Conditional CAR T cells with specificity to oncofetal glycosaminoglycans in solid tumors

Chimeric antigen receptor; CAR T cells; immunotherapy; chondroitin sulfate; 1 oncofetal CS; osteosarcoma; bladder cancer. 2 3

4 Glycosaminoglycans are often deprioritized as targets for synthetic immunotherapy due to the 5 complexity of glyco-epitopes and limited options for obtaining specific subtype-binding. Solid 6 tumors express proteoglycans that are modified with oncofetal chondroitin sulfate (CS), a 7 modification normally restricted to the placenta. Here, we report the design and functionality of 8 conditional chimeric antigen receptor (CAR) T cells with selectivity to oncofetal CS. Following 9 expression in T cells, the CAR could be ‘armed’ with recombinant VAR2CSA lectins (rVAR2) 10 to target tumor cells expressing oncofetal CS. While un-armed CAR T cells remained inactive in 11 the presence of target cells, VAR2-armed CAR T cells displayed robust activation and the ability 12 to eliminate diverse tumor cell types in vitro. Cytotoxicity of the CAR T cells was proportional 13 to the concentration of rVAR2 available to the CAR, offering a potential molecular handle to 14 finetune CAR T cell activity. In vivo , armed CAR T cells rapidly targeted bladder tumors and 15 increased survival of tumor-bearing mice. Thus, our work indicates that cancer-restricted 16 glycosaminoglycans can be exploited as potential targets for CAR T cell therapy. 17 18

19 Chondroitin sulfate (CS) glycosaminoglycans are abundantly present in the placenta where they 20 support growth and motility of trophoblast cells (1-3). Motility is an essential feature of villous 21 trophoblasts that allow them to invade into the uterine tissue during placental implementation (4, 22 5). Plasmodium falciparum malaria parasites express VAR2CSA lectins on the exterior of 23 infected red blood cells, mediating unique binding-specificity to placental-type CS (6-8). In 24 normal physiology, placental-type CS is exclusively present in the placental syncytium and 25 VAR2CSA+ malaria infected erythrocytes therefore only accumulate in the placenta. 26 27 Cell growth and motility are features shared between trophoblasts and tumor cells (9-12) . 28 Perhaps to mimic the features of the placental compartment that support tumor growth, tumor 29 cells — including those in many pediatric and adult solid tumors — re-express placental-type 30 chondroitin sulfate (CS), as a secondary modification to a limited repertoire of proteoglycans (8, 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 3 13-17). Accordingly, recombinant VAR2CSA (rVAR2) can be used to guide therapeutic 1 modalities towards solid tumors when formulated as rVAR2-drug conjugates (8, 15, 18, 19) or 2 bi-specific immune cell engagers (19-21). 3 4 Oncofetal tumor antigens have emerged as potential attractive targets for chimeric antigen 5 receptor (CAR) T cell therapy (22, 23). CARs are genetically engineered synthetic antigen 6 receptors that are able to activate upon antigen recognition, independent of MHC presentation 7 (24, 25). CAR T cell therapy has achieved unprecedented success in treating patients with 8 hematopoietic malignancies, such as acute B-cell lymphoblastic leukaemia and B-cell 9 lymphomas (23, 26-28). However, only a few studies have shown promise for CAR T cell 10 therapy in solid tumors, such as GD2-CAR T cells for relapsed and refractory Neuroblastoma 11 tumors (29), as well as B7H3-, GD2- and IL13RA2-CAR T cells for human Glioma (30-32). 12 CAR T cell therapy remains challenging in solid tumors due to numerous factors including 13 architectural heterogeneity, acquired antigen down-regulation or antigen-loss, or a lack of 14 specific surface tumor antigens (23, 33). Hence, identification of specific antigens in solid 15 tumors is a necessary step for extending the clinical utility of CAR T cell therapy beyond 16 hematopoietic cancers. Until now, CAR T cell therapy has primarily focused on targeting protein 17 antigens expressed on the surface of cancer cells. However, due to the complex challenges of 18 CAR T cell therapy in solid tumors, there is increasing interest in exploring other types of target 19 molecules including carbohydrates, glycolipids, and glycoproteins. For example, targeting the 20 glycosylation component of a protein rather than the protein itself offers potential advantages. 21 First, tumor-specific protein glycoforms can offer increased tumor selectivity and possibly limit 22 off-target effects (34-36). Second, a specific glycosylation moiety or pattern can be present on 23 several different proteoglycans simultaneously across cell populations, including tumor stem 24 cells, which may overcome challenges related to heterogeneity and dormancy (19). Finally, 25 proteins that are not normally glycosylated may be subject to disease-specific glycosylation, 26 thereby increasing the available tumor target reservoir (8, 36-38). In this study, we utilized 27 recombinant VAR2CSA proteins to produce CAR T cells with specificity to oncofetal CS 28 glycosaminoglycans, broadly expressed across various solid tumor cell types. 29 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 4

1 Design and validation of a conditional CAR with selectivity to oncofetal CS 2 glycosaminoglycans 3 Oncofetal CS glycosaminoglycans have been described in multiple solid tumor types including 4 sarcoma, lymphoma, glioma, melanoma, pancreatic cancer, lung cancer, colorectal cancer, breast 5 cancer, prostate cancer, and bladder cancer (2, 8, 13-16, 18-21, 39-41). The oncofetal CS 6 modification can be specifically targeted by rVAR2 proteins that are currently under 7 investigation as vehicles for therapeutic delivery (8, 15, 19-21) and as reagents in liquid biopsy 8 diagnostic applications (14, 16, 40). Indeed, the rVAR2 proteins are specific for oncofetal CS, 9 and binding to tumor cells can be competed with soluble CS ( Supplementary Fig. 1A) (8, 15). 10 In primary tumor specimens, oncofetal CS is found both in the tumor stroma and on cell 11 membranes, and expression generally increases with tumor stage (8, 15, 39). For instance, in 12 bladder cancer patients, oncofetal CS expression is significantly associated with advanced T 13 stage (P=0.0231) and N stage (P=0.0114). ( Supplementary Fig. 1B-D ). Compared to bladder 14 cancer, neuroblastoma tumors express lower amount of oncofetal CS, yet the advanced-stages 15 tumors (II-IV) demonstrate higher expression compared to stage I tumor ( Supplementary Fig. 16 1E-F). Interestingly, high levels of oncofetal CS were associated with poor survival of 17 neuroblastoma patients (Supplementary Fig. 1G), which is a trend also observed in other cancer 18 indications (15, 18). 19 20 With oncofetal CS being a broadly expressed target across solid tumor indications, we decided to 21 explore it as a target for CAR T cell therapy. We first tested the levels of oncofetal CS on naïve 22 and activated human T cells using rVAR2 as the detection reagent to assess the risk of potential 23 CAR-induced T cell self-elimination. Activated and naïve T cells expressed minimal-to-24 undetectable levels of oncofetal CS and the expression was ~20x lower than that detected in 25 human bladder cancer cells (Fig. 1A). 26 27 We next designed a conditional CAR construct that utilized the specificity of rVAR2 for 28 oncofetal CS targeting. The design allowed the CAR to be armed with rVAR2 after expression in 29 the T cell membrane . This un-conventional design was deployed to alleviate inherent problems 30 of expressing a functional VAR2-CAR fusion-protein in human T cells. The conditional CAR 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 5 was comprised of the intracellular activation, co-stimulatory, and transmembrane domains 1 CD3zeta, 41BB, and CD28, fused in-frame with a sequence encoding the split-protein intein 2 domain, SpyCatcher (SpyC), derived from Streptococcus pyogenes fibronectin-binding protein 3 FbaB (Fig. 1B) (42). For context, when the SpyCatcher domain comes into contact with the other 4 half of the split-intein, the so-called SpyTag, they spontaneously form a covalent isopeptide bond 5 that irreversibly links the split-intein components (42, 43). Accordingly, we anticipated this 6 design to allow for the expression of a dormant [SpyC]-CAR in T cells that could then be 7 subsequently armed with rVAR2 genetically fused to a SpyTag, VAR2-[SpyT]. We next 8 transduced human T cells with the chimeric [SpyC]-CAR using lentiviral gene transfer ( Fig. 9 1C). The [SpyC]-CAR sequence includes a Flag-tag that enables determination of CAR 10 expression in T cells. Indeed, [SpyC]-CAR transduction of human T cells was ~90% efficient 11 and was expressed by both CD4 + and CD8 + T cell populations ( Fig. 1D ), with <10% of the 12 population staining negative for Flag (Fig. 1E). 13 14 We next attempted to arm the [SpyC]-CAR T cells with the recombinant VAR2-[SpyT] warhead 15 (Fig. 1F ). The VAR2-[SpyT] recombinant protein contains a V5 tag that enables specific 16 detection of armed CARs via assessment of V5 and Flag double-positive T cells. Here, 68.4% of 17 the [SpyC]-CAR T cells could be armed with the VAR2-[SpyT] protein ( Fig. 1G ), and the 18 spontaneous VAR2-[SpyT][SpyC]-CAR reaction on T cell membrane saturated at ~150 nM 19 VAR2-[SpyT] (Fig. 1H ). In aggregate, these data demonstrate the design and expression of 20 [SpyC]-CAR T cells that can be armed with rVAR2-[SpyT] proteins. 21 22 VAR2-armed CAR T cells activate upon target cell engagement and produce robust 23 cytokine responses 24 We next investigated whether the armed VAR2-[SpyT][SpyC]-CAR T cells became activated 25 upon engagement with oncofetal CS-positive cancer cells. As models of adult epithelial and 26 pediatric mesenchymal tumor types, we used UM-UC-3 muscle-invasive bladder cancer and 27 MG-63 osteosarcoma cells, respectively, as target cell lines for most of experiments. Upon 28 contact with UM-UC-3 and MG-63 tumor cells, VAR2-[SpyT][SpyC]-CAR T cells, but not 29 unarmed [SpyC]-CAR T cells, upregulated the activation markers CD25 and CD69 (Figs. 2A-B). 30 Similar results were obtained with LNCaP (prostate cancer) and U2OS (osteosarcoma) cell lines 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 6 (Supplementary Figs. 2A-B). T cell activation is associated with induction of cytokines that in 1 turn activate other immune cells, such as macrophages and dendritic cells (44). Therefore, as a 2 secondary readout for CAR T cell activation, we examined the expression of key cytokines 3 subsequent to VAR2-[SpyT][SpyC]-CAR T cell engagement with target tumor cells. All cell 4 lines tested were able to trigger a robust upregulation of IFNgamma (IFN γ), IL-2, and TNFalpha 5 (TNFα) secretion by armed VAR2-[SpyT][SpyC]-CAR T cells at orders of magnitude higher 6 levels than that detected in unarmed [SpyC]-CAR T cells or non-transduced T cells (Figs. 2C-D, 7 Supplementary Figs. 2C-D ). Induction of additional cytokines such as IL-4, IL-6, and IL-13 8 was also detected in all the cell lines tested (Supplementary Fig. 3). Combined, these data show 9 that armed VAR2-[SpyT][SpyC]-CAR T cells became activated upon target tumor cell 10 engagement. 11 12 VAR2-[SpyT][SpyC]-CAR T cell cytotoxicity is target-cell type-dependent 13 We next investigated the spatiotemporal relationship between activated VAR2-[SpyT][SpyC]-14 CAR T cells and target cell cytotoxicity. For this, we added VAR2-[SpyT] armed and unarmed 15 [SpyC]-CAR T cells to UM-UC-3 and MG-63 target cells at a 1:1 E:T ratio and imaged the co-16 cultures for 3 days. Armed VAR2-[SpyT][SpyC]-CAR T cells underwent clonal expansion 17 (green cells) upon target cell engagement, while unarmed [SpyC]-CAR T cells remained inactive 18 (Fig. 3A ). Moreover, clonal expansion of the VAR2-[SpyT][SpyC]-CAR T cells efficiently 19 eliminated the target tumor cells (red cells), while cells exposed to unarmed [SpyC]-CAR T cells 20 outgrew the culture by day 3. Notably, VAR2-[SpyT][SpyC]-CAR T cell expansion could be 21 detected in MG-63 cultures at day 1, while similar level of expansion was detected at day 2 in 22 UM-UC-3 ( Fig. 3A ). To further examine potential differences in sensitivity to VAR2-23 [SpyT][SpyC]-CAR T cells amongst different target cell types, we subjected our diverse cell line 24 panel to VAR2-[SpyT][SpyC]-CAR T cells in an E:T-ratio of 1:1 and recorded tumor cell 25 viability over a week. While a decrease in MG-63 cells was observed after 18 hrs, UM-UC-3 26 cells needed 36 hrs of VAR2-[SpyT][SpyC]-CAR T cell exposure before a reduction in cell 27 viability could be detected ( Fig. 3B ). LNCaP and U2OS also required 36 hrs of VAR2-28 [SpyT][SpyC]-CAR T cell exposure to exhibit a reduction in cell viability ( Supplementary Fig. 29 4A). We next plotted the oncofetal CS expression levels of the different cell types ( Fig. 1A and 30 Supplementary Fig. 1A ) against the time required for VAR2-[SpyT][SpyC]-CAR T cells to 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 7 decrease viability of the same cells (Fig. 3B and Supplementary Fig. 4A). The analysis revealed 1 that the level of oncofetal CS expression (MFI) did not directly reflect in vitro efficacy of VAR2-2 [SpyT][SpyC]-CAR T cells ( Fig. 3C ). The same was observed for oncofetal CS target cell 3 expression and expression of key T cell activation markers (i.e., CD25 +/CD69+ and IFN γ) 4 following co-culture ( Supplementary Fig. 4C-D ). Combined, these data show that oncofetal 5 CS-positive tumor cells of different lineages can be targeted and eliminated by VAR2-6 [SpyT][SpyC]-CAR T cells. The data further indicate that the different amounts of oncofetal CS 7 expressed on the target cells all induce sufficient CAR T cell activation after target cell 8 engagement. 9 10 [SpyC]-CAR T cells can be conditionally armed with VAR2-[SpyT] to eliminate tumor cells 11 The [SpyC]-CAR was designed to allow subsequent and conditional arming of [SpyC]-CAR T 12 cells using rVAR2-[SpyT] proteins in a dose dependent manner (Figs. 4A). To substantiate our 13 finding, we evaluated [SpyC]-CAR T cells cytotoxicity to UM-UC-3 and MG-63 target cells 14 after exposure to increasing concentrations of VAR2-[SpyT] (0-200 nM) (Fig. 4A). We observed 15 a concentration-dependent decrease in target cell viability over the week after CAR T cell 16 exposure that reflected the number of VAR2-[SpyT] proteins available for the [SpyC]-CAR T 17 cells during arming ( Fig. 4B ). A similar trend was observed in LNCaP and U2OS cells 18 (Supplementary Fig. 4B ). Notably, UM-UC-3 cells seemed slightly less sensitive to VAR2-19 [SpyT][SpyC]-CAR T cells in the 25-200 nM VAR2-[SpyT] concentration range as compared to 20 MG-63 (Fig. 4B ). To further characterize the difference in sensitivity between the target cell 21 lines, we examined VAR2-[SpyT][SpyC]-CAR T cell cytotoxicity in various effector-to-target 22 cell (E:T) ratios. As expected, higher CAR T cell effectors relative to target cells resulted in 23 greater cytotoxicity; however, UM-UC-3 cells generally required more effector cells than MG-63 24 (Fig. 4C ), further reflecting the less sensitivity of UM-UC-3 to VAR2-[SpyT][SpyC]-CAR T 25 cells ( Fig. 4B-C ). Combined, these data show that [SpyC]-CAR T cells can be conditionally 26 armed with the VAR2-[SpyT] warhead to confer an E:T ratio-dependent cytotoxicity towards 27 target cells. 28 29 VAR2-[SpyT][SpyC]-CAR T cells inhibit tumor growth in vivo and prolong animal 30 survival 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 8 CAR T cell therapy is challenging in solid tumors due to lack of durable efficacy (23, 33). 1 Improvement in efficacy has been pursued by a variety of approaches including co-targeting of 2 immune evasion mechanisms, combining different co-stimulatory domains in the CAR, the use 3 of vaccines that target the CAR epitope, and the use of repeated treatments with short-lived CAR 4 T cells (45). However, none of these approaches have resulted in sufficient increases in efficacy 5 to date. We tested the performance of the VAR2-[SpyT][SpyC]-CAR T cells in a solid tumor 6 xenograft mouse model using UM-UC-3 bladder tumor cells. Since the half-life of the VAR2 7 protein in blood circulation is very short, it is challenging to obtain sufficient exposure to the 8 newly proliferated [SpyC]-CAR T cells in mice. Therefore, instead of injecting additional doses 9 of VAR2-[SpyT] protein to arm the [SpyC]-CARs during clonal expansion, we decided to arm 10 the [SpyC]-CAR T cells in vitro, and then inject several doses of armed CARs into mice. Nude 11 mice were inoculated subcutaneously with 1x10 6 UM-UC-3 cells in their right flank (day 0). At 12 day 7, the mice were ranked by tumor size and evenly distributed into three groups with nine 13 mice per group. Mice were injected intravenously with PBS (Group 1), unarmed [SpyC]-CAR T 14 cells (Group 2), or armed VAR2-[SpyT][SpyC]-CAR T cells (Group 3) on days 7, 10, 13, 16, 15 and 19 (Fig 5A). After the first three injections (day 13), the VAR2-[SpyT][SpyC]-CAR T cells 16 (Group 3) started to reduce tumor growth, which became more pronounced over time ( Fig. 5B) 17 and was statistically significant as compared to unarmed [SpyC]-CAR T cells (Group 2) (Fig. 5C 18 and Supplementary Fig. S5 ). No difference was observed in tumor growth between control 19 groups 1 and 2, indicating no unintentional targeting of tumor cells by unarmed [SpyC]-CAR T 20 cells (Group 2) ( Fig. 5C). In the group treated with VAR2-[SpyT][SpyC]-CAR T cells (Group 21 3), one mouse was tumor-free at humane endpoint of Group 1 and 2 mice, while the remaining 22 mice had various degrees of treatment benefits as compared to the control groups. This translated 23 into increased overall survival of mice treated with VAR2-[SpyT][SpyC]-CAR T cells (Fig. 5D). 24 In summary, these data demonstrate a moderate yet significant effect of VAR2-[SpyT][SpyC]-25 CAR T cells in the treatment of bladder tumors in vivo. 26 27

28 The malarian rVAR2 protein has remarkably high specificity and affinity for oncofetal CS, 29 which is expressed almost exclusively in the placenta and tumors (8). Therefore, targeting 30 oncofetal CS with therapeutic formulations of the VAR2 protein has potential positive 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 9 implications for cancer therapy. Oncofetal CS is present in both early and more advanced disease 1 stages, regardless of tumor origin (15) (18). The concurrent presence of oncofetal CS across 2 different proteins expressed by tumor cells increases the molecular density of the involved 3 glycosaminoglycan and the potential sensitivity to oncofetal CS targeting technologies (8, 13). 4 5 Preclinical studies in cancer and early-phase clinical trials in malaria, have demonstrated the 6 feasibility and safety of VAR2 as a therapeutic agent. A phase I clinical vaccine trial 7 (PAMVAC) in pregnancy-associated malaria demonstrated low immunogenicity of VAR2 in 8 humans when administrated without adjuvants, and acceptable safety profiles (46). In 9 consideration to this subject, VAR2CSA may have co-evolved with humans to exhibit minimal 10 immunogenicity as evidenced by the persistence of malaria endemics today. Moreover, VAR2 11 in formulations as drug-conjugates or bi-specific immune cell engagers (CD3-fusion proteins) all 12 exhibit strong efficacies in both immunocompetent and immunodeficient mouse models with no 13 off-target or immune-related side effects, as well as no organ toxicity (8, 15, 21). 14 15 Since oncofetal CS is widely expressed in solid tumors and the VAR2 protein has been 16 credentialized as a therapeutic vehicle for drug-delivery and immune cell engagement, we 17 proposed that VAR2-directed CAR T cells might show efficacy in solid tumors. To test this idea 18 functionally, and to alleviate issues around the safety of current CAR therapies, we designed a 19 conditional CAR construct that could be armed with a recombinant VAR2 protein produced in 20 bacteria after its expression in T cell plasma membranes. We used the well-defined SpyCatcher-21 SpyTag intein system derived from S. pyogenes (42) as a molecular glue to link bacterial-22 produced recombinant VAR2-[SpyT] with [SpyC]-CARs translated and expressed in primary 23 human T cells. [SpyC]-CAR T cells are indolent as they cannot engage target cells and therefore 24 fail to activate. For activation, the CAR T cells rely on being armed with a SpyTagged binder 25 that can be any molecule ( e.g., proteins, peptides, or an scFv) with specificity to a tumor-26 selective epitope. We used VAR2-[SpyT] protein as the warhead but in principle the [SpyC]-27 CAR T cells could be armed with any binder of choice. Armed VAR2-[SpyT][SpyC]-CAR T 28 cells were able to fully activate upon engagement with oncofetal CS and to eliminate target 29 tumor cells in vitro in a time and dose dependant manner that depended on availability of the 30 VAR2-[SpyT] warhead. In a murine xenograft model of bladder tumor, VAR2-[SpyT][SpyC]-31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 10 CAR T cells were able to curb tumor growth and prolong survival of the mice. However, with 1 only one complete responder, it is clear that our strategy faces similar obstacles as other CAR T 2 approaches in solid tumors that limits CAR T cell efficacy, and further work is required to 3 optimize this approach. 4 5 Upon activation induced proliferation of the VAR2-[SpyT][SpyC]-CAR T, the daughter cells 6 maintain expression of the [SpyC]-CAR but become unarmed as a result of lack of the warhead 7 during proliferation. The cytotoxicity observed therefore comes from the parental VAR2-8 [SpyT][SpyC]-CAR T cells after initial target engagement. This implies that CAR T cell 9 cytotoxicity could potentially be amplified by supplementing excess amounts of VAR2-[SpyT] 10 protein ( i.e., re-arming) during the clonal expansion phase in vivo . This provides a safety 11 measure allowing control over CAR T cell activation based on the availability of VAR2-[SpyT] 12 protein. However, the strategy is limited in vivo by the fact that the serum half-life of rVAR2 13 proteins is <10 min, limiting tumor exposure to the point where the concentration of available 14 VAR2-[SpyT] is likely insufficient for saturating newly proliferated [SpyC]-CAR T cells. In vivo 15 re-arming may be possible with formulations that increases rVAR2 plasma half-life. We also 16 observed that, the degree of oncofetal CS expression on different tumor cells alone does not 17 appear to determine the activity and cytotoxicity of the VAR2-[SpyT][SpyC]-CAR T cells. This 18 indicates that factors beyond pure target expression might contribute to determining tumor cell 19 sensitivity to CAR T cells. The reasons for this observation can be many, however, a simple 20 explanation could be intrinsic differences amongst the cell lines for sensitivity to T cell-mediated 21 cell death. Additional research in this topic is warranted. 22 23 In summary, we have provided proof-of-concept for a conditional CAR T cell approach that can 24 target an oncofetal glycosaminoglycan modification in solid tumors. Adding cancer-specific 25 glycosaminoglycan modifications to the CAR T cell target repertoire provides additional 26 opportunities for immunotherapy in solid tumors. 27 28

29 Cell lines 30 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 11 The cell lines used in this study were maintained in specific culture media tailored to their 1 requirements: T cells were cultured in ImmunoCult XF T cell Expansion Medium supplemented 2 with 100U IL2, UM-UC3 cells were cultured in MEM media supplemented with 1X non-3 essential amino acids, MG-63 cells were cultured in MEM, LNCaP cells were cultured in RPMI, 4 and U2OS cells were cultured in DMEM. All media were supplemented with 10% fetal bovine 5 serum (FBS). Cultures were maintained at 37°C incubator with 5% CO2. Regular testing for 6 mycoplasma contamination was performed, and the cell lines were confirmed to be mycoplasma-7 free prior to use in experiments. 8 To establish stable cell lines expressing nuclear-restricted mKate2 (a far-red fluorescent protein), 9 the NucLight Lentivirus Reagent (Essen bioscience, Cat. 4476) was utilized for transduction. 10 Cells were seeded in 24-well plates 24 hours prior to transduction. The IncuCyte NucLight 11 Lentivirus Reagent was added at a MOI of 3 (= TU/cell), supplemented with 10 μ g/mL 12 protamine sulfate. The plate was incubated at 37°C, 5% CO2 for 24 hours. Media was replaced 13 the next day. After 48 hours, cells were treated with zeocin selection marker (Gibco, Cat. 14 R25001) to select for transduced cells. 15 16 Blood samples and T cell preparation 17 Leukopak from healthy donors was purchased from StemCell Technologies. T cells were 18 separated by negative selection using EasySep™ Human T Cell Isolation Kit (StemCell, Cat. 19 17951). The leukopak samples were prepared by adding an equivalent volume of PBS2 and 20 centrifuging at 500 x g for 10 minutes at room temperature (15 - 25°C) and removing the 21 supernatant. The cells were resuspended to a concentration of 5 x 10 7 cells/mL in PBS2. T cells 22 were isolated according to the manufacturer. Briefly, the samples were transferred to the 23 polystyrene round-bottom tube, 50 µL/mL EasySep™ Human T Cell Isolation Cocktail and 40 24 µL/mL of EasySep™ Dextran RapidSpheres™ were added to the samples and incubated for 5 25 min at RT. The tube was topped up by PBS2, placed in a magnet (StemCell, Cat.18001), and 26 incubated for 3 min. By inverting the magnet, the enriched T cell suspension was transferred into 27 a clean 14 ml tube. The cells were centrifuged and resuspended in 1 mL ImmunoCult™-XF T 28 Cell Expansion medium and counted to adjust to the concentration to 1x10 6 cells/mL. The cells 29 were activated by incubating with 25 µL/mL of human CD3/CD28/CD2 T Cell activator 30 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 12 (StemCell, Cat. 10990) for 3 days at 37 oC with 5% CO 2, and then maintained in media 1 containing 100 U/mL IL-2 (StemCell, Cat. 78036.1) 2 3 The isolated cells were assessed for T cells purity and CD4 and CD8 sub-population proportions 4 before and after virus transduction. T cells were stained with BV421-conjugated anti-CD3 5 (Clone SK7), APC-anti CD4 (Clone OKT4), FITC anti CD8 (Clone SK1), and PE-FLAG (Clone 6 L5) antibodies for 40 minutes, washed and subjected to flow cytometry analysis. Unstained and 7 single-color controls were acquired and used for compensation. 8 9 10 Protein production 11 Recombinant P. falciparum VAR2CSA proteins were produced in E. coli SHuffle cells (NEB) 12 and comprised of the minimal CS-binding region (subunit ID1-ID2a) with a C-terminal V5 tag 13 and 6x-His tag, and an N-terminal SpyTag (8). The recombinant control (rContr) protein is made 14 from the non-CS binding region (DBL4) of VAR2CSA protein with the addition of the C-15 terminal V5 tag (as in (8)). 16 17 VAR2 binding assay 18 Tumor cells were cultured in their appropriate media to reach 70%–80% confluency. To 19 minimize the risk of damage to proteoglycans, the cells were prepared for binding assay using a 20 non-enzymatic cell dissociation solution (Cellstripper, Cat. CA45000-668). For the preparation 21 of T cells, blood-derived T cells were isolated and divided into two parts. The first part was 22 maintained in culture media without T cell activators, as the non-activated T cells. The second 23 group was activated in media supplemented with CD2/CD3/CD28 T cell activator (StemCell, 24 Cat. 10990) (25 μ L/mL) and IL-2 (100 U/mL) for a duration of three days. 25 26 Prior to incubation with VAR2, the cells were washed with PBS containing 2% FBS (PBS2), 27 centrifuged at 350 x g for 5 minutes, and resuspended to a concentration of 1x10 6 cells/mL. 100 28 μ l of the cell suspension was added per well in a 96 well plate, followed by centrifugation to 29 pellet the cells. The supernatant was aspirated, and the cells were resuspended in the protein 30 solutions. The protein solutions were prepared by serial dilutions of rVAR2 diluted in PBS2 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 13 (12.5-200 nM). A cell sample without rVAR2 was used as background for antibody signal. 200 1 nM rContr protein, which is a recombinant non-CS binding region (DBL4) of the full-length 2 VAR2CSA protein (8), was used as a negative control. Binding specificity was tested by the 3 inclusion of a high concentration (400 µg/ml) of purified CSA (Sigma Cat. 27040) which 4 competes for VAR2 binding. For this competition, 200 nM rVAR2 was pre-incubated with CSA 5 before adding to the cells. The plate was incubated for 30 min at 4°C on a shaker. After 6 incubation, the cells were washed twice with PBS2 and then stained for 40 min on ice with an 7 anti-V5 antibody conjugated to FITC (Invitrogen, Cat. R963-25). The cells were washed 3 times 8 with 200 μ l FACS buffer (PBS containing 2% FBS, 2.5 mM EDTA, and 0.05 mM NaN3) and 9 resuspended in FACS buffer containing DAPI (0.1 µg/mL) for gating out the dead cells. Samples 10 were acquired on a FACS Canto II flow cytometer, (BD Biosciences), and the data was analyzed 11 using FlowJo V10.4.2. Unstained and single-color controls were acquired and used for 12 compensation 13 14 15 Generation of CAR T cells 16 Lentivirus particles were generated in HEK293T cells following transfection with the CAR 17 plasmid along with pMD2.G envelope (Addgene, Cat.12259) and psPAX2 packaging plasmids 18 (Addgene, Cat.12260), using extreme Xp transfection reagent. 5x10 6 HEK293T cells were 19 seeded in 10 mL of DMEM media in 10 cm poly-L-lysine coated culture plates. The next day, 20 the media was replaced with 7 mL of fresh media, 3-4 hours before transfection. A total of 25 μ g 21 of plasmid DNA, consisting of 10 μ g library plasmid, 10 μ g envelope plasmid (pMD2.G) and 5 22 μ g packaging plasmid (psPAX2), were diluted in 500 μ L serum-free Opti-MEM (Cat.11058021) 23 in a sterile 1.5 mL tube. X-tremeGENE HP DNA Transfection Reagent (Cat. 6366236001) was 24 added in a 3:1 ratio of reagent to DNA, mixed by pipetting and incubated at room temperature 25 for 25 min. The transfection complex was added to cells dropwise while swirling the plate, and 26 the plate was placed in a CO2 incubator. Viral supernatant was collected 48 h and 72 h post 27 transfection and centrifuged at 350 x g for 10 minutes to remove any cells and debris. The 28 supernatant was filtered through an 0.45 μ m Steriflip and used on the same day or stored at -29 80°C for future use. 30 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 14 To transduce T cells, the isolated human T cells were centrifuged and resuspended in virus 1 supernatant to a concentration of 1x10 6 cells/mL in the presence of 10 μ g/mL protamine sulfate. 2 72 hours after transduction, 2 µg/mL puromycin was added to the media to select for CAR 3 expressing T cells. 4 5 Two weeks after transduction, the CAR expression levels were quantified by flow cytometry 6 with fluorescent-conjugated Flag antibody. Dead cells were excluded by DAPI staining. The data 7 was analyzed using the FlowJo software to calculate the percentage of the live, single cells with 8 CAR expression. 9 10 VAR2-[SpyT][SpyC]-CAR arming on the T cell membrane 11 To assess the optimal quantity of VAR2-[SpyT] required to arm the [SpyC]-CARs on the T cell 12 membrane, a saturation study was performed. Equal numbers of [SpyC]-CAR T cells (1x10 5) 13 were incubated with different concentrations of VAR2-[SpyT] from 0 to 200 nM. The cells were 14 washed three times with PBS2 and stained with FITC-conjugated anti-V5 antibody (at dilution of 15 1:500) and PE conjugated anti Flag antibody (at dilution of 1:100) for 45 min on ice. After 16 staining and washing, cells were resuspended in DAPI-containing FACS buffer for flow 17 cytometry data acquisition. The FlowJo software was utilized for data analysis, initially gating 18 live single cells for CAR expression. Subsequently, the geometric mean fluorescent intensity 19 (MFI) of FITC within the CAR-expressing population was determined to construct the saturation 20 curve. Non-transduced cells were incubated with equivalent amounts of VAR2-[SpyT] and 21 analyzed as the control group. VAR2-[SpyT] arming of the [SpyC]-CAR was also analysed on 22 flowcytometry by assessing the co-localization of the Flag-tag in CAR construct with the V5-tag 23 on VAR2-[SpyT], following the incubation of the cells with either 200 nM of VAR2-[SpyT] or 24 without VAR2. 25 26 Detection of T cell activation markers by flow cytometry 27 The expression of T cell activation markers, CD 25 and CD69, was assessed on effector cells 28 subsequent to encountering their targets. 1x105 tumor cells were co-cultured with 1x105 [SpyC]-29 CAR T cells, in presence (armed CAR) or absence (unarmed CAR) of VAR2-[SpyT]. All 30 samples were prepared in triplicates. The plates were incubated at 37°C overnight, then only the 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 15 suspension cells were harvested, washed, and stained with PE-conjugated anti-Flag (Clone L5), 1 PerCP/Cy5.5-conjugated anti CD25 (Clone BC96, BioLegend, Cat. 302626), and FITC-2 conjugated CD69 (Clone FN50, BioLegend, Cat. 310904) antibodies for 30 min on ice. 3 Unstained and single-color controls were acquired and used for compensation. Following three 4 washes, the samples were resuspended in FACS buffer containing DAPI to exclude the dead 5 cells during subsequent flow cytometry analysis. Unstained and single-color controls were 6 utilized for compensation purpose during analysis using FlowJo software. The cells were first 7 gated for viable single cells that were Flag positive, indicating CAR-expressing cells, and then 8 quantified for up-regulation of CD69 and CD25. 9 10 Cytokine analysis 11 Cytokine production was evaluated using MSD V-plex Proinflammatory Panel 1 Human kit 12 (Meso Scale Discovery, Cat. K15049D) according to the manufacturer’s instruction. Effector and 13 target cells were cocultured at an E:T ratio of 10:1 in 100 μ l media in a 96 well plate and 14 incubated for 48 hours. The CAR was armed with 200nM of VAR2-[SpyT]. Post-incubation, 15 media from each well was collected, centrifuged at 350 x g for 5 min at 4°C to remove cells and 16 debris. Supernatants were collected stored in -80 freezer, and then evaluated for cytokine levels 17 according to the manufacturer’s protocol. Data analyses were performed using Discovery 18 Workbench software. 19 20 In vitro cytotoxicity assay 21 Red fluorescent tumor cells (mKate2+) were co-cultured with [SpyC]-CAR in triplicate at an E:T 22 ratio of 1:1 in the presence or absence of VAR2-[SpyT]. Similar co-cultures involving non-23 transduced T cell, as well as the same number of tumor cells without effector cells were used as 24 controls. The plates were placed in the IncuCyte S3 instrument for scanning every 6 hours for up 25 to 7 days. At each time point, 5 images per well at 10X magnification were collected . Total red 26 area (um2/well) was quantified as a measure of live tumor cells and values were normalized to 27 t=0 measurement. For tracking co-localization of the effector cells with target cells, [SpyC]-CAR 28 T cells were labelled with IncuCyte® CytoLight Rapid Green Reagent. These green-labelled T 29 cells were then co-cultured with red fluorescent tumor cells at a 1:1 ratio in 96 well plates, in the 30 presence or absence of VAR2-[SpyT], and scanned by IncuCyte at 20X magnification for 3 days. 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 16 1 In vivo model 2 Female nude mice (8 weeks old; Jackson Laboratory) were subcutaneously inoculated in their 3 right flanks with 1x10 6 UM-UC-3 cells suspended in PBS and Matrigel® Matrix solution (1:1). 4 At day seven post-inoculation, mice were evenly distributed into three groups by tumor size, 5 with nine mice allocated to each group. The first group received five intravenous (IV) injection 6 of PBS, the second group received five doses of 2.5 million [SpyC]-CAR T cells, and the third 7 group was injected with five doses of 2.5 million VAR2-[SpyT][SpyC]-CAR T cells. The tumor 8 sizes were measured twice per week using a caliper, and tumor volume was calculated by 9 V= /g1849/g1499/g1838/g1499/g1834/g1499/g1868 /g1861 / 6 formula, where w=width of tumor, L=length of tumor and H=height of the 10 tumor. The mice were euthanized when reaching tumor size of 1000 /i2 mm3. Statistical analysis 11 was performed using GraphPad Prism (GraphPad Software). Two-way ANOVA using Tukey’s 12 multiple comparison tests was applied on tumor growth data. Data were presented as mean ± 13 SEM. To quantify the rate of tumor growth over time, we calculated the slope of the tumor 14 growth curve for each individual mouse in each group using linear regression ( Figs. 5C, 15 Supplementary Figs. 5) . The statistical significance of the differences between groups was 16 assessed using one-way ANOVA, followed by Tukey’s multiple comparison test. In order to 17 compare the survival rates between groups, Kaplan-Meier survival curve was generated, utilizing 18 the morbidity of mice as a surrogate for overall survival across time. 19 20 21 22 Author contributions 23 Conceptualization, Nt.K., N.AN, A.S., P.S., SH and M.D.; Methodology, Nt.K., H.Z.O., M.M., 24 N.F., M.E.R., A.M., B.Z., I.M., J.L., F.G., I.N., T.G., S.C., R.D.; Data acquisition and analysis, 25 Nt.K., H.Z.O., S.T., Nr.K., Funding acquisition, M.D., P.S., A.S.; First manuscript draft, Nt.K., 26 S.T., P.S., and M.D., Review & Editing, all authors; Supervision, M.D., N.AN, A.S. 27 28

29 We thank Drs. Sabine Heitzeneder and Crystal Mackall (Stanford, CA) for intellectual input on 30 CAR T cell methodology, and all members of the Daugaard Lab for helpful discussions. We 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 17 thank the funding support from NIH Prostate Cancer PNW-SPORE (1016339, 223493; 5P50 1 CA097186-17); Canadian Institutes of Health Research (CIHR) (PJT-153092); St. Baldrick's 2 Foundation/American Association for Cancer Research/Stand Up to Cancer Pediatric Dream 3 Team Translational Research Grant (to PHS and MD; SU2C-AACR-DT-27-17). Stand Up to 4 Cancer (SU2C) is a division of the Entertainment Industry Foundation , and research grants 5 are administered by the American Association for Cancer Research , the scientific partner of 6 SU2C. Nastaran Khazamipour (Nt.K) was supported by the CIHR-Vanier scholarship (#01353-7 000), UBC Four Year Fellowships (FYF) (#6456), Canadian Urological Association Scholarship 8 Foundation (CUASF-BCC) Research Grant, and the CIHR travel Award- Michael Smith Foreign 9 Study Supplement (#6580) for an internship with Stanford University (CA). AS is supported by 10 NNF Tandem grant (NNF21OC0068192) and NNF Distinguished Innovator grant 11 (NNF22OC0076055); 12 13 Conflicts of Interest 14 M.D. as the corresponding author certifies that all conflicts of interest, including specific 15 financial interests and relationships and affiliations relevant to the subject matter or materials 16 discussed in the manuscript (e.g., employment/affiliation, grants or funding, consultancies, 17 honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or 18 pending), are the following: M.D., A.S., and P.H.S. are co-founders of, and shareholders in, 19 VAR2 Pharmaceuticals. N.A.N., and T.G. are consultants for VAR2 Pharmaceuticals. VAR2 20 Pharmaceuticals is a biotechnology company that specializes in therapeutic development of the 21 VAR2CSA technology ( www.var2pharma.com). The remaining authors declare no conflicts of 22 interest. 23 24 Figure legends 25 Figure 1. Design and validation of a conditional CAR with selectivity to oncofetal CS 26 glycosaminoglycans 27 (A) Naïve and activated human T cells and UM-UC3 tumor cells were incubated, with control 28 protein or different concentrations of V5-tagged VAR2 (12 - 200 nM) protein +/- purified CSA 29 as indicated. Binding of VAR2 was assessed by flow cytometry using anti-V5-FITC. ( B) 30 Illustration of the [SpyC]-CAR containing intracellular CD3zeta, 41BB, and CD28 domains 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 18 fused with an extracellular split-intein SpyCatcher (SpyC) domain. (C) Schematic diagram of the 1 [SpyC]-CAR DNA construct in a lentiviral plasmid. ( D) Isolated human T cells with or without 2 [SpyC]-CAR transduction, were analyzed for CD4 and CD8 expression by flow cytometry. ( E) 3 [SpyC]-CAR T cells were incubated 14 days post-transduction, and expression of the [SpyC]-4 CAR (Flag+) was assessed by flow cytometry. The result is representative of 3 individual 5 donors. ( F) Schematic of [SpyC]-CAR T Cell arming with the recombinant VAR2-[SpyT] 6 protein. ( G) [SpyC]-CAR T cells were mixed with 200nM [SpyT]-VAR2 in triplicate and 7 expression of Flag ([SpyC]-CAR) and V5 ([SpyT]-VAR2) were assessed by flow cytometry. 8 (H) T cells with or without [SpyC]-CAR transduction were incubated with indicated 9 concentrations of VAR2-[SpyT] protein and analyzed for VAR2-[SpyT][SpyC]-CAR assembly 10 by flow cytometry. Error bars shown are mean ± SEM of triplicate wells. All the results are 11 representative of 3 independent experiments. 12 13 Figure 2. VAR2-armed CAR T cells activate upon target cell engagement and produce 14 robust cytokine responses 15 (A-B) Armed and unarmed [SpyC]-CAR T cells were incubated, in triplicate, with (A) UM-UC-16 3 and ( B) MG-63 cells at a 1:1 E:T ratio for 24 hours before analysis for expression of Flag, 17 CD69, and CD25 by flow cytometry. The results are representative of 3 independent 18 experiments. 19 (C-D) Armed and unarmed [SpyC]-CAR T cells were incubated with ( C) UM-UC-3 and (D ) 20 MG-63 cells at a 10:1 E:T ratio in 100 μ l media, for 48 hours and concentrations of the indicated 21 cytokines in the supernatants were quantified. Results are presented as mean ± SEM of three 22 different wells. The statistical significance was determined using one-way ANOVA, Dunnett’s 23 multiple comparison’s test. *, p<0.05; **, p<0.01 ***, p<0.001; ****p<0.0001. 24 25 Figure 3. VAR2-[SpyT][SpyC]-CAR T cell cytotoxicity is target cell type-dependent and 26 does not directly reflect oncofetal CS content 27 (A) [SpyC]-CAR T cells (green) +/- VAR2-[SpyT] protein, were co-cultured with UM-UC-3 and 28 MG-63 target cells (red) at a 1:1 E:T ratio, in triplicate, and monitored over 3 days by IncuCyte. 29 (B) Red fluorescent expressing UM-UC-3 and MG-63 target cells were co-cultured with 30 indicated formulations of T cells and the confluency of tumor cells monitored over time by 31 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 19 IncuCyte. Dashed lines indicate time to VAR2-[SpyT][SpyC]-CAR T cell cytotoxicity. Error 1 bars represent mean ± SEM of triplicate wells. Data from a representative donor of three 2 individual donors is shown. Statistics were calculated for the last timepoint, using one-way 3 ANOVA and Dunnett’s multiple comparisons test ( C) Oncofetal CS expression levels of 4 different cell types were plotted against the time required for VAR2-[SpyT][SpyC]-CAR T cells 5 to induce cytotoxicity. 6 7 Figure 4. [SpyC]-CAR T cells can be conditionally armed with VAR2-[SpyT] to eliminate 8 tumor cells 9 (A) Illustration of the conditional arming of [SpyC]-CAR T cells with increasing amounts of 10 VAR2-[SpyT] protein. ( B) UM-UC-3 and MG-63 target cells (red) were co-cultured with 11 [SpyC]-CAR T cells at a 1:1 E:T ratio in triplicate, with the indicated concentrations of VAR2-12 [SpyT] protein, confluence was assessed as readout of tumor cell viability. Error bars represent 13 mean ± SEM of triplicates. ( C) UM-UC-3 and MG-63 target cells (red) were co-cultured with 14 [SpyC]-CAR T cells +/- VAR2-[SpyT] in indicated E:T ratios and analyzed as in (B). Results are 15 presented as mean ± SEM triplicates. Statistics were calculated, using two-way ANOVA and 16 Dunnett’s multiple comparisons test. Data from a representative of three from 3 individual 17 donors is shown. 18 19 Figure 5. VAR2-CAR T cells suppress solid tumor growth in vivo and prolong animal 20 survival 21 (A) Schematic outline of the experimental design. Nude mice were inoculated with 1x10 6 UM-22 UC-3 tumor cells in their right flank. At day seven the mice were randomized into three groups 23 (n=9). The groups subsequently received five doses of either PBS (G1), 2.5 million [SpyC]-CAR 24 T cells (G2), and 2.5 million VAR2-[SpyT][SpyC]-CAR T cells (G3) by intravenous injection at 25 the indicated times points post-tumor cell inoculation. ( B) Tumor growth curves with the mean 26 tumor volumes of mice in each group. Data presented as mean ± SEM (n = 9 mice). ( C) The 27 slope of tumor growth curve for each individual mouse in each group was calculated using linear 28 regression, and the tumor growth rates was compared between treatment groups. The statistical 29 analysis was performed using ANOVA followed by a Tukey’s multiple comparison’s test. ( D) 30 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 20 Kaplan–Meier plot (left) indicates morbidity as a proxy for overall survival of mice (numbers of 1 mice) over time (right). 2 3 Figure S1. Oncofetal CS expression in solid tumor cell lines and bladder cancer tissue. (A) 4 MG-63, U2OS, LNCaP, and NB16 tumor cell lines were incubated with indicated concentrations 5 of rContr protein or VAR2 (12 - 200 nM) +/- purified CSA and analyzed by flow cytometry 6 using anti-V5-FITC. ( B) Representative H&E and IHC images of normal adjacent urothelium 7 and bladder cancer tissues from two patients. Matched staining images of E-cadherin, as an 8 epithelial marker, in parallel with oncofetal CS (ofCS) detection by VAR2 and anti-V5. ( C) Bar 9 plot of bladder cancer patient tumors (n=64) indicating ofCS expression in relation to T stage. 10 (D) Bar plot of bladder cancer patient tumors (n=63) indicating ofCS expression in relation to N 11 stage. ( E) Representative IHC images of neuroblastoma tumors selected for high and low 12 oncofetal CS expression. (F) Percent ofCS-positive neuroblastoma tumors related to tumor stage. 13 (G) Kaplan-Meier plot indicating overall survival of neuroblastoma patients related to oncofetal 14 CS expression. The scale bar represents 100 μ m. MIBC: muscle-invasive bladder cancer; 15 oncofetal CS: oncofetal chondroitin sulfate. For statistical analysis in the above panels (C: T 16 stage; D: N stage and F: International Neuroblastoma Staging System (INSS)), two-tailed 17 Fisher's exact test was used. 18 19 Figure S2. Activation of VAR2-[SpyT][SpyC]-CAR T cells upon tumor cell engagement. 20 (A-B) Armed and unarmed [SpyC]-CAR T cells were incubated with ( A) LNCaP and (B) U2OS 21 cells at a 1:1 E:T ratio for 24 hours before analyzed for Flag, CD69, and CD25 expression by 22 flow cytometry. The results are representative of 3 independent experiments. (C-D) Armed and 23 unarmed [SpyC]-CAR T cells were incubated in triplicate, with ( C) LNCaP and (D) U2OS cells 24 at a 10:1 E:T ratio in 100 μ l media, for 48 hours and analyzed for the concentration of indicated 25 cytokines in the culture supernatant. Results are presented as mean ± SEM of three different 26 wells. The statistical significance was determined using one-way ANOVA, Dunnett’s multiple 27 comparison’s test. *, p<0.05; **, p<0.01 ***, p<0.001; ****p<0.0001. 28 29 Figure S3: Cytokine responses in co-cultures of effector and target cells. 30 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 21 The concentrations of the indicated cytokines in the culture supernatants were assessed after 48 1 hours of co-culturing between effector T cells formulations and different target cells (i.e., UM-2 UC3, MG-63, LNCaP, and U2OS), at a 10:1 E:T ratio. Data was analyzed with the Discovery 3 Workbench software. Error bars represent mean ± SEM of three different wells. 4 5 Figure S4: Activity of VAR2-[SpyT][SpyC]-CAR T cells after target cell engagement. 6 (A) LNCaP and U2OS target cells (red) were co-cultured in triplicate with indicated 7 formulations of T cells and monitored for one week. Dashed lines indicate time to VAR2-8 [SpyT][SpyC]-CAR T cell cytotoxicity. Error bars represent mean ± SEM of triplicate wells. 9 Data from a representative donor of three individual donors is shown. Statistics were calculated 10 for the last timepoint, using one-way ANOVA and Dunnett’s multiple comparisons test. ( B) 11 LNCaP and U2OS target cells (red) were co-cultured with [SpyC]-CAR T cells at a 1:1 E:T ratio 12 with indicated concentrations of VAR2-[SpyT] protein and analyzed for viability using 13 confluence as the readout. Error bars represent mean ± SEM of triplicate wells. ( C) Percent 14 CD69-positive VAR2-[SpyT][SpyC]-CAR T cells plotted against oncofetal CS expression in 15 indicated target cells. ( D) IFNγ production (pg/ml) in co-cultures of VAR2-[SpyT][SpyC]-CAR 16 T cells and indicated target cells plotted against oncofetal CS expression of the target cells. All 17 data was analyzed by GraphPad Prism Software. 18 19 Figure S5: Linear regression analysis of tumor growth 20 Individual tumor growth curve (blue line) and the slope of the curve (red line) is shown for each 21 mouse treated with PBS, [SpyC]-CAR T cells or VAR2-[SpyT][SpyC]-CAR T cells. 22 23 24

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Anti-melanoma activity of 16 T cells redirected with a TCR-like chimeric antigen receptor. Sci Rep-Uk. 2014;4. 17 26. Deng L, Xiaolin Y, Wu Q, Song X, Li W, Hou Y, et al. Multiple CAR-T cell therapy for 18 acute B-cell lymphoblastic leukemia after hematopoietic stem cell transplantation: A case report. 19 Front Immunol. 2022;13:1039929. 20 27. Denlinger N, Bond D, Jaglowski S. CAR T-cell therapy for B-cell lymphoma. Curr Probl 21 Cancer. 2022;46(1):100826. 22 28. Zhang X, Zhu L, Zhang H, Chen S, Xiao Y. CAR-T Cell Therapy in Hematological 23 Malignancies: Current Opportunities and Challenges. Front Immunol. 2022;13:927153. 24 29. Del Bufalo F, De Angelis B, Caruana I, Del Baldo G, De Ioris MA, Serra A, et al. GD2-25 CART01 for Relapsed or Refractory High-Risk Neuroblastoma. N Engl J Med. 26 2023;388(14):1284-95. 27 30. Majzner RG, Ramakrishna S, Yeom KW, Patel S, Chinnasamy H, Schultz LM, et al. 28 GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. 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Targeting of a 42 Cancer-Associated Lypd3 Glycoform for Tumor Therapy. J Immunother Cancer. 43 2022;10:A1398-A. 44 36. Rossig C, Kailayangiri S, Jamitzky S, Altvater B. Carbohydrate Targets for CAR T Cells 45 in Solid Childhood Cancers. Front Oncol. 2018;8:513. 46 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Khazamipour et al. 24 37. Reily C, Stewart TJ, Renfrow MB, Novak J. Glycosylation in health and disease. Nat Rev 1 Nephrol. 2019;15(6):346-66. 2 38. Mereiter S, Balmana M, Campos D, Gomes J, Reis CA. Glycosylation in the Era of 3 Cancer-Targeted Therapy: Where Are We Heading? Cancer Cell. 2019;36(1):6-16. 4 39. Agerbaek MO, Pereira MA, Clausen TM, Pehrson C, Oo HZ, Spliid C, et al. Burkitt 5 lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria 6 sequestration. Int J Cancer. 2017;140(7):1597-608. 7 40. Clausen TM, Kumar G, Ibsen EK, Orum-Madsen MS, Hurtado-Coll A, Gustavsson T, et 8 al. A simple method for detecting oncofetal chondroitin sulfate glycosaminoglycans in bladder 9 cancer urine. Cell Death Discov. 2020;6:65. 10 41. Al-Nakouzi N, Wang CK, Oo HZ, Nelepcu I, Lallous N, Spliid CB, et al. Reformation of 11 the chondroitin sulfate glycocalyx enables progression of AR-independent prostate cancer. Nat 12 Commun. 2022;13(1):4760. 13 42. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, et al. Peptide tag 14 forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl 15 Acad Sci U S A. 2012;109(12):E690-7. 16 43. Li L, Fierer JO, Rapoport TA, Howarth M. Structural analysis and optimization of the 17 covalent association between SpyCatcher and a peptide Tag. J Mol Biol. 2014;426(2):309-17. 18 44. Curtsinger JM, Mescher MF. Inflammatory cytokines as a third signal for T cell 19 activation. Curr Opin Immunol. 2010;22(3):333-40. 20 45. Foster JB, Barrett DM, Kariko K. The Emerging Role of In Vitro-Transcribed mRNA in 21 Adoptive T Cell Immunotherapy. Mol Ther. 2019;27(4):747-56. 22 46. Mordmüller B, Sulyok M, Egger-Adam D, Resende M, de Jongh WA, Jensen MH, et al. 23 First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted 24 PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria. Clin Infect Dis. 25 2019;69(9):1509-16. 26 27 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint A MFI (x1000)0 2 1 6 8 10 T cells (un-activated) T cells (activated) + + - - - - - - - - - - - - + - 200 12.5 rContr: CSA: rVAR2 (nM): + + - - - - - - - - - - - - + - 200 12.5 Figure 1 D UM-UC-3 0 2 4 6 8 10 + + - - - - - - - - - - - - + - 200 12.5 CD4 56.7% CD8 39.3% APC-CD4 FITC-CD8 FLAG 0.23% PE-FLAG FLAG 88.9% CD4 51.6% CD8 46.1% CD3 99.1% 0 0.5 1.0 1.5 2.0 2.5SSC-A (105) BV421-CD3 0 -103 103 104 105 CD3 99.6% Cell number (%) 90.7% 101 102 103 104105 0 20 40 60 80 100 CAR expression 0 0.5 1.0 1.5 2.0 2.5SSC-A (105) 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 0 0.5 1.0 1.5 2.0 2.5SSC-A (105) BV421-CD3 0 -103 103 104 105 0 0.5 1.0 1.5 2.0 2.5SSC-A (105) PE-FLAG 0 -103 103 104 105 APC-CD40 -103 103 104 105 FITC-CD8 0 -103 103 104 105 Q1 5.8% Q2 <1% Q3 <1% Q4 94.1% T cells [SpyC]-CAR T cells Q1 19.2% Q2 68.4% Q3 7.1% Q4 5.3% Q1 <1% Q2 <1% Q3 74.9% Q4 25.0% CAR (PE) Q1 <1% Q2 <1% Q3 <1% Q4 99.9% Unarmed VAR2-[SpyT] Q1 <1% Q2 <1% Q3 99% Unstained Q1 <1% Q2 <1% Q3 99% 0 -103 103 104 105 0 -103 103 104 105 Q1 Q2 Q3 Q4 Q1 Q2 Q3Q4 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 Q1 Q2 Q3Q4 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 0 -103 103 104 105 rVAR2 ( FITC) CAR (PE) Q1 Q2 Q3Q4 Q1 Q2 Q3Q4 Q1 Q2 Q3Q4 rVAR2 ( FITC) rVAR2 ( FITC) Unstained Unarmed VAR2-[SpyT] F VAR2-[SpyT] (nM) T cells 0 0.5 1.0 1.5 2.0 2.5(MFI x1000) 10 25 50100150 200 T cells[SpyC]-CAR T cells H G Design and validation of a conditional CAR with selectivity to oncofetal CS glycosaminoglycans B C E [SpyC]-CAR T cells + anti-Flag T cells + anti-Flag. T cells Unarmed Armed [SpyC]-CAR T cells VAR2-[SpyT] SpyC CD28 41BB CD3z + was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure 2 MG-63 Q2 Q3 11,4 Q1 24.2 33.2 Q4 31.2 Q1 35.5 Q2 5.18 Q3 12.4 Q4 46.9 0 -103 103 104 105 CD25 (PreCP-cy5.5) CD69 (FITC) Q2 Q3 11.4 0 20 40 60 80 100Normalized (Mode) Armed Unarmed 0 103 104 105 0 103 104 105 0 10 3 104 105-103 0 10 3 104 105-103 CD69 (FITC) Q1 40.7 Q2 1.88 Q3 5.05 Q4 52.4 Q1 30.0 Q2 41.5 Q3 5.83 Q4 22.6 0 -103 103 104 105 CD25 (PreCP-cy5.5) 0 20 40 60 80 100Normalized (Mode)0 103 104 105 0 103 104 105 Armed Unarmed CD69 (FITC) 0 10 3 104 105-103 0 10 3 104 105-103 CD69 (FITC) UM-UC-3 IL-2IFNγ TNFα A Pg/mL ************ IL-2IFNγ TNFα Pg/mL 0 100 200 2000 6000 10000 800000 900000 1000000 0 200 400 5000 20000 40000 400000 900000 1400000 Armed [SpyC]-CAR T cells Unarmed [SpyC]-CAR T cells Non-transduced T cells MG-63 cells ************ ************ ************ ************ ************ Armed [SpyC]-CAR T cells Unarmed [SpyC]-CAR T cells Non-transduced T cells UM-UC-3 cells B C D VAR2-armed CAR T cells activate upon target cell engagement and produce robust cytokine responses CAR T cells Non-transduced T cells CAR (PE) CAR (PE)CAR (PE)CAR (PE) was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure 3 VAR2-[SpyT][SpyC]-CAR T cell cytotoxicity is target cell type-dependent MG-63 UM-UC-3 U2OS LNCaP 0 5 10 15 20 25 30 35 40 2 4 6 8 10 12 0 Time to CAR T efficacy (Hours) Oncofetal CS (MFI x1000) UM-UC-3 cells Day 0Day 1Day 2Day 3 MG-63 cells Target cells Effector cells 0 2 4 6 8 10 UM-UC-3 0 2 4 6 8 0 156 MG-63 VAR2-[SpyT][SpyC]-CAR T cells [SpyC]-CAR T cells Non-trans T cells + VAR2-[SpyT] Non-trans T cells Target cells + VAR2-[SpyT] Target cells Time (Hours) A 0 156 Time (Hours) Unarmed Armed [SpyC]-CAR T cells Unarmed Armed [SpyC]-CAR T cells B 36 18 C [E:T] 1:1 [E:T] 1:1 [E:T] 1:1 [E:T] 1:1 Confluence (Relative to t=0) ***** ***** was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure 4 0 2 4 6 MG-63 10:1 5:1 2.5:1 1:1 1:5 1:10 0:1 0 2 4 6 8 UM-UC-3 E:T ratio 10:1 5:1 2.5:1 1:1 1:5 1:10 0:1 E:T ratio VAR2-[SpyT][SpyC]-CAR T cells [SpyC]-CAR T cells Non-trans T cells +VAR2-[SpyT] Non-trans T cells C [SpyC]-CAR T cells can be conditionally armed with VAR2-[SpyT] to eliminate tumor cells 200 nM VAR2-[SpyT] 100 nM VAR2-[SpyT] 50 nM VAR2-[SpyT] 25 nM VAR2-[SpyT] 12 nM VAR2-[SpyT] Unarmed 0 2 4 6 8 UM-UC-3 0 180 Time (Hours)0 156 0 2 4 6 8 MG-63 Time (Hours) 0 200 Confluence (Relative to t=0) A B [nM] SpyC Confluence (Relative to t=0) CD28 41BB CD3z **** **** **** **** **** **** **** **** **** **** * VAR2-[SpyT] VAR2-[SpyT] was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure 5 0 10 20 30 0 400 800 1200 Time (Days) PBS [SpyC]-CAR VAR2-[SpyT][SpyC]-CAR 0 20 40 60 80 ns PBS 0 15 30 45 0 25 50 75 100 Days after treatment p=0.0465 B D C Tumor volume (mm3) * Slopes (tumor growth) [SpyC]-CAR Survival (%) PBS [SpyC]-CAR A VAR2-CAR T cells curb solid tumor growth in vivo and prolong animal survival UM-UC-3 cells PBS [SpyC]-CAR VAR2-[SpyT][SpyC]-CAR VAR2-[SpyT][SpyC]-CAR VAR2-[SpyT][SpyC]-CAR Day 0 Day 7 Day 10 Day 13 Day 16 Day 19 ** ns 20 22 25 28 31 35 38 42 46 PBS 8 3 3 1 0 0 0 0 0 [SpyC]-CAR 5 3 2 0 0 0 0 0 0 VAR2-[SpyT][SpyC]-CAR 9 8 4 3 2 2 2 2 1 Days a�er treatment ** was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint ofCS E-cadherin Adjacent normal urotheliumBCa Patient 1BCa Patient 2 H&E Bladder cancer Figure S1 ofCS low ofCS high N0 (n=29) N1-3 (n=34) T1-2 (n=12) T3-4 (n=52) 0 50 100ofCS % positivity p=0.0114 0 50 100ofCS % positivity p=0.0231 ofCS low ofCS high NB16 rContr: CSA: rVAR2 (nM): LNCaPU2OS 0 2 4 6 8 10 + + - - - - - - - - - - - - + - 200 12.5 0 1 2 3 + + - - - - - - - - - - - - + - 200 12.5 0 2 4 6 8 + + - - - - - - - - - - - - + - 200 12.5 MG-63 + + - - - - - - - - - - - - + - 200 12.5 0 2 4 6 8 A B C D 10MFI (x1000) Neuroblastoma 0 20 40 60 80 100 Log-rank P=0.0475 Survival (%)0 50 100 I (n=42) II-IV (n=76) P=0.0282 ofCS % positivity ofCS lowofCS high ofCS low ofCS high Followup (days) 0 2000 4000 6000 8000 ofCS low ofCS high E F G was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure S2 CD69 (FITC) 0 10 3 104 105-103 0 10 3 104 105-103 CD69 (FITC) Q1 19.8 Q2 36.3 Q3 16.4 Q4 27.4 Q1 28.5 Q2 9.13 Q3 19.9 Q4 42.5 0 -103 103 104 105 CD25 (PreCP-cy5.5) 0 20 40 60 80 100Normalized (Mode) 0 103 104 105 0 103 104 105 LNCaP Armed Unarmed Q1 32.2 Q2 35.7 Q3 6.42 Q4 25.7 Q1 42.4 Q2 2.64 Q3 6.18 Q4 48.8 CD69 (FITC) 0 10 3 104 105-103 0 10 3 104 105-103 CD69 (FITC) 0 103 104 105 0 103 104 105 Armed Unarmed 0 -103 103 104 105 CD25 (PreCP-cy5.5) 0 20 40 60 80 100Normalized (Mode) U2OS IL-2IFNγ TNFα IL-2IFNγ TNFα Pg/mL 0 250 500 2000 10000 20000 500000 800000 1000000 0 10000 20000 40000 70000 100000 1000000 1400000 1800000************ ************ ************ ********* ********** ********* Armed [SpyC]-CAR T cells Unarmed [SpyC]-CAR T cells Non-transduced T cells LNCaP cells Armed [SpyC]-CAR T cells Unarmed [SpyC]-CAR T cells Non-transduced T cells U2OS cells A C B D Pg/mL CAR T cells Non-transduced T cells CAR (PE) CAR (PE)CAR (PE)CAR (PE) was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure S3 0 1000 2000 3000 4000 5000 TNFα IL-6 0 5 10 15 20 25 IL-4 IL-2 0 1000 2000 3000 4000 5000 IL-13 0 200000 400000 600000 800000 1000000 IFNγ 50 100 150 0 MG-63 UM-UC-3 0 500000 1000000 1500000 0 500 1000 1500 0 10000 20000 30000 40000 0 20 40 60 80 0 50 100 150 200 250 0 5000 10000 15000 0 200000 400000 600000 800000 1000000 0 500 1000 1500 2000 2500 0 5000 10000 15000 20000 0 50 100 150 200 0 10 20 30 0 2000 4000 6000 8000 0 1000 2000 3000 4000 0 10 20 30 0 1000 2000 3000 0 5000 10000 15000 20000 0 500000 1000000 1500000 2000000 0 20000 40000 60000 80000 0 2000 4000 6000 8000Armed [SpyC]-CAR T cells Unarmed [SpyC]-CAR T cells Non-transduced T cells Target cells pg/ml TNFα IL-6IL-4 IL-2 IL-13IFNγ TNFα IL-6IL-4 IL-2 IL-13IFNγ TNFα IL-6IL-4 IL-2 IL-13IFNγ LNCaP U2OS pg/mlpg/mlpg/ml UM-UC-3 UM-UC-3 UM-UC-3 UM-UC-3 UM-UC-3 MG-63 MG-63 MG-63 MG-63 MG-63 LNCaP LNCaP LNCaPLNCaP LNCaP U2OS U2OS U2OS U2OS U2OS was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint LNCaP 0 5 10 15 20 25 U2OS Figure S4 Confluence (Relative to t=0)0 2 4 6 8 0 156 Time (Hours) 0 156 Time (Hours) VAR2-[SpyT][SpyC]-CAR T cells [SpyC]-CAR T cells Non-trans T cells + VAR2-[SpyT] Non-trans T cells Target cells + VAR2-[SpyT] Target cells 36 36 0 2 4 6 8 LNCaP U2OS Time (Hours)0 156 0 2 4 6 8 Time (Hours)0 156 30 32 34 36 38 40 42 44 CD69+ (%) 2 4 6 8 10 12 0 Oncofetal CS (MFI) 2 4 6 8 10 12 0 Oncofetal CS (MFI) INFγ (pg/mL) 0 2 6 8 10 12 144 MG-63 U2OS LNCaP UM-UC-3 UM-UC-3 U2OS MG-63 LNCaP [E:T] 1:1 [E:T] 1:1 Confluence (Relative to t=0) 200 nM VAR2-[SpyT] 100 nM VAR2-[SpyT] 50 nM VAR2-[SpyT] 25 nM VAR2-[SpyT] 12 nM VAR2-[SpyT] Unarmed A B C **** **** D was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint Figure 5 PBS M#1 PBS M#2 PBS M#3 PBS M#4 PBS M#5 PBS M#6 PBS M#7 PBS M#8 PBS M9 VAR2-[SpyT][SpyC]-CAR M#1 VAR2-[SpyT][SpyC]-CAR M#2 VAR2-[SpyT][SpyC]-CAR M#3 VAR2-[SpyT][SpyC]-CAR M#4 VAR2-[SpyT][SpyC]-CAR M#5 VAR2-[SpyT][SpyC]-CAR M#6 VAR2-[SpyT][SpyC]-CAR M#7 VAR2-[SpyT][SpyC]-CAR M#8 VAR2-[SpyT][SpyC]-CAR M#9 [SpyC]-CAR M#1 [SpyC]-CAR M#2 [SpyC]-CAR M#3 [SpyC]-CAR M#4 [SpyC]-CAR M#5 [SpyC]-CAR M#6 [SpyC]-CAR M#7 [SpyC]-CAR M#8 [SpyC]-CAR M#9 PBS M#9 y = 41.191x - 429.33 R² = 0.9222 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 Tumor Volume (mm3) Time (Days) y = 41.114x - 434.86 R² = 0.8507 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 37.932x - 396.37 R² = 0.7903 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 34.441x - 351.65 R² = 0.8039 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 32.238x - 410.71 R² = 0.7412 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 43.215x - 405.61 R² = 0.8439 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 34.256x - 399.01 R² = 0.7877 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 32.712x - 358.43 R² = 0.7772 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 16.635x - 248.85 R² = 0.7311 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 48.418x - 458.84 R² = 0.8575 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 46.308x - 444.19 R² = 0.847 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 43.574x - 409.74 R² = 0.7887 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 65.288x - 647.43 R² = 0.744 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 42.878x - 468.63 R² = 0.8153 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 47.047x - 455.28 R² = 0.84 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 37.647x - 389.05 R² = 0.8182 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 52.812x - 493.77 R² = 0.8379 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 41.543x - 402.02 R² = 0.8515 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 51.23x - 505.2 R² = 0.7776 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 41.972x - 423.57 R² = 0.8021 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 48.266x - 441.92 R² = 0.8733 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 43.585x - 453.98 R² = 0.9089 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 38.703x - 382.44 R² = 0.8115 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 35.153x - 390.36 R² = 0.781 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 45.605x - 485.63 R² = 0.7953 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = 34.098x - 360.43 R² = 0.9294 -400 -200 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) y = -0.0086x + 39.576 R² = 7E-050 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 Tumor Volume (mm3) Time (Days) was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 2, 2024. ; https://doi.org/10.1101/2024.05.29.596014doi: bioRxiv preprint