WO2025221781A2 - Chimeric antigen receptors targeting fgfr4 and/or cd276 and use thereof for the treatment of cancer - Google Patents

Abstract

Chimeric antigen receptors (CARs) and bicistronic chimeric antigen receptors (BiCisCARs) that target fibroblast growth factor receptor 4 (FGFR4), CD276, or both are disclosed. The CARs and BiCisCARs include a hinge and transmembrane domain from either CD28 or CD8 and a co-stimulatory domain from either CD28 or 4-1BB. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine-based activation motifs (ITAMs) of a CD3ζ intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used for the treatment of cancers that express one or both of FGFR4 and CD276.

Description

CHIMERIC ANTIGEN RECEPTORS TARGETING FGFR4 AND/OR CD276 AND USE THEREOF FOR THE TREATMENT OF CANCER

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/634,330, filed April 15, 2024, which is herein incorporated by reference in its entirety.

FIELD

This disclosure concerns chimeric antigen receptors (CARs) and bicistronic CARs targeting one or both of fibroblast growth factor receptor 4 (FGFR4) and CD276, and their use for treating FGFR4-expressing tumors.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under project number ZIA BC 010806 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION OF ELECTRONIC SEQUENCE LISTING

The electronic sequence listing, submitted herewith as an XML file named 4239-111443- O2.xml (164,962 bytes), created on April 8, 2025, is herein incorporated by reference in its entirety.

BACKGROUND

Chimeric antigen receptor (CAR) T-cell therapies targeting cancer-specific antigens have impressive successes in treating refractory and relapsed leukemia and lymphoma (Majzncr and Mackall, Nat Med 25: 1341-1355, 2019; June et al. , Science 359: 1361-1365, 2018). However, they have thus far displayed poor efficacy in treating solid tumors due to several challenges, including heterogenous expression of tumor-associated antigens, loss of target expression under selection pressure, limited T-cell potency, inadequate trafficking, a hostile tumor microenvironment, the propensity of exhaustion and lack of persistence of the CAR T cells (Wagner et al. , Mol Ther 28:2320-2339, 2020; Srivastava and Riddell, J Immunol 200:459-468, 2018; Labanieh et al. , Nat Biomed Eng 2:377 -391 , 2018). Overcoming these hurdles for the treatment of solid tumors remains a significant challenge.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents approximately 3-4% of all childhood cancers (Scheurer et al. , Pizzo and Poplack's pediatric oncology, Eighth Edition, Wolter Kluwer Health, 2021). Fibroblast growth factor receptor 4 (FGFR4) is highly expressed in both PAX3/7-FOXO1 fusion-positive (FP-) and fusion-negative (FN-) RMS, but low or absent in normal tissues (Tian et al. , Cell Rep Med 4(10): 101212, 2023; Taylor et al. , J Clin Invest 119:3395-3407, 2009). Furthermore, FGFR4 is a direct transcriptional target of fusion protein PAX3-FOXO1 (Cao et al., Cancer Res 70:6497-6508, 2010; Gryder et al., Cancer Discov 7:884-899, 2017), driving high expression in FP-RMS. Additionally, -10 % of FN-RMS have activating mutations with high expression of FGFR4 (Chen et al. , Cancer Cell 24:710-724, 2013; Shorn et al., Cancer Discov 4:216-231, 2014; Shorn et al., J Clin Oncol 39:2859-2871, 2021). These characteristics make FGFR4 a tractable molecular target for RMS, including CAR T therapy.

CD276 (B7-H3) is another cell surface protein belonging to immune checkpoint B7 families (Picarda et al., Clin Cancer Res 22:3425-3431, 2016). CD276 is overexpressed on a wide range of human solid tumors including RMS (Vitanza et al., Cancer Discov 13(1): 114-131, 2023; Majzner et al., Clin Cancer Res 25:2560-2574, 2019; Tian et al., J Clin Invest 132(16):el 55621 , 2022), and its overexpression is correlated with tumor progression, metastasis, and poor clinical outcome across a variety of malignancies (Lavoie et al., Cancers (Basel) 13(18):4528, 2021; Yang et al., Int J Biol Sci 16:1767-1773, 2020). Furthermore, PAX3-FOXO1 up-regulates CD276 expression in FP-RMS (Kanayama et al. , Sci Rep 11 : 18802, 2021) and is currently being investigated as a target for CAR T- cell therapy of human cancers (Vitanza et al. , Cancer Discov 13(1):114-131, 2023; Majzner et al., Clin Cancer Res 25:2560-2574, 2019; Tian et al., J Clin Invest 132(16):e 155621 , 2022). Therefore, CD276 is an additional target for CAR T therapy against RMS.

SUMMARY

The present disclosure provides chimeric antigen receptors (CARs) and bicistronic CARs that target FGFR4, CD276, or both. The disclosed CARs and BiCisCARs include a variety of different combinations of the hinge transmembrane (HTM) domain and co-stimulatory domain (CSD) to identify CARs and BiCisCARs with the greatest potency against FGFR4- and/or CD276-expressing tumors. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine -based activation motifs (IT AMs) of a CD3^ intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used for the treatment of cancers that express one or both of FGFR4 and CD276.

Provided herein are FGFR4-targeted CARs that include an extracellular antigen-binding domain that specifically binds FGFR4. In some aspects, the CAR includes: a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a hinge and transmembrane (HTM) domain; an intracellular co-stimulatory domain; and a CD3^ intracellular signaling domain. In some aspects, the HTM is a CD28 HTM domain and the intracellular co-stimulatory domain is a CD28 intracellular co-stimulatory domain. In other aspects, the HTM domain is a CD8 HTM domain and the intracellular co-stimulatory domain is a 4- IBB intracellular co-stimulatory domain. In other aspects, the HTM is a CD28 HTM domain and the intracellular co-stimulatory domain is a 4- IBB intracellular co-stimulatory domain. In some aspects, the CD3^ intracellular signaling domain is a wild-type CD3 intracellular signaling domain. In other aspects, the CD3^ intracellular signaling domain includes at least one amino acid substitution in one or more IT AMs. Nucleic acid molecule and vectors encoding the FGFR4-targeted CARs, and isolated cells expressing the FGFR4-targeted CARs are also provided.

Also provided herein are CD276-targeted CARs that include an extracellular antigen-binding domain that specifically binds CD276. In some aspects, the CAR includes: a VH domain and a VL domain, wherein the VH domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 hinge and transmembrane (HTM) domain; and a 4- IBB intracellular co-stimulatory domain. In some aspects, the CD276-targeted CAR further includes a CD3^ intracellular signaling domain, such as a wild-type CD3^ intracellular signaling domain or a CD3^ intracellular signaling domain having at least one amino acid substitution in one or more IT AMs. Nucleic acid molecule and vectors encoding the CD276-targeted CARs, and isolated cells expressing the CD276-targeted CARs are also provided.

Further provided herein are isolated cells that express a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a FGFR4-targeted CAR as disclosed herein and the second CAR is a CD276-targeted as disclosed herein. In some aspects, the cell is an immune cell, such as a T cell, a B cell, a natural killer (NK) cell, or a monocyte/macrophage. In other aspects, the cell is an induced pluripotent stem cell (iPSC).

Also provided herein are nucleic acid molecules that encode a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a FGFR4-targeted CAR as disclosed herein and the second CAR is a CD276-targeted CAR as disclosed herein. In some aspects of the nucleic acid molecules, the coding sequences for the first CAR and the second CAR are separated by a sequence encoding a 2A site, such as a T2A site. In some examples, the nucleic acid molecule further includes a first leader sequence (such as a CD8 leader sequence) preceding the coding sequence for the first CAR and/or a second leader sequence (such as a GM-CSF leader sequence) preceding the coding sequence for the second CAR. Vectors, such as lentivirus vectors, that include a disclosed nucleic acid molecule are also provided.

Compositions that include a pharmaceutically acceptable carrier and an FGFR4-targeted CAR, a CD276-targeted CAR, a FGFR4/CD276 BiCisCAR, a nucleic acid molecule, a vector or an isolated cell disclosed herein are further provided.

Also provided are methods for treating a FGFR4-expressing and/or CD276-expressing cancer in a subject, and methods of inhibiting tumor growth or metastasis of a FGFR4-expressing and/or CD276-expressing cancer in a subject. In some aspects, the methods include administering to the subject a therapeutically effective amount of a CAR, BiCisCAR, isolated cell, nucleic acid molecule, vector, or composition disclosed herein. In some examples, the cancer expresses both FGFR4 and CD276.

The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1M: Altering the HTM and CSDs significantly improves FGFR4 CAR killing ability against moderate burden RMS orthotopic xenografts. (FIG. 1A) Schematic of FGFR4 CAR designs containing different hinge and transmembrane (HTMs) or co-stimulatory domains (CSD), named FGFR4.8HTM.BBz (left), FGFR4.28HTM.BBz (center), and FGFR4.28HTM.28z (right). (FIG. IB) The cytolytic ability of three FGFR4 CAR T-cells was evaluated in vitro using an xCELLigence Real-Time Cell Analysis (RTCA) against RH30 and RMS559 cells at an E:T ratio of 1 :8. A representative of one of three independent experiments using 3 different donors is shown. Statistical analysis was performed using two-way repeated measures (RM) ANOVA. (FIG. 1 C) IFN-y released by T-cells expressing three FGFR4 CARs following a 72-hour coculture with RH30 or RMS559 cells at an E:T ratio of 1 :8. Means of three independent cocultures are plotted with SD. **p < 0.01 and ****p < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test. (FIG. ID) Schematic of testing CAR T-cells in an RH30 intramuscular (I.M.) xenograft model infused with mock or 2.5E6 FGFR4 CAR T-cells 14 days after tumor inoculation when tumors reached a moderate size (mean tumor size represented by leg volume of 200 mm³). (FIG. IE) Leg volume for testing CAR T-cell efficacy in an RH30 I.M. xenograft model. Each line represents an individual mouse (n = 3 or 5 per group). Two-way RM ANOVA analysis was used to calculate the P values between two groups. ****p < 0.0001; ns, not significant. (FIGS. IF and 1G) RH30 tumor burden, represented by bioluminescent images (FIG. IF) or total flux (photons/second, FIG. 1G), was assessed by an IVIS imaging system. Two-way RM ANOVA analysis was used to calculate the p-value between two groups. ****p <0.0001. (FIG. 1H) Schematic of testing the activity of 2.5E6 FGFR4 CAR T-cells in an RMS559 I.M. xenograft mouse model. (FIG. II) Tumor size was measured as leg volume. Each line represents an individual mouse (n = 4 or 5 per group). ****/) < 0.0001; ns, not significant, by mixed-effects analysis between two groups. (FIGS. 1J and IK) RMS559 tumor burden, represented by bioluminescent images (FIG. 1 J) and total flux (photons/second, FIG. IK), was assessed by an IVIS imaging system. Mixed-effects analysis was used to calculate the -value between the two groups respectively. **p = 0.0026. (FIG. IL) Expansion dynamics of FGFR4 CAR T-cells was accessed by counts in 100 p.1 blood from mice of the RH30 I.M. model treated with 2.5E6 CAR T- cells using flow cytometry at day 10, day 15, day 23, and day 32 (n = 5). Each dot represents an individual mouse. Statistics for comparing two FGFR4 CAR T-cell counts at each time point represent two-way ANOVA with Sidak’s multiple-comparison test. *p < 0.05, ****p < 0.0001. (FIG. IM) Percentage of CAR T-cells expressing or co-expressing exhaustion markers CD39, PD-1 (programmed cell death 1), LAG-3 (lymphocyte activating 3), and TIM-3 (T-cell immunoglobulin and mucin domain-containing protein 3) at day 32 after T-cell infusion into RH30-bearing mice.

FIGS. 2A-2F: Direct targeting and establishment of enhancers at the CD276 locus by PAX3- FOXO1 and MYODI, and heterogenous expression of FGFR4 and CD276 on RMS cell lines, CDXs or PDXs. (FIG. 2A) H3K27ac (top), MYODI (middle), and PAX3-FOXO1 (bottom) ChlP-seq at the CD276 locus in FP-RMS cell lines (red, dark blue, and purple) and FN-RMS cell lines (orange, green). (FIG. 2B) Scatterplot of FGFR4 and CD276 mRNA demonstrated a generally high level of expression for CD276 but more variable expression of FGFR4 in RMS tumors or cells compared to normal tissues. (FIGS. 2C-2F) Flow cytometry was used to measure FGFR4 or CD276 expression on patient-derived RMS cell lines (FIG. 2C) or patient-derived xenografts (PDX) (FIG. 2E) by staining with anti-human FGFR4 antibody (3A11) and anti-human CD276 antibody (MGA271). Co-staining of FGFR4 and CD276 showed heterogeneity of expression of both targets on RMS cells and PDXs. Cell line or PDX IDs and tumor types are listed in the tables (FIGS. 2C and 2E). Quantification of FGFR4 or CD276 molecules on each RMS cell and CDX (FIG. 2D) or PDXs (FIG. 2F) was performed using a phycoerythrin (PE) fluorescence quantitation kit. Dots with error bars show protein expression measured by flow cytometric analysis from 3 independent experiments.

FIGS. 3A-3L: Dual targeting CAR T-cells using CD28 and 4-1BB CSDs exhibit faster tumor killing with persistence and limited exhaustion against an aggressive RMS559 orthotopic model. (FIG. 3 A) Schematic of a CD276 targeting CAR containing a CD8HTM and 4- IBB CSD, referred to as CD276.8HTM.BBz. (FIG. 3B) Leg (tumor) volumes after 2.5E+6 CD276.8HTM.BBz CAR T-cell infusion at day 7 post-RH30 I.M implantation. Each line represents the tumor volume of an individual mouse (n = 6 or 7). ****p < 0.0001, as determined by 2-way RM ANOVA. (FIG. 3C) Representative bioluminescence images of RH30 tumor growth at day 21 post CAR T-cells infusion. (FIG. 3D) Kaplan-Meier survival analysis of mice receiving mock T or CD276.8HTM.BBz treatments. **p - 0.0069 by a log-rank test. (FIG. 3E) Schematic of FGFR4 and CD276 dual targeting BiCisCARs incorporating CD8 or CD28 HTM, or different CSDs, named as FGFR4.8HTM.BBz- CD276.8HTM.BBz, FGFR4.28HTM.BBz-CD276.8HTM.BBz, and FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCARs. (FIG. 3F) Cytolytic activity of two BiCisCAR T-cells against RMS559 at an effector (E): target (T) ratio of 1:10 measured by an xCELLigence RTCA. Representative of 3 independent experiments with T cells from 3 individual donors. Statistical analysis was performed with two-way RM ANOVA. **** p <0.0001 (FIG. 3G) Schematic of testing the efficacy of 2.5E6 BiCisCAR T-cells in an RMS559 I.M. xenograft mouse model. (FIG. 3H) Leg volumes representing tumor burden after CAR T-cell infusion. Each line represents tumor volume from an individual mouse (n = 4 or 5). ****p < 0.0001, as determined by two-way RM ANOVA. (FIGS. 31 and 2J) RMS559 tumor burden measured by bioluminescence images (FIG. 31) and total flux (FIG. 3 J) using IVIS imaging post-BiCisCAR T-cells treatment, ns, not significant, determined by two-way RM ANOVA analysis. (FIG. 3K) The expansion dynamics of BiCisCAR T-cells was analyzed as counts per 100 l blood from mice treated with 2.5E6 CAR T-cells by flow cytometry at day 11, day 15, day 23, and day 32 (n = 5). **p = 0.0023 as determined by two-way RM ANOVA for comparing two BiCisCAR T-cells proliferation dynamics. Statistics for comparing FGFR4 CAR T- cell counts of two groups at each time point was performed as two-way ANOVA with Sidak’s multiple-comparison test. = 0.0004. (FIG. 3L) The fractions of CAR T-cells expressing or coexpressing exhaustion markers CD39, PD-1, LAG-3, and TIM-3 at day 32 after T-cell infusion into the RMS559 I.M. model.

FIGS. 4A-4O: Dual targeting BiCisCAR with two different CSDs shows enhanced expansion, tumor-infiltrating and limited exhaustion. (FIG. 4A) Schematic of testing 1E+6 CAR T- cells in a RMS559 I.M. xenograft NSG mouse model. (FIGS. 4B - 4D) Tumor growth measured by tumor size (FIG. 4B), tumor bioluminescence images (FIG. 4C), and bioluminescence kinetics (FIG. 4D) for RMS559_Luc orthotopic xenograft. *p < 0.05, **p < 0.01, as determined by 2-way RM ANOVA. ns, not significant. (FIG. 4E) Kaplan-Meier survival analysis (n = 5 mice per group). **p < 0.01 ; ns, not significant by Log-rank test. (FIG. 4F) Cell counts of CAR⁺ T-cells (gating from CD45⁺CD3⁺ T-cells) in mouse bloodstream 21 days after CAR T-cell infusion. Statistical analysis was performed using one-way ANOVA with Holm-Sidak’s multiple comparisons tests. (FIG. 4G) Schematic of testing 2.5E+6 CAR T-cells in a JR I.M. xenograft NSG mouse model. (FIGS. 4H and 41) Tumor growth was measured by tumor size (FIG. 4H) or bioluminescence kinetics (FIG. 41) for JR Luc orthotopic xenografts. (FIGS. 4J and 4K) Detection of CAR T-cells (CD45⁺CD3⁺ CAR⁺) in the circulation (FIG. 4J) or tumor (FIG. 4K) from mice by flow cytometry 16 or 21 days after CAR T- cells treatment. Data are shown as individual values (shapes) and the mean ± SE (bar graphs) (n = 5 or 6 mice per group). Data from day 16 are shown as colored shapes; while data from day 21 are shown as black shapes in FIGS. 4K, 4M, 4N, and 40. (FIG. 4L) A representative flow cytometry experiment detects CD39 and Tim-3 expression on CAR⁺ T-cells from JR I.M. xenografts at 21 days after infusion. (FIGS. 4M to 40) Percentages of CD39⁺ (FIG. 4M), Tim-3⁺ (FIG. 4N), and CD39⁺ Tim-3⁺ (FIG. 40) CAR⁺ T-cells in tumor from mice 16 or 21 days after CAR T-cells infusion (n = 5 or 6, mean ± SEM). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGS. 5A-5E: Multimodal single-cell profiling reveals that the FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR exhibits the highest cytotoxicity activity. (FIG. 5A) Workflow of CITE-Seq for simultaneous protein and transcript analysis of tumor-infiltrating CAR T-cells at day 11 post-infusion using a JR IM orthotopic model. ADT, antibody-derived tag; HTO, hashtag oligonucleotide. (FIG. 5B) WNN UMAP visualization of tumor-infiltrating T-cells from mice treated using five CAR T-cells with biological replicates. Each dot represents a single cell and cell clusters are labeled by numbers. The black perimeter lines encircle the cell clusters Cl, C3 and C7, and the percentages of cells in these clusters are shown. (FIG. 5C) The percentages (means + SEM of 2 biological replicates) of each cell subpopulation from five CAR T-cell treated mice. (FIG. 5D) Volcano plot of differentially expressed genes (DEGs) between the 28HTM.28z-8HTM.BBz BiCisCAR and 4 other CAR T-cells infiltrating in JR I.M. xenografts. Genes with an -Logio P (adjusted P, nonparametric Wilcoxon rank-sum test) < 20 and | log2 fold change | > 0.5 are shown in red (n = 18). The top 20 highly expressed genes ranked by average log2 fold change are labeled and they are associated with T-cell cytotoxicity. (FIG. 5E) Heatmap of these top 20 genes expressed in CAR T-cells isolated from JR I.M. xenograft tumors. The FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells exhibited the highest expression of T-cell cytotoxicity genes among all CAR T cells. The colored scale bar represents z-score values for gene expression.

FIGS. 6A-6J: BiCisCAR T-cells overcome heterogeneous expression of FGFR4 and CD276 in vivo. (FIG. 6A) A representative flow-cytometric plot demonstrating the surface expression of FGFR4 and CD276 in RH30 (red), RH30-FGFR4KO (blue), or RH30-CD276KO (orange) cells. The top table shows the means of FGFR4 or CD276 molecules per cell. (FIGS. 6B-6D) Cytotoxicity assays using an xCELEigence RTCA show FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T- cells continued to effectively kill FGFR4KO or CD276KO RH30 cells at an E:T ratio of 1 : 1 . In contrast, FGFR4.28HTM.28z CAR or CD276.8HTM.BBz CAR did not induce cytolysis to FGFR4- KO or CD276KO RH30 cells respectively at an E:T ratio of 1: 1. The orange vertical arrows indicate the time point at which CAR T-cells were added to the plate seeded with the target cells. (FIG. 6E) Schematic of the heterogeneous RH30 I.M model infused with 2.5E6 CAR T-cells on day 14 following RH30-FGFR4KO (right) or RH30-CD276KO (left) tumor inoculation. (FIG. 6F) Depicts representative bioluminescence images of RH30-FGFR4KO or RH30-CD276KO cells growth in the I.M model before and after CAR T-cell treatment. (FIGS. 6G and 6H) Tumor size was monitored over 35 days by measuring leg volume before and after receiving mock or CAR T-cell treatment. Each replicate per group (n = 5) is shown. Two-way repeated measures (RM) ANOVA analysis was used to calculate the p-values between each paired group. (FIGS. 61 and 6J) Bioluminescence kinetics of RH30-FGFR4KO (FIG. 61) or RH30-CD276KO (FIG. 6J) following CAR T-cell treatment, using total flux values (photons per second). Data is presented as means ± SEM, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, as determined by 2-way RM ANOVA.

FIGS. 7A-7E: Presence of both CD28 and 4-1BB CSDs in FGFR4 and CD276 dual-targeting CAR T-cells leads to heightened and sustained T-cell activation signaling. (FIGS. 7A - 70) IFN-y (FIG. 7A), IE-2 (FIG. 7B), and TNF-a (FIG. 70) were released by FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR T-cells following a 20-hour stimulation with plate-coated FGFR4-Fc, CD276-Fc, or both proteins. Data are shown as the mean + SD for 3 independent experiments. The orange dotted line shows the sum of cytokine release after single protein stimulation. Two-way ANOVA Sidak’s multiple comparisons test was performed for difference between two proteins dual stimulation and the sum of single stimulation. **p < 0.01; ****p < 0.0001. (FIG. 7D) Western blot analyses of CAR-CD3^, ZAP70, PLCy I , p65, Akt and Erkl/2 phosphorylation in FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells after CAR stimulation (FGFR4-Fc protein for FGFR4 CAR, CD276-Fc protein for B7-H3, or both proteins for dual CAR activation) in a time course experiment. Numbers under the gel images indicate the ratio of signal intensity obtained with phospho-specific antibodies relative to that of the total protein. Relative values were normalized to one of the unstimulated controls. These results are representative of three independent experiments conducted with distinct T-cell donors. (FIG. 7E) Summary diagram outlining the activation signaling pathway of FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells following dual stimulation with FGFR4 and CD276.

FIGS. 8A-8E: Low dose (2.5E+6) of FGFR4-CAR T-cells containing a CD8 HTM and a 4- 1BB CSD showed no activity in RH30 orthotopic xenografts. (FIG. 8 A) Schematic of RH30 orthotopic, intramuscular, xenograft model infused with mock or 2.5E6 FGFR4.8HTM.BBz CAR T- cells on day 7 post tumor inoculation. (FIG. 8B) tumor size, (FIG. 8C) Bioluminescent images, and (FIG. 8D) total flux of RH30 intramuscular xenografts growth before (day -1) and after infusion with mock T-cells or FGFR4.8HTM.BBz CAR T-cells. Each replicate per group is shown, n = 5. Two-way RM ANOVA analysis is used to calculate the p values between two groups; ns, not significant. (FIG. 8E) Kaplan-Meier survival analysis of mice receiving different treatments is shown.

FIGS. 9A-9H: Phenotypic characterization of three FGFR4 CAR T-cells before infusion into tumor-bearing mice. (FIG. 9A) Representative histogram of 3 FGFR4 CAR expression on T-cell surface assessed by binding to FGFR4-Fc protein after 9 days post-transduction. Mean fluorescent intensity (MFI) of CAR expression in all three FGFR4-CAR constructs and mock T-cells (shown in black) shown in the table. (FIG. 9B) Percentage of CAR positive in all CD45⁺CD3⁺ T-cells measured by flow cytometry at day 9 after manufacturing. Data represent n = 5 independent experiments with 4 different donors, mean ± SEM. Statistics represent one-way ANOVA with Tukey’s multiple comparisons. (FIGS. 9C and 9D) Representative flow cytometry plots characterize phenotypes of CAR-transduced T-cells, including CD4⁺ and CD8⁺ T-cells, stem cell memory (SCM, CD45RA⁺ and CD62L⁺), central memory (CM, CD45RA- and CD62L⁺), effector memory (EM, CD45RA" and CD62L ) and terminally differentiated effector memory (EMRA, CD45RA⁺ and CD62L ) on day 10 after CAR transduction. (FIGS. 9E and 9F) Mean frequencies were plotted for CD4⁺ and CD8⁺ T-cells for FIG. 9D, or memory subsets for FIG. 9D with SEM for 5 independent experiments. (FIGS. 9G and 9H) IL-2 (FIG. 9G) and TNF- a (FIG. 9H) were released by three FGFR4 CAR T-cells following a 72-hour coculture with RH30 or RMS559 cells. Data means are plotted with SD for 3 independent cocultures. **p < 0.01 and ****p < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test. FIGS. 10A-10D: FGFR4-CAR T-cells (10E+6) incorporating a CD8 HTM and 4-1BB CSD showed minimal activity in RMS559 orthotopic xenografts. (FIG. 10A) Schematic of the RMS559 intramuscular xenograft model infused with mock or 10E+6 FGFR4.8HTM.BBz CAR T-cells on day 10 post tumor inoculation. (FIG. 10B) Tumor size and (FIG. 10C) total flux of RMS559 intramuscular xenografts growth before (day -1, day -4) and after infusion with mock T-cells or FGFR4.8HTM.BBz CAR T-cells. Each replicate per group is shown, n = 7. Two-way RM ANOVA analysis is used to calculate the p values between two groups: ns, not significant. (FIG. 10D) Kaplan-Meier survival analysis of mice receiving different treatments is shown.

FIGS. 11A-11H: Phenotypic characterization of FGFR4 CAR T-cells in vivo. (FIGS. 11A- 11C) Percentage of CAR⁺ in CD45⁺CD3⁺ T-cells from blood (FIG. 11A), MFI of surface CAR expression (FIG. 11B), and the percentage of CAR⁺ T-cells in blood singlets (FIG. 11C) from mice of RH30 I.M. model treated with 2.5E6 CAR T-cells analyzed by flow cytometry at day 10, day 15, day 23, and day 32 (n = 5; each replicate is shown). (FIGS. 11D-EF) Percentage of CAR⁺ in CD45⁺CD3⁺ T-cells (FIG. 1 ID), MFI of T-cell surface CAR expression (FIG. HE), and the percentage of CAR⁺ T-cells in the blood (FIG. 1 IF) at day 10, 15, 23, or 32 after CAR T-cell infusion into RMS559 I.M xenograft-bearing mice (n = 5 mice per group). (FIG. 11G) Expansion dynamics of FGFR4 CAR T- cell counts per 100 pl in blood from mice of RMS559 I.M. model treated with 2.5E6 CAR T-cells by flow cytometry. Statistics for comparing two FGFR4 CAR T-cell counts at each time point represent two-way ANOVA with Sidak’s multiple-comparison test. *p < 0.05. (FIG. 11H) Percentage of CAR T-cells expressing or co-expressing CD39, PD-1, LAG-3, and TIM-3 at day 32 after T-cell infusion into RMS559-bearing mice.

FIGS. 12A-12H: Dual targeting CAR T-cells using the same CD8 HTM and 4-1BB CSD construct for FGFR4 and CD276 exhibit reduced tumor killing against RH30 orthotopic model. (FIG. 12 A) Representative histogram of CAR cell surface expression among different BiCisCAR constructs assessed by binding to FGFR4-Fc or CD276-Fc protein after manufacturing for 10 days. MFI of CAR expression in all three BiCisCAR constructs, and mock T-cells (shown in black) are shown in the table. (FIG. 12B) Cytolytic activity of three BiCisCAR T-cells against RH30 at an E:T ratio of 1:10 by an xCELLigence RTCA. Representative of n - 3 independent experiments with n = 3 individual donors. Statistical analysis was performed with two-way RM ANOVA. (FIG. 12C) Schematic of an in vivo model testing the activity of 2.5E6 BiCisCAR T-cells against an RH30 I.M. xenograft. (FIG. 12D) Tumor volumes following CAR T-cell infusion. Data indicates the tumor volume of each mouse (n = 5). ****p < 0.0001, as determined by 2-way RM ANOVA. (FIGS. 12E and 12F) Bioluminescence images (FIG. 12E) and total flux (FIG. 12F) of RH30 tumor growth assessed by IVIS imaging post three BiCisCAR T-cells treatment. Two-way RM analysis was used to calculate the p- value between every two BiCisCAR T-cells groups respectively. (FIG. 12G) Kaplan-Meier survival curve of mice in the above model (n = 5 mice per group); comparisons of survival curves were determined by log-rank test, **p = 0.0035 for mock T versus FGFR4.8HTM.BBz- CD276.8HTM.BBz BiCisCAR, *p = 0.0495 for FGFR4.8HTM.BBz-CD276.8HTM.BBz BiCisCAR versus FGFR4.28HTM.BBz-CD276.8HTM.BBz BiCisCAR or FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR. (FIG. 12H) The cell counts of three BiCisCAR T-cells per 100 pl blood from mice treated with 2.5E6 CAR T-cells were analyzed by flow cytometry at day 35 (n - 5; each replicate is shown). Statistics for every two BiCisCAR T-cell counts represent a two-tail nonparametric test using Mann Whitney test.

FIGS. 13A-13E: (FIG. 13A) Representative tumor BLI images of JR_Luc tumor growth in the orthotopic xenograft model shown in FIG. 4. (FIG. 13B) Representative flow cytometry illustrating CD39 and LAG-3 expression in CAR⁺ T-cells in the blood of mice from the JR I.M. xenograft model, 21 days after infusion of 2.5E+6 CAR T-cells. (FIGS. 13C - 13E) Percentages of CD39⁺ (FIG. 13C), LAG-3⁺ (FIG. 13D), and CD39⁺ LAG-3⁺ (FIG. 13E) in CAR⁺ T-cells circulating in the blood from mice 21 days after CAR T-cell infusion (n = 5 or 6, mean ± SEM). < 0.05, ** < 0.01, *** < 0.001, and ****p < 0.0001, by 1-way ANOVA, with Tukey’s multiple comparisons tests

FIGS. 14A-14C: Cluster annotation based on the top genes of each cluster. (FIG. 14A) UMAP visualization of single cells from five CAR T-cell groups based on WNN assay after removing batch effect among sequencing lanes by SCT. Cells are colored according to CAR T-cell groups. (FIG. 14B) WNN UMAP visualization of a total of 38,108 tumor-infiltrating T-cells from the above model, revealed 13 clusters with different transcriptome and protein profiles. The green dash circle gates out the CD4⁺ T-cells and the purple dash circle frames out CD8⁺ T-cells. (FIG. 14C) Single cell heatmap showing the top 20 differentially expressed genes of each cluster for cell type annotation.

FIG. 15: Violin plots of surface protein marker expression levels among 13 clusters used for clusters annotation.

FIGS. 16A-16C: FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells exhibited an additive or synergistic effect on cytolytic activity. (FIGS. 16A - 16C) Cytolytic activities of FGFR4.28HTM.28z, CD276.8HTM.BBz CARs or FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells were evaluated in vitro with RTCA against RH30 (FIG. 16A), RMS559 (FIG. 16B), or JR (FIG. 16C) cells.

FIGS. 17A-17D: FGFR4 and CD276 dual-targeting CAR T-cells with 4- IBB CSDs demonstrate suboptimal downstream T-cell activation signaling. (FIG. 17A) IFN-y, (FIG. 17B) IL-2 (B), and (FIG. 17C) TNF-a release by FGFR4.28HTM.BBz-CD276.8HTM.BBz BiCisCAR T-cells following a 20-hour stimulation with plate-coated FGFR4-Fc, CD276-Fc, or both proteins. Data are shown as the mean + SD; n = 3 independent stimulation with CAR T-cells. The green dotted line shows the sum of cytokine released after single protein stimulation. Two-way ANOVA Sidak’s multiple comparisons test was performed to statistic the difference between two proteins’ dual stimulation and the sum of single stimulation. ****p < 0.0001. (FIG. 17D) Time course of CAR- CD3C, ZAP70, PLCyl, p65, Akt and Erkl/2 phosphorylation in FGFR4.28HTM.BBz- CD276.8HTM.BBz BiCisCAR or FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells after CAR cross-linking (FGFR4-Fc protein for FGFR4 CAR, CD276-Fc protein for B7-H3, or both proteins for dual CAR cross-linking) measured by Western blot analysis. Numbers under the gels represent the ratio of the intensity of the signal obtained with phospho-specific antibodies relative to the total. Relative values were normalized to one of the unstimulated controls. Representative of three independent experiments with different T-cell donors.

FIG. 18: Schematic showing the location of the three IT AM domains in CD3^. The modified CD3 (1XX) includes two Y to F amino acid substitutions in each of ITAM 2 and ITAM3. The amino acid sequences of wild-type human CD3^ and CD3^(1XX) are also shown.

FIG. 19: Schematic representation of FGFR4 and CD276 CAR constructs. (1 - 3) FGFR4 CARs with variations in hinge and transmembrane (HTM) regions, co-stimulatory domains (CSD), or CD3^ signaling calibration. (4 - 5) CD276 CARs without or with CD3^ calibration. (6 - 10) Dualtargeting FGFR4/CD276 CARs (BiCisCARs) with different CD3C calibrations, including CD3C deletion or ITAM mutations in the CD3C domain. In 1XX CARs, the two tyrosine (Y) residues within the respective ITAM are mutated to phenylalanine (F) residues for the indicated IT AMs.

FIG. 20: Transduction efficiency of FGFR4+ CAR or CD276+ CAR T-cells. Representative contour plots showing CAR surface expression across different CAR constructs, assessed by binding to FGFR4-Fc or CD276 protein on day 9 post-manufacturing. CAR T-cells were generated from T cells of donor PB251028_02.

FIGS. 21A-21C: Cytolytic activity of FGFR4 and CD276 CAR T cells against RH30 cells following CD3^ calibration. (FIG. 21 A) Cytolytic activity of all FGFR4 and CD276 CAR T-cell constructs against RH30 cells at an effector- to-tar get (E: T) ratio of 1:5, measured using the IncuCyte Live-Cell Analysis System. Data are presented as mean ± SEM. CAR T cells were manufactured using T cells from donor PB241008_01. (FIG. 21B) Cytolytic activity of single-targeting FGFR4 or CD276 CAR T cells, showing that CD3C calibration reduced their killing efficiency. (FIG. 21C) Cytolytic activity of dual-targeting FGFR4/CD276 CAR T cells, demonstrating that CD3C calibration enhanced their cytotoxic potency.

FIGS. 22A-22C: Cytolytic activity of FGFR4 and CD276 CAR T cells against RMS559 cells following CD3C calibration. (FIG. 22A) Cytolytic activity of all FGFR4 and CD276 CAR T-cell constructs against RMS559 cells at an E:T ratio of 1:10, measured using the IncuCyte Live-Cell Analysis System. Data are presented as mean ± SEM. CAR T cells were manufactured using T cells from donor PB251028_02. (FIG. 22B) Cytolytic activity of single-targeting FGFR4 or CD276 CAR T cells, showing that CD3(^ calibration reduced their killing efficiency. (FIG. 22C) Cytolytic activity of dual-targeting FGFR4/CD276 CAR T cells, demonstrating that C 3L calibration enhanced their cytotoxic potency and led to more efficient tumor cell elimination. SEQUENCES

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the 3 All VH domain.

SEQ ID NO: 2 is the amino acid sequence of the 3 All VL domain.

SEQ ID NOs: 3-8 are the amino acid sequences of the 3A11 CDRs (IMGT).

SEQ ID NO: 9 is the amino acid sequence of the MGA271 VH domain.

SEQ ID NO: 10 is the amino acid sequence of the MGA271 VL domain.

SEQ ID NOs: 11-16 are the amino acid sequences of the MGA271 CDRs (IMGT).

SEQ ID NO: 17 is a nucleotide sequence encoding FGFR4(LH).28HTM.BBz CAR.

SEQ ID NO: 18 is the amino acid sequence of FGFR4(LH).28HTM.BBz CAR.

SEQ ID NO: 19 is a nucleotide sequence encoding

FGFR4(LH).28HTM.BBz/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 20 is the amino acid sequence of FGFR4(LH).28HTM.BBz/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 21 is a nucleotide sequence encoding FGFR4(LH).28HTM.28z(lXX) CAR.

SEQ ID NO: 22 is the amino acid sequence of FGFR4(LH).28HTM.28z(lXX) CAR.

SEQ ID NO: 23 is a nucleotide sequence encoding CD276.8HTM.BB CAR.

SEQ ID NO: 24 is the amino acid sequence of CD276.8HTM.BB CAR.

SEQ ID NO: 25 is a nucleotide sequence encoding FGFR4(LH).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR.

SEQ ID NO: 26 is the amino acid sequence of

FGFR4(LH).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR.

SEQ ID NO: 27 is a nucleotide sequence encoding FGFR4(LH).28HTM.28z/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 28 is the amino acid sequence of FGFR4(LH).28HTM.28z/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 29 is a nucleotide sequence encoding FGFR4(LH).28HTM.28z(lXX)/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 30 is the amino acid sequence of FGFR4(LH).28HTM.28z(lXX)/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 31 is a nucleotide sequence encoding FGFR4(LH).8HTM.BBz CAR.

SEQ ID NO: 32 is the amino acid sequence of FGFR4(LH).8HTM.BBz CAR.

SEQ ID NO: 33 is a nucleotide sequence encoding FGFR4(LH).28HTM.28z CAR. SEQ ID NO: 34 is the amino acid sequence of FGFR4(LH).28HTM.28z CAR.

SEQ ID NO: 35 is a nucleotide sequence encoding FGFR4(LH).28HTM.28z/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 36 is the amino acid sequence of FGFR4(LH).28HTM.28z/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 37 is a nucleotide sequence encoding FGFR4(HL).8HTM.BBz CAR.

SEQ ID NO: 38 is the amino acid sequence of FGFR4(HL).8HTM.BBz CAR.

SEQ ID NO: 39 is a nucleotide sequence encoding FGFR4(HL).28HTM.BBz CAR.

SEQ ID NO: 40 is the amino acid sequence of FGFR4(HL).28HTM.BBz CAR.

SEQ ID NO: 41 is a nucleotide sequence encoding

FGFR4(HL).28HTM.BBz/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 42 is the amino acid sequence of FGFR4(HL).28HTM.BBz/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 43 is a nucleotide sequence encoding FGFR4(HL).28HTM.28z CAR.

SEQ ID NO: 44 is the amino acid sequence of FGFR4(HL).28HTM.28z CAR.

SEQ ID NO: 45 is a nucleotide sequence encoding FGFR4(HL).28HTM.28z(lXX) CAR.

SEQ ID NO: 46 is the amino acid sequence of FGFR4(HL).28HTM.28z(lXX) CAR.

SEQ ID NO: 47 is a nucleotide sequence encoding

FGFR4(HL).28HTM.28z/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 48 is the amino acid sequence of FGFR4(HL).28HTM.28z/CD276.8HTM.BBz BiCisCAR.

SEQ ID NO: 49 is a nucleotide sequence encoding

FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR.

SEQ ID NO: 50 is the amino acid sequence of

FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR.

SEQ ID NO: 51 is a nucleotide sequence encoding FGFR4(HL).28HTM.28z/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 52 is the amino acid sequence of FGFR4(HL).28HTM.28z/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 53 is a nucleotide sequence encoding

FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 54 is the amino acid sequence of FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BB BiCisCAR.

SEQ ID NO: 55 is a nucleotide sequence encoding a CD8 leader sequence.

SEQ ID NO: 56 is a nucleotide sequence encoding a 3A11 scFv (VL-linker-VH).

SEQ ID NO: 57 is a nucleotide sequence encoding a 3A11 scFv (VH-linker-VL).

SEQ ID NO: 58 is a nucleotide sequence encoding a MGA271 scFv (VL-linker-VH). SEQ ID NO: 59 is a nucleotide sequence encoding a CD28 HTM.

SEQ ID NO: 60 is a nucleotide sequence encoding a CD8 HTM

SEQ ID NO: 61 is a codon-optimized nucleotide sequence encoding a CD8 HTM.

SEQ ID NO: 62 is a nucleotide sequence encoding a 4-1BB.

SEQ ID NO: 63 is a codon-optimized nucleotide sequence encoding a 4- IBB.

SEQ ID NO: 64 is a nucleotide sequence encoding a CD28 co-stimulatory domain.

SEQ ID NO: 65 is a nucleotide sequence encoding a CD3^ signaling domain.

SEQ ID NO: 66 is a nucleotide sequence encoding CD3^(1 XX).

SEQ ID NO: 67 is a nucleotide sequence encoding a T2A site.

SEQ ID NO: 68 is a nucleotide sequence encoding a GM-CSF leader sequence.

SEQ ID NO: 69 is a nucleotide sequence encoding truncated EGFR (tEGFR).

SEQ ID NO: 70 is the amino acid sequence of a CD8 leader sequence.

SEQ ID NO: 71 is the amino acid sequence of a 3A11 scFv (VL-linker VH).

SEQ ID NO: 72 is the amino acid sequence of a 3A11 scFv (VH-linker-VL).

SEQ ID NO: 73 is the amino acid sequence of a MGA271 scFv ( VL-linker- VH).

SEQ ID NO: 74 is the amino acid sequence of a CD28HTM.

SEQ ID NO: 75 is the amino acid sequence of a CD8HTM.

SEQ ID NO: 76 is the amino acid sequence of 4-1BB.

SEQ ID NO: 77 is the amino acid sequence of a CD28 co-stimulatory domain.

SEQ ID NO: 78 is the amino acid sequence of a CD3^ signaling domain

SEQ ID NO: 79 i: s the amino acid sequence of CD3 (1XX).

SEQ ID NO: 80 is the amino acid sequence of a T2A site.

SEQ ID NO: 81 is the amino acid sequence of a GM-CSF leader sequence.

SEQ ID NO: 82 i: s the amino acid sequence of tEGFR.

SEQ ID NO: 83 is a codon-optimized nucleotide sequence encoding CD8 HTM.

SEQ ID NO: 84 is a codon-optimized nucleotide sequence encoding 4-1BB.

SEQ ID NOs: 85-86 are codon-optimized nucleotide sequences encoding CD3^.

SEQ ID NO: 87 is a codon-optimized nucleotide sequence encoding CD3^(1 XX).

SEQ ID NOs: 88-89 are barcode sequences.

SEQ ID NO: 90 is a nucleotide sequence encoding CD276.8HTM.BBz CAR.

SEQ ID NO: 91 is the amino acid sequence of CD276.8HTM.BBz CAR.

SEQ ID NO: 92 is a nucleotide sequence encoding CD276.8HTM.BBz(lXX) CAR.

SEQ ID NO: 93 is the amino acid sequence of CD276.8HTM.BBz(lXX) CAR. DETAILED DESCRIPTION

I. Introduction

Second generation CAR designs allow for extensive customization with selection of singlechain variable fragment (scFv) binder, hinge transmembrane (HTM), and intracellular co-stimulatory domains (CSDs) which collectively determine the activation threshold and signaling properties of a CAR (Feucht et al., Nat Med 25:82-88, 2019; Heitzeneder et al., Cancer Cell 40:53-69, 2022; Majzner et al., Cancer Discov 10:702-723, 2020; Rafiq et al. , Nat Rev Clin Oncol 17:147-167, 2020). An FGFR4-targeting CAR, based on a second-generation CAR design with a CD8 HTM and a 4-1BB CSD, is being developed for a phase I clinical trial (Tian et al., Cell Rep Med 4(10): 101212, 2023). Despite its potent antitumor activities, the therapeutic effect of this FGFR4 CAR in stress conditions, such as with a low infused CAR T-cells to tumor burden ratio, is unclear. The present disclosure examined the efficacy of the previously reported FGFR4 CAR T-cells under stress conditions and sought to improve their potency by replacing HTM and CSD domains with those of CD28. Replacing the 4- IBB with a CD28 CSD resulted in a more rapid tumor eradication.

Despite improvement of single CAR's potency, multi-targeting CARs potentially prevent tumor escape resulting from heterogeneous expression of antigens or loss of target expression under selection pressure (Tian et al., J Clin Invest 132(16):el55621 , 2022; Hirabayashi et al., Nat Cancer 2:904-918, 2021), a phenomenon observed after CAR therapies targeting single antigen (Ruella et al., Comput Struct Biotechnol J 14:357-362, 2016; Majzner and Mackall, Cancer Discov 8: 1219-1226, 2018). However, the optimal strategy for developing dual-targeting CARs has not yet been established, especially for solid tumors. Bivalent constructs are more potent and relatively easy to use compared to co-transduction or co-infusion (Tian et al., J Clin Invest 132(16):el55621, 2022; Fernandez de Larrea et al., Blood Cancer Discov 1:146-154, 2020; Shalabi et al., Blood 140:451-463, 2022). It has been demonstrated that a bicistronic “OR” CAR construct encoding two single targeting CAR cassettes performed better than a bivalent CAR construct containing two tandem single-chain variable fragments (scFvs), partly due to the maintenance of structural integrity of assembled antigenbinding moieties (Tian et al., J Clin Invest 132( 16) :el55621 , 2022; Shalabi et al., Blood 140:451-463, 2022). Furthermore, the optimal combination of T-cell CSDs from two different CAR cassettes in a bicistronic CAR construct needs to be tested, despite a common perception in the field that dual CD28 and 4-1BB co-stimulation may provide an ideal T-cell activation signaling (Lavoie el al., Cancers (Basel) 13(18):4528, 2021; Fernandez de Larrea et al., Blood Cancer Discov 1: 146-154, 2020; Shalabi et al., Blood 140:451-463, 2022). Therefore, disclosed herein are bicistronic CARs (BiCisCAR) against both FGFR4 and CD276, having different HTM and CSDs, and a comparison of their therapeutic efficacy in several RMS mouse models. A BiCisCAR using both CD28 and 4- IBB CSDs demonstrated synergistic cytokine production and cytotoxicity through a robust activation of downstream T-cell receptor (TCR) signaling pathways. T-cells expressing this BiCisCAR also showed persistence and limited exhaustion, indicating that this bicistronic CAR engineering strategy not only addresses heterogeneous expression of target antigens but also provides a robust CAR for treating RMS.

To further improve the therapeutic efficacy of the single and bicistronic CARs, modifications were made to the immunoreceptor tyrosine-based activation motif (IT AM) domains of CD3^. In some aspects, tyrosine (Y) to phenylalanine (F) substitutions were introduced into the second and third ITAMs (see FIGS. 18-19).

II. Abbreviations

ACC adrenocortical carcinoma

ADT antibody-derived tag

ARMS alveolar rhabdomyosarcoma

BiCisCAR bicistronic chimeric antigen receptor

Bp base pair

CAR chimeric antigen receptor

CDX cell line derived xenograft

ChIP chromatin immunoprecipitation

CM central memory

CSD co-stimulatory domain

DSRCT desmoplastic small round cell tumor

E:T effector to target ratio

EM effector memory

ERMS embryonal rhabdomyosarcoma

FACS fluorescence activated cell sorting

FBS fetal bovine serum

FGFR4 fibroblast growth factor receptor 4

FN-RMS fusion-negative rhabdomyosarcoma

FOXO 1 forkhead box protein 01

FP-RMS fusion-positive rhabdomyosarcoma

GvHD graft versus host disease

HBE hepatoblastoma

HCC hepatocellular carcinoma

HTM hinge and transmembrane

IFN interferon

IL interleukin

I.M. intramuscular I.P. intraperitoneal

ITAM immunoreceptor tyrosine -based activation motif

IU international unit

KO knockout

LAG-3 lymphocyte-activation gene 3

MFI mean fluorescent intensity

MOI multiplicity of infection

MYODI myogenic differentiation 1

NSG NOD scid gamma

PAX-3 paired box gene 3

PBMC peripheral blood mononuclear cell

PD-1 programmed cell death protein 1

PDX patient-derived xenograft

PE phycoerythrin

RBC red blood cell

RMS rhabdomyosarcoma scFv single-chain variable fragment

SCM stem cell memory

TCR T cell receptor

TIL tumor infiltrating lymphocyte

TIM-3 T cell immunoglobulin and mucin-domain containing 3

TNF tumor necrosis factor

VH variable heavy

VL variable light

WNN weighted nearest neighbors

YST yolk sac tumor

III. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

4-1BB: A co-stimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4-1BB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response. An exemplary amino acid sequence of 4- IBB is set forth herein as SEQ ID NO: 76.

Administration: To provide or give a subject an agent, such as a CAR provided herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

Adrenocortical carcinoma (ACC): A type of cancer in which malignant cells form in the outer layer of the adrenal gland. ACC is also referred to as cancer of the adrenal cortex.

Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen (such as FGFR4 or CD276). Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) domain and the variable light (VL) domain, respectively. Together, the VH domain and the VL domain are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn/aligncdr/abrsa.php).

A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.

A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies.

A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species (such as mouse).

A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

Binding affinity: Affinity of an antibody (or CAR) for an antigen. In one aspect, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another aspect, binding affinity is measured by an antigen/antibody dissociation rate. In another aspect, a binding affinity is measured by a competition radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In other aspects, antibody affinity is measured by flow cytometry, surface plasmon reference, or biolayer interferometry (BLI). An antibody that “specifically binds” an antigen (such as FGFR4) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens. In some examples, a CAR (such as an FGFR4-targeted CAR provided herein) specifically binds to a target (such as a FGFR4) with a binding constant that is at least 10³ M^-1 greater, 10⁴M ’ greater or 10⁵ M’¹ greater than a binding constant for other molecules in a sample or subject. In some examples, a CAR has an equilibrium constant (KD) of 5 llM or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a CAR binds to a target, such as FGFR4, with a binding affinity of at least about 1 x 10‘⁶ M, at least about 0.5 x 10‘⁶ M, at least about 1 x 10‘⁷ M, at least about 0.5 x 10‘⁷ M, at least about 1 x 10'⁸ M, at least about 0.5 x 10'⁸ M, at least about 1 x 10'⁹ M, at least about 0.5 x 10'⁹ M, or at least about 0.1 x 10'⁹. In certain aspects, a specific binding agent that binds to its target has a dissociation constant (Kd) of <1000 nM, <750 nM, 500 nM, <250 nM, <100 nM, <50 nM, <25 nM, <10 nM, <5 nM, <2.5 nM, <1 nM, <0.5 nM, <0.25 nM, <0.01 nM, or <0.001 nM (e.g., 10'⁶M or less, e.g., from 10'⁶M to 10 ¹⁰M, e.g., from 10‘¹⁰ M to 10'¹² M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs), which is based on BLI technology. In some examples, Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.).

Brain cancer or tumor: A type of cancer or tumor that develops from brain tissue. Brain cancers include, but are not limited to, neuroblastoma, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytoma, choroid plexus carcinoma, ependymoma and pineoblastoma.

Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma and mucinous carcinoma. Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2/neu protein. Triple negative breast cancer is also called ER-negative PR-negative HER2/neu-negative breast cancer.

CD276: An immune checkpoint molecule that is expressed in the stroma of most or all solid tumors and may also be expressed by solid tumor cells. This protein is a member of the B7 superfamily of co-stimulatory molecules. CD276 is also known as B7H3.

CD276-expressing cancer: A cancer that expresses or overexpresses CD276. Examples of CD276-expressing cancers include, but are not limited to, liver cancers (such as hepatocellular carcinoma), pancreatic cancers, kidney cancers, bladder cancers, cervical cancers, esophageal cancers, prostate cancers, breast cancers, ovarian cancers, colon cancers, lung cancers, brain cancers (such as neuroblastoma or glioblastoma), pediatric cancers (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing’s sarcoma), melanoma and mesothelioma (see, for example, Seaman et al., Cancer Cell 31 (4) :501-505, 2017). In some instances, a CD276-expressing cancer refers to a cancer in which CD276 is expressed in the tumor stroma, and may also be expressed by the tumor cells.

Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a FGFR4-expressing tumor, CD276-expressing tumor, or both. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al. , Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.}'. Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby- Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody or CAR that binds FGFR4, CD276, or both, used in combination with a radioactive or chemical compound. In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as anti-PDl or anti-PDL1 (<?.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (<?.g., bevacizumab), or combinations thereof (<?.g., anti-PD-1 and anti-CTLA-4).

Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a scFv) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3 . Typically, CARs are comprised of an antigen-binding moiety, a hinge and transmembrane domain (HTM) and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3L or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, CD8, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and/or DAP10. In some examples, the CAR is multispecific (such as bispecific) or bicistronic. A multispecific CAR is a single CAR molecule comprised of at least two antigen-binding domains (such as scFvs) that each bind a different antigen or a different epitope on the same antigen (see, for example, US 2018/0230225). For example, a bispecific CAR refers to a single CAR molecule having two antigen-binding domains that each bind a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules that are linked by a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both of the antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO/2018/213337). Colon cancer: A type of cancer that develops in the colon or the rectum. The most common type of colon cancer (also known as “colorectal cancer”) is colorectal adenocarcinoma, which accounts for approximately 95% of all colon cancers. Adenocarcinomas develop in the cells lining the inside of the colon and/or rectum. Other types of colorectal cancers include gastrointestinal carcinoid tumors, metastatic colorectal cancer, primary colorectal lymphoma (a type of nonHodgkin’s lymphoma), gastrointestinal stromal tumors (classified as a sarcoma and arising from interstitial cells of Cajal), leiomyosarcoma (arising from smooth muscle cells) and colorectal melanoma.

Complementarity determining region (CDR): Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site. The light and heavy chains of an Ig each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2 and HCDR3, respectively.

Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the activity or affinity of a protein, such as the affinity of an antibody or CAR to FGFR4, CD276, or both. For example, a monoclonal antibody or CAR that specifically binds FGFR4, CD276, or both can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the FGFR4 polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody or CAR specifically binds FGFR4, CD276, or both. Non-conservative substitutions are those that reduce an activity or binding to FGFR4, CD276, or both.

Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Degenerate variant: A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

Desmoplastic small round cell tumor (DSRCT): A type of tumor that grows in the abdomen and pelvic area. DSRCT is typically a soft tissue sarcoma, which is a type of cancer that forms in the connective tissue of the body, such as fat, muscles, tendons, blood vessels and nerves. This type of cancer is rare and typically occurs in young white males between the ages of 10 and 30.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a polypeptide, such as FGFR4.

Fibroblast growth factor receptor (FGFR): A family of tyrosine kinase receptors activated by fibroblast growth factors (FGF), comprising extracellular immunoglobulin-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain. The family includes at least four members: FGFR1, FGFR2, FGFR3, and FGFR4. FGFR4 is involved in the regulation of several pathways, including cell proliferation, cell differentiation, cell migration, lipid metabolism, bile acid biosynthesis, vitamin D metabolism, glucose uptake, and phosphate homeostasis. The FGFR4 protein is composed of an extracellular region having three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment, and a cytoplasmic tyrosine kinase domain. Exemplary sequences for FGFR4 are publicly available such as under NCBI Gene ID 2264.

FGFR4-expressing cancer: Any type of cancer that expresses or overexpresses FGFR4. Exemplary FGFR4-expressing cancers include, but are not limited to, rhabdomyosarcoma (RMS; such as alveolar RMS or embryonal RMS), lung cancer, liver cancer (such as hepatocellular carcinoma or hepatoblastoma), breast cancer, pancreatic cancer, prostate cancer, desmoplastic small round cell tumor, adrenocortical carcinoma, and gastric adenocarcinoma.

Framework region: Amino acid sequences interposed between CDRs. Framework regions include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.

Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins.

Gastric adenocarcinoma: An adenocarcinoma of the stomach (also known as stomach cancer). Gastric adenocarcinoma begins in the mucus-producing cells in the innermost lining of the stomach.

Heterologous: Originating from a separate genetic source or species.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an “antigenspecific response”). In one aspect, an immune response is a T cell response, such as a CD4⁺ response or a CD8⁺ response. In another aspect, the response is a B cell response, and results in the production of specific antibodies.

Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antibodies or CARs) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a drug or a detectable label. In some aspects herein, the linker connects a VH domain to a VL domain of an scFv (such as an scFv targeting FGFR4 or CD276).

Liver cancer: Any type of cancer occurring in liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC), which develops in hepatocytes. Other types of liver cancer include cholangiocarcinoma, which develops in the bile ducts; liver angiosarcoma, which is a rare form of liver cancer that begins in the blood vessels of the liver; and hepatoblastoma (HBL), which is a very rare type of liver cancer found most often in children.

Lung cancer: Cancer that forms in tissues of the lung, usually in the cells lining air passages. Most cancers that begin in the lung are carcinomas. The two primary types of lung carcinoma are small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Subclasses of NSCLC include adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma.

Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.”

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Ovarian cancer: Cancer that forms in tissues of the ovary. Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Another type of ovarian cancer is stromal cell cancer, which originates in cells that release hormones and connect the different structures of the ovaries.

Pancreatic cancer: A disease in which malignant cells are found in the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer. The vast majority (-94%) of pancreatic cancers are exocrine tumors. Exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (IPMN), and mucinous cystadenocarcinoma. In some aspects, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also referred to as islet cell tumors, are classified by the type of hormones they produce. Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide) and nonfunctional islet cell tumor.

Pediatric cancer: A cancer that develops in children ages 0 to 14. The major types of pediatric cancers include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing’s sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors (such as neuroblastoma and medulloblastoma), Wilm’s tumor, non-Hodgkin lymphoma, and retinoblastoma.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the CARs and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

Prostate Cancer: A malignant tumor, generally of glandular origin, of the prostate. Prostate cancers include adenocarcinomas and small cell carcinomas. Many prostate cancers express prostate specific antigen (PSA).

Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

Rhabdomyosarcoma (RMS): A soft tissue malignant tumor of skeletal muscle origin. The most common primary sites for rhabdomyosarcoma are the head and neck (e.g., parameningeal, orbit, pharyngeal, etc.), the genitourinary tract, and the extremities. Other less common primary sites include the trunk, chest wall, the abdomen (including the retroperitoneum and biliary tract), and the perineal/anal region. There are at least two types of RMS; the most common forms are alveolar RMS (ARMS) and embryonal histological RMS (ERMS). Approximately 20% of children with rhabdomyosarcoma have the ARMS subtype. An increased frequency of this subtype is noted in adolescents and in patients with primary sites involving the extremities, trunk, and perineum/perianal region. ARMS is associated with chromosomal translocations encoding a fusion gene involving FKHR on chromosome 13 and members of the PAX family. The embryonal subtype is the most frequently observed subtype in children, accounting for approximately 60-70% of rhabdomyosarcomas of childhood. Tumors with embryonal histology typically arise in the head and neck region or in the genitourinary tract, although they may occur at any primary site. ERMS is characterized by a younger age at diagnosis, loss of heterozygosity, and altered genomic imprinting.

Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well-known. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Homologs and variants of a VL or a VH of an antibody that specifically binds FGFR4, CD276, or both, or a fragment thereof are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. A skilled person will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals, such as dogs, cats, horses, pigs, and cows. In some aspects, a subject is a human with an FGFR4-expressing cancer. In some aspects, a subject is a human with a CD276-expressing cancer. In some aspects, a subject is a human with a cancer that expresses both FGFR4 and CD276.

Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount of a CAR or bicistronic CAR necessary to inhibit or suppress growth of a tumor. In one aspect, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor (such as an FGFR4-expressing cancer, CD276-expressing cancer, or both), such as reduce a tumor size and/or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and/or reduce the number and/or size/volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size/volume/number prior to treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as an FGFR4-expressing cancer, CD276-expressing cancer, or both), such as increase survival time by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, for example as compared to a survival time compared to a subject with no treatment or a different treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as an FGFR4-expressing cancer, CD276-expressing cancer, or both), such as increase survival time by at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months, for example as compared a survival time compared to a subject with no treatment or a different treatment. In some aspects, combinations of these affects are achieved. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.

Yolk sac tumor (YST): A type of cancer that begins in germ cells. YSTs occur most often in the ovary or testicle but may also occur in other parts of the body, such as the chest, abdomen, and brain. These tumors are the most common malignant germ cell tumor in children.

IV. Antibody, CAR and BiCisCAR Sequences for Targeting FGFR4 and CD276

Disclosed herein are chimeric antigen receptors (CARs) and bicistronic CARs (BiCisCARs) that target FGFR4, CD276 (also known as B7-H3), or both. The FGFR4-targeted CARs have an antigen-binding domain based on antibody FGFR4-specific monoclonal antibody 3A11 (see, e.g., WO 2017/049296). The CD276-targeted CARs have an antigen-binding domain based on CD276-specific antibody MGA271, also known as enoblituzumab (see, e.g., WO 2021/207171). The amino acid sequences of the VH and VL domains of the 3 All and MGA271 antibodies, along with their respective CDR sequences (as determined by IMGT), are provided below. Also provided below are nucleotide and amino acid sequences of exemplary FGFR4 CAR constructs, CD276 CAR constructs, and FGFR4/CD276 BiCisCAR constructs.

A. Antibody sequences

VH domain of 3A11 (SEQ ID NO: 1)

QVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAY NQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYYGSDYDYWGQGTTLTVSS

VL domain of 3A11 (SEQ ID NO: 2)

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGV PDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK Table 1. CDR sequences of antibody 3A11 (IMGT)

VH domain of MGA271 (SEQ ID NO: 9)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYAD

TVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS

VL domain of MGA271 VL (SEQ ID NO: 10)

DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS

GSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK

Table 2. CDR sequences of antibody MGA271 (IMGT)

B. CAR and BiCisCAR sequences

For each of the CAR and BiCisCAR nucleic acid sequences listed below, the name of the construct and the 5' to 3' order of the components of the CAR/BiCisCAR arc provided. The tables following the amino acid sequences of each construct list the nucleotide and amino acid positions of each component of the construct.

FGFR4(LH).2

CD8 Leader- 3 leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcagcggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccat gtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattcct tgctagtaacagtggcctttattattttctgggtgaagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcagacg actcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgcgaactgcgcgtcaagttctcacggtccgccgacgcc cccgcatatcaacagggccagaatcagctctacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcggacg cgacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaacgaactccagaaagacaagatggcggaagcctac tcagaaatcgggatgaagggagagcggaggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacctacg atgccttgcatatgcaagcactcccaccccggtctagagctaaacgctctgggtctggtgaaggacgaggtagccttcttacgtgcggagacgtgg aggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgattccaaggaaggtttgcaatg gaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtataagtggagatcttcacattttg ccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgttaaagaaataacggggtttttg ctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaagcagcatggtcaattctccct tgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataagcggcaacaagaatttgtgcta tgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgagaacagctgcaaagccaccgg ccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaacgtctcaagaggccgcgaat gcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccctgaatgtttgccccaggctat gaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtgaaaacctgcccggccggagtt atgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgtacatatgggtgtaccggtcctg gacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctcttggtggttgctctcggcatag gtctttttatgtaa

FGFR4(LH).28HTM.BBz CAR (SEQ ID NO: 18)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSR AKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSI NATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTD LHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKL FGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL EGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENN TLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGL FM

Table 3. FGFR4.28HTM.BBz CAR components

FGFR4(LH).28HTM.BBz/CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 19)

CD8 Leader- 3A11 VL-VH-CD28HTM-4-lBB-CD3^-T2A-GM-CSF leader-MGA271 VL-VH-

CD8HTM-4-lBB-CD3 atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccatta tccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgct attccttgctagtaacagtggcctttattattttctgggtgaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC

GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG

CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTATAACGAGCTGCAGAAGGACAAG

ATGGCAGAAGCCTATAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC

ATGACGGCCTCTATCAGGGCCTGAGCACCGCCACCAAAGATACCTATGATGCCCTCCACA

TGCAGGCTCTGCCTCCCAGATAA

FGFR4.28HTM.BBz/CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 20)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL

KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG

GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK

QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN

YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF

WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE

EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP

QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSR

AKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVG

DRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQ

PEDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGG

SLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNS

LYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIA

SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE

EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL

YQGLSTATKDTYDALHMQALPPR

Table 4. FGFR4.28HTM.BBz/CD276.8HTM.BBz BiCisCAR components FGFR4(LH).2

CD8 Leader- 3 leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcagcggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccat gtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattcct tgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgg gcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCA CATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCgaaggacgaggtagcct tcttacgtgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgatt ccaaggaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtata agtggagatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgtta aagaaataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaa gcagcatggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataag cggcaacaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgag aacagctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaa cgtctcaagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccc tgaatgtttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtga aaacctgcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgta catatgggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctct tggtggttgctctcggcataggtctttttatgtaa

FGFR4.28HTM.28z (1XX) CAR (SEQ ID NO: 22)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRA KRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSIN ATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDL HAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNT LVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLF M Table 5. FGFR4.28HTM.28z(lXX) CAR components

CD276.8HTM.BB CAR (SEQ ID NO: 23)

CD8 leader-MGA271 VL-VH-CD8HTM-4-1BB-T2A-GM-CSF leader-tEGFR atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcccggccgGATATCCAGCTGACCCAGTC

CCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCA

GAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGGCGC

TGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGTTCTCCGGCTCCGGCTC

TGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTA

CTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCA

AGGGAGGCGGAGGTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAGGTGCAGCT

GGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC

CTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGG

ACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACACCGT

GAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGA

ACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAATATC

TACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTCCGCG

GCCGCAaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgccctgaggcgtgcc ggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgacatctacatctgggcgcccttggccgggacttgtggggtcc ttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactca agaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgTCTCGGGCTAAACGCTCTGGAAG

CGGCGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTA

TGCTGCTGCTTGTTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGAT

TCCAAGGAAGGTTTGCAATGGAATCGGTATAGGGGAGTTTAAGGATTCACTTAGCATAA

ACGCTACTAATATTAAACACTTCAAAAACTGTACGAGTATAAGTGGAGATCTTCACATTT

TGCCGGTTGCATTCCGAGGCGATTCATTCACCCACACGCCACCGCTTGACCCACAAGAAT

TGGATATTCTTAAAACCGTTAAAGAAATAACGGGGTTTTTGCTCATTCAAGCGTGGCCAG

AAAATCGCACTGACCTCCATGCTTTCGAGAACCTGGAGATTATAAGAGGACGAACTAAG

CAGCATGGTCAATTCTCCCTTGCTGTGGTCAGCCTGAACATCACCAGTCTTGGTTTGCGGT

CCCTCAAGGAAATTTCAGATGGAGATGTCATCATAAGCGGCAACAAGAATTTGTGCTAT

GCAAATACCATAAACTGGAAAAAACTGTTTGGCACTTCCGGCCAGAAAACCAAGATTAT

TTCAAATCGGGGCGAGAACAGCTGCAAAGCCACCGGCCAGGTTTGTCATGCCTTGTGCTC

TCCGGAGGGCTGTTGGGGGCCAGAACCCAGGGACTGCGTCAGTTGCAGAAACGTCTCAA

GAGGCCGCGAATGCGTTGACAAGTGTAACCTCCTTGAGGGCGAGCCACGAGAGTTTGTT

GAGAACAGCGAGTGTATACAATGTCACCCTGAATGTTTGCCCCAGGCTATGAATATAACC TGCACAGGCCGCGGGCCTGATAACTGCATCCAGTGTGCTCATTACATAGATGGACCTCAC TGTGTGAAAACCTGCCCGGCCGGAGTTATGGGAGAAAACAACACTCTGGTGTGGAAATA

CGCTGATGCAGGCCACGTGTGCCACCTTTGTCACCCGAATTGTACATATGGGTGTACCGG

TCCTGGACTTGAAGGTTGCCCTACCAATGGCCCTAAAATACCCAGTATCGCAACTGGCAT

GGTAGGCGCTCTTCTCTTGCTCTTGGTGGTTGCTCTCGGCATCGGTCTTTTTATGTAA

CD276.8HTM.BB CAR (SEQ ID NO: 24)

MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPG

KAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLE

IKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKG

LEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYG

SRLDYWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCELSRAKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGE

FKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW

PENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTI

NWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECV

DKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGV

MGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVV

ALGIGLFM

Table 6. CD276.8HTM.BB CAR components

FGFR4(LH).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR (SEQ ID NO: 25)

CD8 Leader- 3A11 leader-MGA271 VL-

VH-CD8HTM-4-l atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccatta tccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgct attccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgc cccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCT TCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC

AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTTTAACGAGCTGCAGAAGGACAAG ATGGCAGAAGCCTTTAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC ATGACGGCCTCTTTCAAGGCCTGAGCACCGCCACCAAAGATACCTTTGATGCCCTCCACA TGCAGGCTCTGCCTCCCAGATAA

FGFR4.28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR (SEQ ID NO: 26)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL

KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG

GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK

QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN

YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF

WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD

FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP

QEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRA

KRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGD

RVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGS

LRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSL

YLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE

YDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQ

GLSTATKDTFDALHMQALPPR Table 7. FGFR4.28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR components

FGFR4(LH).28HTM.28z/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 27)

CD8 Leader- 3A11 VL-VH-CD28HTM-CD28-CD3^-T2A-GM-CSF leader-MGA271 VL-VH-

CD8HTM-4-1BB atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccatta tccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgct attccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgc cccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGC AGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTT GTTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATA TCCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCA CATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGC AAGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGG TTCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAG GACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGC ACCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTG CAGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGC GCCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGC AAGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGAC ACCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCA GATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGA

ATATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTT CCGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtag gcccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctg ctctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacag gaggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgtaa

FGFR4.28HTM.28z/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 28)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD

FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSR AKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVG DRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGG SLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNS

LYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Table 8. FGFR4.28HTM.28z/CD276.8HTM.BB BiCisCAR components FGFR4(LH).2 NO: 29)

CD8 Leader- 3 SF leader-MGA271 VL-

VH-CD8HTM atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccatta tccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgct attccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgc cccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCT TCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgtaa

FGFR4.28HTM.28z(lXX)/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 30)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKTSRVEAEDLGVYYCWQGTHFPQTFGG GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRA KRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGD RVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGS LRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSL

YLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Table 9. FGFR4.28HTM.28z(lXX)/CD276.8HTM.BB BiCisCAR

FGFR4(LH).8HTM.BBz (SEQ ID NO: 31)

CD8 Leader- 3A11 VL-VH-CD8HTM-4-lBB-CD3z-T2A-GM-CSF leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcagcggccgcaactaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccct ctccttgcgccccgaggcttgccgcccggccgcgggtggagccgtgcatacccgggggctggactttgcctgcgacatctacatttgggccccgc tggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctttactgcaagaggggccggaagaagctgctttacatcttcaagcagccgtt catgcggcccgtgcagacgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgcgaactgcgcgtcaagtt ctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctctacaacgagctgaacctgggaaggagagaggagtacgacgtgct ggacaagcgacgcggacgcgacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaacgaactccagaaaga caagatggcggaagcctactcagaaatcgggatgaagggagagcggaggaggggaaagggtcacgacgggctgtaccagggactgagcacc gccactaaggatacctacgatgccttgcatatgcaagcactcccaccccggtctagagctaaacgctctgggtctggtgaaggacgaggtagcctt cttacgtgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgatt ccaaggaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtata agtggagatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgtta aagaaataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaa gcagcatggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataag cggcaacaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgag aacagctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaa cgtctcaagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccc tgaatgtttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtga aaacctgcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgta catatgggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctct tggtggttgctctcggcataggtctttttatgtaa

FGFR4(LH).8HTM.BBz (SEQ ID NO: 32)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN YYGSDYDYWGQGTTLTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL

DFACDTYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYTFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SRAKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDS LSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENR

TDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWK KLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGEN NTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGI GLFM

Table 10. FGFR4(LH).8HTM.BBz

FGFR4(LH).28HTM.28z (SEQ ID NO: 33)

CD8 Leader- 3A11 VL-VH-CD28HTM- CD28-CD3z-T2A-GM-CSF leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcagcggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccat gtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattcct tgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgg gcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA CATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCgaaggacgaggtagcct tcttacgtgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgatt ccaaggaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtata agtggagatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgtta aagaaataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaa gcagcatggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataag cggcaacaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgag aacagctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaa cgtctcaagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccc tgaatgtttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtga aaacctgcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgta catatgggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctct tggtggttgctctcggcataggtctttttatgtaa

FGFR4(LH).28HTM.28z (SEQ ID NO: 34)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSR AKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSI NATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTD LHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKL FGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL EGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENN TLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGL FM*

Table 11. FGFR4(LH).28HTM.28z

FGFR4(LH).28HTM.28z/CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 35)

CD8 Leader-3 All VL-VH-CD28HTM-CD28-CD3z-T2A-GM-CSF leader-MGA271 VL-VH-

CD8HTM-4-lBB-CD3z atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggcccgatgttgtgatgacccagactccacttactttgtcg gttaccattggacaaccagcctccatctcttgca gtc agtcagagcctcttagat gtgatggagagacatatttga ttg ttgttaaagaggcca ggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtggatcagggacagatttcacact gaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaa atcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttcaactggagcagtctggggctgagctggtcaggc ctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatggcctgga atggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctccagcaca gcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactactggggcc aaggcaccactctcacagtctcctcaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccatta tccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgct attccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgc cccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTATAACGAGCTGCAGAAGGACAAG ATGGCAGAAGCCTATAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC

ATGACGGCCTCTATCAGGGCCTGAGCACCGCCACCAAAGATACCTATGATGCCCTCCACA

TGCAGGCTCTGCCTCCCAGATAA

FGFR4(LH).28HTM.28z/CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 36)

MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLL

KRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGG

GTKLEIKGGGGSGGGGSGGGGSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVK

QTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGN

YYGSDYDYWGQGTTLTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF

WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD

FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP

QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSR

AKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVG

DRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQ

PEDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGG

SLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNS

LYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIA

SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE

EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR

Table 12. FGFR4(LH).28HTM.28z /CD276.8HTM.BBz BiCisCAR components

FGFR4(HL).8HTM.BBz (SEQ ID NO: 37)

CD8 Leader- 3A11 VH-VL -CD8HTM-4-lBB-CD3z-T2A-GM-CSF leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaagcggccgcaactaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccct ctccttgcgccccgaggcttgccgcccggccgcgggtggagccgtgcatacccgggggctggactttgcctgcgacatctacatttgggccccgc tggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctttactgcaagaggggccggaagaagctgctttacatcttcaagcagccgtt catgcggcccgtgcagacgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgcgaactgcgcgtcaagtt ctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctctacaacgagctgaacctgggaaggagagaggagtacgacgtgct ggacaagcgacgcggacgcgacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaacgaactccagaaaga caagatggcggaagcctactcagaaatcgggatgaagggagagcggaggaggggaaagggtcacgacgggctgtaccagggactgagcacc gccactaaggatacctacgatgccttgcatatgcaagcactcccaccccggtctagagctaaacgctctgggtctggtgaaggacgaggtagcctt cttacgtgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgatt ccaaggaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtata agtggagatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgtta aagaaataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaa gcagcatggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataag cggcaacaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgag aacagctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaa cgtctcaagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccc tgaatgtttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtga aaacctgcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgta catatgggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctct tggtggttgctctcggcataggtctttttatgtaa

FGFR4(HL).8HTM.BBz (SEQ ID NO: 38)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSR AKRSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSI NATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTD LHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKL FGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL EGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENN TLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGL FM

Table 13. FGFR4(HL).8HTM.BBz CAR components

FGFR4(HL).28HTM.BBz CAR (SEQ ID NO: 39)

CD8 Leader- 3A11 VH-VL-CD28HTM-4-lBB-CD3z-T2A-GM-CSF leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaagcggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatg tgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattcctt gctagtaacagtggcctttattattttctgggtgaagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcagacga ctcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgcgaactgcgcgtcaagttctcacggtccgccgacgccc ccgcatatcaacagggccagaatcagctctacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcggacgc gacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaacgaactccagaaagacaagatggcggaagcctact cagaaatcgggatgaagggagagcggaggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacctacga tgccttgcatatgcaagcactcccaccccggtctagagctaaacgctctgggtctggtgaaggacgaggtagccttcttacgtgcggagacgtgga ggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgattccaaggaaggtttgcaatgg aatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtataagtggagatcttcacattttgc cggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgttaaagaaataacggggtttttgc tcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaagcagcatggtcaattctccctt gctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataagcggcaacaagaatttgtgctat gcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgagaacagctgcaaagccaccgg ccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaacgtctcaagaggccgcgaat gcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccctgaatgtttgccccaggctat gaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtgaaaacctgcccggccggagtt atgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgtacatatgggtgtaccggtcctg gacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctcttggtggttgctctcggcatag gtctttttatgtaa

FGFR4(HL).28HTM.BBz CAR (SEQ ID NO: 40)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAK

RSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINA

TNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH

AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFG

TSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEG

EPREFVENSECIQCHPECEPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTE

VWKYADAGHVCHECHPNCTYGCTGPGEEGCPTNGPKIPSIATGMVGAEEEEEVVAEGIGEF M*

Table 14. FGFR4(HL).28HTM.BBz CAR components

FGFR4(HL).28HTM.BBz/CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 41)

CD8 Leader- 3A11 VH-VL -CD28HTM-4-lBB-CD3z-T2A-GM-CSF leader-MGA271 VL-VH-

CD8HTM-4-lBB-CD3z atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattat ccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgcta ttccttgctagtaacagtggcctttattattttctgggtgaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaa ctactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTATAACGAGCTGCAGAAGGACAAG ATGGCAGAAGCCTATAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC ATGACGGCCTCTATCAGGGCCTGAGCACCGCCACCAAAGATACCTATGATGCCCTCCACA TGCAGGCTCTGCCTCCCAGATAA

FGFR4(HL).28HTM.BBz/CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 42)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT

PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY

GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL

DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW

VLVVVGGVLACYSLLVTVAFI1FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAK

RSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDR

VTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSL

RLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLY

LQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQ

PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK

QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY

DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ

GLSTATKDTYDALHMQALPPR

Table 15. FGFR4(HL).28HTM.BBz/CD276.8HTM.BBz BiCisCAR components

FGFR4(HL).28HTM.28z (SEQ ID NO: 43)

CD8 Leader- 3A11 VH-VL -CD28HTM- CD28-CD3z-T2A-GM-CSF leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccglctattactgtacaagagggaattactacgglagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaagcggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatg tgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattcctt gctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggg cccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA CATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCgaaggacgaggtagcct tcttacgtgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgatt ccaaggaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtata agtggagatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgtta aagaaataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaa gcagcatggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataag cggcaacaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgag aacagctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaa cgtctcaagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccc tgaatgtttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtga aaacctgcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgta catatgggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctct tggtggttgctctcggcataggtctttttatgtaa

FGFR4(HL).28HTM.28z (SEQ ID NO: 44)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW

VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAK

RSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINA

TNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH

AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFG

TSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEG

EPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTL

VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLF

M*

Table 16. FGFR4(HL).28HTM.28z components

FGFR4(HL).28HTM.28z (1XX) CAR (SEQ ID NO: 45)

CD8 Leader-3A11 VH-VL -CD28HTM-CD28-CD3z(lXX)-T2A-GM-CSF leader-tEGFR atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaagcggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatg tgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattcctt gctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggg cccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCA CATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCgaaggacgaggtagcct tcttacgtgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgatt ccaaggaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtata agtggagatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgtta aagaaataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaa gcagcatggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataag cggcaacaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgag aacagctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaa cgtctcaagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccc tgaatgtttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtga aaacctgcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgta catatgggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctct tggtggttgctctcggcataggtctttttatgtaa

FGFR4(HL).28HTM.28z (1XX) CAR (SEQ ID NO: 46)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL DSDGETYLNWLLKRPGQSPKRL1YLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRAKR SGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINAT NIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHA FENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTS GQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEP REFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVW KYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

Table 17. FGFR4(HL).28HTM.28z (1XX) CAR components FGFR4(HL).28HTM.28z /CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 47)

CD8 Leader- 3A11 VH-VL -CD28HTM-CD28-CD3z-T2A-GM-CSF leader-MGA271 VL-VH-

CD8HTM-4-lBB-CD3z atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattat ccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgcta ttccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgcc ccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTATAACGAGCTGCAGAAGGACAAG ATGGCAGAAGCCTATAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC ATGACGGCCTCTATCAGGGCCTGAGCACCGCCACCAAAGATACCTATGATGCCCTCCACA TGCAGGCTCTGCCTCCCAGATAA

FGFR4(HL).28HTM.28z /CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 48)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT

PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY

GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL

DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAK

RSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDR

VTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSL

RLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLY

LQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQ

PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK

QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY

DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ

GLSTATKDTYDALHMQALPPR*

Table 18. FGFR4(HL).28HTM.28z /CD276.8HTM.BBz BiCisCAR components

FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR (SEQ ID NO: 49)

CD8 Leader- 3A11 VH-VL -CD28HTM-CD28-CD3z(lXX)-T2A-GM-CSF leader-MGA271 VL-

VH-CD8HTM-4-lBB-CD3z(lXX) atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattat ccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgcta ttccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgcc ccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCT

TCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA

AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG

ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA

CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA

TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC

CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC

GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTTTAACGAGCTGCAGAAGGACAAG ATGGCAGAAGCCTTTAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC

ATGACGGCCTCTTTCAAGGCCTGAGCACCGCCACCAAAGATACCTTTGATGCCCTCCACA TGCAGGCTCTGCCTCCCAGATAA

FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR (SEQ ID NO: 50)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT

PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY

GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL

DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW

VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRAKR

SGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDRV

TITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLR

LSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYL

QMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQP

LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ

PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGL

STATKDTFDALHMQALPPR Table 19. FGFR4(HL).28HTM.28z(lXX)/CD276.8HTM.BBz(lXX) BiCisCAR components

FGFR4(HL).28HTM.28z/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 51)

CD8 Leader-3A11 VH-VL -CD28HTM-CD28-CD3z-T2A-GM-CSF leader-MGA271 VL-VH-

CD8HTM-4-1BB atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattat ccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgcta ttccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgcc ccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA

TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgTAA

FGFR4(HL).28HTM.28z/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 52)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT

PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY

GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL

DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

WQGTHFPQTFGGGTKLE1KAAA1EVMYPPPYLDNEKSNGT11HVKGKHLCPSPLFPGPSKPFW

VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAK

RSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDR

VTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSL

RLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLY

LQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQ

PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK

QPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Table 20. FGFR4(HL).28HTM.28z/CD276.8HTM.BB BiCisCAR components FGFR4(HL).28HTM.28z (1XX)/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 53)

CD8 Leader- 3A11 VH-VL -CD28HTM-CD28-CD3z(lXX)-T2A-GM-CSF leader-MGA271 VL-

VH-CD8HTM-4-1BB atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccccaggttcaactggagcagtctggggctgagctg gtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgggtgaagcagacacctgtgcatg gcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagacaaatcctc cagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagggaattactacggtagtgactatgactact ggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcggcagcggtggaggagggagcgatgttgtgatgacc cagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggagagacatatttga attggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccggttcactggcagtgga tcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattttcctcaaacgttcggt ggaggcaccaagctggaaatcaaaGCGGCCGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattat ccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgcta ttccttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgcc ccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCT TCACATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCA GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTG TTACAAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATAT CCAGCTGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCAC ATGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCA AGGCCCCTAAGGCGCTGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGT TCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCA CCAAGCTGGAAATCAAGggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGC AGCTGGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCG CCGCCTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACA CCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAG ATGAACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAA TATCTACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTC CGGATCCacaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggc ccgcagctggaggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgct ctccctggtcattaccctctactgcaagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacagga ggaggacggctgctcctgccggttccccgaggaggaggagggcggctgcgagctgTAA

FGFR4(HL).28HTM.28z (1XX)/CD276.8HTM.BB BiCisCAR (SEQ ID NO: 54)

MALPVTALLLPLALLLHAARPQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQT

PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYY

GSDYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLL

DSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

WQGTHFPQTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW

VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRAKR

SGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDRV

TITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLR

LSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYL

QMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQP

LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ

PFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Table 21. FGFR4(HL).28HTM.28z (1XX)/CD276.8HTM.BB BiCisCAR components

CD276.8HTM.BBz CAR (SEQ ID NO: 90)

CD8 leader-MGA271 VL-VH-CD8HTM-4-lBB-CD3z-T2A-GM-CSF leader-tEGFR atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcccggccgGATATCCAGCTGACCCAGTC CCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCA

GAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGGCGC TGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGTTCTCCGGCTCCGGCTC TGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTA CTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCA AGGGAGGCGGAGGTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAGGTGCAGCT

GGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGG ACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACACCGT GAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGA ACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAATATC

TACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTCCGCG

GCCGCAaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgccctgaggcgtgcc ggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgacatctacatctgggcgcccttggccgggacttgtggggtcc ttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactca agaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGA

CGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAA GATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCAC ATGCAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCAGAGG

AAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTGTTAC

AAGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGAAGGTTTGC

AATGGAATCGGTATAGGGGAGTTTAAGGATTCACTTAGCATAAACGCTACTAATATTAA

ACACTTCAAAAACTGTACGAGTATAAGTGGAGATCTTCACATTTTGCCGGTTGCATTCCG

AGGCGATTCATTCACCCACACGCCACCGCTTGACCCACAAGAATTGGATATTCTTAAAAC

CGTTAAAGAAATAACGGGGTTTTTGCTCATTCAAGCGTGGCCAGAAAATCGCACTGACCT

CCATGCTTTCGAGAACCTGGAGATTATAAGAGGACGAACTAAGCAGCATGGTCAATTCT

CCCTTGCTGTGGTCAGCCTGAACATCACCAGTCTTGGTTTGCGGTCCCTCAAGGAAATTT

CAGATGGAGATGTCATCATAAGCGGCAACAAGAATTTGTGCTATGCAAATACCATAAAC

TGGAAAAAACTGTTTGGCACTTCCGGCCAGAAAACCAAGATTATTTCAAATCGGGGCGA

GAACAGCTGCAAAGCCACCGGCCAGGTTTGTCATGCCTTGTGCTCTCCGGAGGGCTGTTG

GGGGCCAGAACCCAGGGACTGCGTCAGTTGCAGAAACGTCTCAAGAGGCCGCGAATGCG

TTGACAAGTGTAACCTCCTTGAGGGCGAGCCACGAGAGTTTGTTGAGAACAGCGAGTGT

ATACAATGTCACCCTGAATGTTTGCCCCAGGCTATGAATATAACCTGCACAGGCCGCGGG

CCTGATAACTGCATCCAGTGTGCTCATTACATAGATGGACCTCACTGTGTGAAAACCTGC

CCGGCCGGAGTTATGGGAGAAAACAACACTCTGGTGTGGAAATACGCTGATGCAGGCCA

CGTGTGCCACCTTTGTCACCCGAATTGTACATATGGGTGTACCGGTCCTGGACTTGAAGG

TTGCCCTACCAATGGCCCTAAAATACCCAGTATCGCAACTGGCATGGTAGGCGCTCTTCT

CTTGCTCTTGGTGGTTGCTCTCGGCATCGGTCTTTTTATGtaa

CD276.8HTM.BBz CAR (SEQ ID NO: 91)

MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPG

KAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLE

IKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKG

LEWVAY1SSDSSAIYYADTVKGRFT1SRDNAKNSLYLQMNSLRDEDTAVYYCGRGREN1YYG

SRLDYWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAK

RSGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINA

TNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH

AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFG

TSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEG

EPREFVENSEC1QCHPECLPQAMN1TCTGRGPDNCIQCAHY1DGPHCVKTCPAGVMGENNTL

VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLF

M

Table 22. CD276.8HTM.BBz CAR components

CD276.8HTM.BBz(lXX) CAR (SEQ ID NO: 92)

CD8 leader-MGA271 VL-VH-CD8HTM-4-1BB- CD3z(lXX)-T2A-GM-CSF leader-tEGFR atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcccggccgGATATCCAGCTGACCCAGTC

CCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCA

GAACGTGGACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGGCGC

TGATCTACTCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGTTCTCCGGCTCCGGCTC

TGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTA

CTGCCAGCAGTACAACAACTACCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCA

AGGGAGGCGGAGGTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAGGTGCAGCT

GGTCGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC

CTCCGGCTTCACCTTCTCCTCCTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGG

ACTGGAATGGGTGGCCTACATCTCCTCCGACTCCTCCGCCATCTACTACGCCGACACCGT

GAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGA

ACTCCCTGCGGGACGAGGACACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAATATC

TACTACGGCTCCCGGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTCCGCG

GCCGCAaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgccctgaggcgtgcc ggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgacatctacatctgggcgcccttggccgggacttgtggggtcc ttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactca agaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgAGGGTGAAATTCTCCAGATCCGC CGATGCCCCTGCATATCAGCAGGGACAGAATCAGCTGTACAATGAGCTGAACCTCGGCC

GCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGG

CAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTTTAACGAGCTGCAGAAGGACAAG

ATGGCAGAAGCCTTTAGCGAGATCGGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCC

ATGACGGCCTCTTTCAAGGCCTGAGCACCGCCACCAAAGATACCTTTGATGCCCTCCACA

TGCAGGCTCTGCCTCCCAGATCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCAGAGGA

AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTGTTACA

AGCCTTTTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGAAGGTTTGCA

ATGGAATCGGTATAGGGGAGTTTAAGGATTCACTTAGCATAAACGCTACTAATATTAAAC

ACTTCAAAAACTGTACGAGTATAAGTGGAGATCTTCACATTTTGCCGGTTGCATTCCGAG

GCGATTCATTCACCCACACGCCACCGCTTGACCCACAAGAATTGGATATTCTTAAAACCG

TTAAAGAAATAACGGGGTTTTTGCTCATTCAAGCGTGGCCAGAAAATCGCACTGACCTCC

ATGCTTTCGAGAACCTGGAGATTATAAGAGGACGAACTAAGCAGCATGGTCAATTCTCC

CTTGCTGTGGTCAGCCTGAACATCACCAGTCTTGGTTTGCGGTCCCTCAAGGAAATTTCA

GATGGAGATGTCATCATAAGCGGCAACAAGAATTTGTGCTATGCAAATACCATAAACTG

GAAAAAACTGTTTGGCACTTCCGGCCAGAAAACCAAGATTATTTCAAATCGGGGCGAGA

ACAGCTGCAAAGCCACCGGCCAGGTTTGTCATGCCTTGTGCTCTCCGGAGGGCTGTTGGG

GGCCAGAACCCAGGGACTGCGTCAGTTGCAGAAACGTCTCAAGAGGCCGCGAATGCGTT

GACAAGTGTAACCTCCTTGAGGGCGAGCCACGAGAGTTTGTTGAGAACAGCGAGTGTAT

ACAATGTCACCCTGAATGTTTGCCCCAGGCTATGAATATAACCTGCACAGGCCGCGGGCC

TGATAACTGCATCCAGTGTGCTCATTACATAGATGGACCTCACTGTGTGAAAACCTGCCC

GGCCGGAGTTATGGGAGAAAACAACACTCTGGTGTGGAAATACGCTGATGCAGGCCACG

TGTGCCACCTTTGTCACCCGAATTGTACATATGGGTGTACCGGTCCTGGACTTGAAGGTT

GCCCTACCAATGGCCCTAAAATACCCAGTATCGCAACTGGCATGGTAGGCGCTCTTCTCT

TGCTCTTGGTGGTTGCTCTCGGCATCGGTCTTTTTATGtaa CD276.8HTM.BBz(lXX) CAR (SEQ ID NO: 93)

MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPG

KAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLE

IKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKG

LEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYG

SRLDYWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRAKR

SGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINAT

NIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHA

FENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTS

GQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEP

REFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVW

KYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

Table 23. CD276.8HTM.BBz(lXX) CAR components

C. Nucleotide sequences of CAR and BiCisCAR components

CD8 leader sequence (SEQ ID NO: 55) atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccc

3A11 VL-linker-VH (SEQ ID NO: 56)

Gatgttgtgatgacccagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatg gagagacatatttgaattggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgaccgg ttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggcacacattt tcctcaaacgttcggtggaggcaccaagctggaaatcaaaggaggcggaggttccggtggcggcggcagcggtggaggagggagccaggttc aactggagcagtctggggctgagctggtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgca ctgggtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttcaagggcaa ggccatactgactgcagacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagagg gaattactacggtagtgactatgactactggggccaaggcaccactctcacagtctcctca 3A11 VH-linker-VL (SEQ ID NO: 57)

Caggttcaactggagcagtctggggctgagctggtcaggcctggggcttcagtgacgctgtcctgcaaggcttcgggctacacatttactgactat gaaatgcactgggtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctgaaactggtggtactgcctacaatcagaagttca agggcaaggccatactgactgcagacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgta caagagggaattactacggtagtgactatgactactggggccaaggcaccactctcacagtctcctcaggaggcggaggttccggtggcggcgg cagcggtggaggagggagcgatgttgtgatgacccagactccacttactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtc agagcctcttagatagtgatggagagacatatttgaattggttgttaaagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactgg actctggagtccctgaccggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttatta ttgctggcaaggcacacattttcctcaaacgttcggtggaggcaccaagctggaaatcaaa

MGA271 VL-VH (SEQ ID NO: 58) gatatccagctgacccagtccccctccttcctgtctgcctccgtgggcgacagagtgaccatcacatgcaaggcctcccagaacgtggacaccaac gtggcctggtatcagcagaagcctggcaaggcccctaaggcgctgatctactccgcctcctaccgctactccggcgtgccttcccggttctccggct ccggctctggcaccgacttcaccctgaccatctccagcctgcagcctgaggacttcgccacctactactgccagcagtacaacaactaccctttcac cttcggccagggcaccaagctggaaatcaagggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcgaggtgca gctggtcgagtctggcggaggactggtgcagcctggcggctccctgagactgtcttgcgccgcctccggcttcaccttctcctccttcggcatgcac tgggtccgccaggctccaggcaagggactggaatgggtggcctacatctcctccgactcctccgccatctactacgccgacaccgtgaagggca ggtlcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaactccctgcgggacgaggacaccgccgtgtactactgcggcag aggccgggagaatatctactacggctcccggctggattattggggccagggcaccaccgtgaccgtgtcttcc

CD28 HTM (SEQ ID NO: 59) attgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttc ccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagtaacagtggcctttattattttctgggtg

CD8 HTM (SEQ ID NO: 60)

Accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgccctgaggcgtgccggccagcgg cggggggcgcagtgcacacgagggggctggacttcgcctgtgacatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtca ctggttatcaccctttactgc

CD8 HTM codon optimized- 1 (SEQ ID NO: 61) acaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggcccgcagctgg aggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgctctccctggtca ttaccctctactgc

CD8 HTM codon optimized-2 (SEQ ID NO: 83) actaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccctctccttgcgccccgaggcttgccgcccggccgcg ggtggagccgtgcatacccgggggctggactttgcctgcgacatctacatttgggccccgctggccggcacttgcggcgtgctcctgctgtcgctg gtcatcaccctttactgc

4-1BB co-stimulatory domain (SEQ ID NO: 62)

Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttc cagaagaagaagaaggaggatgtgaactg

4-1BB co-stimulatory domain codon optiinized-l (SEQ ID NO: 63) aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcagacgactcaggaagaggacggatgctcgtgcagat tccctgaggaggaagaggggggatgcgaactg 4-1BB co-stimulatory domain codon optimized-2 (SEQ ID NO: 84) aagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacaggaggaggacggctgctcctgccgg ttccccgaggaggaggagggcggctgcgagctg

CD28 co-stimulatory domain (SEQ ID NO: 64) aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccc caccacgcgacttcgcagcctatcgctcc

CD3 signaling domain (SEQ ID NO: 65) cgcgtcaagttctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctctacaacgagctgaacctgggaaggagagaggag tacgacgtgctggacaagcgacgcggacgcgacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaacgaac tccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcggaggaggggaaagggtcacgacgggctgtaccaggg actgagcaccgccactaaggatacctacgatgccttgcatatgcaagcactcccaccccgg

CD3 codon optimized- 1 (SEQ ID NO: 85)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCT

CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA

GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

CD3 codon optimized-2 (SEQ ID NO: 86)

AGGGTGAAATTCTCCAGATCCGCCGATGCCCCTGCATATCAGCAGGGACAGAATCAGCT GTACAATGAGCTGAACCTCGGCCGCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCG GCAGAGATCCCGAGATGGGAGGCAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTA TAACGAGCTGCAGAAGGACAAGATGGCAGAAGCCTATAGCGAGATCGGAATGAAGGGC

GAAAGAAGGCGGGGCAAGGGCCATGACGGCCTCTATCAGGGCCTGAGCACCGCCACCA AAGATACCTATGATGCCCTCCACATGCAGGCTCTGCCTCCCAGA

CD3 (1XX) signaling domain (SEQ ID NO: 66) agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagagga gtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaact gcagaaagataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttttccaaggtct cagtacagccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgc

CD3 (1XX) signaling domain codon optimized (SEQ ID NO: 87)

AGGGTGAAATTCTCCAGATCCGCCGATGCCCCTGCATATCAGCAGGGACAGAATCAGCT GTACAATGAGCTGAACCTCGGCCGCAGGGAGGAGTATGACGTCCTGGATAAAAGGCGCG GCAGAGATCCCGAGATGGGAGGCAAGCCTAGGCGGAAGAATCCCCAGGAGGGCCTGTTT AACGAGCTGCAGAAGGACAAGATGGCAGAAGCCTTTAGCGAGATCGGAATGAAGGGCG

AAAGAAGGCGGGGCAAGGGCCATGACGGCCTCTTTCAAGGCCTGAGCACCGCCACCAAA GATACCTTTGATGCCCTCCACATGCAGGCTCTGCCTCCCAGA

T2A (SEQ ID NO: 67) gaaggacgaggtagccttcttacgtgcggagacgtggaggaaaacccaggaccc GM-CSF leader (SEQ ID NO: 68) atgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgattccaagg tEGFR (SEQ ID NO: 69) aaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtataagtgga gatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgttaaagaaa taacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaagcagcat ggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataagcggcaac aagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgagaacagctg caaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaacgtctcaa gaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccctgaatgttt gccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtgaaaacctgc ccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgtacatatgggt gtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctcttggtggttg ctctcggcataggtctttttatgtaa

D. Amino acid sequences of CAR and BiCisCAR components

CD8 leader amino acid (SEQ ID NO: 70)

MALPVTALLLPLALLLHAARP

3A11 VL-linker-VH (SEQ ID NO: 71)

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLLKRPGQSPKRLIYLVSKLDSGVP DRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKGGGGSGGGGSGGG GSQVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTA YNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYYGSDYDYWGQGTTLTVSS

3A11 VH-linker-VL (SEQ ID NO: 72)

QVQLEQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYN QKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGNYYGSDYDYWGQGTTLTVSSGGG GSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGETYLNWLLKRPGQSPKR LIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK

MGA271 VL-VH (SEQ ID NO: 73)

DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADT VKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS

CD28 HTM (SEQ ID NO: 74)

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF wv

CD8 HTM (SEQ ID NO: 75)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV

ITLYC 4-1BB co-stimulatory domain (SEQ ID NO: 76)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD28 co-stimulatory domain (SEQ ID NO: 77)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

CD3 signaling domain (SEQ ID NO: 78)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 (1XX) signaling domain (SEQ ID NO: 79)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFN ELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

T2A (SEQ ID NO: 80)

EGRGSLLTCGDVEENPGP

GM-CSF leader (SEQ ID NO: 81)

MLLLVTSLLLCELPHPAFLLIPR tEGFR (SEQ ID NO: 82)

KVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVK EITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISG NKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPH CVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGM VGALLLLLVVALGIGLFM

V. CARs and BiCisCARs Targeting FGFR4 and/or CD276

Disclosed herein are FGFR4-targeted chimeric antigen receptors (CARs), CD276-targeted CARs, and bicistronic CARs (BiCisCARs) that target both FGFR4 and CD276. The disclosed CARs and BiCisCARs include different combinations of the hinge transmembrane (HTM) domain and co- stimulatory domain (CSD) to identify CARs and BiCisCARs with the greatest potency against FGFR4- and/or CD276-expressing tumors. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine-based activation motifs (ITAMs) of a CD C intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used, for example, for the treatment of cancers that express one or both of FGFR4 and CD276.

A. FGFR4-targeted CARs

Provided herein are FGFR4-targeted CARs that include an extracellular antigen-binding domain that specifically binds FGFR4, thereby targeting the CAR to FGFR4-expressing cells. In some aspects, the extracellular antigen-binding domain of the CAR includes a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2. In some aspects, the CAR further includes a hinge and transmembrane (HTM) domain; an intracellular co-stimulatory domain; and a CD3C intracellular signaling domain.

In some aspects, the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and/or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. In some examples, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1; and/or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2 and includes the CDR1 , CDR2 and CDR3 sequences of SEQ ID NO: 2. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1; and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.

In some aspects of the FGFR4-targeted CAR, the extracellular antigen-binding domain of the CAR further includes a linker separating the VH domain and the VL domain. In some examples, the extracellular antigen-binding domain includes in the N-terminal to C-terminal orientation: VH domain-linker- VL domain. In other examples, the extracellular antigen-binding domain includes in the N-terminal to C-terminal orientation: VL domain-linker- VH domain.

In some aspects, the VH domain and/or the VL domain include human framework sequences.

In some aspects, the CAR includes a CD28 HTM domain and a CD28 co-stimulatory domain. In some examples, the amino acid sequence of the CD28 HTM is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74. In particular examples, the amino acid sequence of the CD28 HTM includes or consists of SEQ ID NO: 74. In some examples, the amino acid sequence of the CD28 intracellular co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 77. In particular examples, the amino acid sequence of the CD28 intracellular co- stimulatory domain includes or consists of SEQ ID NO: 77.

In some aspects, the CAR includes a CD28 HTM and a 4- IBB co-stimulatory domain. In some examples, the amino acid sequence of the CD28 HTM is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 74. In particular examples, the amino acid sequence of the CD28 HTM includes or consists of SEQ ID NO: 74. In some examples, the amino acid sequence of the 4-1BB co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In particular examples, the amino acid sequence of the 4-1BB co-stimulatory domain includes or consists of SEQ ID NO: 76.

In some aspects, the CD3^ intracellular signaling domain is a wild-type CD3L such as a wildtype human CD3^. In some examples, the amino acid sequence of the CD3q intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 78. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain includes or consists of SEQ ID NO: 78.

In other aspects, the CD3^ intracellular signaling domain includes one or more modifications, such as amino acid substitutions. For example, the modifications can result in an alteration in function or signaling activity of the CD3C intracellular signaling domain.

In some aspects, the CD3^ intracellular signaling domain includes a first, a second, and a third immunoreceptor tyrosine -based activation motif (IT AM) (see FIG. 18), wherein at least one tyrosine is substituted with a phenylalanine in the second and/or third IT AM. In some examples, the second IT AM includes two tyrosine to phenylalanine substitutions or the third IT AM includes two tyrosine to phenylalanine substitutions. In some examples, the second IT AM and the third IT AM each have two tyrosine to phenylalanine substitutions. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 79. In specific nonlimiting examples, the amino acid sequence of the CD3^ intracellular signaling domain includes or consists of SEQ ID NO: 79.

In some aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 22-490 of SEQ ID NO: 18, residues 22-489 of SEQ ID NO: 22, residues 22-489 of SEQ ID NO: 34, residues 22-490 of SEQ ID NO: 40, residues 22-489 of SEQ ID NO: 44, or residues 22-489 of SEQ ID NO: 46. In some examples, the amino acid sequence of the CAR includes or consists of residues 22-490 of SEQ ID NO: 18, residues 22-489 of SEQ ID NO: 22, residues 22-489 of SEQ ID NO: 34, residues 22-490 of SEQ ID NO: 40, residues 22-489 of SEQ ID NO: 44, or residues 22-489 of SEQ ID NO: 46.

Also provided are isolated cells expressing an FGFR4-targeted CAR disclosed herein. In some aspects, the cell is an immune cell or an induced pluripotent stem cell (iPSC). In some examples, the immune cell is a T cell, a B cell, a natural killer (NK) cell or a monocyte/macrophage.

Further provided are nucleic acid molecules encoding an FGFR4- targeted CAR disclosed herein. In some aspects, the nucleic acid molecule includes or consists of nucleotides 64-1470 of SEQ ID NO: 17, nucleotides 64-1467 of SEQ ID NO: 21, nucleotides 64-1467 of SEQ ID NO: 33, nucleotides 64-1470 of SEQ ID NO: 39, nucleotides 64-1467 of SEQ ID NO: 43, or nucleotides 64- 1467 of SEQ ID NO: 45, or a degenerate variant thereof. In some aspects, the nucleic acid molecule further includes a first leader sequence preceding the coding sequence of the extracellular antigen- binding domain, such as a CD8 leader sequence (e.g., a CD8 leader sequence including or consisting of SEQ ID NO: 55). In some aspects, the nucleic acid molecule further includes a nucleic acid sequence encoding a truncated epidermal growth factor receptor (tEGFR), such as a tEGFR that includes or consists of SEQ ID NO: 69. In some aspects, the nucleic acid molecule further includes a second leader sequence preceding the tEGFR coding sequence, such as a GM-CSF leader sequence (e.g., a GM-CSF leader sequence including or consisting of SEQ ID NO: 68).

In some aspects of the FGFR4-targeted CARs, the nucleic acid molecule includes the following orientation in the 5’ to 3’ direction: the first leader sequence, the coding sequence for the extracellular antigen-binding domain, the coding sequence for the HTM, the coding sequence for the co-stimulatory domain, the coding sequence for the intracellular signaling domain, a 2A site, the second leader sequence, and the coding sequence for tEGFR. In some examples, the 2A site is a T2A site (such as a T2A site set forth as SEQ ID NO: 67).

In several aspects, the nucleic acid molecule encoding the FGFR4-targeted CAR has a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 43, or SEQ ID NO: 45, or a degenerate variant thereof. In some examples, the nucleic acid molecule has a nucleotide sequence that includes or consists of SEQ ID NO: 17, SEQ ID NO: 21 , SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 43, or SEQ ID NO: 45, or a degenerate variant thereof.

Also provided are vectors that include a nucleic acid molecule encoding an FGFR4-targeted CAR disclosed herein. In some aspects, the vector is a viral vector, such as a lentiviral vector. Isolated cells that include a nucleic acid molecule or vector disclosed herein are further provided. The cells can be, for example, immune cells (e.g., T cells, B cells, NK cells or monocytes/macrophages) or iPSCs.

B. CD276-targeted CARs

Also provided herein are CD276-targeted CARs that include an extracellular antigen-binding domain that specifically binds CD276, thereby targeting the CAR to CD276-expressing cells. In some aspects, the extracellular antigen-binding domain of the CAR includes a VH domain and a VL domain, wherein the VH domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10. In some aspects, the CAR further includes a CD8 HTM domain and a 4- IBB intracellular co-stimulatory domain. In some aspects, the CD276-targeted CAR further includes a CD3^ intracellular signaling domain.

In some aspects, the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13: and/or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In some examples, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10. In particular examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 10.

In some aspects of the CD276-targeted CAR, the VH domain and the VL domain include human framework sequences.

In some aspects, the amino acid sequence of the CD8 HTM domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 75. In some examples, the amino acid sequence of the CD8 HTM domain includes or consists of SEQ ID NO: 75.

In some aspects, the amino acid sequence of the 4- IBB co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In some examples, the amino acid sequence of the 4-1 BB co-stimulatory domain includes or consists of SEQ ID NO: 76.

In some aspects of the CD276-targeted CARs, the CD3^ intracellular signaling domain is a wild-type CD3^, such as a wild-type human CD3^. In some examples, the amino acid sequence of the CD3^ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 78. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain includes or consists of SEQ ID NO: 78.

In other aspects of the CD276-targeted CARs, the CD3^ intracellular signaling domain includes one or more modifications, such as amino acid substitutions. For example, the modifications can result in an alteration in function or signaling activity of the CD3^ intracellular signaling domain.

In some aspects, the CD3^ intracellular signaling domain includes a first, a second, and a third IT AM (see FIG. 18), wherein at least one tyrosine is substituted with a phenylalanine in the second and/or third IT AM. In some examples, the second ITAM includes two tyrosine to phenylalanine substitutions or the third ITAM includes two tyrosine to phenylalanine substitutions. In some examples, the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 79. In specific non-limiting examples, the amino acid sequence of the CD3C intracellular signaling domain includes or consists of SEQ ID NO: 79. In some aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 541-1012 of SEQ ID NO: 20. In some examples, the amino acid sequence of the CAR includes or consists of residues 541-1012 of SEQ ID NO: 20.

In other aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 22-379 of SEQ ID NO: 24. In some examples, the amino acid sequence of the CAR includes or consists of residues 22- 379 of SEQ ID NO: 24.

In other aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 540-1011 of SEQ ID NO: 26. In some examples, the amino acid sequence of the CAR includes or consists of residues 540-1011 of SEQ ID NO: 26.

In other aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 22-491 of SEQ ID NO: 91. In some examples, the amino acid sequence of the CAR includes or consists of residues 22- 491 of SEQ ID NO: 91.

In other aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 22-491 of SEQ ID NO: 93. In some examples, the amino acid sequence of the CAR includes or consists of residues 22- 491 of SEQ ID NO: 93.

Further provided are isolated cells that express a CD276-targeted CAR disclosed herein. In some aspects, the cell is an immune cell (such as a T cell, B cell, NK cell, or monocyte/macrophage) or an iPSC.

Nucleic acid molecules that encode a CD276-targeted CAR disclosed herein are also provided. In some aspects, the nucleic acid molecule includes or consists of nucleotides 1621-3036 of SEQ ID NO: 19, nucleotides 64-1137 of SEQ ID NO: 23, nucleotides 1618-3033 of SEQ ID NO: 25, nucleotides 64-1470 of SEQ ID NO: 17, nucleotides 64-1467 of SEQ ID NO: 21, nucleotides 64-1467 of SEQ ID NO: 33, nucleotides 64-1470 of SEQ ID NO: 39, nucleotides 64-1467 of SEQ ID NO: 43, nucleotides 64-1467 of SEQ ID NO: 45, nucleotides 64-1473 of SEQ ID NO: 90, or nucleotides 64- 1473 of SEQ ID NO: 92, or a degenerate variant thereof. In some aspects, the nucleic acid molecule further includes a first leader sequence preceding the coding sequence of the extracellular antigenbinding domain, such as a CD8 leader sequence (e.g., a CD8 leader sequence including or consisting of SEQ ID NO: 55). In some aspects, the nucleic acid molecule further includes a nucleic acid sequence encoding a tEGFR, such as a tEGFR that includes or consists of SEQ ID NO: 69. In some aspects, the nucleic acid molecule further includes a second leader sequence preceding the tEGFR coding sequence, such as a GM-CSF leader sequence (e.g., a GM-CSF leader sequence including or consisting of SEQ ID NO: 68). In some aspects of the FGFR4-targeted CARs, the nucleic acid molecule includes the following orientation in the 5’ to 3’ direction: the first leader sequence, the coding sequence for the extracellular antigen-binding domain, the coding sequence for the HTM, the coding sequence for the co-stimulatory domain, the coding sequence for the intracellular signaling domain, a 2A site, the second leader sequence, and the coding sequence for tEGFR. In some examples, the 2A site is a T2A site (such as a T2A site set forth as SEQ ID NO: 67). In a particular aspect, the nucleotide sequence of the nucleic acid molecule includes or consists of SEQ ID NO: 23, or a degenerate variant thereof. In other particular aspects, the nucleotide sequence of the nucleic acid molecule includes or consists of SEQ ID NO: 90 or SEQ ID NO: 92, or a degenerate variant thereof.

Also provided are vectors that include a nucleic acid molecule encoding a CD276-targeted CAR disclosed herein. In some aspects, the vector is a viral vector, such as a lentiviral vector. Isolated cells that include a nucleic acid molecule or vector disclosed herein are further provided. The cells can be, for example, immune cells (e.g., T cells, B cells, NK cells or monocytes/macrophages) or iPSCs.

C. FGFR4-targeted and CD276-targeted BiCisCARs

Further provided herein are isolated cells that express a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is an FGFR4-targeted CAR disclosed herein and the second CAR is a CD276-targeted disclosed herein. In some aspects, the cell is an immune cell, such as a T cell, a B cell, a NK cell, or a monocyte/macrophage. In other aspects, the cell is an iPSC.

Also provided herein are nucleic acid molecules that encode a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a FGFR4-targeted CAR as disclosed herein and the second CAR is a CD276-targeted CAR as disclosed herein.

In some aspects of the nucleic acid molecules, the coding sequences for the first CAR and the second CAR are separated by a 2A site sequence, such as a T2A site. In some examples, the nucleotide sequence of the T2A site includes or consists of SEQ ID NO: 67.

In some aspects, the nucleic acid molecule encoding the BiCisCAR further includes a first leader sequence preceding the coding sequence for the first CAR and/or a second leader sequence preceding the coding sequence for the second CAR. In some examples, the first leader sequence is a CD8 leader sequence, such as the CD8 leader sequence set forth as SEQ ID NO: 55. In some examples, the second leader sequence is a GM-CSF leader sequence, such as the GM-CSF leader sequence set forth as SEQ ID NO: 68.

In some aspects, the nucleotide sequence encoding the BiCisCAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53. In some examples, the nucleotide sequence encoding the BiCisCAR includes or consists of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.

In some aspects, the nucleic acid molecules are operably linked to a promoter.

Also provided are vectors that include a BiCisCAR nucleic acid molecule disclosed herein. In some aspects, the vector is a lentivirus vector.

Isolated cells that include a BiCiSCAR nucleic acid molecule or vector are further provided. In some aspects, the cell is an immune cell or an iPSC. In some examples, the immune cell is a T cell, a B cell, a NK cell or a monocyte/macrophage.

E. Compositions and Methods of Use

Compositions that include a pharmaceutically acceptable carrier and an FGFR4-targeted CAR, a CD276-targeted CAR, a FGFR4/CD276 BiCisCAR, a nucleic acid molecule, a vector or an isolated cell disclosed herein are further provided. CAR and BiCisCAR compositions are further described in section VI.

Also provided are methods for treating a FGFR4-expressing and/or a CD276-expressing cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a CAR, BiCisCAR, isolated cell, nucleic acid molecule, vector, or composition disclosed herein. Further provided are methods of inhibiting tumor growth or metastasis of a FGFR4-expressing and/or a CD276-expressing cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a CAR, BiCisCAR, isolated cell, nucleic acid molecule, vector, or composition disclosed herein. In some aspects of these methods, the FGFR4-expressing cancer is a rhabdomyosarcoma (RMS), a lung cancer, a liver cancer, a breast cancer, a pancreatic cancer, a prostate cancer, a desmoplastic small round cell tumor (DSRCT), a yolk sac tumor (YST), an adrenocortical carcinoma (ACC), or a gastric adenocarcinoma. In some aspects of these methods, the CD276-expressing cancer is a liver cancer (such as hepatocellular carcinoma), a pancreatic cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer (such as neuroblastoma or glioblastoma), a pediatric cancer (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing’s sarcoma), a melanoma, or a mesothelioma. In some examples, the subject has a cancer that expresses both FGFR4 and CD276. Methods of using the disclosed CAR and BiCisCAR compositions are further described in section VII.

VI. CAR and BiCisCAR Compositions

Compositions are provided that include an FGFR4-targeted and/or CD276-targeted CAR or BiCisCAR (such as a nucleic acid/vector encoding an FGFR4-targeted and/or CD276-targeted CAR or BiCisCAR, or cells expressing an FGFR4-targeted and/or CD276-targeted CAR or BiCisCAR), in a pharmaceutically acceptable carrier. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The CAR/BiCisCAR composition can be formulated for systemic or local (such as intra-tumor) administration. In some aspects, the CAR/BiCisCAR composition is formulated for parenteral administration, such as intravenous administration.

The compositions for administration can include a solution of the CAR/BiCisCAR in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

The compositions that include a CAR/BiCisCAR can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the compositions can be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Treatment can involve daily or multidaily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the j udgment of the administering practitioner.

Typical dosages of the CAR/BiCisCAR compositions or additional agents can range from about 0.01 to about 30 mg/kg, such as from about 0.1 to about 10 mg/kg. In some examples, the dosage is at least about 0.1 mg/kg, at least about 0.2 mg/kg, at least about 0.3 mg/kg, at least about 0.4 mg/kg, at least about 0.5 mg/kg, at least about 1 mg/kg, at least about 4 mg/kg, at least about 3 mg/kg, at least about 5 mg/kg, at least about 6 mg/kg, at least about 7 mg/kg, at least about 8 mg/kg is at least about 9 mg/kg, at least about 10 mg/kg, at least about 11 mg/kg, at least about 12 mg/kg, at least about 13 mg/kg, at least about 14 mg/kg, at least about 15 mg/kg, at least about 16 mg/kg, at least about 17 mg/kg, at least about 18 mg/kg, at least about 19 mg/kg, at least about 20 mg/kg, at least about 21 mg/kg, at least about 22 mg/kg, at least about 23 mg/kg, at least about 24 mg/kg at least about 25 mg/kg, at least about 26 mg/kg, at least about 27 mg/kg, at least about 28 mg/kg, at least about 29 mg/kg, or at least about 30 mg/kg. In particular examples, the subject is administered a CAR/BiCisCAR or composition thereof, or additional agent(s), on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the CAR/BiCisCAR, composition or additional agent(s) for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

In some aspects, a CAR/BiCisCAR or composition is administered intravenously, subcutaneously or by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some aspects, the initial period of treatment (e.g., administration of the therapeutic agent daily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In a related aspect, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain aspects, the dosing regimen of the therapeutic agent is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 weeks followed by 1 , 2, 3 or 4 weeks off.

The compositions disclosed herein can also be administered by other routes, including via inhalation, oral, topical or intraocular. In some examples, the composition is administered via fine- needle.

CAR/BiCisCAR compositions may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The CAR/BiCisCAR solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RITUXAN™ in 1997. CAR/BiCisCAR compositions can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.

Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 Ltm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 inn in diameter and are administered subcutaneously or intramuscularly. See, for example. Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

Polymers can be used for ion-controlled release of the CAR/BiCisCAR compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (e.g., see Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins such as CARs/BiCisCARs (Ijntema et al., Int. J. Pharm.112:215-224, 1994). In yet another aspect, liposomes are used for controlled release (Betageri et al.. Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

VII. Methods of Use

The CAR/BiCisCAR compositions disclosed herein can be administered to slow or inhibit the growth of tumor cells, inhibit the metastasis of tumor cells, and/or increase the survival of a subject having a tumor, such as an FGFR4-expressing tumor and/or a CD276-expressing tumor, such as solid tumors. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, increase the survival of a subject having a tumor, and/or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a cancer that expresses FGFR4, such as, but not limited to RMS (e.g., ARMS or ERMS), a lung cancer, a liver cancer (e.g., HCC or HBL), a breast cancer, a pancreatic cancer, a prostate cancer, desmoplastic small round cell tumor (DSRCT), a yolk sac tumor (YST), an adrenocortical carcinoma (ACC), or a gastric adenocarcinoma. In some aspects, the subject has been diagnosed with a cancer that expresses CD276, such as but not limited to, a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, melanoma or mesothelioma.

Provided herein is a method of treating a FGFR4-expressing cancer in a subject by administering to the subject a therapeutically effective amount of an FGFR4-targeted CAR or BiCisCAR composition disclosed herein. Also provided herein is a method of inhibiting tumor growth or metastasis of a FGFR4-expressing cancer in a subject by administering to the subject a therapeutically effective amount of an FGFR4-targeted CAR or BiCisCAR composition disclosed herein. In some aspects, the FGFR4-expressing cancer is RMS (e.g., ARMS or ERMS), a lung cancer, a liver cancer e.g., HCC or HBL), a breast cancer, a pancreatic cancer, a prostate cancer, DSRCT, YST, ACC, or a gastric adenocarcinoma.

Also provided herein is a method of treating a CD276-expressing cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-targeted CAR or BiCisCAR composition disclosed herein. Further provided herein is a method of inhibiting tumor growth or metastasis of a CD276-expressing cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-targeted CAR or BiCisCAR composition disclosed herein. In some aspects, the CD276-expressing cancer is a pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.

In some aspects of the methods, the cancer expresses both FGFR4 and CD276.

A therapeutically effective amount of a FGFR4-targeted or CD276-targeted CAR/BiCisCAR composition disclosed herein can depend upon the severity of the disease, the type of disease, and the general state of the patient’s health. A therapeutically effective amount of the CAR/BiCisCAR composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

Administration of the CAR/BiCisCAR compositions disclosed herein can also be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical resection of a tumor). Any suitable anti-cancer agent can be administered in combination with the CAR/BiCisCAR compositions disclosed herein, such as administered prior to, concurrently with, or following administration of the CAR/BiCisCAR composition. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L- asparaginase).

Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and lluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.

EXAMPLES

The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

The Examples below describe studies to improve the efficacy of CAR T-cells against solid tumors and to develop principles for a dual targeting CAR using RMS as a model. The results showed that replacing the CD8 hinge and transmembrane domain (HTM) and the 4- IBB co- stimulatory domain (CSD) of a previously developed clinical grade FGFR4 targeting CAR with CD28 HTM and CSD resulted in increased cytotoxicity in vitro and improved anti-tumor efficacy in vivo.

Simultaneous targeting of two tumor-associated cell surface antigens by a bicistronic CAR is a therapeutic strategy to tackle the heterogenous expression of target antigens and avoid immune escape in solid tumors (Tian et al., J Clin Invest 132(16):el55621, 2022). Expression levels of FGFR4 in RMS demonstrated heterogeneity at both the mRNA and protein levels. Subsequently, CD276 was identified as a second cell-surface antigen, which was directly targeted by PAX3-FOXO1 in FP-RMS, and regulated by MYODI, with the establishment of a super enhancer and associated with high expression in the majority of FN- and FP-RMS. Thus, these data supported the rationale for dual targeting of both FGFR4 and CD276 as an effective approach for RMS treatment.

Three dual-targeting BiCisCARs were evaluated, combining different FGFR4-targeting constructs with a CD276-targeting CAR. The BiCisCAR using FGFR4-targeting CAR with a CD8HTM and a 4- IBB CSD did not show any advantage in an RH30 orthotopic xenograft model, partially due to weak T-cell expansion or persistence. Although the other two BiCisCAR variants exhibited comparable tumor clearance in RH30 and RMS559 IM tumors, the FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR, incorporating different CSDs, not only cleared tumors more rapidly but also exhibited robust antitumor activity in a stressful RMS559 orthotopic model with only 1 million CAR T-cells administered. This BiCisCAR also outperformed the other CARs in another aggressive, JR, EM. model, suggesting the combination of CD28 and 4- IBB CSDs may be a more effective strategy for a dual-targeting CAR design. Third-generation single CARs containing both CD28 and 4- IBB CSDs in tandem have been reported to be inferior compared with their second- generation counterparts in clinical trials (Enblad et al., Clin Cancer Res 24:6185-6194, 2018; Ramos el al., Mol Ther 26:2727-2737 , 2018), possibly due to steric hindrance of cis-CSDs interfering with the interactions of co-activators required for optimal T-cell activation and survival. Although recent findings demonstrated that patients with high-risk refractory or relapsed neuroblastoma responded well to the third generation GD2-CART01 cells (Del Bufalo et al. , N Engl J Med 388: 1284-1295, 2023), third generation CARs targeting single antigens do not address the challenge of heterogenous target antigen expression. Therefore, the dual-targeting BiCisCAR design strategy disclosed herein not only provides both CD28 and 4- IBB co-stimulation signaling for improved T-cell activation, but also allows for simultaneous targeting of two tumor-associated antigens to prevent potential tumor escape.

In the Examples below, the mechanism behind the observed synergistic signaling resulting from both CARs concurrently engaging with their cognate antigens was also investigated. The results showed that activation of the CD28 CSD of FGFR4 CAR induced higher phosphorylation of cascade signaling molecules, including pZAP70-pPLCyl-pAKT-mTORCl and pZAP70-pPLCyl-pERKl/2- AP-1, consistent with rapid antitumor effects (Zhao et al., Cancer Cell 28:415-428, 2015; Salter et al. , Sci Signal 1 l(544):eaat6753, 2018; Sun et al. , Cancer Cell 37:216-225, 2020). Concurrently, activation of 4- IBB CSD of CD276 CAR increased phosphorylation of p65, subsequently activating the NF-KB pathway, critical for sustaining CAR T-cell persistence (Kawalekar et al. , Immunity 44:380-390, 2016). Therefore, the disclosed BiCisCAR T-cells combine the advantages of both CD28 and 4- IBB signaling when engaging their cognate antigens, resulting in a stronger, sustained, and complementary TCR activation through mTORCl, AP-1, and NF-KB signaling pathways. Additionally, through a multi-modal single-cell analysis of CAR T-cells isolated from tumors in animal models, a high proportion of effector T-cells and effector memory T-cells expressing high levels of cytotoxic genes (GZMK, GZMA, GZMH, GZMB, GNLY, PRF1, and NKG7) in T-cells expressing FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR was observed. This could explain the potency of the BiCisCAR, consistently displaying the fastest tumor shrinkage or clearance throughout the study, including the eradication of RMS559 tumors with just 1 million CAR T-cells.

Example 1: Materials and Methods

This example describes the materials and experimental procedures for the studies described in Examples 2-9.

Cell lines, cell culture, CDX and PDX tumors

Human RMS cell lines RH30, RMS559, SCMC, RH5, RH4, RD, and CTR cells were cultured in Dulbecco Modified Eagle Medium (DMEM, Quality Biological), supplemented with 10% FBS (Gibco, Life Technologies), 10 mM HEPES, 100 U/mL penicillin, 100 pg/ml streptomycin and 2 mM L-glutamine (Gibco, Life technologies). The JR and BIRCH cell lines were cultured in RPMI1640, supplemented with 10% FBS (Gibco, Life Technologies), 100 U/mL penicillin, 100 jrg/ml streptomycin, and 2 mM L-glutamine. All cell lines used were STR DNA fingerprinted to confirm their identity every 6 months and regularly tested to be mycoplasma negative by the My co Alert kit (Lonza).

The RMS cell lines RH30, RMS559, and JR were stably transduced with a lentiviral vector encoding the GFP-Firefly-Luciferase gene for the monitoring of tumor burden by IVIS imaging and implanted into NSG mice for tumor study which were named cell line derived xenografts (CDX). FGFR4- or C 276-KO RH30 cell lines were generated by CRISPR/Cas9 gene-editing technology and subcloned by screening clones with comparable levels of CD276 or FGFR4 respectively as parental cell lines.

Patient-derived xenograft tumors for antigen density quantification were obtained from St. Jude Children's research hospital (Memphis, Tennessee, USA) and then expanded in NSG mice. After dissecting the tumor from mice, tumors were processed into homogenous single-cell suspensions via mechanical dissociation using a gentle MACS dissociator (Miltenyi), passage through a 70-micron filter, and followed by 2 wash steps with PBS. The PDX samples used in this study are xenograft SJRHB013759_Xl (FP-RMS), RMS-ZH003 (FP-RMS), SJRHB013758 (FN-RMS), RMS-ZH010 (FN-RMS), SJRHB000026_X2 (FN-RMS), SJRHB013_X (FN-RMS), and SJRHB015720_Xl (MYODI Mutant RMS).

Generation of CAR Constructs

All CAR constructs were generated using the pELPS lentiviral transfer vector with an EF-la promoter. FGFR4-targeting CARs were generated using the FGFR4-specific single-chain variable fragment (3A11 scFv), the CD8a or CD28 hinge and transmembrane domain (HTM), the CD28 or 4- 1BB co-stimulatory domain (CSD) and CD3C intracellular domain (FGFR4.8HTM.BBz previously described (Tian et al., Cell Rep Med 4( 10) : 101212, 2023), FGFR4.28HTM.BBz and FGFR4.28HTM.28z). CD276-specific CARs were generated using the MGA271 scFv provided by MacroGenics (Rockville, MD), the CD8a HTM domain, the 4-1 BB CSDs and CD3^ intracellular domain (CD276.8HTM.BBz).

The dual targeting CAR constructs were generated by individually combining the CAR cassette encoding the FGFR4.8HTM.BBz, FGFR4.28HTM.BBz or FGFR4.28HTM.28z with the CD276.8HTM.BBz CAR linked with a P2A-sequence peptide (FGFR4.8HTM.BBz- CD276.8HTM.BBz, FGFR4.28HTM.BBz-CD276.8HTM.BBz, and FGFR4.28HTM.28z- CD276.8HTM.BBz). These constructs were generated using restriction cloning (Rapid T4 DNA Ligation Kit, Takara) or using the Seamless Cloning and Assembly Kit (GeneArt, Invitrogen) according to manufacturer instructions. Any homologous sequences were codon-wobbled to avoid recombination for these bicistronic CAR constructs.

Lentiviral preparation, transduction, and expansion of human T-cells

The aforementioned single targeting CARs or BiCisCAR-encoding lentiviral supernatant was produced by transient transfection of the Lenti-X-293T lentiviral packaging cell line with the corresponding CAR plasmids, using the previously described method (Qin et al. , Mol Ther Oncolytics 11:127-137, 2018). Concentrated lentivirus for transduction of human T-cells was prepared as previously described (Tian etal., J Clin Invest 132(16):el55621, 2022). Briefly, buffy coats from healthy donors were obtained from the blood bank of NIH for isolation of peripheral blood mononuclear cells (PBMCs) using Histopaque®-1.077gm/mL (Sigma, Cat# 10771) according to the manufacturer’s instructions. PBMCs were activated with CD3 and CD28 microbeads at a ratio of 1:1 (Dynabeads Human T-Expander CD3/CD28, Thermo Fisher Scientific, Cat# 11141D) in AIM-V media (Invitrogen) containing 40 lU/mL recombinant IL-2 (rIL-2, Clinigen Inc.) and 5% heat- inactivated FBS for 48 hours. Then activated PBMCs were transduced with CAR-expressing lentiviral at a multiplicity of infection (MOI) of 14 twice, followed by CD3/CD28 beads removal, and cell expansion in fresh AIM-V media with 5% heat-inactivated FBS and 200 ZU/mL rIL-2. Culture media was changed every 2~3 days until harvest on day 9 or 10. Mock T-cells, also called un-transduced T- cells (UTD) were treated the same as transduced T-cells except during the transduction procedure.

ChlP-Seq data Analysis

Previously published ChlP-seq data for histone mark H3K27ac, transcript factor MYODI and fusion gene PAX3-FOXO1 in RMS tumors and cell lines (RH4 and RH30 for FP-RMS; CTR and RD for FN-RMS) were used in the analysis (Gryder et al., Cancer Discov 7:884-899, 2017; Gryder et al. , Nat Genet 51:1714-1722, 2019). All reads were mapped to human genome build h l9 using BWA, and indexed BAMs were converted to compressed TDF format at 25 bp bin resolution after reads extension to the median fragment length (-200 bp extended past each mapped single end of 75 bp reads). Files were visualized in an IGV viewer (online at software.broadinstitute.org/software/igv/download).

Quantitation of FGFR4 or CD276 molecules on RMS cells or PDXs

Staining for FGFR4 or CD276 expression on patient-derived RMS cell lines was performed with mouse anti -human FGFR4 antibody (clone 3A11, in-house) or recombinant humanized antihuman CD276 antibody (clone MGA271, Creative Biolabs, Cat# TAB-117CL), followed by incubation with PE-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (Biolegend, Cat# 405307) or R-Phycoerythrin AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, Fey Fragment specific (Jackson ImmunoResearch Laboratories, Cat# 109-116-170). FGFR4 or CD276 surface molecules per cell were calculated after subtracting background signal emanating from a respective isotype control antibody (Clone MG1-45 for mouse IgGl isotype control antibody, Clone QA16A12 for human IgGl antibody, Biolegend) by the Quantibrite PE Quantitation Kit (BD Biosciences, Cat# 340495) according to the manufacturer's protocol.

For quantification of cell surface FGFR4 and CD276 on RMS CDXs and PDXs, xenografted tumors were implanted into the flank of 5-8 week-old female NSG mice after thawing and washing. When the tumor engraftment grew up to 100 - 2000 mm³ measured by a caliper, xenograft tumor tissue was mechanically dissociated by the flat end of a plunger after harvesting and passed through 100 pm cell strainer followed by two wash steps with PBS to obtain a homogenous single cell suspension. Single tumor cells were processed immediately for staining as forementioned for cell lines. Dead/live dye was used for identifying viable tumor cells.

Cytotoxicity Assay of CAR T-cells xCELLigence® real-time cell analysis (RTCA) was used to test the killing ability of CAR T- cells against tumor cells as previously described (Tian el al., J Clin Invest 132(16):el55621, 2022). Briefly, 1E+4 of human RMS cell lines (RH30, RMS559, JR, FGFR4KO, or CD276KO RH30 cells) were separately seeded on an E-Plate 16 (ACEABiosciences). After cell settling down for 4 hours, effector CAR T-cells were added into the corresponding wells at different E:T ratios as indicated in figure legends. Then tumor cell number was continuously monitored for additional 20, 44, or 68 hours and CAR T-cell -mediated cell death was indicated by a decrease in cell number. Data were analyzed using RTCA Software 2.0 (Acea Biosciences) with normalization to that before CAR T-cells addition (about 4 hours after tumor cell addition). At the endpoint, cells were spun down, and supernatant was collected for cytokine production measurements using V-PLEX Custom Human Biomarkers Proinflammatory Panel 1 (Human IFN-y, Human IL-2, and Human TNF-a, Meso Scale Discovery) following manufacturer’s instructions.

Xenograft mouse models

Five to eight week-old female in-house bred NSG mice (NOD.Cg-PrkdcscidI12rgtm- IWjl/SzJ; NCI CCR Animal Resource Program, NCI Biological Testing Branch) were used for animal experiments. Animals bearing engrafted tumors were randomized into cohorts to ensure a similar mean tumor burden/group based on bioluminescent flux[P/s] values and caliper measurements at study enrollment. Animals were euthanized upon showing symptoms of graft versus host diseases (GvHD), including not feeding, lack of activity, abnormal grooming behavior, hunched back posture, or when any diameter of tumor xenografts reached 1.7 cm as the endpoint.

For orthotopic RMS models, luciferase labeled RMS cells (1E+6 of RH30, 2E+6 of RMS559, or 3E+6 of JR, as indicated in the figure legend) were resuspended in Matrigel (Corning) after washing and intramuscularly injected into NSG mice as previously described ⁷. At 7 days or 14 days after tumor implantation, 1E+6 or 2.5E+6 CAR-positive T-cells were intravenously injected via tail vein. Tumor engraftment and growths were followed by leg volume measurement using a caliper twice every week and bioluminescence imaging on an IVIS spectrum instrument (Caliper Life Science, Hopkinton, MA, USA) every week. Tumor volumes were calculated as Leg volume = n(length X width²)/6. For bioluminescence imaging, mice were intraperitoneally injected (i.p.) with 3 mg D-luciferin (PerkinElmer) and imaged 20 minutes later. Bioluminescent signal flux was quantified as photons per second per square centimeter per steradian (photons/s/cnr/sr) with Living Image software (PerkinElmer, Waltham, MA, USA). All mice were monitored every other day for signs of toxicity. Mice were euthanized when either length or width reached/exceeded 1.7 cm or an animal displayed signs of toxicity including excessive weight loss due to tumor burden or GvHD.

Mice were bled via submandibular vein at specific intervals (day 10, 15, 23, or 32) to measure circulating T-cell frequency and/or phenotype. At the time of euthanasia, blood, spleen, or tumors were collected for CAR T-cell analyses. Immunophenotyping staining of circulating, splenic T-cells and TILs by flow cytometry

Red blood cells (RBCs) were first removed from 100-200 p.L peripheral blood samples using RBC lysis buffer (BioLegend, Cat# 420301). Spleen or xenograft tumor tissue was mechanically minced or dissociated separately by the flat end of a plunger after harvesting and individually passed through 70 pm or 100 pm cell strainer followed by two wash steps with FACS buffer (DPBS supplemented with 2% heat inactivated FBS and 2 mM EDTA) to obtain a homogenous single cell suspension. Then cells were stained with the following antibodies specific to human antigens for 30 minutes in the dark at 4°C: CD45-FITC (BioLegend, Clone HI30), CD62L-Percp/Cy5.5 (BioLegend, Clone DREG-56), Tim-3-PE-Cy7 (BioLegend, Clone F38-3E2), CD4-PE-Dazzle594 (BioLegend, Clone A161A1), CD8-APC (BD, Clone BW135/80), CD3-AF700 (BioLegend, Clone OKT3), CD39- APC-Cy7 (BioLegend, Clone Al), LAG3-BV421 (BioLegend, Clone 11C3C65), CD45RA-Brilliant Violet 605 (BioLegend, Clone H1100), PD-1-BV711 (BioLegend, Clone EH12.2H7), or CD8- BUV737 (BD, Clone SKI). Cells were washed 3 times with FACS buffer before flow cytometry analysis. Cells were gated for viable (fixable viability dye Ghost Dye™ Violet 510, TONBO, Cat#13- 0870-T100) and singlet cells (SSC-W/SSC-H) before assessment of antigen expression. The absolute CAR T-cell counts in the blood or spleen from tumor -bearing NSG mice were quantified using CountBright Absolute Counting beads (Invitrogen, Cat# C36995) on an LSR Fortessa or FACSymphony A5 (BD Bioscience).

CAR expression was assessed by flow cytometry after incubation with soluble, recombinant, human FGFR4-Fc Chimera Protein (Abeam, Cat# ab83999) for FGFR4-CAR T-cells or Biotinylated Human B7-H3 (41g) / B7-H3b Protein (Aero Biosystem, Cat# B73-H82F5) for CD276 CAR T-cells, then followed by incubation with R-Phycoerythrin AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, Fey Fragment specific (Jackson ImmunoResearch Laboratories, Cat# 109-116-170) and R- Phycoerythrin-conjugated streptavidin or AF647 conjugated Fab specific for human IgG-Fc (Jackson ImmunoResearch Laboratories, Cat# 109-607-008) respectively. Both CAR binders’ expression of BiCisCAR T-cells was assessed using a combination of both detection reagents as indicated for individual figures. Data were collected with an LSR Fortessa or FACSymphony A5 (BD Bioscience) and analyzed by FlowJo v 10.7.2 software.

Single-cell analysis of CAR TILs using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq)

TILs were purified from RMS JR xenografts 11 days after T-cell administration using 40% and 70% percoll (Cytiva, Cat# 17089102) gradient separation. Viable human CD45⁺ lymphocytes were separated by sorting on a BD FACS Aria Fusion or a BD FACSAria UV. After washing with CITE-seq antibody staining buffer (DPBS with 0.05% BSA), sorted CD45⁺ lymphocytes were counted, resuspended at a concentration of 1 E+6/50pL, and stained with TotalSeq-C human “hashtag” antibodies (hashtag 5 or hashtag 6) for each mouse, allowing identification of different replicates from each group in the analysis. Next, the above TILs were individually stained with a cocktail of a TotalSeq-C human lyophilized panel (BioLegend) of 17 surface proteins, including CD3 (Clone UCHT1), CD4 (Clone RPA-T4), CD8 (Clone RPA-T8), CD45RA (Clone HI100), CD45RO (Clone UCHL1), CD27 (Clone 0323), CD95 (Clone DX2), CD62L(Clone DREG-56), CD25 (Clone BC96), CD137 (Clone 4B4-1), LAG-3 (Clone 11C3C65), CD39 (Clone Al), PD-1 (Clone EH12.2H7), TIM-3 (Clone F38-2E2), Mouse IgGl, K isotype (Clone MOPC-21), Mouse IgG2a, K isotype (Clone MOPC- 173), Mouse IgG2b, K isotype (Clone MPC-11). After 3 washes, TILs were resuspended in PBS and counted, and then replicates in each group were combined before proceeding immediately to singlecell immune profiling using a Chromium Single Cell 5’ Solution v2 platform system (lOx Genomics). lOx Genomics 5’ single-cell gene expression and cell-surface protein tag (CITE-seq) libraries were prepared as instructed by the lOx Genomics user guides. Libraries were sequenced on an Illumina NextS eq 500.

Reads were aligned with human reference genome version GRCh38 and CAR binder scFv region sequences and quantified with Cell Ranger 7.0.1 (lOx Genomics) using the standard workflow, which allowed for gating on CAR⁺ T-cells. The Cell Ranger raw output was imported into R (v4.2.1) using the Seurat package (v 4.3.0). Following filters were applied using the subset function to select high-quality live human single CAR T-cells: nFeature RNA > 400 & nFeature RNA < 8000; percent mitochondrial reads < 20%. Single-cell protein data (representing the quantification of antibody- derived tags (ADTs) in CITEseq data) was normalized by the DSB method as previously reported (Tian et al., J Clin Invest 132(16):el55621, 2022; Mule et al., Nat Commun 13:2099, 2022), which removes technical noise associated with unbound antibodies. The data matrices from 5 experimental groups, a total of 10 samples, were merged and then scaled and transformed with the SCTransform pipeline (Hafemeister and Satija, Genome Biol 20:296, 2019) to remove batch effect among different lanes. Weighted nearest neighbors (WNN) analysis of both merged RNA and ADT modalities was performed to obtain a UMAP reduction based on the above WNN graph, and a total of 21 clusters were generated with resolution = 0.4 (Hao et al., Cell 184:3573-3587, 2021). Clusters were defined by common markers analysis (Seurat FindAllMarkers with min.pct = 0.25 and logfe. threshold = 0.25) and protein markers expression (FIG. 15). Clusters 12, 13, 15, and 19 of very few cells were combined into cluster 0 and annotated as CD4⁺ Activated T-cells, because they expressed similar RNA and protein profiling despite their different TCR clone sequences. Similarly, clusters 16 and 17 were merged into cluster 1 and defined as CD8+ Early Tern (GZMK⁺CD27⁺). Cells in cluster 4 exhibited high expression of mitochondria genes, while cells in cluster 8 had low nCounts, nFeatures and high percentage of mitochondria genes. Therefore, both cluster 4 and 8 were removed from the analysis due to their low quality.

A differential gene expression assay was performed between FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR and the other four CAR groups to identify the genes listed specifically and highly expressed on the TILs of FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR group. Data matrix was first normalized using a SCTransform (SCT) assay. Then differentially expressed genes were identified by the ‘FindMarkers’ function in Seurat with parameter logfc. threshold = 0 using the non-parametric Wilcoxon rank sum test.

Cytokine production stimulated by FGFR4 or CD276 proteins

Serial diluted FGFR4-Fc or CD276-Fc chimera protein were first coated on 96-well flat bottom cell-culture plates overnight at 4°C. After washing once with PBS, 1E+4 CAR⁺ T-cells were plated in 200 pl AIM-V medium (Thermo Fisher, Cat# 12055091) per well for 20 hours. Activation of CAR T cells was performed in triplicates for each experimental condition. Culture supernatants were collected and measured for the production of IFNy, IL-2, and TNF-a by V-PLEX Custom Human Biomarkers Proinllammatory Panel 1 kit.

Activation of CAR T-cells for western-blotting assay

To assess signaling through the CAR, 2E+6 of BiCisCAR T-cells after starving for 4 hours were placed on ice for 5 minutes, and then 1 pg/ml of human FGFR4-Fc Chimera Protein (Abeam, Cat# ab83999) 1 pg/ml of Human B7-H3 (4Ig)-Fc Chimera Protein (Aero Biosystem, Cat# B73- H82F5) were added and incubated for 30 minutes on ice. Subsequently, cross-linking was induced by adding 5 pg/ml of AF647 conjugated Fab specific for human IgG-Fc (Jackson ImmunoResearch Laboratories, Cat# 109-607-008) to the indicated tubes and incubated at 37°C for the indicated periods. At the end of the period of stimulation, cells were quenched on ice for 5 minutes and spun at 4°C, then cell pellets were resuspended in 100 pL of Pierce RIPA buffer (Thermo Fisher Scientific, Cat# 89900) supplemented with Halt protease/phosphatase inhibitors (Thermo Fisher Scientific, Cat# 78442).

Western blot analysis was performed as previously described (Tian et al., Cell Rep Med 4(10): 101212, 2023; Hirabayashi et al., Nat Cancer 2:904-918, 2021). Briefly, cells were lysed by sonication, incubated on ice for 30 minutes, and then lysates were centrifuged for 20 minutes, the supernatant removed, and protein concentration quantified using a BCA (Promega) assay. Twenty pg of lysate protein was resolved on 4-12% Bis-Tris gels (Invitrogen, Cat# NP0336) and transferred to PVDF membrane, blocked in 5% nonfat milk in Tris-buffered saline and Tween-20 (TBS-T). Membranes were incubated at 4°C overnight in the following primary antibodies purchased from Cell Signaling Technology: anti-phospho-CD3g (Y142) (Clone EP265(2)Y, Cat# lb68235, Abeam), anti- CD3C (Clone 6C10.2, Cat# SC-1239, Santa Cruz Biotechnology); anti-phospho-Zap-70 (Tyr319)/Syk (Tyr352) (Clone 65E4, Cat #2717), anti-Zap-70 (Clone L1E5, Cat #2709); anti-Phospho-PLCyl (Tyr783) (Clone D6M9S, Cat #14008), anti-PLCy I (Clone D9H10, Cat#569O); anti-phospho-ERKl/2 (T202/Y204) (Clone D13.14.4E, Cat# 4370S), anti-ERKl/2 (Cat# 9102S); anti-phospho-Akt (Ser473) (D9E, 4060S) and anti-Akt (pan) (C67E7, 4691S) (Cell Signaling), anti-phospho-NF-KB p65 (Ser536) (Clone 93H1, Cat# 3033), anti-NF-KB p65 (Clone D14E12, Cat# 8242). After 3 washing three times in TBS-T, membranes were then incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (both Santa Cruz) at a dilution of 1 to 5,000 and developed with ECL Prime (Amersham, Cat# RPN2232) on the gel station (Bio-Rad). Densitometric analysis of the phosphorylation- specific antibodies was performed using ImageLab software. Phosphorylation levels were determined by calculating the ratio of the intensity of the signal obtained with phospho -specific antibodies relative to the total. Relative values were normalized to one of the untreated controls in every gel.

Statistics

GraphPad Prism 8 software was used for graphing and data analysis. Log-rank statistical tests were used for survival analyses. An unpaired, 2-tailed Student’s t-test was used to calculate the significant difference between 2 groups. An ordinary 1-way ANOVA was used for multiple groups comparisons. A 2-way ANOVA was used for in vitro cytokine production data, in vivo tumor growth curves, and bioluminescence signal analysis, -values of less than 0.05 was considered statistically significant. The statistical analysis method is also described in the individual figure legends.

Example 2: Altering HTM or CSD significantly improves FGFR4 CAR activity against RMS orthotopic xenografts

It was previously reported that 10 million FGFR4 CAR T-cells with a CD8a HTM and a 4- 1BB CSD (FGFR4.8HTM.BBz, FIG. 1 A, left) could effectively eradicate an aggressive RH30 FP- RMS orthotopic intramuscular xenografts in NSG mice (Tian et al., Cell Rep Med 4(10): 101212, 2023). However, under stress conditions with only 2.5 million CAR T-cells, they are unable to eliminate tumors in this model (FIG. 8). Since modifications of CAR HTM and CSD may enhance CAR activities (Heitzeneder et al. , Cancer Cell 40:53-69, 2022; Majzner et al. , Cancer Discov 10:702-723, 2020), these domains of the original FGFR4.8HTM.BBz CAR were substituted with a CD28 HTM (FGFR4.28HTM.BBz) or together with a CD28 CSD domain (FGFR4.28HTM.28z) (FIG. 1 A). Their efficacy was tested on two RMS xenograft models, RH30 (FP-RMS), and a more aggressive RMS559 (FN-RMS with a FGFR4 V550L activation mutation; McKinnon et al., Oncogene 37:2630-2644, 2018). The different domains did not alter the CD4/CD8 ratio or memory T- cell differentiation phenotypes of the CAR T-cells (FIGS. 9A-9F). FGFR4.28HTM.BBz showed similar in vitro tumor-killing ability and cytokine production as the original FGFR4.8HTM.BBz CAR T-cells, while FGFR4.28HTM.28z demonstrated significantly increased cytotoxicity (FIG. IB) and cytokine production compared to the other designs (FIG. 1C; FIGS. 9G and 9H). In an RH30 orthotopic mouse model, only 2.5 million of FGFR4.28HTM.BBz or FGFR4.28HTM.28z CAR T- cells could mediate complete responses with significant survival benefits (FIGS. ID to 1G), demonstrating their superiority over the original FGFR4-CAR in these stress conditions. In addition, FGFR4.28HTM.28z CAR exhibited better tumor control than FGFR4.28HTM.BBz CAR in the more aggressive orthotopic model of RMS559 cells. Two out of five mice treated with FGFR4.28HTM.28z CAR had a complete response (FIGS. 1H-1K), whereas 10 million of FGFR4.8HTM.BBz CAR T- cells failed to show therapeutic benefit (FIGS. 10A-10D).

The expansion and propensity for exhaustion of the CAR T-cells expressing the different CSDs in mouse blood was next investigated. FGFR4.28HTM.BBz showed increased expansion and persistence compared to FGFR4.28HTM.28z at day 23 post-CAR T-cells infusion in the RH30 model (FIG. IL; FIGS. 11A-11C) or at day 32 in the RMS559 IM model (FIGS. 11D-11G). FGFR4.28HTM.28z exhibited higher expression of the T-cell exhaustion markers such as CD39, PD- 1, LAG-3, and Tim-3 (FIG. IM, FIG. 11H), in keeping with previous reports that CD28 CSD-based CAR T-cells are susceptible to exhaustion (Roselli et al., J Immunother Cancer 9(10):e003354, 2021; Wijewarnasuriya et al., Cancer Immunol Res 8:732-742, 2020; Philipson et al., Sci Signal 13(625):eaay8248, 2020; Long et al., Nat Med 21:581-590, 2015). Taking these data together, the CD28 HTM domain imparted a greater tumor-killing activity than a CD8 HTM in the second- generation CAR design with a 4-1BB CSD. In addition, while CD28 CSD was linked with a shorter persistence and increased T-cell exhaustion compared to 4- IBB CSD constructs, an FGFR4 CAR using both CD28 HTM and CSD demonstrated significantly enhanced anti-tumor activity, leading to extended survival compared to the 4-lBB-based CARs in a more aggressive RMS559 model.

Example 3: Identification and quantification of surface antigens FGFR4 and CD276 in RMS cells

The limited efficacy of improved FGFR4 CAR against RMS559 tumor may be due to heterogenous expression of FGFR4 expression on RMS. Therefore, identifying another tumor- associated antigen was sought to achieve better tumor control by preventing immune escape. CD276 has been reported as a potential downstream target of PAX3-FOXO1 in FP-RMS (Kanayama et al., Sci Rep 11:18802, 2021). Reanalysis of chromatin immunoprecipitation (ChIP) sequencing data (Gryder et al., Cancer Discov 7:884-899, 2017; Gryder et al., Nat Genet 51:1714-1722, 2019) confirmed CD276 as a target of PAX3-FOXO1 in FP-RMS cell lines (RH4 and RH30), and readily detected both H3K27ac marks and MYODI bindings at the enhancers in the CD276 locus for both FP-RMS and FN-RMS cell lines including RH4, RH30, CTR and RD (FIG. 2A), suggesting active expression of CD276 in RMS cells. Therefore, like FGFR4, CD276 is a biologically relevant cell surface immune target for RMS.

Using RNA-Seq data for human RMS tumors (n = 98) and cell lines (n = 33), it was confirmed that FGFR4 and CD276 were highly expressed in RMS compared to 147 normal tissues with significant heterogeneity among tumor samples (FIG. 2B). To determine whether this expression pattern is consistent with their protein level, the cell surface expression of FGFR4 and CD276 on RMS cell lines and patient-derived xenografts (PDXs) was quantified using flow cytometry (FIGS. 2C to 2F). On average, RMS cell lines and cell-line-derived xenografts (CDX) expressed 6,150 (range 211-15,115) FGFR4 molecules/cell and 41,577 (range 2,893-67,786) CD276 molecules/cell respectively (FIG. 2D). Additionally, there were on average 1,614 (range 132- 6,266), and 3,957 (range 1,908-6,169) molecules/cell for FGFR4 and CD276 respectively on 7 RMS PDXs (FIGS. 2E and 2F). Notably, there was considerable intratumor and interpatient variability in the expression of both antigens in all RMS samples (FIGS. 2D and 2F). Thus, these data indicate that targeting both antigens simultaneously may be more effective to prevent immune escape in CAR T-cell therapies for RMS.

Example 4: Dual targeting FGFR4 and CD276 BiCisCARs eradicate RH30 and RMS559 orthotopic tumors

Given high CD276 expression on RMS cells, the efficacy of a previously published CD276- targeting construct (CD276.8HTM.BBz; FIG. 3A) was tested against RMS in an RH30 I.M. xenograft model. The results showed that CD276 CAR T-cells significantly shrank tumors, however, they could not eliminate tumors in all treated mice (FIGS. 3B-3D).

To address the heterogeneous expression of target antigens that may lead to resistance, 3 bicistronic CARs (BiCisCARs) targeting both FGFR4 and CD276 were generated using 3 FGFR4 single CARs (FGFR4.8HTM.BBz, FGFR4.28HTM.BBz, and FGFR4.28HTM.28z) in combination with the CD276 CAR (CD276.8HTM.BBz) (FIG. 3E) and then tested their properties. These BiCisCARs use a cleavable linker, allowing co-expression of both CARs in the same T-cell (Tian et al., J Clin Invest 132(16):el55621, 2022). All three BiCisCAR T-cells had similar FGFR4 and CD276 binding capacity (FIG. 12A) and exhibited a similar high tumor-killing ability to RH30 in vitro at an effector: target (E: T) ratio of 1:10 (FIG. 12B). However, FGFR4.8HTM.BBz-CD276.8HTM.BBz BiCisCAR, a dual targeting CAR sharing the same CD8 HTM and 4- IBB CSD for both FGFR4 and CD276 CAR cassettes, only delayed tumor progression compared to mock T-cells in an RH30 orthotopic xenograft model (FIGS. 12C-12G). A reduced ability of T-cells transduced with this CAR for expansion or persistence was observed compared to the other two BiCisCARs (FIG. S5H). In contrast, both FGFR4.28HTM.BBz-CD276.8HTM.BBz and FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCARs completely eradicated tumors in all mice, and the latter showed a more rapid tumor elimination (FIGS. 12C-12G).

To further evaluate these two more effective BiCisCARs, aggressive RMS559 cell in vitro and in vivo models were used where FGFR4 single CAR T-cells showed limited antitumor activity. The FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells demonstrated superior tumor-killing activity than those of FGFR4.28HTM.BBz-CD276.8HTM.BBz in vitro (FIG. 3F). When tested in vivo, T-cells expressing FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR exhibited faster tumor eradication, increased persistence, and reduced expression of exhaustion markers such as CD39, PD- 1, and LAG-3 (FIGS. 3G-3L). Example 5: FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR showed superior expansion, tumor infiltration, and limited exhaustion under stress conditions

These CARs including two FGFR4-targeting CARs (FGFR4.28HTM.BBz and FGFR4.28HTM.28z), one CD276-targeting CAR (CD276.8HTM.BBz), and two BiCisCARs (FGFR4.28HTM.BBz-CD276.8HTM.BBz and FGFR4.28HTM.28z-CD276.8HTM.BBz) were next tested in a high stress model where only 1 million CAR T cells were used to treat large RMS559 orthotopic xenografts (FIG. 4A). Two FGFR4-targeting CARs and FGFR4.28HTM.BBz- CD276.8HTM.BBz BiCisCAR showed minimal antitumor activity, and CD276-targeting CAR cleared tumors in 2 out of 5 mice. By contrast, FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR demonstrated the highest potency, controlling tumors in 4 out of 5 mice, accompanied by a more substantial cell expansion (FIGS. 4B-4F).

Because of very high level expression of CD276 on both RH30 and RMS559 cells and dosedependence on their antigen target density on tumors for CAR T-cell activity (Heitzeneder et al., Cancer Cell 40:53-69, 2022), the performance of these CARs were examined using another aggressive FP-RMS RMS cell JR expressing lower CD276 (8,066 ± 123 molccules/ccll in cell line; 2,893 ± 234 molecules/cell in xenograft) (FIG. 2D). Luciferase-labeled JR cells were intramuscularly injected in NSG mice and then treated with 2.5 million CAR T-cells 13 days later (FIG. 4G). Similar to the results in the aforementioned RMS559 model under stressed conditions, the two single targeting FGFR4 CAR T-cells failed to control tumor growth (FIG. 4H). FGFR4.28HTM.BBz- CD276.8HTM.BBz BiCisCAR T-cells only had modest antitumor activity, worse than the single CD276 targeting CAR (FIGS. 4H and 41). Nevertheless, both the CD276.8HTM.BBz single CAR and FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells controlled tumor and significantly increased survival, with the BiCisCAR demonstrating a swifter tumor clearance (FIGS. 4H and 41; FIG. 13 A).

T-cell expansion, tumor infiltration, and exhaustion are pivotal factors that influence therapeutic efficacy of CAR T-cells (Rafiq et al., Nat Rev Clin Oncol 17:147-167, 2020; Moon et al. , Clin Cancer Res 20:4262-4273, 2014; Ghorashian et al., Nat Med 25:1408-1414, 2019; Caruana et al., Nat Med 21:524-529, 2015). To gain a deeper insight into these characteristics of our CARs, an analysis was conducted of tumor-infiltrating CAR T-cells as well as circulating CAR T-cells in the blood on day 16 and 21 using flow cytometry, prior to tumor clearance. Remarkably, the FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells exhibited the highest CAR T-cell count in the bloodstream and within tumors when compared to the other four CARs including CD276.8HTM.BBz (FIGS. 4J and 4K). Furthermore, the proportion of T-cells transduced with FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR that expressed exhaustion markers was significantly lower within tumor (CD39 and Tim-3) and in the blood (CD39 and LAG-3) compared to those observed with other CARs (FIGS. 4L-4O; FIGS. 13B-13E). These findings are consistent with the enhanced and rapid antitumor activity of this BiCisCAR. Consequently, FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR surpassed all other single -targeting CARs and dual-targeting BiCisCARs, showing the most robust expansion and the least exhaustion.

Example 6: Multimodal single-cell profiling reveals distinct phenotype of FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR T-cells, confirming its functional superiority over other CAR T- cells

To gain insight into the molecular underpinnings of the superior antitumor effect exhibited by FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells, Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq) was performed using 14 protein markers (Table 24) on tumorinfiltrating T-cells (TILs) isolated from JR tumor-bearing mice 11 days after CAR T-cells treatment. This time point coincided with the onset of maximal infiltration of TILs, just prior to observable tumor shrinkage (FIG. 5A). After filtering and batch effect correction, high-quality data was obtained for 38,108 tumor-infiltrating T-cells from 10 mice, which identified 13 distinct cell populations using weighted nearest-neighbor (WNN) analysis (see Example 1) of integrated protein and transcriptome data (FIGS. 14A and 14B). These cell populations include CD8⁺ T-cells (clusters 1 , 2, 3, 5, 7, 10, and 14), CD4⁺ T-cells (clusters 0 and 9), CD4⁺ or CD8⁺ mixture T-cells (cluster 6), y5 T-cells (cluster 11), and natural killer (NK)-like T-cells (cluster 18) (FIGS. 14B and 14C; FIG. 15). Expression signatures of these clusters allowed for annotating each T-cell subtype (Table 25, FIG. 14C and FIG. 15).

Table 24. Antibody-oligo conjugates directed against T-cell antigens and cell hashing antibodies were used in CITE-seq assay

Table 25. Clusters Annotation TILs exhibited notable differences of subtypes among mice treated with various CAR T-cells (FIGS. 5B and 5C). For example, CD276.8HTM.BBz CAR T-cells demonstrated the highest fraction of activated T effector cells (C6_Activated Teff, IFNG⁺) in both CD4⁺ and CD8⁺ compartments (FIG. 5C). Interestingly, the FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR displayed a higher proportion of CD8⁺ effector memory cells and granzyme-positive effector T-cells (Cl + C3 + C7) within the CD8⁺ T-cell populations, as depicted in FIGS. 5B and 5C. Concurrently, tumors treated with CD276.8HTM.BBz single CAR and FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T cells exhibited lower frequencies of activated CD4⁺ T-cells (FIGS. 5B and 5C). These findings are consistent with the established differentiation program of T-cell responses following activation (activated T^IFN-y producing activated Teff^ cytotoxic T-cell) (Cui and Kaech, Immunol Rev 236:151-166, 2010; Harty et al., Nat Rev Immunol 8:107-119, 2008; Seder and Ahmed, Nat Immunol 4:835-842, 2003; Lawrence and Braciale, J Immunol 173:1209-1218, 2004). Furthermore, to identify genes that were preferentially expressed in tumor-infiltrating T-cells expressing FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR, a differential expression analysis was performed (FIG. 5D). This analysis revealed the overexpression of genes such as perforin and multiple granzymes, which are associated with antitumor activity and are considered hallmarks of cytotoxic CD8⁺ T-cells (FIG. 5E). Therefore, these data demonstrate that FGFR4.28HTM.28z- CD276.8HTM.BBz BiCisCAR T-cells rapidly expanded to generate effector and memory subsets in vivo, providing an explanation for their heightened potency against the aggressive RMS559 and JR solid RMS tumors.

Example 8: BiCisCAR T-cells overcome heterogeneous expression of FGFR4 and CD276 in vivo

To assess the BiCisCAR’s capacity to address the heterogeneous expression of FGFR4 and CD276 in RMS, CRISPR-KO clones were created for FGFR4 or CD276 in RH30 cells (FIG. 6A). As anticipated, single CAR T-cells could not eliminate RH30 cells lacking the target antigen. In contrast, FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells demonstrated consistent cytotoxicity regardless of the presence or absence of either FGFR4 or CD276 (FIGS. 6B-6D).

To model heterogeneous expression of tumor antigens in an in vivo model, bilateral orthotopic xenografts with RH30 FGFR4-KO (right leg) and CD276-KO (left leg) were established (FIG. 6E). In accordance with the in vitro data, mice treated with single CAR T-cells exhibited selective tumor shrinkage or eradication only in the presence of the corresponding target antigen. In contrast, dual targeting CAR T-cells effectively eliminated tumors on both sides (FIGS. 6F-6J). This result not only affirmed the specificity of the single CARs but also underscored the efficacy of BiCisCAR in eradicating tumors with heterogenous antigen expression. Example 9: Presence of both CD28 and 4-1BB CSDs in dual-targeting CAR T-cells leads to heightened CAR signal strength, sustained T-cell activation signaling

The data described herein, in conjunction with findings from previous studies (Cappell and Kochenderfer, Nat Rev Clin Oncol 18:715-727, 2021; Zhao et al., Cancer Cell 28:415-428, 2015; Philipson et al., Sci Signal 13(625):eaay8248, 2020; Long et al., Nat Med 21:581-590, 2015; Kawalekar et al., Immunity 44:380-390, 2016), indicate that CAR T-cells with a CD28 CSD exhibit rapid expansion and increased anti-tumor activity but are linked to shorter persistence and a greater tendency for T-cell exhaustion compared to those with a 4- IBB CSD. The T-cells expressing dual targeting FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR demonstrated the highest level of tumor killing, T-cell expansion, persistence, and lowest degree of exhaustion. This suggests an additive or potentially synergistic effect resulting from the presence of both CD28 and 4- IBB CSDs in the same T-cell. To quantify the activity of the BiCisCAR, its cytotoxicity was first compared to single CARs in vitro. T-cells expressing the FGFR4.28HTM.28z BiCisCAR exhibited superior cytotoxic activity than the single-targeting CARs against RH30, RMS559, and JR cells which express high levels of FGFR4 but varying levels of CD276 (FIG. 16). The cytokine production was then measured in activated BiCisCAR T-cells following stimulation with serially diluted plate-coated proteins containing FGFR4 or CD276 or both antigens. The production of all three cytokines, IFN-y, IL-2, and TNF-a, displayed synergistic behavior when both CAR binders engaged their respective antigens for the FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells at the antigen concentration above 0.00625 pM (FIGS. 7A-7C). In contrast, FGFR4.28HTM.BBz-CD276.8HTM.BBz BiCisCAR T-cells exhibited lower cytokine levels following dual activation compared to CD276 activation alone at antigen concentrations <0.01 pM, with only weak synergistic effects on IFN-y and IL-2 production and an additive effect for TNF-a production even at higher antigen concentration (> 0.025 pM) (FIGS. 17A-17C). Hence, these data indicate BiCisCAR T-cells possessing both CD28 and 4-1BB CSDs exhibit a strong synergistic effect for cytokine production, whereas the BiCisCAR using the same 4- IBB CSD domain primarily showed additive or modest synergistic effects at higher antigen densities.

The activation of TCR triggers signaling cascades that ultimately influence cell fate by regulating cytokine production, cell survival, proliferation, and differentiation (Gaud et al. , Nat Rev Immunol 18:485-497, 2018; Hwang et al., Exp Mol Med 52:750-761, 2020). It was hypothesized that dual targeting CARs with different CSDs activate distinct signaling pathways, resulting in optimal signaling strength for robust T-cell activation. To test this hypothesis, the downstream phosphorylation of the two BiCisCARs was examined in T-cells stimulated by soluble FGFR4-Fc, CD276-Fc, or both antigens at various time points. Western blotting revealed higher phosphorylation levels of proximal signaling proteins (pCD3^-CAR, pZAP70, and pPLCyl) and distal signaling proteins (pAKT and pERKl/2) in FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells following FGFR4 stimulation compared to CD276 stimulation for 5 or 15 minutes (FIG. 7D). In addition, sustained phosphorylation of most of these signaling proteins was observed at 30 minutes post-stimulation in the presence of both antigens compared to single antigen stimulation (FIG. 7D). Notably, a higher level of p65 phosphorylation was also observed, which is associated with 4- IBB signaling (Dai et al. , Front Immunol 11 :539654, 2020), in CD276 CAR T-cells compared to CD28- based FGFR4 CAR T-cells at 5 minutes after antigen stimulation (FIG. 7D). Dual stimulation in FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells further prolonged p65 phosphorylation to at least 30 minutes post-stimulation (FIG. 7D). In contrast, the two 4-lBB-based CAR cassettes in FGFR4.28HTM.BBz-CD276.8HTM.BBz BiCisCAR T-cells showed lower levels of PLCyl, AKT, and ERK1/2 phosphorylation stimulated by FGFR4 or CD276 protein for 5 minutes (FIG. 17D). Additionally, the FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR exhibited higher activation of PLCyl, AKT, and ERK1/2 phosphorylation compared to FGFR4.28HTM.BBz-CD276.8HTM.BBz BiCisCAR T-cells upon dual stimulation with both FGFR4 and CD276 (FIG. 17D). Thus, the presence of two different CSDs in the BiCisCAR T-cells likely initiates complementary signaling pathways downstream of TCR, including AKT, ERK1/2, and p65 (FIG. 7E). Activation of these pathways enhanced T-cell activation response and may consequently augment CAR T cell antitumor activity.

Example 10: CD3c calibration of FGFR4/CD276 BiCisCAR constructs

This example describes optimization of BiCisCAR constructs by calibrating CD3^ to enhance cytolytic potential.

Lentiviral preparation and CAR T-cells manufacturing

The CAR-encoding lentiviral supernatant was produced by a transient transfection of the Lenti-X-293T lentiviral packaging cell line with corresponding CAR plasmids. Concentrated lentivirus for transduction of human T-cells was prepared as previously described (Tian et al., J Clin Invest 132(16):el55621, 2022). Briefly, buffy coats from healthy donors were obtained from the NIH blood bank for peripheral blood mononuclear cells (PBMC) isolation using Histopaque®- 1.077 gm/mL (Sigma, Cat# 10771) according to the manufacturer’s instructions. PBMCs were activated with CD3 and CD28 microbeads at a ratio of 1: 1 (Dynabeads Human T-Expander CD3/CD28, Thermo Fisher Scientific, Cat# 11141D) in AIM-V media (Invitrogen) containing 40 lU/mL recombinant IL-2 (rIL-2, Clinigen Inc.) and 5% heat-inactivated FBS for 48 hours. Then activated PBMCs were transduced with CAR-expressing lentivirus at a multiplicity of infection (MOI) of 20 twice, followed by CD3/CD28 beads removal. T cells were expanded in fresh AIM-V media with 5% heat-inactivated FBS, 20 ng/mL rIL-7 and 10 ng/ml rIL-15. Culture medium was changed every 2 - 3 days until harvest on day 9 or 10. Mock T-cells, also referred to as un-transduced T-cells (UTD), were treated the same as CAR-transduced T-cells, except that the transduction procedure was excluded.

Cytotoxicity assay of CAR T-cells

GFP-expressing RMS cell lines (RH30, RMS559) were plated at a density of 1 x 10⁴ cells per well in 100 |1L of RPMI medium in a 96-well flat clear-bottom white TC-treated plate (Corning). Four hours later, 100 |1L of CAR T-cells were added to the designated wells at an effector-to-target (E:T) ratio of 1:5 for RH30 or 1:10 for RMS559. GFP fluorescence was monitored every 2 hours for 72 hours using the IncuCyte ZOOM system to track live tumor cells. The GFP-positive area per well, representing the remaining tumor cells, was normalized to the measurement taken immediately before the addition of CAR T cells.

Results

Following the generation of FGFR4- and CD276-targeting CAR T cells (FIG. 19), we assessed transduction efficiency by staining with recombinant human FGFR4 or CD276 protein (FIG. 20). FGFR4 single-target CAR T cells, including FGFR4.28HTM.BBz, FGFR4.28HTM.28z, and FGFR4.28HTM.28z(1 XX), specifically bound to FGFR4. While CD276 single-target CAR T cells, including CD276.8HTM.BBz and CD276.8HTM.BBz(lXX), exhibited selective binding to CD276. In contrast, all FGFR4/CD276 dual-targeting CAR T cells bound both antigens, confirming successful co-expression (FIG. 20). These findings indicate comparable expression levels of the individual CAR constructs within dual-targeting configurations.

Next, the in vitro cytolytic activity of the CAR T cells against RH30 (FIGS. 21A-21C) and RMS559 (FIGS. 22A-22C) cells following CD3^ calibration was assessed using the IncuCyte Live- Cell Analysis System. Among single-target CAR T cells, FGFR4 CARs incorporating CD28 costimulation (FGFR4.28HTM.28z) showed superior cytotoxicity compared to those containing 4-1BB (FGFR4.28HTM.BBz) in both RH30 and RMS559 assays (FIGS. 21B and 22B). However, introducing ITAM mutations into the CD3C domain (FGFR4.28HTM.28z(lXX)) significantly impaired cytotoxic function (FIG. 21B), consistent with the signaling redundancy between CD28 and CD3^, where attenuated CD3c activity compromises overall T-cell activation (Feucht et al., Nat Med 25:82-88, 2019). Likewise, CD276 CARs with ITAM mutations (CD276.8HTM.BBz(lXX)) exhibited reduced killing capacity against both RH30 and RMS559 cells (FIGS. 21B and 22B), further supporting the importance of intact CD3 signaling for effective T-cell function.

As demonstrated in the Examples above, dual- targeting BiCisCAR T cells that incorporate distinct co-stimulatory domains (FGFR4.28HTM.28z-CD276.8HTM.BBz) outperformed those using uniform co-stimulation (FGFR4.28HTM.BBz-CD276.8HTM.BBz) in cytotoxicity assays against RH30 and RMS559 (FIGS. 21C and 22C). Further optimization via CD3c calibration significantly enhanced the cytolytic potency of dual CAR T cells. Specifically, deletion of CD3C in the second CAR cassette or incorporation of IT AM mutations in one or both CARs markedly improved cytotoxic function (FIG. 21C). The most effective configuration combined ITAM mutations in the FGFR4 CAR with CD3c deletion in the CD276 CAR, resulting in the highest levels of tumor cell killing across both models (FIGS. 21C and 22C). These results demonstrate that fine-tuning CD3C signaling within dual- targeting CAR constructs can significantly enhance their therapeutic efficacy.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A chimeric antigen receptor (CAR), comprising: an extracellular antigen-binding domain that specifically binds fibroblast growth factor receptor 4 (FGFR4), comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a hinge and transmembrane (HTM) domain, comprising a CD28 HTM domain or a CD8 HTM domain; an intracellular co-stimulatory domain, comprising a CD28 intracellular co-stimulatory domain or a 4- IBB intracellular co-stimulatory domain; and a CD3^ intracellular signaling domain.

2. The CAR of claim 1, wherein: the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and/or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

3. The CAR of claim 1 or claim 2, wherein: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 1 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1; and/or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 2 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2.

4. The CAR of any one of claims 1-3, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1; and/or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 2.

5. The CAR of claim 1 or claim 2, wherein the VH domain and the VL domain comprise human framework sequences.

6. The CAR of any one of claims 1-5, comprising: a CD28 HTM domain and a CD28 co-stimulatory domain; or a CD28 HTM and a 4- IBB co-stimulatory domain.

7. The CAR of any one of claims 1-6, wherein: the amino acid sequence of the CD28 HTM is at least 90% identical to SEQ ID NO: 74, or comprises or consists of SEQ ID NO: 74; wherein the amino acid sequence of the CD28 intracellular co-stimulatory domain is at least 90% identical to SEQ ID NO: 77, or comprises or consists of SEQ ID NO: 77; or the amino acid sequence of the 4- IBB co-stimulatory domain is at least 90% identical to SEQ ID NO: 76, or comprises or consists of SEQ ID NO: 76.

8. The CAR of any one of claims 1-7, wherein the amino acid sequence of the CD3 intracellular signaling domain is at least 90% identical to SEQ ID NO: 78, or comprises or consists of SEQ ID NO: 78.

9. The CAR of any one of claims 1-7, wherein the CD3^ intracellular signaling domain comprises a first, a second, and a third immunoreceptor tyrosine -based activation motif (IT AM), wherein at least one tyrosine is substituted with a phenylalanine in the second and/or third ITAM.

10. The CAR of claim 9, wherein: the second ITAM comprises two tyrosine to phenylalanine substitutions; the third ITAM comprises two tyrosine to phenylalanine substitutions; or the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions.

11. The CAR of claim 9 or claim 10, wherein the amino acid sequence of the CD3^ intracellular signaling domain is at least 90% identical to SEQ ID NO: 79, or comprises or consists of SEQ ID NO: 79.

12. The CAR of any one of claims 1-11, wherein: the amino acid sequence of the CAR is at least 90% identical to residues 22-490 of SEQ ID NO: 18, residues 22-489 of SEQ ID NO: 22, residues 22-489 of SEQ ID NO: 34, residues 22-490 of SEQ ID NO: 40, residues 22-489 of SEQ ID NO: 44, or residues 22-489 of SEQ ID NO: 46; or the amino acid sequence of the CAR comprises or consists of residues 22-490 of SEQ ID NO: 18, residues 22-489 of SEQ ID NO: 22, residues 22-489 of SEQ ID NO: 34, residues 22-490 of SEQ ID NO: 40, residues 22-489 of SEQ ID NO: 44, or residues 22-489 of SEQ ID NO: 46.

13. A chimeric antigen receptor (CAR), comprising: an extracellular antigen-binding domain that specifically binds CD276, comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 hinge and transmembrane (HTM) region; and a 4- IBB intracellular co-stimulatory domain.

14. The CAR of claim 13, further comprising a CD3^ intracellular signaling domain.

15. The CAR of claim 13 or claim 14, wherein:

VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and/or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

16. The CAR of any one of claims 13-15, wherein: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 9 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 10 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10.

17. The CAR of any one of claims 13-16, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10.

18. The CAR of any one of claims 13-15, wherein the VH domain and the VL domain comprise human framework sequences.

19. The CAR of any one of claims 13-18, wherein: the amino acid sequence of the CD8 HTM domain is at least 90% identical to SEQ ID NO: 75, or comprises or consists of SEQ ID NO: 75; or the amino acid sequence of the 4- IBB co-stimulatory domain is at least 90% identical to SEQ ID NO: 76, or comprises or consists of SEQ ID NO: 76.

20. The CAR of any one of claims 14-19, wherein the amino acid sequence of the CD3^ intracellular signaling domain is at least 90% identical to SEQ ID NO: 78, or comprises or consists of SEQ ID NO: 78.

21. The CAR of any one of claims 14-19, wherein the CD3^ intracellular signaling domain comprises a first, a second, and a third immunoreceptor tyrosine-based activation motif (IT AM), wherein at least one tyrosine is substituted with a phenylalanine in the second and/or third ITAM.

22. The CAR of claim 21, wherein: the second ITAM comprises two tyrosine to phenylalanine substitutions; the third ITAM comprises two tyrosine to phenylalanine substitutions; or the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions.

23. The CAR of claim 21 or claim 22, wherein the amino acid sequence of the CD3 intracellular signaling domain is at least 90% identical to SEQ ID NO: 79, or comprises or consists of SEQ ID NO: 79.

24. The CAR of any one of claims 13-23, wherein: the amino acid sequence of the CAR is at least 90% identical to residues 541-1012 of SEQ ID NO: 20; the amino acid sequence of the CAR is at least 90% identical to residues 22-379 of SEQ ID NO: 24; the amino acid sequence of the CAR is at least 90% identical to residues 540-1011 of SEQ ID NO: 26; the amino acid sequence of the CAR comprises or consists of residues 541-1012 of SEQ ID NO: 20; the amino acid sequence of the CAR comprises or consists of residues 22-379 of SEQ ID NO: 24; the amino acid sequence of the CAR comprises or consists of residues 540-1011 of SEQ ID NO: 26; the amino acid sequence of the CAR comprises or consists of residues 22-491 of SEQ ID NO: 91; or the amino acid sequence of the CAR comprises or consists of residues 22-491 of SEQ ID NO: 93.

25. An isolated cell expressing the CAR of any one of claims 1-24.

26. A nucleic acid molecule encoding the CAR of any one of claims 1-24.

27. The nucleic acid molecule of claim 26, comprising or consisting of: nucleotides 64-1470 of SEQ ID NO: 17, nucleotides 1621-3036 of SEQ ID NO: 19, nucleotides 64-1467 of SEQ ID NO: 21, nucleotides 64-1137 of SEQ ID NO: 23, or nucleotides 1618- 3033 of SEQ ID NO: 25, nucleotides 64-1467 of SEQ ID NO: 33, nucleotides 64-1470 of SEQ ID NO: 39, nucleotides 64-1467 of SEQ ID NO: 43, nucleotides 64-1467 of SEQ ID NO: 45, nucleotides 64-1473 of SEQ ID NO: 90, or nucleotides 64-1473 of SEQ ID NO: 92, or a degenerate variant thereof; or

SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 90, or SEQ ID NO: 92, or a degenerate variant thereof.

28. An isolated cell expressing a first chimeric antigen receptor (CAR) targeting FGFR4 and a second CAR targeting CD276, wherein the first CAR comprises the CAR of any one of claims 1-20, and the second CAR comprises: an extracellular antigen-binding domain that specifically binds CD276, comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 hinge and transmembrane (HTM) domain; and a 4- IBB intracellular co-stimulatory domain.

29. The isolated cell of claim 28, wherein the second CAR further comprises a CD3^ intracellular signaling domain.

30. The isolated cell of claim 28 or claim 29, wherein for the second CAR: the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and/or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

31. The isolated cell of any one of claims 28-30, wherein for the second CAR: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 9 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 10 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10.

32. The isolated cell of any one of claims 28-31, wherein for the second CAR: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10.

33. The isolated cell of any one of claims 28-30, wherein for the second CAR, the VH domain and the VL domain comprise human framework sequences.

34. The isolated cell of any one of claims 28-33, wherein: the amino acid sequence of the CD8 HTM is at least 90% identical to SEQ ID NO: 75, or comprises or consists of SEQ ID NO: 75; or the amino acid sequence of the 4- IBB intracellular co-stimulatory domain is at least 90% identical to SEQ ID NO: 76, or comprises or consists of SEQ ID NO: 76.

35. The isolated cell of any one of claims 29-34, wherein the amino acid sequence of the CD3C intracellular signaling domain is at least 90% identical to SEQ ID NO: 78, or comprises or consists of SEQ ID NO: 78.

36. The isolated cell of any one of claims 29-34, wherein the CD3^ intracellular signaling domain comprises a first, a second, and a third immunorcccptor tyrosine-based activation motif

(IT AM), wherein at least one tyrosine is substituted with a phenylalanine in the second and/or third ITAM.

37. The isolated cell of claim 36, wherein: the second ITAM comprises two tyrosine to phenylalanine substitutions; the third ITAM comprises two tyrosine to phenylalanine substitutions; or the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions.

38. The isolated cell of claim 36 or claim 37, wherein the amino acid sequence of the CD3C intracellular signaling domain is at least 90% identical to SEQ ID NO: 79, or comprises or consists of SEQ ID NO: 79.

39. The isolated cell of any one of claims 28-38, wherein: the amino acid sequence of the second CAR is at least 90% identical to residues 541-1012 of SEQ ID NO: 20, or comprises or consists of residues 541-1012 of SEQ ID NO: 20; the amino acid sequence of the second CAR is at least 90% identical to residues 22-379 of SEQ ID NO: 24, or comprises or consists of SEQ ID NO: 24; or the amino acid sequence of the second CAR is at least 90% identical to residues 540-1011 of SEQ ID NO: 26, or comprises or consists of residues 540-1011 of SEQ ID NO: 26.

40. A nucleic acid molecule encoding a first chimeric antigen receptor (CAR) targeting FGFR4 and a second CAR targeting CD276, wherein the first CAR comprises the CAR of any one of claims 1-20, and the second CAR comprises: an extracellular antigen-binding domain that specifically binds CD276, comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 hinge and transmembrane (HTM) domain; and a 4- IBB intracellular co-stimulatory domain.

41. The nucleic acid molecule of claim 40, wherein the second CAR further comprises a CD3C intracellular signaling domain.

42. The nucleic acid molecule of claim 40 or claim 41, wherein for the second CAR: the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and/or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

43. The nucleic acid molecule of any one of claims 40-42, wherein for the second CAR: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 9 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 10 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10.

44. The nucleic acid molecule of any one of claims 40-43, wherein for the second CAR: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10.

45. The nucleic acid molecule of any one of claims 40-42, wherein for the second CAR, the VH domain and the VL domain comprise human framework sequences.

46. The nucleic acid molecule of any one of claims 40-45, wherein: the amino acid sequence of the CD8 HTM is at least 90% identical to SEQ ID NO: 75, or comprises or consists of SEQ ID NO: 75; or the amino acid sequence of the 4- IBB intracellular co-stimulatory domain is at least 90% identical to SEQ ID NO: 76, or comprises or consists of SEQ ID NO: 76.

47. The nucleic acid molecule of any one of claims 40-46, wherein the amino acid sequence of the CD3^ intracellular signaling domain is at least 90% identical to SEQ ID NO: 78, or comprises or consists of SEQ ID NO: 78.

48. The nucleic acid molecule of any one of claims 40-46, wherein the CD3^ intracellular signaling domain comprises a first, a second, and a third immunoreceptor tyrosine-based activation motif (IT AM), wherein at least one tyrosine is substituted with a phenylalanine in the second and/or third IT AM.

49. The nucleic acid molecule of claim 48, wherein: the second IT AM comprises two tyrosine to phenylalanine substitutions; the third ITAM comprises two tyrosine to phenylalanine substitutions; or the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions.

50. The nucleic acid molecule of claim 48 or claim 49, wherein the amino acid sequence of the CD3C intracellular signaling domain is at least 90% identical to SEQ ID NO: 79, or comprises or consists of SEQ ID NO: 79.

51. The nucleic acid molecule of any one of claims 40-50, wherein: the amino acid sequence of the second CAR is at least 90% identical to residues 541-1012 of SEQ ID NO: 20, or comprises or consists of residues 541-1012 of SEQ ID NO: 20; the amino acid sequence of the second CAR is at least 90% identical to residues 22-379 of SEQ ID NO: 24, or comprises or consists of residues 22-379 of SEQ ID NO: 24; or the amino acid sequence of the second CAR is at least 90% identical to residues 540-1011 of SEQ ID NO: 26, or comprises or consists of residues 540-1011 of SEQ ID NO: 26.

52. The nucleic acid molecule of any one of claims 40-51, wherein the coding sequences for the first CAR and the second CAR are separated by a sequence encoding a 2A site.

53. The nucleic acid molecule of any one of claims 40-52, comprising or consisting of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53.

54. The nucleic acid molecule of any one of claims 26, 27 and 40-53, operably linked to a promoter.

55. A vector comprising the nucleic acid molecule of any one of claims 26, 27 and 40-54.

56. The vector of claim 55, which is a lentivirus vector.

57. An isolated cell comprising the nucleic acid molecule of any one of claims 26, 27 and 40-54 or the vector of claim 55 or claim 56.

58. The isolated cell of any one of claims 25, 28-39 and 57, wherein the cell is an immune cell or an induced pluripotent stem cell (iPSC).

59. The isolated cell of claim 58, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell or a monocyte/macrophage.

60. A composition comprising a pharmaceutically acceptable carrier and the CAR of any one of claims 1-24, the isolated cell of any one of claims 25 and 57-59, nucleic acid molecule of any one of claims 26-54, or the vector of claim 55 or claim 56.

61. A method of treating a FGFR4-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of the CAR of any one of claims 1-24, the isolated cell of any one of claims 25 and 57-59, nucleic acid molecule of any one of claims 26-54, the vector of claim 55 or claim 56, or the composition of claim 60, thereby treating the cancer.

62. A method of inhibiting tumor growth or metastasis of a FGFR4-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of the CAR of any one of claims 1-24, the isolated cell of any one of claims 25 and 57-59, nucleic acid molecule of any one of claims 26-54, the vector of claim 55 or claim 56, or the composition of claim 60, thereby inhibiting tumor growth or metastasis of the cancer.

63. The method of claim 61 or claim 62, wherein the FGFR4-expressing cancer is a rhabdomyosarcoma (RMS), a lung cancer, a liver cancer, a breast cancer, a pancreatic cancer, a prostate cancer, a desmoplastic small round cell tumor (DSRCT), a yolk sac tumor (YST), an adrenocortical carcinoma (ACC), or a gastric adenocarcinoma.

64. The method of any one of claims 61-63, wherein the FGFR4-expressing cancer also expresses CD276.