Host-cell Interactions of Engineered T cell Micropharmacies

Genetically engineered, cytotoxic, adoptive T cells localize to antigen positive cancer cells inside patients, but tumor heterogeneity and multiple immune escape mechanisms have prevented the eradication of most solid tumor types. More effective, multifunctional engineered T cells are in development to overcome the barriers to the treatment of solid tumors, but the interactions of these highly modified cells with the host are poorly understood. We previously engineered prodrug-activating enzymatic functions into chimeric antigen receptor (CAR) T cells, endowing them with an orthogonal killing mechanism to conventional T-cell cytotoxicity. These drug-delivering cells, termed Synthetic Enzyme-Armed KillER (SEAKER) cells, demonstrated efficacy in mouse lymphoma xenograft models. However, the interactions of an immunocompromised xenograft with such complex engineered T cells are distinct from those in an immunocompetent host, precluding an understanding of how these physiologic processes may affect the therapy. Here, we also expand the repertoire of SEAKER cells to target solid-tumor melanomas in syngeneic mouse models using specific targeting with TCR-engineered T cells. We demonstrate that SEAKER cells localize specifically to tumors, and activate bioactive prodrugs, despite host immune responses. We additionally show that TCR-engineered SEAKER cells are efficacious in immunocompetent hosts, demonstrating that the SEAKER platform is applicable to many adoptive cell therapies.


Results
The SEAKER platform is compatible with primary murine T cells.
The SEAKER platform has been shown to be efficacious in human B cell lymphoma models (7). However, these xenograft mouse models using NOD-SCID gamma (NSG) mice lack an endogenous adaptive immune system and NK cells, which may affect SEAKER cell kinetics and persistence. Syngeneic models are useful in understanding the behavior of these complex cells for clinical translation. To understand key issues of kinetics, biodistribution, and immunogenicity of the SEAKER platform in immunocompetent hosts, we developed a syngeneic SEAKER cell system. Gene cassettes that included either the secreted β-lactamase (β-Lac) or carboxypeptidase G2 (CPG2) enzymes upstream of GFP via P2A cleavage sites were designed ( Figure 1A, Supplemental Figure 1A). The best signal sequences for each enzyme were determined empirically and used in this study (7). We transduced OT-1 T cells, which recognize a chicken ovalbumin peptide (OVA) presented on H-2Kb. Both β-Lac ( Figure 1B) and CPG2 (Supplemental Figure 1B) constructs were successfully transduced into primary murine OT-1 T cells, to provide β-Lac OT-1 SEAKER cells and CPG2 OT-1 SEAKER cells for further evaluation herein. A threshold 25% transduction efficiency was set for all experiments unless otherwise noted.
We have previously demonstrated that the β-Lac enzyme cleaves a Ceph-AMS prodrug to release the potent cytotoxic nucleoside analog AMS (7). Rapidly dividing cells are more susceptible to AMS (7,21). To evaluate the ability of β-Lac OT-1 SEAKER cells to activate Ceph-AMS, spent culture media from these cells was collected and incubated with 180 nM Ceph-AMS and EL-4 tumor cells. While the Ceph-AMS prodrug alone was non-toxic, combination with pre-conditioned media from the murine OT-1 SEAKER cells led to substantial cytotoxicity against the EL-4 target cells, demonstrating that the enzyme was fully functional ( Figure 1C). administration began on day 1 at either 3 or 4 mg/kg twice a day (BID) for three doses ( Figure 1F). Mice treated with β-Lac OT-1 SEAKERs and the Ceph-AMS prodrug demonstrated the lowest change in tumor progression across two replicate experiments ( Figure 1G, replicates shown in Supplemental Figure 5). These data demonstrated that murine T cells are capable of producing enough enzyme in vivo to unmask prodrug and delay tumor growth.
A similar experiment was performed using CPG2 OT-1 SEAKER cells and the corresponding AMS-Glu prodrug (50 mg/kg, BID for 12 doses) (Supplemental Figure 6A). In this experiment, 2 out of 4 mice treated with the combination CPG2 SEAKERs and the AMS-Glu prodrug had a durable response showing no evidence of tumor for weeks (Supplemental Figure 6B-D). The CPG2-AMS-Glu combination showed no overt systemic toxicity in this model (Supplemental Figure 6E) while the β-Lac-Ceph-AMS combination did exhibit significant systemic toxicity and mouse weight loss (Supplemental Figure 5C,H). Because β-Lac has fast enzyme kinetics for conversion of Ceph-AMS into AMS (7), we hypothesized that peritoneal β-Lac caused immediate conversion of the prodrug upon peritoneal injection, leading to systemic leakage of the active AMS drug. On the other hand, CPG2 has slower enzyme kinetics for conversion of its cognate prodrug AMS-Glu (7), and use of this slower system may be beneficial in scenarios where systemic drug leakage is of concern, thus mitigating drug toxicity. Nonetheless, these proof-of-concept experiments demonstrated that both enzyme-prodrug systems had antitumor efficacy in a syngeneic mouse model. However, these experiments utilized IP injected SEAKER cells and prodrugs, which simplified the need for specific T cell homing and localization to the tumor.

Antigen specific SEAKER cells localized to melanoma tumors
Models in which SEAKER cells must traffic and localize to solid tumor masses should better demonstrate the benefits of the SEAKER platform, as compared to peritoneal or in situ delivery models. Therefore, to better understand antigen specific SEAKER cell localization in solid tumors, we performed a time course of β-Lac OT-1 SEAKER cell localization to B16F10 melanoma tumors expressing the SIINFEKL ovalbumin peptide (B16-SIIN) in C57BL/6 mice. B16-SIIN tumors were engrafted subcutaneously (SC) on day -7.
Cyclophosphamide was injected at 100 mg/kg IP to precondition mice for adoptive T cell transfer on day -1.
β-Lac OT-1 SEAKERs were engrafted intravenously (IV) on day 0. Mice were serially euthanized on days 2, 5, 7, 10 and 14, and tumors and spleens were harvested and analyzed by flow cytometry (Figure 2A). OT-1 T cells have a CD45.1 congenic marker, allowing us to track the total OT-1 T cells as well as the transduced OT-1 SEAKER cells within the same animal. Both untransduced OT-1 T cells and OT-1 SEAKER cells exhibited a peak expansion at day 5 and began contracting by day 7 in the tumor (Figure 2B-E). As anticipated, OT-1 T cells localized at 10-fold lower frequencies in the spleen than tumor, highlighting the specificity of antigen specific T-cell localization. Interestingly, we observed a more pronounced contraction of OT-1 SEAKER cells as compared to untransduced T cells beginning at day 10 ( Figure 2F). Nonetheless, the kinetics of the OT-1 SEAKER cells matched that of wild-type OT-1 T cells during the first week of the response, indicating functional fidelity. To further investigate the fitness of β-Lac OT-1 SEAKERs, we compared the tumor control to mock transduced OT-1 T cells at a suboptimal dose of 3x10 6 cells per mouse. Tumor kinetics were similar between β-Lac OT-1 SEAKERs and mock transduced OT-1 T cells, demonstrating a transient control of tumor as compared to PBS treated mice ( Figure 2G,H).
T cells naturally localize to antigen-positive tissues, but also localize to secondary lymphoid organs (23), which might cause off-target toxicity in the SEAKER platform, due to prodrug unmasking. To understand whether SEAKER cells localize to off-target tissues, we treated B16-SIIN-bearing mice and harvested tumors, major secondary lymphoid organs (draining lymph nodes, bone marrow and spleen), blood and the lungs at day 6 post β-Lac OT-1 SEAKER cell engraftment ( Figure 3A). As anticipated, the β-Lac OT-1 SEAKER cells localized at high percentages in the tumor, but were present only at very low frequencies in off-target organs ( Figure 3B-E).
Taken together, these findings demonstrated that SEAKER cells localize specifically to tumors and were rarely . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint found in off-target secondary lymphoid organs during the peak expansion, which indicates a reduced risk of off-target toxicity.

SEAKER cells deliver functional synthetic enzymes to tumors
To study the pharmacokinetics of SEAKER cells in vivo, we used Gaussia luciferase (gLuc), which enables the bioluminescent imaging (BLI) of antigen specific T cells in both tumor and healthy tissues (Supplemental (24). To track the exact localization of enzyme secreting cells, we designed a β-Lac OT-1 SEAKER construct that includes a membrane-anchored gLuc, and mCherry. These cells were used in the B16-SIIN model described above and mice were serially imaged by BLI ( Figure 4A-B). In accordance with the flow cytometric analysis of SEAKER cells (Figure 2), we observed a peak in expansion at day 5 post T cell engraftment followed by a contraction of detectable cells ( Figure 4C-D). Administration of rhIL-2 at 4.5 x 10 5 IU/mouse/day for 6 days to stimulate T cell growth further did not impact SEAKER cell expansion ( Figure 4D). Similar expansion kinetics were seen in the analogous CPG2 OT-1 SEAKER models (Supplemental Figure 10).
Taken together, these results demonstrated that SEAKER cell kinetics can be tracked longitudinally using BLI. Importantly, two separate methods of kinetic tracking (flow cytometry and BLI) both demonstrated the same kinetics, despite introduction of the foreign gLuc protein.
Although localization of SEAKER cells to antigen-positive tumors was robust, it was not yet evident whether these cells were delivering functional enzymes to tumors in the immunocompetent hosts. Thus, we harvested tumors from mice treated with β-Lac OT-1 SEAKER cells and extracted the soluble protein fraction ( Figure   4E). When treated with the gLuc substrate coelenterazine, only tumor homogenates from the β-Lac-gLuc-mCherry OT-1 SEAKER-treated mice had any detected BLI signal ex vivo ( Figure 4F). We then mixed tumor homogenate extracts with nitrocefin to assess β-Lac enzyme function directly. Both the tri-cistronic β-Lac-gLuc-mCherry OT-1 SEAKER and bi-cistronic β-Lac-GFP OT-1 SEAKER treated mice had significantly more enzyme activity than control mice in the tumor homogenates ( Figure 4G), and the bi-cistronic vector resulted in higher levels of expression. These results showed that the level of secretion of β-Lac enzyme in vivo can be fine-tuned through the number of 2a elements. Each additional 2a site on the vector decreases the production efficiency of each genetic element. Importantly, levels of enzyme activity were sufficient to convert substrate ex vivo, indicating the feasibility of the SEAKER-prodrug approach in immunocompetent hosts. We also showed that targeted delivery of β-Lac enzyme was achievable in a peritoneal ovarian tumor model using the anti-MUC16 CAR model (Supplemental Figure 11). This demonstrated that the SEAKER platform can deliver enzymes using both CAR and TCR-based antigen targeting mechanisms to a variety of anatomical locations in immunocompetent hosts.

SEAKER cells synergize with prodrug to delay melanoma progression
Having established that SEAKER cells are functional in vivo and localize specifically to B16-SIIN tumors, we designed an efficacy model to test whether β-Lac OT-1 SEAKER cells can be potentiated with the non-toxic Ceph-AMS prodrug to delay tumor growth. Consistent with a previous report (25), direct treatment with the AMS parent drug induced pronounced systemic toxicity in mice, manifested by rapid weight loss, and had only a modest anti-tumor effect against B16 at the maximum tolerated dose of 0.1 mg/kg (Supplemental Figure 12).
To determine whether the SEAKER platform could improve anti-tumor efficacy and decrease toxicity, B16-SIIN tumor-bearing mice in our syngeneic model were engrafted with β-Lac OT-1 SEAKER cells on day 0, then treated with Ceph-AMS on days 4-8 (4 mg/kg, BID for 10 doses) ( Figure 5A). Mice treated with β-Lac OT-1 SEAKER cells and prodrug demonstrated delayed tumor growth as measured by digital calipers ( Figure   5B). Furthermore, survival of mice given SEAKER cells and prodrug was significantly enhanced compared to mice treated with SEAKER cells or prodrug alone ( Figure 5C). Importantly, we observed no overt toxicity as . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint evidenced by no decrease in weight of mice treated with SEAKER plus prodrug ( Figure 5D). These results showed that the SEAKER platform is efficacious against melanoma solid tumors without the characteristic toxicity associated with direct administration of the AMS parent drug, due to local generation at the tumor.

SEAKER cells survive prodrug treatment in the tumor
One potential limitation of the SEAKER platform is that the SEAKER cells may themselves be subject to the cytotoxic effects of the prodrug they unmask. To assess this issue, we used our trackable SEAKER cells in the SC efficacy model established above ( Figure 5) and imaged for the presence of the T cells every day during the prodrug dosing regimen ( Figure 6A). Surprisingly, groups of mice given the SEAKER cells alone or SEAKERs plus prodrug demonstrated identical T-cell pharmacokinetics during the dosing schedule ( Figure 6B-C). We speculate that the SEAKER cells may survive because the prodrug is administered at the end of the T cell expansion period, when the T cells are presumably slowing proliferation. This may explain the unexpected resistance of SEAKER cells to the active cytotoxic drug unmasked in vivo.

Assessing the immunogenicity of the β-Lac SEAKER cells in immunocompetent hosts
The bacterial origin of SEAKER enzymes raises the possibility that they will be immunogenic in immunocompetent hosts, potentially preventing the cellular or enzymatic functions. Mice treated with β-Lac OT-1 SEAKERs mounted a humoral response to the enzyme that peaked at day 5 post T cell injection, which followed a similar kinetic time course to that of the SEAKER cells themselves (Supplemental Figure 13A Importantly, when those same serum samples were incubated with recombinant β-Lac and nitrocefin substrate, equivalent levels of substrate cleavage resulted from enzymes treated with naive mouse serum or β-Lac OT-1 SEAKER-treated mouse serum (Supplemental Figure 13F-G). This indicates that, although the antibodies that bind β-Lac are induced, they do not block the function of the enzyme.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Multiple rounds of adoptive T cell transfer might increase anti-tumor efficacy and T cell localization in the tumor. However, immune responses against adoptive T cells have been documented after single injections, preventing additional rounds of cell administration (8). We assessed whether SEAKER cells could be readministered in previously-treated mice to explore the impacts of retreatment upon the pharmacokinetics of the cells. B16-SIIN tumor-bearing mice received a preconditioning dose of cyclophosphamide at 100 mg/kg IP at day -8 then a dose of either PBS or β-Lac-GFP OT-1 SEAKER cells at day -7. Mice were then stratified into two further groups: one cyclophosphamide dose or three consecutive cyclophosphamide doses at 100 mg/kg IP. gLuc+ SEAKER cells were then engrafted into all groups on day 0 and imaged day 5 post engraftment ( Figure   6D). Mice that had not been pretreated engrafted the SEAKERs by day 5. In contrast, four of five mice treated with two successive SEAKER doses and one cyclophosphamide dose rejected the second SEAKER cell engraftment ( Figure 6E-F). However, mice who received three doses of cyclophosphamide prior to the second adoptive transfer showed enhanced engraftment of the second SEAKER administration ( Figure 6E-F).
Furthermore, primary engraftment of wild-type OT-1 T cells or CPG2 OT-1 SEAKER cells had no impact on subsequent engraftment of β-Lac OT-1 SEAKER cells (Supplemental Figure 14). These results demonstrated that the host immune responses to SEAKER cells can be overcome through simple pharmacological manipulation. Taken together, these studies show that the SEAKER-produced enzymes are still functional despite being bound by antibodies, and that multiple rounds of SEAKER cell treatment can be achieved with stringent preconditioning, similar to what is used in humans clinically.

Discussion
We have examined the kinetics, biodistribution, and efficacy of complex, cargo-delivering, adoptive T cells in immunocompetent hosts bearing solid tumors, revealing both opportunities and potential challenges. The results presented demonstrate the feasibility and efficacy of adoptive T cell micropharmacies in syngeneic systems.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Traditionally optimized for their cytotoxic capacity, T cells can now be used for consistent, large-scale, highly-localized cargo delivery. This will allow for effective therapeutic killing, even for poorly expressed, downregulated, or heterogenous antigens within a tumor with limited access. The current study presents four conceptual advances for adoptive T cell micropharmacies: 1) cargo delivery and efficacy in an immunocompetent system, 2) cargo delivery and efficacy in solid-tumor models, 3) expansion of the target repertoire of SEAKER cells to intracellular antigens using TCRs, and 4) circumvention of the SEAKER and enzyme immunogenicity issues.
Syngeneic tumor models enable the study of adoptive T cells in immunologically competent hosts. In contrast, xenograft models in NSG mice lack common gamma chain-dependent immune cells, such as T cells, B cells, and NK cells (26,27). Furthermore, innate immune function in these models is altered. As a consequence, secondary lymphoid organ formation is also altered. Competition for pro-survival cytokines is largely non-existent, which allows adoptively transferred human cells to maintain homeostatic proliferation in the absence of antigen, confounding pharmacokinetic and biodistribution studies (19,20). Critically, anti-mouse reactive human cells selectively expand in xenograft models, and induce graft-versus-host disease at late timepoints (28). Investigation of true T cell pharmacokinetics is not feasible in these models. Syngeneic mouse models circumvent all of the caveats of xenograft models. The OT-1 immunocompetent SEAKER models developed in this study provided systems in which to test important pharmacokinetic parameters of SEAKER cells in advance of clinical translation. In the context of a complete immune system, SEAKER cells are still able to localize at high concentrations to antigen-positive tumors and to deliver cargo with minimal off-tumor localization.
Solid tumors present additional challenges to effective adoptive T cell therapy in comparison to hematopoietic cancers. T cell penetration in solid tumors is often aberrant, due to altered tumor extracellular matrix, trapping by suppressive immune cells, and necrotic acellular cores (29,30). Solid tumors also express antigens that are . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint found on vital healthy tissues. This leads to toxicities that limit the effectiveness of many solid tumor therapies. Additionally, solid tumors are notoriously heterogenous. Many tumor-specific antigens are only present in a subset of cells within the tumor, leading to inevitable escape after adoptive T cell therapy. The B16-SIIN tumor model is highly aggressive, with high resistance to T cell-mediated killing in vivo. Addition of the SEAKER enzyme/prodrug system led to delayed solid tumor growth and improved survival without the characteristic systemic toxicity of chemotherapeutics, even in the absence of substantial T cell killing. The SEAKER platform is a modular small-molecule delivery platform that takes advantage of intrinsic T cell function to deliver therapeutic cargo to tumors. Antigen stimulation increases and localizes therapeutic payload . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint to tumor via two mechanisms. Firstly, stimulated T cells are retained in their target tissues and undergo proliferation. This leads to high tumor concentrations of T cells, while trace numbers of cells localize to off-target tumors (Figure 2, Figure 3). Secondly, activated T cells express more retroviral transgenes (33). T cell activation leads to activation of retroviral promoter LTRs and expression of downstream genes (34). Although we used constitutively expressed enzymes, these two mechanisms localize the enzyme to its target tissue. When tumor, SEAKER cells, and prodrug are all injected IP, toxicity in the mice is observed, due to systemic leakage of unmasked AMS from the peritoneal cavity. However, in a more clinically relevant adoptive transfer model using IV administered SEAKER cells and a solid subcutaneous tumor, anti-tumor benefit was observed without the characteristic toxicity of AMS. The SEAKER platform has the highest therapeutic window when T cells must traffic into the tumor from circulation. Once in the target tissue, any cell in the vicinity of unmasked drug will be susceptible, offering the possibility to kill antigen negative cells and stromal cells. Future iterations of the SEAKER platform may also include T cell activation inducible promoters, such as NFAT, to further control the delivery of payloads.
The potential for targeted delivery of synthetic cargo to tissues has applications outside of cancer.
Enzyme-prodrug therapy could be used in a variety of diseases to deliver drugs directly to the affected tissues.
In the current study, we expanded the application of SEAKER cells to include both CAR T cells and TCR T cells, demonstrating the wider translational potential of targeted micropharmacies. Cellular micropharmacy-directed therapy could be used in a variety of disease states, such as inflammatory or autoimmune conditions, to deliver potent immunosuppressives to target tissues, sparing systemic immunosuppression. Critically, a barrier to further application of the SEAKER platform in inflammatory disease states is the need for cells that do not kill their target. This will require further engineering and exploration of other cell types as targeted micropharmacies.
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Recombinant proteins
CPG2 and β-Lac proteins were produced and purified by GenScript as previously described (7). Constructs contain C-terminal hemagglutinin (HA) and His6 epitope tags and were purified by nickel affinity chromatography.

Generation of Retroviral Vectors and Producer Cell lines
To generate murine SEAKER cells, the gene encoding β-lactamase (β-Lac) and carboxypeptidase G2 (CPG2) were cloned into the SFG gamma retroviral vector (generously provided by the R. Brentjens lab, MSKCC) alongside a P2A self-cleaving peptide and green fluorescent protein (GFP) or alongside the CAR constructs for the anti-murine CD19 scFv or the 4H11 anti-MUC16 scFv with murine CD28 and CD3ζ genes.
Absorbance at 320 nm was recorded on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and decrease in UV signal signified substrate cleavage.

T cell Isolation and Modification
Mouse T cells were isolated from spleens of naïve mice by mechanical disruption using a 100 µm cell strainer. Splenocytes were collected and red blood cells were lysed using ACK lysis buffer to remove red blood  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Enzyme-linked immunosorbent assay (ELISA) analysis.

Sandwich ELISAs were performed on 96-well Immulon HBX plates (Thermo Fisher Scientific). A mouse IgG
anti-β-Lac antibody (clone: 3E11.G3, Thermo Fisher Scientific) was used to capture recombinant β-Lac and primary murine serum samples were used as primary antibody. A polyclonal anti-mouse IgG HRP antibody was used as detection antibody (Novus Biologicals, Littleton, Colorado, USA). Protein was detected using TMB (3,3′,5,5′-tetramet hylbenzidine) substrate (Thermo Fisher Scientific) and H 2 SO 4 acid quench, and read on a SpectraMax M2 plate reader. Data were analyzed with SoftMax Pro software.

Cytotoxicity assays
T-cell and prodrug cytotoxicity assays with secreted enzymes were performed after 24 to 48 hours using was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint was detected in a Wallac EnVision Multilabel reader (Perkin Elmer). Target lysis was determined as (1-(RLUsample)/(RLUmax))x100.

In vivo experiments
All experiments were performed in compliance with all relevant ethical regulations and in accordance with MSKCC IACUC protocol 96-11-044. All mice were included in the analyses and no attrition was noted.
Mice were 6 to 12 weeks old and weighed 18-30 grams when treated. Both male and female mice were used.
Female mice were randomized into groups to allow balance in groups for tumor growth before treatment. Male mice were maintained with their initial littermates to avoid fighting. Experiments were not blinded, but results were confirmed by blinded third parties. Experiments were replicated 2 or more times as indicated in the legends.All BLI was performed using a Xenogen IVIS Spectrum and analyzed using Living Image software . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint B16F10 SIINFEKL melanoma models: C57/BL6 mice were engrafted SC with 1 x 10 6 B16F10 melanoma cells engineered to express the SIINFEKL peptide (B16-SIIN) on day -7 (generously provided by the Andrea Schietinger Lab, MSKCC). Tumors were engrafted using a 1:1 mixture with matrigel. On day -1, mice were treated IP with 100 mg/kg cyclophosphamide (Sigma-Aldrich, St. Louis, Missouri). On day 0, mice were engrafted IV via retro-orbital injection with 1-3 x 10 6 bulk murine T cells.

B16F10 SIINFEKL OT-1 T cell Kinetics and localization:
Experimental parameters described in "B16F10 SIINFEKL melanoma models" above were used. Tumors were For lymphoid organ SEAKER cell experiments, mice were harvested on day 6. Tumor and spleens were harvested as described above. Inguinal lymph nodes and lungs were removed and mechanically dissociated through a 100 µm strainer into RPMI-1640. Bone marrow was collected by removing femurs and crushing them . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint with mortar and pestle in RPMI-1640. Blood samples were collected through cheek bleeds into heparinized tubes. ACK lysing buffer was used to lyse blood cells for 5 minutes. Collected cells were washed in PBS and prepared for flow cytometry as described.

B16F10 SIINFEKL OT-1 β-Lac Enzyme Activity Model
Experimental parameters described in "B16F10 SIINFEKL melanoma models'' above were used. On day 5 post T cell engraftment, tumor samples were harvested through a cell strainer into PBS to solubilize the cell-free proteins. Samples were centrifuged at 12,000g to remove all debris and clarified supernatant fluids were run in the nitrocefin assay.

B16F10 SIINFEKL OT-1 Efficacy Model
Experimental parameters described in "B16F10 SIINFEKL melanoma models'' above were used. Prodrug  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ;     . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Supplemental Figure 9. PMEL T cells home to gp100 antigen positive B16F10 tumors. (A) Schematic where C57BL/6 mice received SC engraftment of 1 x 10 6 B16F10 cells at day -7. Cyclophosphamide was injected IP at day -1. PMEL gLuc T cells were then engrafted and T cells were imaged serially through injection of 100ug coelentrazine. (B) Images of experiment (A). Due to retro-orbital injection, non-specific signal may be noted near the eyes.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Supplemental Figure 11. β-lac-mMUC16 SEAKER cells deliver β-Lac to peritoneal ovarian tumors. (A) C57BL/6 mice were engrafted with 3 x 10 6 ID8 ovarian tumor cells IP at day -21. Mice were preconditioned with 100 mg/kg cyclophosphamide at day -1. On day 0, 3 x 10 6 β-Lac-mMUC16 SEAKERs were engrafted retro-orbitally. Peritoneal lavages were performed on day 7 for flow cytometry and nitrocefin enzyme activity. (B) Flow cytometry of myc staining for CAR+ T cells. (C) Quantification of (B). (D) Flow cytometry of cleaved CCF2 staining, indicating β-Lac activity. (E) Quantification of (D). (F) Cell pellets from peritoneal lavages were lysed and mixed with nitrocefin. A graph of raw absorbance at 490 nm is displayed. (G) Supernatant from the peritoneal lavages were mixed with nitrocefin and raw absorbance at 490 nm was measured.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Supplemental Figure 13. β-Lac SEAKER cells elicit a humoral immune response in C57BL/6 mice. (A) Mice were engrafted with 1 x 10 6 B16F10 cells SC at day -7, 100 mg/kg of cyclophosphamide on day -1, and β-Lac PMEL T cells on day 0. Sera were collected at indicated time points. (B-D) Serum samples were tested for reactivity to recombinant untagged β-Lac at day 2 (B), day 5 (C), and day 7 (D). Raw absorbance is depicted at 450 nm. (E) Average serum binding was normalized to naive mice at the 1:30 serum dilution. (F) Schematic depicting β-Lac enzyme functional assay in which recombinant β-Lac was mixed with serum samples and nitrocefin was added to assess change in activity. (G) Graph depicting raw absorbance values for recombinant β-Lac incubated with indicated serum samples and concentrations.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint Supplemental Figure 14. β-Lac SEAKER cells can be re-engrafted with CPG2 SEAKER cells. (A) C57BL/6 mice were engrafted with 1 x 10 6 B16F10 SIINFEKL cells SC at day -14, pretreated with 100 mg/kg cyclophosphamide at day -8 and treated with 3 x 10 6 indicated OT-1 cells retro-orbitally on day -7. Mice were retreated with 100 mg/kg cyclophosphamide on day -1 and re-engrafted with trackable gLuc+ β-Lac SEAKER cells on day 0. Mice were imaged serially. (B) T cell bioluminescent imaging from experiment (A). (C) Quantification of background adjusted BLI values from (B).
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.04.05.535717 doi: bioRxiv preprint