human PSMA antibody (clone LNI-17, mouse IgG1, κ, BioLegend 342504)\nLine 543-545:\nAnti-human CD3-APC (clone OKT3; mouse IgG2a, κ; BioLegend 317317)\nLine 553: Rat anti-CD19 (clone 6OMP31; Thermo Fisher 53-0194-82; 1:500)\n4. In Fig2c, details regarding the generation of 25%,50%,100% CD19 loss cell lines are not required.\nResponse: Briefly, the 25%, 50%, 75%, and 100% CD19 loss cells were generated by mixing CD19-knockout Nalm-6 cells (0% CD19+) with wild-type CD19⁺ Nalm-6 cells (100% CD19+) at the defined ratios of cell number to simulate varying levels of antigen escape. We have included these details in Materials and Methods (line 474-477).\n5. In Fig3 and the verbiage in line 127-144 appears to be irrelevant to the topic of this manuscript. Also, although interesting, it is not clear how plausible it is for a CAR T therapy to engineer T cells with four transgenes for inducible CAR expression by Tam and light.\nResponse: The intent of Fig. 3 and lines 127-144 is to introduce and characterize the drug‑gated light activation (DGLA) safety module, which we use to mitigate potential off‑tumor cytotoxicity of the PAT CAR therapy. We have revised the text to make the contents more aligned with the topic of the manuscript (line 128-144).\nIndeed, as the reviewer pointed out, the overall transduction efficiency of DGLA-PAT CAR T cells was low (~10-20 %), which caused difficulties for in vivo studies using this design. Therefore, we have adopted a second design, the DGLA-sPAT CAR T (Fig. 4), to demonstrate the in vivo therapeutic effect of DGLA and PAT CAR. In this design, the DGLA-related transgenes are specifically delivered into tumor cells which can achieve high efficiency (~85% induction rate with the small size of tCD19 antigen, Fig. 4e), while T cells are engineered with synNotch and the PAT CAR reporter cassette, similar to the conventional synNotch design of which the potential for CAR T therapy has been well-demonstrated by previous studies (Morsut et al., Cell, 2016; Roybal et al., Cell, 2016).\n6. In Fig6, direct evidence for in situ AAV-injection and light-induced tCD19 expression in MDA tumor is not provided (although the concept is interesting). The PEbody CAR-T cell infiltration in tumor has not been thoroughly characterized nor has their function.\nResponse: We appreciate your constructive suggestions. In the revised manuscript we have included immunohistochemistry study confirming tCD19 expression in AAV‑injected, light‑stimulated MDA tumors (Fig. S8c, line 264). This directly demonstrates successful intratumoral AAV transduction and light‑dependent induction of the tCD19 target used for PEbody CAR engagement.\nPEbody CAR‑T cell infiltration: we previously showed tumor infiltration of PEbody CAR‑T cells following tail‑vein delivery using a PEbody CAR and firefly luciferase reporter cassette (Gal4UAS-Pmin-PEbody PAT CAR-P2A-Fluc, Fig. 5c-e and Fig. S4b in revised manuscript). In the revision, we further quantified intratumoral human T cells by flow cytometry of dissociated tumor tissue using anti‑human CD3 antibody and PE staining, confirming PEbody CAR‑T cell presence within the tumor microenvironment (Fig. S6b).\nFunctional characterization: the cytotoxic function of PEbody CAR‑T cells is presented in Fig. 1c–f and Fig. S1b–d. We have performed more characterization of PEbody CAR T cells including up‑regulation of activation markers CD25 and CD69 (Fig. S1f) and secretion of cytokines IFN‑γ, TNF‑α, and IL‑2 (Fig. S1g) upon target antigen engagement.\nCollectively, these additions provide direct validation of in situ AAV delivery and light‑induced tCD19 expression and infiltration of PEbody CAR‑T cells, and substantiate their functional activity.\nReviewer #3 (Remarks to the Author)\nThis is a timely article reporting the development of a monobody based PE targeting CAR (PAT) that allows for T cell reactivity to be directed to tumor antigens via PE-conjugated antibodies. The authors demonstrate the ability of PAT T cells to kill tumor cells in a specific manner based on the presence of PE-conjugated antibodies. They further improve the CAR safety profile by engineering the PAT under a light/tamoxifen controlled Cre recombinase (DGLA-PAT CAR), which restricts CAR activity to local illuminated areas and requires the presence of tamoxifen. The authors also engineered a synNotch regulated PAT (sPAT), whereby the PAT expression is induced by priming of the SynNotch antigen. Most notably, the authors devised a CAR-antigen pairing system, whereby tumor antigen expression is controlled by the DGLA system and T cell reactivity is controlled by the sPAT modules (DGLA-sPAT). In this strategy, tumor cells can be vaccinated with an inducible antigen that can then “train” T cells to react specifically at the tumor site through the synNotch module. In murine flank models of human breast cancer, the authors show MDA cells engineered or vaccinated with the DGLA inducible CD19 cassette, express the target antigen only under illumination and with tamoxifen treatment. Administration of sPAT T cells and PE-conjugated PD-L1 antibody resulted in tumor regression only in tumors that were illuminated with light. These efforts are framed in the context of antigen escape, tumor heterogeneity, and safety concerns arising from clinical experience of standard CAR-T therapy, particularly for solid tumors. The impact and novelty of the manuscript is well suited for Nature Communications. The following revision experiments/edits would strengthen the manuscript:\n1. In cytotoxicity assays (Figure 1 and Figure 3), the authors should include a conventional CAR control as a benchmark. It is unclear whether the inducible CAR formats reported in this study are of similar potency as standard CARs used in the field. The authors do a good job of motivating the clinical relevance of the study (i.e. addresses antigen heterogeneity, safety, and the biocompatibility of PE), but having a benchmark for potency is also needed to assess the clinical relevance. In addition to tumor killing, measuring the levels of cytokine secretion (i.e. IFNy, IL-2) from tumor cocultures is appropriate for assessing T cell functionality.\nResponse: We really appreciate your suggestion. We agree that benchmarking against a conventional CAR and assessing cytokine secretion are important for evaluating clinical relevance. In the revised manuscript, we have added conventional CAR controls to the cytotoxicity assays (Fig. 1c,e and Fig. 3d,g in the revised manuscript). We also included ELISA measurements of key secreted cytokines including IFN‑γ, TNF‑α, and IL‑2 after tumor co‑cultures (Fig. S1g), providing functional readouts complementary to target‑cell killing.\n2. Similar to comment 1, Mock (untransduced) T cells are the appropriate negative controls in cytotoxicity assays that allow the reader to assess the leaky or non-specific activity of the system and should be included.\nResponse: Mock controls using untransduced T cells have been included in the cytotoxicity assays (Fig. 1c,e and Fig. 3d,g in the revised manuscript) and noted in figure legend.\n3. In Figure 3C, are the cells gated on transgene+? It is unclear if the relatively low induction of CD19 CAR-T (~15%) is because of inefficiencies of the inducible system or if it is due to low transduction efficiencies. The authors should clarify these details.\nResponse: We thank you for the suggestion. For the assays in Fig. 3c, we used FACS-sorted T cells containing the transgenes of the inducible system. Thus, the result reflects transgene positive cells rather than variability from mixed transduction efficiencies. The ~15% induction likely arises from limited expression of the DGLA regulator components in primary T cells, where transgene expression is tightly regulated. This phenomenon is consistent with our previous findings, where we observed that the efficiency of induction is dependent on regulator expression levels in T cells (Huang et al., Sci Adv, 2020). To address this limitation, we subsequently adopted an alternative strategy where the DGLA system is delivered into tumor cells rather than T cells (Fig. 4a), which allows for stronger expression of tCD19 antigen and significantly improved induction efficiency (~85%, Fig. 4e).\n4. The cytotoxicity data presented in this study are normalized. The authors should indicate what the plots were normalized to.\nResponse: We appreciate your suggestion for clarification. The cytotoxicity data were normalized to tumor-only control wells without T cells. Details regarding this normalization method have been added to the figure legends and Materials and Methods section in the revised manuscript (line 460-461).\n5. In Figure 5E, it is unclear if the increase in fluc signal is driven by T cell expansion or synNotch activation since there is no constitutive and orthogonal luciferase that was coexpressed in the T cells. Did the authors administer PE conjugated antibody in this experiment? Adequate clarification or additional experiments should be provided to help interpret the source of the increasing fluc signal.\nResponse: We thank you for this insightful question. In the experiment shown in Figure 5E, PE-conjugated antibody was administered one day after T cell injection. We believe that the initial Fluc signal observed is primarily driven by synNotch activation of the injected T cells homing to the tCD19+ tumor site, rather than T cell expansion per se. This is because T cell expansion would require activation signaling, which in our system depends on the synNotch-mediated induction of PEbody CAR followed by its engagement with the target antigen via the corresponding PE-conjugated antibody. It is also important to note that in our synNotch-inducible Fluc design, any newly-generated T cells (resulting from expansion) would still require synNotch activation to express Fluc. Therefore, while T cell expansion may contribute to the overall signal increase over time, T cell expansion alone may not increase the Fluc signal without the synNotch activation. We have clarified this point in the revised manuscript (line 224-225) to ensure accurate interpretation of these results.\n6. The authors should add a reference for the following study that demonstrated an inducible CAR regulated by tamoxifen activated recombinase.\nChakravarti, Deboki, et al. \"Inducible gene switches with memory in human T cells for cellular immunotherapy.\" ACS synthetic biology 8.8 (2019): 1744-1754.\nResponse: We thank you for bringing this relevant reference to our attention. We have added this reference in the revised manuscript (line 279-280) to acknowledge this prior work on inducible CAR regulation.\nReferences\n1. Allen, M. E., et al. An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics. ACS Synth Biol 8, 2359-2371 (2019).\n2. Cabrera, A., et al. The sound of silence: Transgene silencing in mammalian cell engineering. Cell Syst 13, 950-973 (2022).\n3. Chappaz, S. and D. Finke The IL-7 signaling pathway regulates lymph node development independent of peripheral lymphocytes. J Immunol 184, 3562-3569 (2010).\n4. Di Stasi, A., et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365, 1673-1683 (2011).\n5. Flugel, C. L., et al. Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours. Nat Rev Clin Oncol 20, 49-62 (2023).\n6. Gendler, S. J. MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia 6, 339-353 (2001).\n7. Holliday, D. L. and V. Speirs Choosing the right cell line for breast cancer research. Breast Cancer Res 13, 215 (2011).\n8. Huang, Z., et al. Engineering light-controllable CAR T cells for cancer immunotherapy. Sci Adv 6, eaay9209 (2020).\n9. Lee, S. Y., et al. CD8(+) chimeric antigen receptor T cells manufactured in absence of CD4(+) cells exhibit hypofunctional phenotype. J Immunother Cancer 11 (2023).\n10. Morgan, R. A., et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18, 843-851 (2010).\n11. Morsut, L., et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780-791 (2016).\n12. Movahedin, M., et al. Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen. Glycobiology 27, 677-687 (2017).\n13. Park, A. K., et al. Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumors. Sci Transl Med 12 (2020).\n14. Roybal, K. T., et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419-432 e416 (2016).\n15. Slamon, D. J., et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177-182 (1987).\n16. Terakura, S., et al. Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells. Blood 119, 72-82 (2012).\n17. Wu, Y., et al. Control of the activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed Eng 5, 1336-1347 (2021).\nWe thank the reviewers for their thorough evaluation of our manuscript and their constructive feedback. We have conducted additional experiments extensively and addressed all comments and suggestions, which have helped us improve the clarity and quality of our work. Below, we provide detailed responses to each reviewer's comments and describe the revisions made to address their concerns. We believe these changes have significantly strengthened the manuscript.\nReviewer #1 (Remarks to the Author)\nIn this study, the authors developed a novel CAR that can be programmed with PE-conjugated antibodies to target multiple user-defined antigens, which allows for overcoming antigen escape and tumor heterogeneity. In this CAR-T system, by integrating PAT CAR and DGLA gene expression, DGLA-PAT CAR can confine the CAR T activation in the local tumor tissue and reduce off-target toxicity. this is a meaningful study. However, there are some experiments and controls that detract from the significance of this work. The following suggestions also should be taken into account.\n1. The authors emphasize the local infiltration and activation of CAR-T and speculate that the expression of CAR-T diminishes after it migrates out of the tumor tissue. In animal models, it is recommended to supplement data related to the detection of CAR-T in peripheral blood, spleen, and draining lymph nodes after CAR-T treatment. In addition, it is recommended to add safety-related studies, such as liver and kidney function tests, as well as pathologic evaluation of important organs.\nResponse: We appreciate this constructive suggestion and have added new data on CAR-T cell detection and safety-related studies. Briefly, we performed detection of DGLA-PAT CAR-T cells in peripheral blood and spleen after CAR-T treatment (Fig. S6a). The draining lymph nodes cannot be located due to their under development in NSG mice (Chappaz et al., J Immunol, 2010). The non-activated T cells, via anti-human CD3 antibody staining, are readily detectable in these locations, but none of them were activated as shown by the negative signals in PAT-CAR staining (Fig. S6a). Consistent with our deign, the DGLA-PAT CAR-T cells were significantly activated in tumor region evidenced by the clear PAT CAR staining (Fig. S6b), confirming the intended tumor-specific activation.\nHistopathology study, via H&E staining, of heart, lung, spleen, kidney, liver, and brain after CAR-T treatment (Fig. S7a), as well as liver and kidney functional tests were further performed (Fig. S7b). No treatment-related lesions or toxicities are observed. These results verified the safety of our approach. The Materials and Methods (line 555-575) and Results (line 249-254) sections have been updated accordingly.\n2. In Figure 2b, Isotype as a control should be added to verify the knockdown effect of CD19 in Nalm-6 cells.\nResponse: Thank you for pointing this out. We have now included the isotype control staining result to verify the effect of CD19-knockout in the Nalm-6 CD19-KO cell line (Fig. S2a, line 112-113). The isotype control profile has also been included in the revised Fig. 2B (CD19 staining histogram), and noted in the figure legend.\n3. CD19 is a lineage marker for B cells, and the authors applied CD19+/PSMA-, as well as CD19-/PSMA+ prostate cancer PC-3 cells in their experiments, which required the detection of CD19 as well as PSMA expression in PC-3 cells\nResponse: We have now validated the CD19 and PSMA antigen profiles of the engineered PC-3 cell lines and included the data in the revised manuscript (Fig. S2b-c, line 122-123).\n4. In Figure 3, the authors constructed Tam-light induced PAT CAR-T and verified the ability to target kill target cells. However, the result does not seem to be sufficiently confirmed to attenuate the off-tumor toxic response to CAR-T therapy.\nResponse: We appreciate your insightful feedback regarding the evaluation of off-tumor toxicity attenuation in our study. In response, we would like to clarify two important points:\nFirst, we have demonstrated in Fig. 3g-h that the cytotoxicity of DGLA PAT CAR-T cells can be effectively regulated through external stimuli such as light. These results indicate that the cytotoxicity of the engineered DGLA CAR-T cells can be precisely confined at the tumor site, supporting the potential for reducing off-tumor toxicity through on-demand activation such as light stimulation on tumor regions as shown in Fig. 5c-f and Fig. 6.\nSecond, due to the low efficiency of delivering all the three gene cassettes into primary T cells, it would be difficult to perform a direct in vivo validation with this initial design. Recognizing these limitations, we subsequently transitioned to an alternative design that is more suitable for in vivo evaluation (Fig. 4a-b), as detailed in the later sections of our manuscript (Fig. 5c-f, Fig. 6, and Fig. S10), which demonstrated attenuated off-tumor toxic response to CAR-T therapy.\n5. Figure S3b, Lack of statistical analysis results for left and right side tumors before treatment. From the results of Day15, before treatment, the left side tCD19+ MDA tumors were smaller than the right side tCD19- MDA tumors, and the results of pre-treatment statistical analysis were required to ensure the reliability of the results. In addition, considering the possible effects of gene editing on MDA-MD-231 cells, the effects of expressing tCD19 on the proliferation, invasion and migration of MDA-MD-231 need to be verified.\nResponse: We appreciate the reviewer’s concern regarding potential baseline differences in tumor size prior to CAR-T-cell treatment. We have performed the statistical analysis on tumor growth comparing the left and right sides, each normalized to their respective Day 7 measurements, to evaluate any pre-treatment imbalance. As shown in Fig. 5f (associated with IVIS images of Fig. S4c in the revised manuscript), there was no statistically significant difference between the left side (tCD19+) and right side (tCD19-) MDA-MB-231 tumors prior to CAR-T-cell injection (noted as “ns” in Fig. 5f, p > 0.05). A significant divergence emerged only after CAR-T treatment, confirming that the therapeutic effect, rather than an initial size bias, accounts for the observed difference on tumor growth.\nRegarding the potential effects of gene editing and tCD19 expression on MDA-MB-231 cell behavior, we respectfully note that tCD19 is a truncated, non-signaling surface marker that has been extensively validated as a clinically safe selection marker (Di Stasi et al., N Engl J Med, 2011; Terakura et al., Blood, 2012; Lee et al., J Immunother Cancer, 2023). It lacks intracellular signaling domains and has no known impact on cell proliferation, migration, or invasion. This inert nature has been demonstrated in multiple cell types, including T cells and hematopoietic stem cells, without affecting their biological behavior (Park et al., Sci Transl Med, 2020). We have also performed a comparison of proliferation, migration, and invasion between tCD19+ and tCD19- MDA-MB-231 lines, and observed no significant difference (Fig. S4a, line 217-218). Therefore, the use of tCD19 in our study is unlikely to alter the intrinsic properties of MDA-MB-231 cells.\n6. In figure 3h, the results showed a difference in cytotoxicity between the non-induced and induced groups in CD19- Nalm-6 cells (100% loss of CD19 antigen). However, the non-induced group were normal activated T cells and the induced group were CAR-T cells that could target CD19. It is theoretically implausible that there is a difference in cytotoxicity between these two groups in CD19- cells.\nResponse: We thank you for this critical note. As correctly noted, CD19- Nalm-6 cells (with complete loss of CD19 expression) should not exhibit differential sensitivity to non-induced T cells versus induced PEbody CAR-T cells, as the antigen is absent. The small but statistically significant difference observed in the initial experiment is possibly attributable to experimental variation rather than true antigen-specific cytotoxicity. To clarify this point, we have re-conducted this assay with more biological repeats (n = 4). The updated results, now included in the revised Fig. 3h, show no significant difference between these two groups, consistent with the PEbody CAR design.\n7. The activation of CAR-T cells requires more detailed characterization, such as activation markers and cytokines.\nResponse: We thank you for this important suggestion. To provide a more comprehensive characterization of CAR-T cell activation, we have performed additional experiments to characterize surface activation markers and cytokine release. Specifically, we analyzed the expression of CD69 and CD25 by flow cytometry (Fig. S1f), and measured the secretion of key cytokines including IFN-γ, TNF-α, and IL-2 via ELISA (Fig. S1g). These results further confirm the antigen-specific activation of CAR-T cells and are now included in the revised manuscript (line 101-103).\nReviewer #2 (Remarks to the Author)\nThis manuscript from Ziliang Huang and colleagues described a strategy of T cell engineering for cancer therapy, which combines the applications of a PE-binding protein domain, the synNotch-regulated gene expression system, the tamoxifen-induced protein translocation system, and the light-responsive protein dimerization system. The authors aim to use the presented system to solve the current problems in T cell-mediated cancer therapy including the antigen loss, tumor heterogenicity, off-tumor toxicity, etc. They clearly showed that their DGLA-sPAT CAR system could work in experimental models based on CD19, PD-L1 as model antigens.\nBelow are concerns that if addressed could expand the significance of the studies and offer clarification on certain points.\n1. PEbody is generated by directed evolution. It’s not clear whether PEbody can also bind cell/tissue in host, which means using it in a CAR might be risky for an on-target/off-PE effect. If this is the first case of using it in a CAR, there could great value in investigating this attribute in the present study.\nResponse: We thank you for highlighting this important point. In the current work, we have assessed the binding specificity of PEbody when used as the antigen binding motif in CAR against several human cell lines originated from different tissues including blood (Nalm-6, Fig. 1c), breast (MCF-7, MDA-MB-231, Fig. 1f), and prostate (PC-3, Fig. 1f). Our results showed that when PE-conjugated antibody is absent, there is no binding/killing against the target cells, indicating the binding of PEbody towards host cells/tissues is minimal. To further address the potential concern of “on-target/off-PE” effects in vivo, we performed histological studies of important organs, including heart, lung, spleen, kidney, liver, and brain (Fig. S7a), as well as liver and kidney functional assays (Fig. S7b), after PEbody PAT CAR T treatment. Our results revealed no signs of tissue damage or inflammation, supporting the conclusion that PEbody CAR-T cells do not exhibit detectable off-PE toxicity in host tissues. These new results have been included in the revised manuscript (line 252-254).\n2. In the DGLA-PAT CAR design, tumor antigen is induced by light and Tamoxifen, PAT CAR expression is induced by synNotch recognition of induced tumor antigen, PAT CAR-T killing is induced by PE-antibodies. The concern is that due to multiple gatekeeping events, the eventual percentage of cancer-fighting CAR-T cells seems to be on the lower side of transferred CAR-T cells. According to Fig3c, e, f, the CAR induction is around 15-20%, activation is around 20%. Can the authors articulate or speculate as to why only a subset of these CARs are activated?\nResponse: We appreciate your thoughtful comment regarding the proportion of activated CAR-T cells in the DGLA-PAT CAR design. As noted, Fig. 3c, e, f reflect results from the DGLA-PAT CAR T system, in which PAT CAR expression is directly induced in T cells via light- and tamoxifen-dependent transcription, without involvement of the synNotch mechanism. The observed CAR expression (~15-20%) and subsequent activation (~20%) are influenced by several factors. First, the efficiencies of tamoxifen-induced nuclear translocation and light-induced pMag-nMag dimerization are inherently variable in primary human T cells, and may constrain the overall induction rate (Allen et al., ACS Synth Biol, 2019). Second, the induction of large genetic cargos, such as the CAR construct, tends to be less efficient compared to smaller reporters like GFP, as we have observed in previous studies (Wu et al., Nat Biomed Eng, 2021). Consistently, the induction efficiency of the PEbody CAR (~1200 bp) is higher than that of CD19CAR (~1600 bp) (Fig. 3e vs 3c). Third, expression levels of exogenous optogenetic components (e.g., pMag and nMag) are tightly regulated in T cells, further limiting the maximal response to light stimulation (Huang et al., Sci Adv, 2020).\nTo address these limitations, we later implemented the DGLA-sPAT CAR T design, in which tamoxifen and light are used to induce antigen expression in tumor cells rather than directly driving CAR expression (Fig. 4a,b). This approach achieves a much higher antigen induction efficiency (~86%, partly attributed to the small tCD19 size, as shown in Fig. 4d,e), which then activates PEbody PAT CAR expression in T cells via a synNotch mechanism with substantially improved efficiency (Fig. 4f). The modularity and scalability of the DGLA-sPAT design allow for better control and higher activation efficiency in engineered CAR-T cells.\n3. For the concept of “training center” tumor, the initial step is T cells expressing synNotch recognize either endogenous or DGLA-induced antigen, which means CAR-T cell activity is still dependent on one antigen, and antigen loss or downregulation will likely make such CAR-T therapy nonresponsive. Can the authors please address (defend or refute this assertion) this important issue either experimentally or in text.\nResponse: We thank you for raising this important point regarding potential antigen loss and its impact on CAR-T responsiveness in the “training center” tumor strategy. In the DGLA-sPAT CAR T design, the expression of the tumor antigen that serves as the input for synNotch activation is not reliant on endogenous antigen expression, regulation, or evasion, but rather induced synthetically via the DGLA system, which is activated by tamoxifen and light with high efficiency (as shown in Fig. 4d,e). Once triggered, this induction results in stable and constitutive expression of the synthetic antigen on the tumor cell surface.\nFurthermore, the DGLA gene cassette is stably integrated in the genome of tumor cells at a much higher copy number than that of typical endogenous antigen genes. This makes antigen loss or downregulation much more unlikely, thereby mitigating a major cause of resistance commonly seen in conventional CAR-T therapies. Since this is a synthetic and controllable gene cassette, we can also integrate genomic insulators flanking the inducible gene in the future for the stable maintenance of synthetic gene expression (Cabrera et al., Cell Syst, 2022).\nTaken together, the DGLA-sPAT CAR T system is designed to be resilient against antigen escape, as the “training center” antigen is not subjected to the selective pressures that typically drive immune evasion through loss of endogenous targets. We have added this part in Discussion of the revised manuscript (line 356-362).\n4. In Fig 6, it seems that a clear interpretation of these data would require evaluating the effects of blue light treatment and AAV infection in one tumor side, control groups without sPAT CAR T cells.\nResponse: We thank you for raising this important point. To directly assess the potential effects of blue light exposure and AAV delivery independent of sPAT CAR T cell activity, we performed an additional in vivo control study using a bilateral tumor model (Fig. S9). In this study, one tumor received both blue light illumination and AAV administration, while the contralateral tumor received no treatment (Fig. S9a,b). No sPAT CAR T cells were administered in this experiment. The results showed no significant difference in tumor growth between the treated and untreated sides (Fig. S9c,d), indicating that blue light and AAV delivery alone do not induce tumor regression. These findings, included in line 270-272 of the revised manuscript, support the conclusion that the therapeutic effects observed in Fig. 6 are specifically attributable to sPAT CAR T cell activity, rather than nonspecific effects of the light or vector delivery.\n5. Overall, the in vivo CAR T immunotherapy studies need to be strengthened. Although experiments described in Fig6 showed that DGLA-PAT CAR strategy could work to initiate tumor control based on inducible tCD19 expression and anti-PD-L1 treatment, there is no evidence that DGLA-PAT CAR is better than traditional CAR-T strategy in tumor control. And although the concept is novel, there is concern that given that the DGLA-PAT CAR strategy is more complicated in cell engineering, stricter in tumor killing initiation, possibly resulting in less tumor-fighting CAR T cells, a parallel comparison between DGLA-PAT CAR and traditional CAR is recommended. Can the authors please address (defend or refute this assertion) this important issue either experimentally or in text.\nResponse: We appreciate your comment regarding the need to evaluate the DGLA-PAT CAR strategy in comparison to traditional CAR-T therapies. Compared to traditional CAR-T approaches, the DGLA-sPAT CAR design offers two major advantages.\n(1) Spatially confined CAR-T activation. Traditional CAR-T therapies often suffer from on-target/off-tumor toxicity, especially in solid tumors where target antigens may also be expressed at low levels on healthy tissues (Morgan et al., Mol Ther, 2010; Flugel et al., Nat Rev Clin Oncol, 2023). Particularly, in a bilateral model, one side representing the target tumor and the other mimicking normal tissue with a similar antigen profile, traditional CAR-T cells injected at the target tumor side still trafficked to and killed the distal tumor mimicking normal tissue expressing similar antigens, modeling potential off-tumor effects (Wu et al., Nat Biomed Eng, 2021). In contrast, the DGLA-sPAT CAR system enables precise spatial control of cytotoxicity via light-directed local induction of synthetic tumor antigens (Fig. 5c-e), allowing T cell activation confinement strictly within illuminated tumor regions (Fig. 5f, Fig. 6). This spatial gating offers a path to improved safety of DGLA-sPAT CAR over the traditional CAR T therapy.\n(2) Antigen flexibility to overcome tumor heterogeneity and escape. Traditional CARs are hardwired to recognize a single predefined antigen, which can lead to tumor relapse due to antigen heterogeneity or antigen loss. Our strategy enables dynamic antigen control, allowing CAR-T cells to be reprogrammed to respond to different or multiple antigens either simultaneously or sequentially introducing corresponding antibodies. This capability, as demonstrated in Fig. 1c,f (antigen switching), Fig. 2d,e (tumor heterogeneity), and Fig.2c and Fig. 3h (antigen escape), provides a modular platform to adapt CAR-T function in response to evolving tumor phenotypes.\nIn addition, we performed a direct head-to-head comparison between DGLA-sPAT CAR-T cells and conventional CAR-T cells using a PSMA⁺ tumor model (Fig. S10a), in which human PSMA was also expressed in the mouse liver via lentiviral transduction (Fig. S10b-d). This enables evaluation of both antitumor efficacy and on-target off-tumor toxicity (OTOT). As shown in Fig. S10, both DGLA-sPAT CAR-T and conventional CAR-T cells efficiently controlled tumor growth (Fig. S10e-g), demonstrating that the DGLA-sPAT design does not compromise antitumor potency. However, mice receiving conventional anti-PSMA CAR-T cells exhibited marked systemic toxicity, including significant weight loss (Fig. S10h), elevated inflammatory cytokines (Fig. S10i-k), increased serum alanine aminotransferase levels (Fig. S10l), and pronounced lymphocytic infiltration with hepatocyte damage in the liver (Fig. S10m). In contrast, none of these OTOT-associated toxicities were observed in DGLA-sPAT-treated mice, whose organs remained histologically normal and showed no immune infiltration. Thus, our results demonstrate that the DGLA-sPAT CAR platform maintains potent tumor control while substantially improving safety through spatially restricted CAR activation, an advantage over traditional CAR-T approach in the revised manuscript (line 274-290).\nIn summary, compared with traditional CAR-T approaches, DGLA-sPAT CAR offers two key advantages: programmable multi-antigen control to address heterogeneity and antigen escape, and light-gated spatial confinement of cytotoxicity to reduce on-target/off-tumor toxicity. We have emphasized this point in Discussion in the revised manuscript (line 333-336).\n6. Only one tumor type, the MDA cell line, was used for in vivo tumor therapy study. To support the concept of “versatile” therapy strategy, multiple tumor types, antigens and antibodies need to be tested in more than one tumor model in vivo.\nResponse: We appreciate your suggestion regarding the need to demonstrate the versatility of our therapeutic strategy across multiple tumor models. To this end, we have conducted an additional in vivo experiment using a different tumor type: the prostate cancer cell line PC-3 with prostate-specific membrane antigen PSMA (Fig. S5). In this model, NSG mice were inoculated bilaterally with the engineered PC-3 cells with DGLA-inducible tCD19 (Fig. S5a). After systemic administration of sPAT CAR T cells via tail vein injection, we delivered an anti-PSMA-PE antibody to guide CAR-T killing (Fig. S5b). Consistent with our previous findings, the DGLA-sPAT CAR T system effectively controlled tumor growth, demonstrating high antigen specificity and minimal off-target effects (Fig. S5c,d). Importantly, the contralateral tumors, representing tissues with a similar antigen expression profile but without DGLA-induced target antigen presentation, showed no significant regression. These results support the generalizability and adaptability of our approach to different tumor types, antigens, and antibody-PE conjugates, further reinforcing the versatility of the DGLA-sPAT CAR T platform. We have included this result (line 247-249) and updated the Materials and Methods (line 537-553) in the revised manuscript.\n7. The description of the results is not clear. In the spirit of rigor and transparency, it is critical that experimental details be provided.\nResponse: We thank you for emphasizing the importance of clarity and transparency in data presentation. In response, we have revised the manuscript to include more and substantial experimental details in Results and Materials and Methods sections. These updates clarify key aspects of the study design, experimental procedures, and data interpretation to ensure rigor and reproducibility. We believe these additions will help readers more clearly understand and evaluate the findings presented. Particularly, we have provided more details for the experiments below:\nActivation markers and cytokine measurements (line 456-464)\nIn vitro killing assays (line 466-476)\nCRISPR-Cas9 knockout of CD19 gene (line 478-487)\nAntigen escape model experiments (line 489-492)\nTumor migration and infiltration assays (line 506-513)\nKidney and liver functional tests (line 515-521)\nIn vivo T cell homing and activation (line 537-553)\nEx vivo flow cytometry and frozen-section histology/IHC (line 555-575)\nMinor concerns,\n1. The surface expression of PEbody PAT CAR in T cells is not confirmed. This is important to know as this will impact signaling and ultimately efficacy.\nResponse: We thank you for pointing out this important consideration. To confirm surface expression of the PEbody PAT CAR in T cells, we have performed live cell imaging of transduced T cells using EGFP as marker to verify the surface expression of PEbody CAR (Fig. S1a, line 88 in the revised manuscript). The results clearly demonstrate that the PEbody PAT CAR is expressed on the T cell surface, supporting its proper localization and potential for antigen recognition and signaling.\n2. In Fig1f, it seems that MCF-7 is not sensitive to killing by anti-HER2 and anti-MUC1. Please elaborate in the discussion.\nResponse: We thank you for the opportunity to further clarify our findings regarding MCF‐7 cell sensitivity to PEbody CAR‐mediated killing. In our system, the PEbody CAR T cells utilize antibodies to bridge between the CAR and tumor antigens such as HER‐2 and MUC1 on the target cells. The efficacy of this approach is highly dependent on the expression level and conformation of these antigens on tumor cells. MCF‐7 cells are well documented to express low levels of HER‐2 (Slamon et al., Science, 1987; Holliday et al., Breast Cancer Res, 2011). Consequently, the low HER‐2 density on MCF‐7 cells results in fewer available binding sites for the anti‐HER2 adapter, thereby reducing the effective engagement of the PEbody CAR T cells. Similarly, although MCF‐7 cells do express MUC1, the antibody adapter’s ability to mediate effective CAR T cell activation could be influenced by the antigen’s conformation and glycosylation status. Gendler provided an in‐depth analysis of MUC1 structure and highlighted how its glycosylation can modulate antibody binding (Gendler, J Mammary Gland Biol Neoplasia, 2001), while epitope accessibility and post‐translational modifications of MUC1 have been reported to affect the efficacy of antibody‐mediated targeting (Movahedin et al., Glycobiology, 2017). Thus, the suboptimal presentation of MUC1 on MCF‐7 cells likely contributes to the reduced cytotoxic response observed with anti‐MUC1 adapter‐mediated targeting.\nIn summary, the adapter-based mechanism of our PEbody CAR T cells is critically dependent on both the density and conformational state of the target antigens. The inherently low HER‐2 expression and the less favorable presentation of MUC1 on MCF‐7 cells explain the limited cytotoxicity observed in our experiments. We have incorporated these points in Discussion (line 364-377) to provide a clearer rationale for the observed differential sensitivity of MCF‐7 cells.\n3. Information of the antibodies used are not provided.\nResponse: We have now included detailed information for the antibodies used in Materials and Methods section, including clone names, sources, and catalog numbers to ensure clarity and reproducibility.\nLine 457-458:\nPE-conjugated anti-human CD19 antibody (clone HIB90, mouse IgG1, κ, BioLegend 302208)\nLine 461-464:\nAPC-conjugated anti-human CD69 antibody (clone FN50, mouse IgG1, κ, Biolegend 310910)\nAPC-conjugated anti-human CD25 antibody (clone BC96, mouse IgG1, κ, Biolegend 302609)\nLine 471-475:\nCD19 (clone HIB19, mouse IgG1, κ, BioLegend 302208)\nCD20 (clone 2H7, mouse IgG2b, κ, BioLegend 302306)\nCD38 (clone HB-7, mouse IgG1, κ, BioLegend 356604)\nMUC1 (clone 16A, mouse IgG1, λ, BioLegend 355604)\nHER2 (clone 24D2, mouse IgG1, κ, BioLegend 324406)\nPD-L1 (clone 29E.2A3, mouse IgG2b, κ, BioLegend 329706)\nPSMA (LNI-17, mouse IgG1, κ, BioLegend 342504)\nLine 484-485:\nCD19 antibody for flow cytometry (clone HIB19, mouse IgG1, κ, Biolegend 302222)\nLine 485-486:\nIsotype control (mouse IgG1, κ, BioLegend 400130)\nLine 560-562:\nAnti-human CD3-APC (clone OKT3; mouse IgG2a, κ; BioLegend 317317)\nLine 570: Rat anti-CD19 (clone 6OMP31; Thermo Fisher 53-0194-82; 1:500)\n4. In Fig2c, details regarding the generation of 25%,50%,100% CD19 loss cell lines are not required.\nResponse: Briefly, the 25%, 50%, 75%, and 100% CD19 loss cells were generated by mixing CD19-knockout Nalm-6 cells (0% CD19+) with wild-type CD19⁺ Nalm-6 cells (100% CD19+) at the defined ratios of cell number to simulate varying levels of antigen escape. We have included these details in Materials and Methods (line 489-492).\n5. In Fig3 and the verbiage in line 127-144 appears to be irrelevant to the topic of this manuscript. Also, although interesting, it is not clear how plausible it is for a CAR T therapy to engineer T cells with four transgenes for inducible CAR expression by Tam and light.\nResponse: The intent of Fig. 3 and lines 127-144 is to introduce and characterize the drug‑gated light activation (DGLA) safety module, which we use to mitigate potential off‑tumor cytotoxicity of the PAT CAR therapy. We have revised the text to make the contents more aligned with the topic of the manuscript (line 128-144).\nIndeed, as the reviewer pointed out, the overall transduction efficiency of DGLA-PAT CAR T cells was low (~10-20 %), which caused difficulties for in vivo studies using this design. Therefore, we have adopted a second design, the DGLA-sPAT CAR T (Fig. 4), to demonstrate the in vivo therapeutic effect of DGLA and PAT CAR. In this design, the DGLA-related transgenes are specifically delivered into tumor cells which can achieve high efficiency (~85% induction rate with the small size of tCD19 antigen, Fig. 4e), while T cells are engineered with synNotch and the PAT CAR reporter cassette, similar to the conventional synNotch design of which the potential for CAR T therapy has been well-demonstrated by previous studies (Morsut et al., Cell, 2016; Roybal et al., Cell, 2016).\n6. In Fig6, direct evidence for in situ AAV-injection and light-induced tCD19 expression in MDA tumor is not provided (although the concept is interesting). The PEbody CAR-T cell infiltration in tumor has not been thoroughly characterized nor has their function.\nResponse: We appreciate your constructive suggestions. In the revised manuscript we have included immunohistochemistry study confirming tCD19 expression in AAV‑injected, light‑stimulated MDA tumors (Fig. S8c, line 264). This directly demonstrates successful intratumoral AAV transduction and light‑dependent induction of the tCD19 target used for PEbody CAR engagement.\nPEbody CAR‑T cell infiltration: we showed tumor infiltration of PEbody CAR‑T cells following tail‑vein delivery using a PEbody CAR and firefly luciferase reporter cassette (Gal4UAS-Pmin-PEbody PAT CAR-P2A-Fluc, Fig. 5c-e and Fig. S4b in revised manuscript). In the revision, we further quantified intratumoral human T cells by flow cytometry of dissociated tumor tissue using anti‑human CD3 antibody and PE staining, confirming PEbody CAR‑T cell presence within the tumor microenvironment (Fig. S6b).\nFunctional characterization: the cytotoxic function of PEbody CAR‑T cells is presented in Fig. 1c–f and Fig. S1b–d. We have performed more characterization of PEbody CAR T cells including up‑regulation of activation markers CD25 and CD69 (Fig. S1f) and secretion of cytokines IFN‑γ, TNF‑α, and IL‑2 (Fig. S1g) upon target antigen engagement.\nCollectively, these additions provide direct validation of in situ AAV delivery and light‑induced tCD19 expression and infiltration of PEbody CAR‑T cells, and substantiate their functional activity.\nReviewer #3 (Remarks to the Author)\nThis is a timely article reporting the development of a monobody based PE targeting CAR (PAT) that allows for T cell reactivity to be directed to tumor antigens via PE-conjugated antibodies. The authors demonstrate the ability of PAT T cells to kill tumor cells in a specific manner based on the presence of PE-conjugated antibodies. They further improve the CAR safety profile by engineering the PAT under a light/tamoxifen controlled Cre recombinase (DGLA-PAT CAR), which restricts CAR activity to local illuminated areas and requires the presence of tamoxifen. The authors also engineered a synNotch regulated PAT (sPAT), whereby the PAT expression is induced by priming of the SynNotch antigen. Most notably, the authors devised a CAR-antigen pairing system, whereby tumor antigen expression is controlled by the DGLA system and T cell reactivity is controlled by the sPAT modules (DGLA-sPAT). In this strategy, tumor cells can be vaccinated with an inducible antigen that can then “train” T cells to react specifically at the tumor site through the synNotch module. In murine flank models of human breast cancer, the authors show MDA cells engineered or vaccinated with the DGLA inducible CD19 cassette, express the target antigen only under illumination and with tamoxifen treatment. Administration of sPAT T cells and PE-conjugated PD-L1 antibody resulted in tumor regression only in tumors that were illuminated with light. These efforts are framed in the context of antigen escape, tumor heterogeneity, and safety concerns arising from clinical experience of standard CAR-T therapy, particularly for solid tumors. The impact and novelty of the manuscript is well suited for Nature Communications. The following revision experiments/edits would strengthen the manuscript:\n1. In cytotoxicity assays (Figure 1 and Figure 3), the authors should include a conventional CAR control as a benchmark. It is unclear whether the inducible CAR formats reported in this study are of similar potency as standard CARs used in the field. The authors do a good job of motivating the clinical relevance of the study (i.e. addresses antigen heterogeneity, safety, and the biocompatibility of PE), but having a benchmark for potency is also needed to assess the clinical relevance. In addition to tumor killing, measuring the levels of cytokine secretion (i.e. IFNy, IL-2) from tumor cocultures is appropriate for assessing T cell functionality.\nResponse: We really appreciate your suggestion. We agree that benchmarking against a conventional CAR and assessing cytokine secretion are important for evaluating clinical relevance. In the revised manuscript, we have added conventional CAR controls to the cytotoxicity assays (Fig. 1c,e and Fig. 3d,g in the revised manuscript). We also included ELISA measurements of key secreted cytokines including IFN‑γ, TNF‑α, and IL‑2 after tumor co‑cultures (Fig. S1g), providing functional readouts complementary to target‑cell killing.\nTo further strengthen this benchmarking, we conducted an in vivo head-to-head comparison between DGLA-sPAT CAR-T cells and conventional CAR-T cells using a PSMA⁺ tumor model (Fig. S10a). Both CAR formats achieved comparable tumor control (Fig. S10e-g), demonstrating that the DGLA-sPAT design preserves full antitumor potency in vivo. Additionally, in this model, human PSMA was also ectopically expressed in the mouse liver via tail-vein lentiviral transduction (Fig. S10b-d) to enable assessment of on-target off-tumor (OTOT) effects. As expected, mice treated with conventional CAR-T cells exhibited pronounced systemic activation, including significant weight loss (Fig. S10h), elevated inflammatory cytokines (Fig. S10i-k), increased serum alanine aminotransferase (ALT, Fig. S10l), a standard indicator of hepatocellular injury, and apparent lymphocytic infiltration with liver tissue damage (Fig. S10m). In contrast, DGLA-sPAT CAR-T cells maintained robust tumor-killing activity without inducing OTOT-associated inflammation or tissue injury, underscoring their preserved functionality together with substantially improved safety. Together, the existing in vitro benchmark data combined with the direct in vivo comparison confirm that the DGLA-PAT/DGLA-sPAT inducible CAR-T systems maintain potency comparable to conventional CAR-T cells, while offering a significant safety advantage through spatially restricted activation. We have clarified these points in the revised manuscript (line 274-290).\n2. Similar to comment 1, Mock (untransduced) T cells are the appropriate negative controls in cytotoxicity assays that allow the reader to assess the leaky or non-specific activity of the system and should be included.\nResponse: Mock controls using untransduced T cells have been included in the cytotoxicity assays (Fig. 1c,e and Fig. 3d,g in the revised manuscript) and noted in the corresponding figure legends.\n3. In Figure 3C, are the cells gated on transgene+? It is unclear if the relatively low induction of CD19 CAR-T (~15%) is because of inefficiencies of the inducible system or if it is due to low transduction efficiencies. The authors should clarify these details.\nResponse: We thank you for the suggestion. For the assays in Fig. 3c, we used FACS-sorted T cells containing the transgenes of the inducible system. Thus, the result reflects transgene positive cells rather than variability from mixed transduction efficiencies. The ~15% induction likely arises from limited expression of the DGLA regulator components in primary T cells, where transgene expression is tightly regulated. This phenomenon is consistent with our previous findings, where we observed that the efficiency of induction is dependent on regulator expression levels in T cells (Huang et al., Sci Adv, 2020). To address this limitation, we subsequently adopted an alternative strategy where the DGLA system is delivered into tumor cells rather than T cells (Fig. 4a), which allows for stronger expression of tCD19 antigen and significantly improved induction efficiency (~85%, Fig. 4e).\n4. The cytotoxicity data presented in this study are normalized. The authors should indicate what the plots were normalized to.\nResponse: We appreciate your suggestion for clarification. The cytotoxicity data were normalized to tumor-only control wells without T cells. Details regarding this normalization method have been added to the figure legends and Materials and Methods section in the revised manuscript (line 475-476).\n5. In Figure 5E, it is unclear if the increase in fluc signal is driven by T cell expansion or synNotch activation since there is no constitutive and orthogonal luciferase that was coexpressed in the T cells. Did the authors administer PE conjugated antibody in this experiment? Adequate clarification or additional experiments should be provided to help interpret the source of the increasing fluc signal.\nResponse: We thank you for this insightful question. In the experiment shown in Figure 5E, PE-conjugated antibody was administered one day after T cell injection. We believe that the initial Fluc signal observed is primarily driven by synNotch activation of the injected T cells homing to the tCD19+ tumor site, rather than T cell expansion per se. This is because T cell expansion would require activation signaling, which in our system depends on the synNotch-mediated induction of PEbody CAR followed by its engagement with the target antigen via the corresponding PE-conjugated antibody. It is also important to note that in our synNotch-inducible Fluc design, any newly-generated T cells (resulting from expansion) would still require synNotch activation to express Fluc. Therefore, while T cell expansion may contribute to the overall signal increase over time, T cell expansion alone may not increase the Fluc signal without the synNotch activation. We have clarified this point in the revised manuscript (line 224-228) to ensure accurate interpretation of these results.\n6. The authors should add a reference for the following study that demonstrated an inducible CAR regulated by tamoxifen activated recombinase.\nChakravarti, Deboki, et al. \"Inducible gene switches with memory in human T cells for cellular immunotherapy.\" ACS synthetic biology 8.8 (2019): 1744-1754.\nResponse: We thank you for bringing this relevant reference to our attention. We have added this reference in the revised manuscript (line 294-296) to acknowledge this prior work on inducible CAR regulation.\nReferences\n1. Allen, M. E., et al. An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics. ACS Synth Biol 8, 2359-2371 (2019).\n2. Cabrera, A., et al. The sound of silence: Transgene silencing in mammalian cell engineering. Cell Syst 13, 950-973 (2022).\n3. Chappaz, S. and D. Finke The IL-7 signaling pathway regulates lymph node development independent of peripheral lymphocytes. J Immunol 184, 3562-3569 (2010).\n4. Di Stasi, A., et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365, 1673-1683 (2011).\n5. Flugel, C. L., et al. Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours. Nat Rev Clin Oncol 20, 49-62 (2023).\n6. Gendler, S. J. MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia 6, 339-353 (2001).\n7. Holliday, D. L. and V. Speirs Choosing the right cell line for breast cancer research. Breast Cancer Res 13, 215 (2011).\n8. Huang, Z., et al. Engineering light-controllable CAR T cells for cancer immunotherapy. Sci Adv 6, eaay9209 (2020).\n9. Lee, S. Y., et al. CD8(+) chimeric antigen receptor T cells manufactured in absence of CD4(+) cells exhibit hypofunctional phenotype. J Immunother Cancer 11 (2023).\n10. Morgan, R. A., et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18, 843-851 (2010).\n11. Morsut, L., et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780-791 (2016).\n12. Movahedin, M., et al. Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen. Glycobiology 27, 677-687 (2017).\n13. Park, A. K., et al. Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumors. Sci Transl Med 12 (2020).\n14. Roybal, K. T., et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419-432 e416 (2016).\n15. Slamon, D. J., et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177-182 (1987).\n16. Terakura, S., et al. Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells. Blood 119, 72-82 (2012).\n17. Wu, Y., et al. Control of the activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed Eng 5, 1336-1347 (2021).\nWe thank the Editor and the Reviewers for their careful evaluation of our manuscript and for the constructive and encouraging comments. We have incorporated the requested new data and revised the manuscript accordingly. Below, we provide a point-by-point response to each comment, with changes indicated in the revised version.\nReviewer #1 (Remarks to the Author):\nI thank the authors for their comprehensive responses to my previous comments and for the additional experiments provided, which have effectively addressed my concerns.\nSpecifically, the following:\n1. Supplementary data on CAR-T cell detection in peripheral blood and spleen, as well as safety experiments including liver and kidney function tests and histopathological studies, strongly support the tumor-local activation characteristics and safety of this system.\n2. The addition of isotype controls in the CD19 knockout experiment enhances the reliability of the results.\n3. The validation of CD19 and PSMA expression in engineered PC-3 cells is now complete.\n4. The explanation of the design change from DGLA-PAT to DGLA-sPAT and related in vivo experiments effectively address questions regarding the control of off-tumor toxicity.\n5. Statistical analysis of pre-treatment tumor size and verification that tCD19 expression has no effect on cell behavior eliminate my concerns about potential experimental design.\n6. The re-performed CD19-negative cytotoxicity experiment showed no difference between the two groups, consistent with theoretical expectations, making the conclusions more reliable.\nIn summary, the revised paper shows improvements in experimental completeness and clarity of argumentation. The method proposed in this study is innovative in improving the specificity and safety of CAR-T therapy.\nI believe the authors have responded carefully to the reviewers' comments, and the paper meets the publication requirements; therefore, I recommend acceptance.\nResponse: We sincerely thank the reviewer for the thorough evaluation and the positive and encouraging comments. We are pleased that the additional experiments and clarifications have adequately addressed the reviewer’s concerns.\nReviewer #2 (Remarks to the Author):\nAll in all, the authors did a great job at addressing my (and it seems like the other reviewers') previous concerns. I commend them for their effort to address my concerns experimentally. This research is now much more robust. Overall, very nice work.\nResponse: We thank the reviewer for the positive feedback and are pleased that the revisions have addressed the concerns and strengthened the manuscript.\nReviewer #3 (Remarks to the Author):\nPlease add the ELISA data for conventional CAR in Figure S1G.\nOtherwise, the revised manuscript has addressed my concerns and I recommend it for publication.\nResponse: We thank the reviewer for the suggestion and have added the requested ELISA data for conventional CAR in Supplementary Figure S1G of the revised manuscript (line 105).\nResponse to Reviewers\nWe thank the Editor and the Reviewers for their careful evaluation of our manuscript and for the constructive and encouraging comments. Below, we provide a point-by-point response to each comment.\nReviewer #3 (Remarks to the Author):\nThe authors have appropriately addressed my concerns in the revised manuscript. I recommend it for publication and congratulate the reviewers on the excellent work.\nResponse: We sincerely thank Reviewer #3 for the positive evaluation and encouraging comments. We appreciate the reviewer’s recognition of our revisions and are grateful for the support of our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for their comprehensive responses to my previous comments and for the additional experiments provided, which have effectively addressed my concerns.\nSpecifically, the following:\n1. Supplementary data on CAR-T cell detection in peripheral blood and spleen, as well as safety experiments including liver and kidney function tests and histopathological studies, strongly support the tumor-local activation characteristics and safety of this system.\n2. The addition of isotype controls in the CD19 knockout experiment enhances the reliability of the results.\n3. The validation of CD19 and PSMA expression in engineered PC-3 cells is now complete.\n4. The explanation of the design change from DGLA-PAT to DGLA-sPAT and related in vivo experiments effectively address questions regarding the control of off-tumor toxicity.\n5. Statistical analysis of pre-treatment tumor size and verification that tCD19 expression has no effect on cell behavior eliminate my concerns about potential experimental design.\n6. The re-performed CD19-negative cytotoxicity experiment showed no difference between the two groups, consistent with theoretical expectations, making the conclusions more reliable.\nIn summary, the revised paper shows improvements in experimental completeness and clarity of argumentation. The method proposed in this study is innovative in improving the specificity and safety of CAR-T therapy.\nI believe the authors have responded carefully to the reviewers' comments, and the paper meets the publication requirements; therefore, I recommend acceptance.", "reviewer_2": "All in all, the authors did a great job at addressing my (and it seems like the other reviewers') previous concerns. I commend them for their effort to address my concerns experimentally. This research is now much more robust. Overall, very nice work.", "reviewer_3": "Please add the ELISA data for conventional CAR in Figure S1G.\nOtherwise, the revised manuscript has addressed my concerns and I recommend it for publication."}, "author_response": "We thank the Editor and the Reviewers for their careful evaluation of our manuscript and for the constructive and encouraging comments. We have incorporated the requested new data and revised the manuscript accordingly. Below, we provide a point-by-point response to each comment, with changes indicated in the revised version."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have appropriately addressed my concerns in the revised manuscript. I recommend it for publication and congratulate the reviewers on the excellent work."}, "author_response": "We sincerely thank Reviewer #3 for the positive evaluation and encouraging comments. We appreciate the reviewer’s recognition of our revisions and are grateful for the support of our work."}]

Decision letter