Chimeric antigen receptor therapy for treating solid tumors

Abstract

Provided herein are novel anti-effector moiety antibodies or antigen-binding domains thereof, and CARs (with or without one or more booster elements) containing such effector moiety antigen-binding domains, as well as host cells expressing the receptors, and nucleic acid molecules encoding the receptors, and methods for their use in patient-specific immunotherapy that can be used to treat solid tumor cancers and other diseases and conditions.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 393,088, filed July 28, 2022, and U.S. Provisional Patent Application No. 63 / 433,632, filed December 19, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] Disclosure Areas This application relates to the field of cancer, and in particular to compositions encoding functional chimeric antigen receptors and methods of use thereof in patient-specific immunotherapy for treating solid tumors.

[0003] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. This ASCII copy, created in 2022 [[XXX]], is entitled Sequence Listing.txt and is [[XXX]] kilobytes in size.

[0004] Background of the Invention Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year, making it the second leading cause of death after cardiovascular disease, accounting for approximately one in four deaths. Solid tumors account for the vast majority of these deaths. While significant advances have been made in medical treatment of certain cancers, the five-year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrollably, making them extremely difficult to treat.

[0005] Chimeric antigen receptors are hybrid molecules that contain three essential units: (1) an extracellular antigen-binding motif, (2) a linking / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is typically engineered to resemble a single-chain variable fragment (scFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, including receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Tandem, triple, or quadruple targeting domains can be constructed by sequentially linking multiple antigen-binding motifs and attaching them to the CAR hinge, transmembrane domain, and intracellular sequence. Alternative cell types for CAR expression (e.g., NK, NKT, iNKT, or gamma-delta T cells) are also under development (Brown CE et al., Clin Cancer Res. 2012; 18(8):2199-209; Lehner M et al., PLoS One. 2012; 7(2):e31210). Significant challenges remain in defining the most active T cell populations to transduce with CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0006] The linking motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or it can be designed as an extended, flexible linker. Structural motifs, such as those derived from the IgG constant domain, can be used to distance the scFv-binding domain from the T cell plasma membrane. This may be important for some tumor targets, where the binding domain is particularly close to the tumor cell surface membrane (e.g., disialoganglioside GD2; Orentas et al., unpublished). To date, the signaling motif used in CARs has always included the CD3-ζ chain, because this core motif is a key signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was also used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009;183(9):5563-74). With the advent of new technologies that activate T cells with beads linked to anti-CD3 and anti-CD28 antibodies, the presence of the canonical "signal 2" from CD28 no longer needs to be encoded by the CAR itself. When using bead activation, third-generation vectors were not superior to second-generation vectors in in vitro assays and conferred no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013;121(7):1165-74; Kochenderfer JN et al., Blood. 2012;119(12):2709-20).This is supported by the clinical success of second-generation CD28 / CD3-ζ (Lee DW et al., American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD19-specific CARs in the CD137 / CD3-ζ signaling mode (Porter DL et al., N Engl J Med. 2011;365(8):725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members, such as OX40, can also provide important sustained signals in CAR-transduced T cells (Yvon E et al., Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which the CAR T-cell population is cultured.

[0007] Chimeric antigen receptor (CAR) T-cell therapy is a promising approach for treating both hematological and solid tumors; however, while the desired therapeutic benefit has not been achieved in solid tumors, it has proven highly effective in treating hematological malignancies, with several CAR T products approved by the U.S. Food and Drug Administration (FDA) for B-cell malignancies and multiple myeloma (Gill S, et al., Blood Rev. 2016; 30(3):157-1671; Victor E. et al., J Immunol April 1, 2021, 206(7):1561-1568; Wagner J, et al., Mol Ther. 2020 Nov 4; 28(11):2320-2339; He C, et al., Cancers. 2020; 12(7):196). CAR cells are engineered receptors for antigens that redirect the specificity and function of T lymphocytes and other immune cells to intended tumor targets (Sadelain M, et al., Cancer Discov. 2013;3:388-98). Engineered CAR T molecules redirect immune activity to desired antigens and, depending on the quantity and quality of this interaction, can produce a sustained, desired effect on tumor cells. Solid tumors present challenges for current CAR T targeting approaches. Challenges with this therapeutic modality include tumor antigen escape, poor persistence of engineered CAR molecules, and reduced efficacy within the solid tumor environment. Furthermore, CAR T cell-mediated toxicity resulting in cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity (ICANS), as well as off-target and on-target extratumoral CAR reactivity, have hindered further advances in CAR therapy in solid tumors. Optimization of CAR design remains largely empirical, and small modifications to the modular design can have a significant impact on a particular treatment (Guedan S, et al., Mol Ther Methods Clin Dev. 2018 Dec 31;12:145-156).Co-expression of multiple CAR molecules in the same effector cell and optimization of CAR structure and costimulatory domains may be feasible and may improve CAR T effector function and persistence (Schneider D, et al., Sci Transl Med. 2021 Mar 24; 13(586)). Furthermore, third-generation CAR T cells that combine the signaling capabilities of two costimulatory domains may lead to improved CAR survival, expansion, and efficacy (Subklewe M, et al., Transfus Med Hemother. 2019; 46(1):15-24; Maria-Luisa Schubert, MD, et al., Blood. 2019. 134(Supplement_1)).

[0008] Tumor antigen escape and tumor target heterogeneity are common causes of CAR therapy failure, and recent studies suggest that this may be a particularly important factor in the treatment of solid tumors (Majzner RG, and Mackall CL., Cancer Discov. 2018 Oct;8(10):1219-1226). Single-targeted CAR therapy has been shown to be effective in treating various cancers, including B-ALL and multiple myeloma, but relapse rates can be as high as 60% (Walsh Z, et al., Curr Hematol Malig Rep 14,451-459 (2019)). Simultaneous targeting of multiple antigens reduces the risk of relapse and resistance (Schneider D, et al., Sci Transl Med. 2021 Mar 24;13(586)). To circumvent this outcome, several options have been clinically tested, including sequential treatment with mono-CARs or co-infusion of CD19 and CD22 CARs, but ultimately proved ineffective (Shalabi H, et al., Haematologica Italy. 2018;103:e215-8). Ongoing or completed studies of multitargeted CARs have focused on various combinations of CD19, CD20, CD22, HER2, TSLPR, and IL-13Rα2 (Walsh, Z. et al., Curr Hematol Malig Rep 14, 451-459 (2019); Shalabi H, et al., Haematologica Italy. 2018; 103: e215-8; Bielamowicz K, et al., Neuro-Oncology. 2018; 20(4): 506-18; Han X, et al., J Hematol Oncol 12, 128 (2019)). Further clinical evidence is needed to determine which multitargeted CAR therapy is most effective.

[0009] As previously mentioned, the efficacy of CAR therapy can be limited by the short persistence of CAR T cells, which can eventually be overwhelmed by rebound tumor burden, particularly in solid tumors. The use of interleukin (IL)-7 and CCR2b in vivo experiments has proven effective, demonstrating both improved persistence and improved antitumor activity in neuroblastoma and melanoma models (Guangchao Li, et al., Frontiers in Oncology. 2021;11:2021). Studies on hematological cancers have shown that multitargeting and costimulation by combining CARs with chimeric costimulatory receptors (CCRs) is an effective method to increase cytotoxicity efficiency and durability, thereby preventing recurrence of tumor clones and ultimately improving clinical outcomes of CAR T cell treatment (Katsarou A, et al., Sci Transl Med. 2021 Dec 8; Vol. 13 (No. 623); Pietrobon, V., et al., Int. J. Mol. Sci. 2021, Vol. 22, 10828).

[0010] Thus, while CARs may be considered capable of inducing T cell activation in a manner similar to endogenous T cell receptors, the clinical application of CAR-based technologies has to date been limited by significant obstacles, including in vivo expansion of CAR+ T cells, rapid cell loss after infusion, insufficient clinical activity, and recurrence of the underlying disease or condition. Many of these issues arise from tumor target heterogeneity and tumor-mediated resistance to therapy, including the influence of tumor microenvironment and tumor stromal factors, and may be addressed by engineering CAR T cells.

[0011] Solid tumors present a challenging environment for CARs, including an immunosuppressive environment characterized by physical, functional, and dynamic barriers that impede T cell function. The tumor microenvironment (TME) can prove challenging for successful CAR function and targeting. Tumors can employ strategies to resist the targeted effects of CARs by increasing the production of inhibitory cytokines (Lindo L, et al., Front Immunol. 2021 Feb 10; Vol. 11: 618387). To combat this increasingly hostile environment, research on "armored" CARs has been developed. Alabanza et al. (Front Immunol. 2022 Feb 9; Vol. 13: 832645) used this approach by co-expressing a dominant-negative form of TGFβ receptor II ("armored") on BCMA-targeted CAR T cells to resist the inhibitory effects of TGFβ in the bone marrow niche of multiple myeloma. This resulted in functional persistence despite prolonged exposure to TGFβ in an animal model of multiple myeloma. The TME contains diverse cell populations, making targeting difficult. Yeku et al. (2017) in ovarian cancer demonstrated that previous CAR T cell therapies targeting the folate receptor for ovarian cancer have met with limited success in clinical trials, in part due to the effects of immunosuppressive cytokines such as IL-4, IL-6, LIF, IL-10, TGFβ, myeloid-derived suppressor cells, tumor-associated macrophages (TAMs), and regulatory T cells, which suppress the efficacy of targeted CARs. By generating IL-12-armed CAR T cells, the inventors demonstrated that treatment can overcome the inhibitory microenvironment, altering the ascites cytokine and TAM microenvironment, and overcoming PD-L1-mediated inhibition (Yeku OO, et al., Sci Rep 7, 10541 (2017)).

[0012] In addition to the challenging TME, the tumor stromal barrier poses a challenge for effective CAR penetration. Solid tumors have a dense extracellular matrix (ECM) formed by cancer-associated fibroblasts (CAFs), which inhibits T cell penetration deep into the tumor, thereby abolishing continuous contact between tumor cells and CAR-T cells (Zhang, BL et al., Sci. China Life Sci. 2016, Vol. 59 (No. 4), 340-348). One approach is to promote ECM degradation, thereby enabling effective infiltration of CAR-T cells into the solid tumor matrix. Engineering hyaluronidase (HAase) and the checkpoint-blocking antibody α-PDL1 on the surface of CAR-T cells has been shown to enhance tumor penetration and antitumor efficacy in solid tumors (Yangyang Zhao, et al., ACS Central Science 2022, Vol. 8 (No. 5), 603-614). Similarly, an approach to engineer CAR to express the enzyme heparanase (HPSE) showed improved ability to disrupt ECM (Caruana I, et al., Nat Med. 2015 May;21(5):524-529).

[0013] Along with the various strategies discussed above aimed at improving the durability and efficacy of CAR therapy, measures to improve the safety profile of CAR T therapy are also being developed. Widespread adoption and application of CAR T therapy is limited by numerous challenges, including tumor lysis syndrome, neurotoxicity syndrome, and cytokine release syndrome. Cytokine release syndrome (CRS) is a systemic inflammatory response induced by T cell activation. CRS is primarily caused by activated CAR T cells significantly increasing the secretion of pro-inflammatory factors (e.g., IL-6, IFN-γ, and TNF-α) by immune cells, disrupting the balance between pro- and anti-inflammatory responses (Hay KA, et al., Blood. 2017;130:2295-306). The use of suicide genes to prevent excessive off-target activity and improve the safety of CAR T cells is becoming increasingly important. Suicide genes, as regulatory genes, can be coexpressed with CAR constructs and induce cell death when activated by additional agents such as drugs or antibodies. By design, the best suicide gene activation agents are characterized by biological inactivity, adequate bioavailability and biodistribution profiles, and negligible or no toxicity (Jones BS, et al., Front Pharmacol. 2014;5:254). Proof of concept was demonstrated by Kao et al. (2019) in a study using a truncated epidermal growth factor receptor (EGFRt) as a suicide gene system co-delivered with an anti-CD19 CAR. Both in vitro and in vivo analyses demonstrated favorable results (Kao Roy L, et al., Human Gene Therapy. Apr 2019;413-428). Clinical evaluation of these strategies is currently underway, but they offer promising prospects for safer CAR T therapeutic strategies.

[0014] Thus, there is an urgent and long-felt need in the art to discover compositions and methods for the treatment of cancer using CAR-based therapies that can exhibit cancer-specific therapeutic properties without the aforementioned drawbacks.

[0015] The present invention provides a method for the development of CAR-mediated T cell proliferation and proliferation in vivo, comprising one or more of the following features: i) high surface expression on transduced T cells; ii) high cytolytic potential and in vivo expansion and persistence of transduced T cells; iii) multitargeting to overcome antigen escape; iv) arming to overcome immunosuppression in the TME; v) cytokine stimulatory elements to promote cytokine-autonomous T cell stimulation resulting in enhanced antitumor cytotoxicity, expansion, memory formation, cytokine secretion, and persistence; vi) overcoming the physical barrier of the tumor stroma / extracellular matrix (ECM) and enhancing CAR proliferation and proliferation in vivo; These ongoing unmet needs are addressed by providing a digestive enzyme that allows tumor penetration of T, and vii) a CAR, or a boosted CAR composition that exhibits an on-switch or off-switch to control expression of a co-expressed functional "booster" element, targetable antigens such as CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD 38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM, folate binding protein, folate receptor Folate receptor alpha (FOLR1), folate receptor beta (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, pl85, IL-2 receptor, interleukin-1 receptor accessory protein (IL1RAP), EGFRvIII(de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, ανβ3, WT1, LMP2, HPV E6, HPVE7, Her-2 / neu, non-mutant p53, NY-ESO-1, MelanA / MART 1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, mutant p53, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYP1B 1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES 1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL1, MUC1, MUC16 / CA125, MAGE A4, MAGEC2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, C LDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl 86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endoglin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPRC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c Therapeutic methods using such boosted CARs are provided that can be used to treat solid tumors, including tumors expressing VISTA, LRRC5, LRRC15, or any combination thereof or fragment thereof, as well as other diseases and / or conditions that express a CAR-associated target, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to any of the foregoing, or any combination thereof, or other antigens, or any combination thereof.

[0016] Summary of the Invention The present invention provides a novel anti-effector partial antibody or its antigen-binding domain, and a chimeric antigen receptor (CAR) containing such an effector partial antigen-binding domain, as well as a host cell (e.g., T cell) expressing the receptor and a nucleic acid molecule encoding the receptor.The CAR may consist of a single molecule expressed on the surface of an effector cell, or may consist of a CAR composed of an effector cell-expressed signal transduction module and a soluble targeting module (when the soluble targeting module is combined with the cell-expressed signal transduction module, a complete functional CAR is formed).The CAR exhibits high surface expression on transduced T cells, and has high cytolytic activity and in vivo expansion and persistence of transduced T cells.Methods of using the disclosed CAR, host cell, and nucleic acid molecule are also provided, for example, for treating cancer in a subject.

[0017] In its broadest aspect, provided herein are novel chimeric antigen receptors (CARs) comprising a CAR construct with a main effector moiety followed by one or more 2A sequences, in frame to one or more additional "booster" elements for improved function, including enhanced tumor penetration, to improve the therapeutic efficacy of CAR-T cells in solid tumors, hematological tumors, autoimmune diseases, genetic diseases, or other relevant indications.

[0018] In yet another broad aspect, provided herein are novel chimeric antigen receptors (CARs) comprising boosted CARs, in which functionally co-expressed boosted CAR elements are expressed with high transduction efficiency from a single multicistronic vector, thereby simplifying CAR production and release and reducing costs for market introduction. In one aspect, the boosted CAR composition exhibits one or more of the following characteristics: i) high surface expression on transduced T cells; ii) multitargeting to overcome antigen escape; iii) one or more armamentarium elements to overcome immunosuppression in the TME; iv) one or more cytokine-stimulating elements (e.g., including, but not limited to, secretion of chemoattractant receptors and / or chemotactic molecules) to promote cytokine-autonomous T cell stimulation, resulting in enhanced antitumor cytotoxicity, expansion, memory formation, cytokine secretion, and persistence; v) overcoming the physical barrier of the tumor stroma / extracellular matrix (ECM) and enhancing CAR expression. and vi) one or more digestive enzymes that enable tumor penetration of T cells; vi) one or more pro-inflammatory immune activators; and vii) one or more on-switches or off-switches for controlling expression of the CAR, wherein the boosted CAR achieves high levels of cytolytic activity and persistence to promote in vivo expansion of the transduced T cells, and persistence of patient-specific anti-tumor T cells, resulting in tumor stabilization, reduction, elimination, or remission of cancer or autoimmune disease, alloimmune disease, or autoaggressive disease, or prevention or amelioration of recurrence of cancer or autoimmune disease, alloimmune disease, or autoaggressive disease, or a combination thereof, in a patient-specific manner.

[0019] In yet another broad aspect, the novel chimeric antigen receptors (CARs) provided herein can comprise single-targeting, tandem-targeting, or multi-targeting CAR constructs (including those in DuoCAR format), or any combination thereof.

[0020] In certain embodiments, the novel boosted CAR is under the control of one or more constitutive, tissue-specific, or inducible promoters, or any combination thereof.

[0021] In certain embodiments, the one or more switches include a tag, a kill switch, an on switch, an off switch, and / or an adapter switch, or any combination thereof.

[0022] In certain embodiments, novel chimeric antigen receptors (CARs) are provided: single, tandem, multi-targeting CARs, and DuoCARs (with or without one or more booster elements) used to transduce effector cells for the treatment of solid and hematological tumors and other diseases with the following target antigens (e.g., but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3, etc.). TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD 38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM, folate binding protein, folate receptor Folate receptor alpha (FOLR1), folate receptor beta (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, pl85, IL-2 receptor, interleukin-1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, ανβ3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART 1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGEAl, MAGE A3, CYP1B 1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES 1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endoglin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPRC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15, or any combination thereof, or fragment thereof, is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any of the foregoing, or any combination thereof).

[0023] In certain embodiments, effector cells include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, dendritic cells (DCs), gamma delta T cells, monocytes, macrophages, stem cells, and induced pluripotent stem (iPS) cells.

[0024] In yet another broad aspect, one or more of the above-identified novel boost chimeric antigen receptors (CARs) provided above with respect to SEQ ID NOs: 151-256 may comprise single-targeting, tandem-targeting, or multi-targeting CAR constructs (including those in DuoCAR format), or any combination thereof.

[0025] For each of the various aspects and embodiments of single, tandem, multi-targeting CARs, and DuoCARs, CAR constructs (with or without one or more booster elements) specifically contemplated herein, the nucleotide sequence encoding the functional CAR (with or without one or more booster elements) comprises the nucleotide sequence of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 245, 247, 249, 251, 253, 255, or a combination thereof.

[0026] For each of the various aspects and embodiments of single, tandem, multi-targeting CARs, and DuoCARs, CAR constructs (with or without one or more booster elements) specifically contemplated herein, each vector encodes a functional CAR (with or without one or more booster elements) comprising the amino acid sequence of SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, 256, or combinations thereof.

[0027] In yet another broad aspect, one or more of the above-identified novel boost chimeric antigen receptors (CARs) provided above with respect to SEQ ID NOs: 127-149 may comprise single-targeting, tandem-targeting, or multi-targeting CAR constructs (including those in DuoCAR format), or any combination thereof.

[0028] For each of the various aspects and embodiments, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN antibody or fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149.

[0029] For each of the various aspects and embodiments, an isolated polynucleotide encoding a fully human anti-HER2, FOLR1, MUC16, CD276, EGFR, GD2, NKGD2 antibody or fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, and 141.

[0030] For each of the various aspects and embodiments, provided herein are novel single, tandem, DuoCAR, or multitargeting CAR molecules (with or without one or more booster elements) comprising at least one extracellular antigen-binding domain comprising an anti-ROR1 and / or anti-MSLN antigen-binding domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain. For each of the various aspects and embodiments, provided herein are novel single, tandem, DuoCAR, or multitargeting CAR molecules (with or without one or more booster elements) comprising at least one extracellular antigen-binding domain comprising an anti-HER2, FOLR1, MUC16, CD276, EGFR, GD2, and / or NKGD2 antigen-binding domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, and 141; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain. For each of the various aspects and embodiments, provided herein are novel single, tandem, DuoCAR, or multitargeting CAR molecules (with or without one or more booster elements) comprising at least one extracellular antigen-binding domain comprising an anti-ROR1 and / or anti-MSLN antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain.

[0031] For each of the various aspects and embodiments, provided herein are novel single, tandem, DuoCAR, or multitargeting CAR molecules (with or without one or more booster elements) comprising at least one extracellular antigen-binding domain comprising an anti-HER2, FOLR1, MUC16, CD276, EGFR, GD2, and / or NKGD2 antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, and 142; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain.

[0032] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, and 149.

[0033] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, and 150.

[0034] In one aspect, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeted chimeric antigen receptor (CAR) (with or without one or more boosting elements) comprising, from N-terminus to C-terminus, at least one anti-ROR1 and / or anti-MSLN antigen binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149; at least one transmembrane domain; and at least one intracellular signaling domain.

[0035] In one aspect, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeted chimeric antigen receptor (CAR) (with or without one or more boosting elements) comprising, from N-terminus to C-terminus, at least one anti-ROR1 and / or anti-MSLN antigen binding domain encoded by a nucleotide sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one transmembrane domain, and at least one intracellular signaling domain.

[0036] In one embodiment, the targeting domain of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and contains an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149, connected to an additional binding tag or epitope, while the effector cell expression component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is connected to a tag or epitope expressed on a soluble single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) module. contains a binding domain specifically directed to bind to an epitope, e.g., specific binding to the cell-binding component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) on the soluble component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) forms a fully functional single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) structure.

[0037] In yet another embodiment, an isolated nucleic acid molecule encoding a single, tandem, Duo, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the encoded single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) extracellular anti-ROR1 and / or anti-MSLN antigen binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to ROR1 and / or MSLN.

[0038] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular anti-ROR1 and / or anti-MSLN antigen binding domain is connected to the transmembrane domain by a linker domain.

[0039] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the encoded anti-ROR1 and / or anti-MSLN extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0040] In one aspect, the single, tandem, DuoCAR, or multi-targeting CAR provided herein (with or without one or more boosting elements) further comprises a linker or spacer domain.

[0041] In one embodiment, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), in which an extracellular anti-ROR1 and / or anti-MSLN antigen binding domain, an intracellular signaling domain, or both, is connected to a transmembrane domain by a linker or spacer domain.

[0042] In one embodiment, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded linker domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19, or CD28 and is linked to a transmembrane domain.

[0043] In another embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0044] In yet another aspect, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeting, chimeric antigen receptor (CAR) construct, wherein the CAR comprises at least one extracellular antigen-binding domain comprising an anti-MSLN and / or anti-ROR1 antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain, wherein the T cells are from a human with cancer or an autoimmune disease, an alloimmune disease, or an autoaggressive disease. The cancer is, inter alia, a blood cancer such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0045] In another aspect, a method of generating T cells containing single, tandem, DuoCAR, or multi-targeting CAR constructs (hereinafter "CAR-T cells") (with or without one or more booster elements) is provided. The method includes transducing T cells with a vector or nucleic acid molecule encoding the disclosed CARs that specifically bind to MSLN and / or ROR1, thereby generating CAR-T cells.

[0046] In yet another aspect, a method of generating a population of RNA-engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements) into cells of a subject, thereby generating single, tandem, DuoCAR, or multi-targeted CAR cells (with or without one or more booster elements).

[0047] In yet another aspect, a method is provided for diagnosing a disease, disorder, or condition associated with expression of MSLN and / or ROR1 on a cell, the method comprising: a) contacting the cell with a human anti-MSLN and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; and b) detecting the presence of MSLN and / or ROR1, wherein the presence of MSLN and / or ROR1 diagnoses, detects, a disease, disorder, or condition associated with expression of MSLN and / or ROR1.

[0048] In another embodiment, a method of inhibiting MSLN- and / or ROR1-dependent T cell inhibition is provided, the method comprising contacting a cell with a human anti-MSLN- and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150. In one embodiment, the cell is selected from the group consisting of an MSLN- and / or ROR1-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0049] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed single, tandem, or multi-targeting CARs (with or without one or more booster elements).

[0050] In another embodiment, a method of treating or preventing cancer in a mammal is provided, the method comprising administering to the mammal one or more of the disclosed single, tandem, or multitargeting CARs (with or without one or more booster elements) in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed single, tandem, or multitargeting CARs (with or without one or more booster elements) that specifically bind to MSLN and / or ROR1 and / or one or more of the aforementioned antigens under conditions sufficient to form an immune complex between the antigen-binding domain on the single, tandem, or multitargeting CAR (with or without one or more booster elements) and the extracellular domain of MSLN and / or ROR1, and / or one or more of the aforementioned antigens in the subject.

[0051] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a single, tandem, or multi-targeting CAR (with or without one or more booster elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more booster elements) comprises at least one MSLN and / or ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 144, 146, 148, and 150, or any combination thereof; at least one transmembrane domain; and at least one intracellular signaling domain, wherein the persistent population of engineered T cells, or a progeny population of T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0052] In yet another aspect, there is provided a kit for generating the chimeric antigen receptor T cells described above, or for preventing, treating, or ameliorating any of the cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in a subject described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, and instructions for use of the kit.

[0053] In one aspect of the present invention, immunotherapy compositions are provided comprising single, tandem, DuoCAR, or multitargeted CARs (with or without one or more booster elements) that can be used to transduce autologous lymphocytes to generate active patient-specific anti-tumor lymphocyte cell populations that can be directly infused back into the patient to promote in vivo expansion, persistence of patient-specific anti-tumor T cells that result in tumor stabilization, cancer reduction, elimination, remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0054] In one embodiment, a pharmaceutical composition is provided in which at least one transmembrane domain of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0055] It will be understood that single, tandem, DuoCAR, or multitargeting CARs (with or without one or more booster elements), host cells, nucleic acids, and methods are useful beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

[0056] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and arrangements of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0057] [Figure 1] The structure of the boosted CAR is shown. The boosted CAR consisted of a CAR molecule followed by a 2A sequence in frame with the boosting element. The CAR molecules represented mono-CARs and multi-targeting tandem or dual CARs. Various boosting elements were added, including cytokines (membrane-bound IL7), arming (TGFβRIIdn), suicide tags (tEGFR), extracellular matrix enzymes (ECM), chemokine receptors (CXCL8, CCL2), and stromal targeting molecules (FAP). [Figure 2A]Figure 2A shows the mIL7-armed ROR1 and / or MSLN CAR constructs and their surface expression on transduced primary T cells. MonoCARs armed with membrane-bound IL7 (mIL7) contained an ROR1 or MSLN scFv binding domain, an IgG4 or CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB or CD28 costimulatory domain, a CD3ζ activation domain, followed by a 2A peptide, in frame with membrane-bound IL7. Tandem-boosted CAR constructs comprised an MSLN-ROR1 tandem scFv targeting domain, an IgG4 short hinge, a CD8 or CD28 transmembrane domain, a single 4-1BB or tandem CD28_4-1BB costimulatory domain, a CD3ζ activation domain, and a 2A sequence linked to mIL7. The DuoCAR constructs contained a mono-ROR1 CAR followed by a 2A sequence and a mono-MSLN CAR with a different costimulatory or transmembrane domain in frame with the 2A peptide linked to mIL7. The mono-ROR or MSLN CAR and tandem CAR were included as control constructs. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding ROR1 and / or MSLN CAR constructs. Transduced T cells were assayed for CAR surface expression by staining with ROR1 Fc and / or MSLN His followed by flow cytometry with anti-Fc-AF647 or anti-His APC (Figure 2B). The percentage of ROR1 CAR expression in T cells transduced with the constructed CARs encoding ROR1 binders is plotted. (Figure 2C) The percentage of MSLN CAR expression was quantified in T cells transduced with the CAR-containing MSLN-binding agent. The mean fluorescence intensity of ROR1-binding agent expression (Figure 2D) and MSLN-binding agent expression (Figure 2E) is shown in bar graphs. Data represent one independent experiment from two different donors. [Figure 2B]Figure 2A shows the mIL7-armed ROR1 and / or MSLN CAR constructs and their surface expression on transduced primary T cells. MonoCARs armed with membrane-bound IL7 (mIL7) contained an ROR1 or MSLN scFv binding domain, an IgG4 or CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB or CD28 costimulatory domain, a CD3ζ activation domain, followed by a 2A peptide, in frame with membrane-bound IL7. Tandem-boosted CAR constructs comprised an MSLN-ROR1 tandem scFv targeting domain, an IgG4 short hinge, a CD8 or CD28 transmembrane domain, a single 4-1BB or tandem CD28_4-1BB costimulatory domain, a CD3ζ activation domain, and a 2A sequence linked to mIL7. The DuoCAR constructs contained a mono-ROR1 CAR followed by a 2A sequence and a mono-MSLN CAR with a different costimulatory or transmembrane domain in frame with the 2A peptide linked to mIL7. The mono-ROR or MSLN CAR and tandem CAR were included as control constructs. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding ROR1 and / or MSLN CAR constructs. Transduced T cells were assayed for CAR surface expression by staining with ROR1 Fc and / or MSLN His followed by flow cytometry with anti-Fc-AF647 or anti-His APC (Figure 2B). The percentage of ROR1 CAR expression in T cells transduced with the constructed CARs encoding ROR1 binders is plotted. (Figure 2C) The percentage of MSLN CAR expression was quantified in T cells transduced with the CAR-containing MSLN-binding agent. The mean fluorescence intensity of ROR1-binding agent expression (Figure 2D) and MSLN-binding agent expression (Figure 2E) is shown in bar graphs. Data represent one independent experiment from two different donors. [Figure 2C]Figure 2A shows the mIL7-armed ROR1 and / or MSLN CAR constructs and their surface expression on transduced primary T cells. MonoCARs armed with membrane-bound IL7 (mIL7) contained an ROR1 or MSLN scFv binding domain, an IgG4 or CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB or CD28 costimulatory domain, a CD3ζ activation domain, followed by a 2A peptide, in frame with membrane-bound IL7. Tandem-boosted CAR constructs comprised an MSLN-ROR1 tandem scFv targeting domain, an IgG4 short hinge, a CD8 or CD28 transmembrane domain, a single 4-1BB or tandem CD28_4-1BB costimulatory domain, a CD3ζ activation domain, and a 2A sequence linked to mIL7. The DuoCAR constructs contained a mono-ROR1 CAR followed by a 2A sequence and a mono-MSLN CAR with a different costimulatory or transmembrane domain in frame with the 2A peptide linked to mIL7. The mono-ROR or MSLN CAR and tandem CAR were included as control constructs. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding ROR1 and / or MSLN CAR constructs. Transduced T cells were assayed for CAR surface expression by staining with ROR1 Fc and / or MSLN His followed by flow cytometry with anti-Fc-AF647 or anti-His APC (Figure 2B). The percentage of ROR1 CAR expression in T cells transduced with the constructed CARs encoding ROR1 binders is plotted. (Figure 2C) The percentage of MSLN CAR expression was quantified in T cells transduced with the CAR-containing MSLN-binding agent. The mean fluorescence intensity of ROR1-binding agent expression (Figure 2D) and MSLN-binding agent expression (Figure 2E) is shown in bar graphs. Data represent one independent experiment from two different donors. [Figure 2D]Figure 2A shows the mIL7-armed ROR1 and / or MSLN CAR constructs and their surface expression on transduced primary T cells. MonoCARs armed with membrane-bound IL7 (mIL7) contained an ROR1 or MSLN scFv binding domain, an IgG4 or CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB or CD28 costimulatory domain, a CD3ζ activation domain, followed by a 2A peptide, in frame with membrane-bound IL7. Tandem-boosted CAR constructs comprised an MSLN-ROR1 tandem scFv targeting domain, an IgG4 short hinge, a CD8 or CD28 transmembrane domain, a single 4-1BB or tandem CD28_4-1BB costimulatory domain, a CD3ζ activation domain, and a 2A sequence linked to mIL7. The DuoCAR constructs contained a mono-ROR1 CAR followed by a 2A sequence and a mono-MSLN CAR with a different costimulatory or transmembrane domain in frame with the 2A peptide linked to mIL7. The mono-ROR or MSLN CAR and tandem CAR were included as control constructs. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding ROR1 and / or MSLN CAR constructs. Transduced T cells were assayed for CAR surface expression by staining with ROR1 Fc and / or MSLN His followed by flow cytometry with anti-Fc-AF647 or anti-His APC (Figure 2B). The percentage of ROR1 CAR expression in T cells transduced with the constructed CARs encoding ROR1 binders is plotted. (Figure 2C) The percentage of MSLN CAR expression was quantified in T cells transduced with the CAR-containing MSLN-binding agent. The mean fluorescence intensity of ROR1-binding agent expression (Figure 2D) and MSLN-binding agent expression (Figure 2E) is shown in bar graphs. Data represent one independent experiment from two different donors. [Figure 2E]Figure 2A shows the mIL7-armed ROR1 and / or MSLN CAR constructs and their surface expression on transduced primary T cells. MonoCARs armed with membrane-bound IL7 (mIL7) contained an ROR1 or MSLN scFv binding domain, an IgG4 or CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB or CD28 costimulatory domain, a CD3ζ activation domain, followed by a 2A peptide, in frame with membrane-bound IL7. Tandem-boosted CAR constructs comprised an MSLN-ROR1 tandem scFv targeting domain, an IgG4 short hinge, a CD8 or CD28 transmembrane domain, a single 4-1BB or tandem CD28_4-1BB costimulatory domain, a CD3ζ activation domain, and a 2A sequence linked to mIL7. The DuoCAR constructs contained a mono-ROR1 CAR followed by a 2A sequence and a mono-MSLN CAR with a different costimulatory or transmembrane domain in frame with the 2A peptide linked to mIL7. The mono-ROR or MSLN CAR and tandem CAR were included as control constructs. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding ROR1 and / or MSLN CAR constructs. Transduced T cells were assayed for CAR surface expression by staining with ROR1 Fc and / or MSLN His followed by flow cytometry with anti-Fc-AF647 or anti-His APC (Figure 2B). The percentage of ROR1 CAR expression in T cells transduced with the constructed CARs encoding ROR1 binders is plotted. (Figure 2C) The percentage of MSLN CAR expression was quantified in T cells transduced with the CAR-containing MSLN-binding agent. The mean fluorescence intensity of ROR1-binding agent expression (Figure 2D) and MSLN-binding agent expression (Figure 2E) is shown in bar graphs. Data represent one independent experiment from two different donors. [Figure 3A]Figure 3 shows the cytotoxicity of ROR1 and / or MSLN CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using the ROR1+ MSLN+ tumor line OVCAR3 with (Figure 3A) a CAR containing the ROR1 scFv, (Figure 3B) a CAR containing the MSLN scFv, and (Figure 3C) the ROR1-MSLN- tumor line HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment from two different donors. [Figure 3B] Figure 3 shows the cytotoxicity of ROR1 and / or MSLN CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using the ROR1+ MSLN+ tumor line OVCAR3 with (Figure 3A) a CAR containing the ROR1 scFv, (Figure 3B) a CAR containing the MSLN scFv, and (Figure 3C) the ROR1-MSLN- tumor line HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment from two different donors. [Figure 3C]Figure 3 shows the cytotoxicity of ROR1 and / or MSLN CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using the ROR1+ MSLN+ tumor line OVCAR3 with (Figure 3A) a CAR containing the ROR1 scFv, (Figure 3B) a CAR containing the MSLN scFv, and (Figure 3C) the ROR1-MSLN- tumor line HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment from two different donors. [Figure 4A] Figure 4 shows the relative in vitro potency of ROR1 and / or MSLN CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines. CAR T cells and tumor cells were cocultured overnight at 10 different effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. Relative potency compared to the ROR1 CAR LTG2529 was calculated using a nonlinear EC50 shift, where x is a logarithmic concentration function in GraphPad Prism. The relative potency of each construct targeting the following ROR1+ MSLN+ tumor lines was plotted as a bar graph: (Figure 4A) OVCAR-3, (Figure 4B) NCI-H226, and (Figure 4C) CAPAN-1. Data represent one independent experiment from one to two different donors. [Figure 4B]Figure 4 shows the relative in vitro potency of ROR1 and / or MSLN CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines. CAR T cells and tumor cells were cocultured overnight at 10 different effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. Relative potency compared to the ROR1 CAR LTG2529 was calculated using a nonlinear EC50 shift, where x is a logarithmic concentration function in GraphPad Prism. The relative potency of each construct targeting the following ROR1+ MSLN+ tumor lines was plotted as a bar graph: (Figure 4A) OVCAR-3, (Figure 4B) NCI-H226, and (Figure 4C) CAPAN-1. Data represent one independent experiment from one to two different donors. [Figure 4C] Figure 4 shows the relative in vitro potency of ROR1 and / or MSLN CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines. CAR T cells and tumor cells were cocultured overnight at 10 different effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. Relative potency compared to the ROR1 CAR LTG2529 was calculated using a nonlinear EC50 shift, where x is a logarithmic concentration function in GraphPad Prism. The relative potency of each construct targeting the following ROR1+ MSLN+ tumor lines was plotted as a bar graph: (Figure 4A) OVCAR-3, (Figure 4B) NCI-H226, and (Figure 4C) CAPAN-1. Data represent one independent experiment from one to two different donors. [Figure 5A]Figure 5 shows CAR T cytokine release against the NCI-H226 lung cancer cell line. CAR T cell culture supernatants were assessed either alone or after overnight incubation with ROR1+MSLN+ NCI-H226 target cells at 10 different E:T ratios. Cytokine production was analyzed by ELISA for (Figure 5A) IFNγ, (Figure 5B) TNFα, and (Figure 5C) IL-2. Mean ± SEM of two technical replicates. Data represent one experiment performed in technical triplicates from one donor and three independent experiments from separate donors. [Figure 5B] Figure 5 shows CAR T cytokine release against the NCI-H226 lung cancer cell line. CAR T cell culture supernatants were assessed either alone or after overnight incubation with ROR1+MSLN+ NCI-H226 target cells at 10 different E:T ratios. Cytokine production was analyzed by ELISA for (Figure 5A) IFNγ, (Figure 5B) TNFα, and (Figure 5C) IL-2. Mean ± SEM of two technical replicates. Data represent one experiment performed in technical triplicates from one donor and three independent experiments from separate donors. [Figure 5C] Figure 5 shows CAR T cytokine release against the NCI-H226 lung cancer cell line. CAR T cell culture supernatants were assessed either alone or after overnight incubation with ROR1+MSLN+ NCI-H226 target cells at 10 different E:T ratios. Cytokine production was analyzed by ELISA for (Figure 5A) IFNγ, (Figure 5B) TNFα, and (Figure 5C) IL-2. Mean ± SEM of two technical replicates. Data represent one experiment performed in technical triplicates from one donor and three independent experiments from separate donors. [Figure 6A]The expression of membrane-bound IL7 and its functionality in sustaining CAR T cell growth after IL-2 withdrawal are shown (Figure 6A). Membrane-bound IL7 expression was determined by Western blotting using an IL7 antibody followed by a goat anti-mouse HPR-conjugated secondary antibody. GAPDH measured with anti-GAPDH and goat anti-rabbit secondary antibodies were included as loading controls. CAR T cells were transduced with lentivirus encoding ROR1 and / or MSLN CAR constructs at an MOI of 20, with or without mIL7. Transduced CAR T cells were washed and cultured at 1e6 / ml using TexMACS medium without IL-2 supplementation for long-term target cell stimulation. Cell expansion (Figure 6B) and T cell size (Figure 6C) were monitored weekly for 2-3 weeks until no further cell expansion was observed. Data represent one independent experiment from two separate donors. [Figure 6B] The expression of membrane-bound IL7 and its functionality in sustaining CAR T cell growth after IL-2 withdrawal are shown (Figure 6A). Membrane-bound IL7 expression was determined by Western blotting using an IL7 antibody followed by a goat anti-mouse HPR-conjugated secondary antibody. GAPDH measured with anti-GAPDH and goat anti-rabbit secondary antibodies were included as loading controls. CAR T cells were transduced with lentivirus encoding ROR1 and / or MSLN CAR constructs at an MOI of 20, with or without mIL7. Transduced CAR T cells were washed and cultured at 1e6 / ml using TexMACS medium without IL-2 supplementation for long-term target cell stimulation. Cell expansion (Figure 6B) and T cell size (Figure 6C) were monitored weekly for 2-3 weeks until no further cell expansion was observed. Data represent one independent experiment from two separate donors. [Figure 6C]The expression of membrane-bound IL7 and its functionality in sustaining CAR T cell growth after IL-2 withdrawal are shown (Figure 6A). Membrane-bound IL7 expression was determined by Western blotting using an IL7 antibody followed by a goat anti-mouse HPR-conjugated secondary antibody. GAPDH measured with anti-GAPDH and goat anti-rabbit secondary antibodies were included as loading controls. CAR T cells were transduced with lentivirus encoding ROR1 and / or MSLN CAR constructs at an MOI of 20, with or without mIL7. Transduced CAR T cells were washed and cultured at 1e6 / ml using TexMACS medium without IL-2 supplementation for long-term target cell stimulation. Cell expansion (Figure 6B) and T cell size (Figure 6C) were monitored weekly for 2-3 weeks until no further cell expansion was observed. Data represent one independent experiment from two separate donors. [Figure 7A] The time to 50% target cell killing (KT50) (Figure 7A) and relative potency of MSLN CAR T cells before and after IL-2 deprivation (Figure 7B) are shown. MSLN CARs carrying mIL7 D0245 and ROR2 / MSLN DuoCARs carrying mIL7 D0282 were cultured for 69 days in TexMACS medium without IL-2 supplementation. The cytotoxicity of CARs D0245 and D0282 was measured using the ROR1+MSLN+ pancreatic cancer cell line AsPC-1 with an xCELLigence RTCA instrument. MSLN CARs D0181, D0245, and D0282 without IL-2 deprivation were included as controls. CAR T cells and target cells were cocultured at an ET ratio of 2:1. Specific target lysis percentage was assessed by inhibition of electron flow. KT50 represents the co-incubation time required to achieve 50% target cell lysis. Relative efficacy was calculated based on the KT50 of MSLN CAR D0181 without IL-2 depletion. Data represent one independent experiment from two separate donors. [Figure 7B]The time to 50% target cell killing (KT50) (Figure 7A) and relative potency of MSLN CAR T cells before and after IL-2 deprivation (Figure 7B) are shown. MSLN CARs carrying mIL7 D0245 and ROR2 / MSLN DuoCARs carrying mIL7 D0282 were cultured for 69 days in TexMACS medium without IL-2 supplementation. The cytotoxicity of CARs D0245 and D0282 was measured using the ROR1+MSLN+ pancreatic cancer cell line AsPC-1 with an xCELLigence RTCA instrument. MSLN CARs D0181, D0245, and D0282 without IL-2 deprivation were included as controls. CAR T cells and target cells were cocultured at an ET ratio of 2:1. Specific target lysis percentage was assessed by inhibition of electron flow. KT50 represents the co-incubation time required to achieve 50% target cell lysis. Relative efficacy was calculated based on the KT50 of MSLN CAR D0181 without IL-2 depletion. Data represent one independent experiment from two separate donors. [Figure 8A]In vitro characterization of the TGFβRIIdn-boosted MSLN CAR is shown. (Figure 8A) MSLN CAR D0181 consisted of the MSLN scFv binding domain, CD8 hinge and transmembrane domains, 41BB costimulatory domain, and CD3ζ activation domain. The boosted CAR D0211 was constructed from a mono-MSLN CAR, a 2A peptide linker, in-frame to the dominant-negative TGFβ receptor II (TGFβRIIdn). Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the MSLN CAR D0181 and boosted MSLN CAR D0211 constructs. (Figure 8B) CAR surface expression was assessed by flow cytometry using MSLN-His followed by anti-His-APC staining. Expression of TGFβRIIdn was determined by biotinylated TGFβR and streptavidin-PE staining. Histogram overlays of UTD, CAR D0181, and D0211 are shown on the right. (Figure 8C) Luciferase-based cytotoxicity assays were performed using the MSLN+ tumor lines NCI-H226, A431-MSLN, and MSLN-A431. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. (Figure 8D) CAR T cell culture supernatants were evaluated after overnight incubation with MSLN + NCI-H226 target cells at 10 different E:T ratios. Cytokine production of IFNγ and TNFα was analyzed by ELISA. Mean ± SEM of two technical replicates. Data represent one independent experiment from four separate donors. (Figure 8E) Kinetic killing assay testing the functionality of TGFβRIIdn-armed, boosted MSLN and ROR1 CAR T cells against the AsPc-1 tumor cell line in the presence or absence of TGFβ. [Figure 8B]In vitro characterization of the TGFβRIIdn-boosted MSLN CAR is shown. (Figure 8A) MSLN CAR D0181 consisted of the MSLN scFv binding domain, CD8 hinge and transmembrane domains, 41BB costimulatory domain, and CD3ζ activation domain. The boosted CAR D0211 was constructed from a mono-MSLN CAR, a 2A peptide linker, in-frame to the dominant-negative TGFβ receptor II (TGFβRIIdn). Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the MSLN CAR D0181 and boosted MSLN CAR D0211 constructs. (Figure 8B) CAR surface expression was assessed by flow cytometry using MSLN-His followed by anti-His-APC staining. Expression of TGFβRIIdn was determined by biotinylated TGFβR and streptavidin-PE staining. Histogram overlays of UTD, CAR D0181, and D0211 are shown on the right. (Figure 8C) Luciferase-based cytotoxicity assays were performed using the MSLN+ tumor lines NCI-H226, A431-MSLN, and MSLN-A431. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. (Figure 8D) CAR T cell culture supernatants were evaluated after overnight incubation with MSLN + NCI-H226 target cells at 10 different E:T ratios. Cytokine production of IFNγ and TNFα was analyzed by ELISA. Mean ± SEM of two technical replicates. Data represent one independent experiment from four separate donors. (Figure 8E) Kinetic killing assay testing the functionality of TGFβRIIdn-armed, boosted MSLN and ROR1 CAR T cells against the AsPc-1 tumor cell line in the presence or absence of TGFβ. [Figure 8C]In vitro characterization of the TGFβRIIdn-boosted MSLN CAR is shown. (Figure 8A) MSLN CAR D0181 consisted of the MSLN scFv binding domain, CD8 hinge and transmembrane domains, 41BB costimulatory domain, and CD3ζ activation domain. The boosted CAR D0211 was constructed from a mono-MSLN CAR, a 2A peptide linker, in-frame to the dominant-negative TGFβ receptor II (TGFβRIIdn). Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the MSLN CAR D0181 and boosted MSLN CAR D0211 constructs. (Figure 8B) CAR surface expression was assessed by flow cytometry using MSLN-His followed by anti-His-APC staining. Expression of TGFβRIIdn was determined by biotinylated TGFβR and streptavidin-PE staining. Histogram overlays of UTD, CAR D0181, and D0211 are shown on the right. (Figure 8C) Luciferase-based cytotoxicity assays were performed using the MSLN+ tumor lines NCI-H226, A431-MSLN, and MSLN-A431. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. (Figure 8D) CAR T cell culture supernatants were evaluated after overnight incubation with MSLN + NCI-H226 target cells at 10 different E:T ratios. Cytokine production of IFNγ and TNFα was analyzed by ELISA. Mean ± SEM of two technical replicates. Data represent one independent experiment from four separate donors. (Figure 8E) Kinetic killing assay testing the functionality of TGFβRIIdn-armed, boosted MSLN and ROR1 CAR T cells against the AsPc-1 tumor cell line in the presence or absence of TGFβ. [Figure 8D]In vitro characterization of the TGFβRIIdn-boosted MSLN CAR is shown. (Figure 8A) MSLN CAR D0181 consisted of the MSLN scFv binding domain, CD8 hinge and transmembrane domains, 41BB costimulatory domain, and CD3ζ activation domain. The boosted CAR D0211 was constructed from a mono-MSLN CAR, a 2A peptide linker, in-frame to the dominant-negative TGFβ receptor II (TGFβRIIdn). Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the MSLN CAR D0181 and boosted MSLN CAR D0211 constructs. (Figure 8B) CAR surface expression was assessed by flow cytometry using MSLN-His followed by anti-His-APC staining. Expression of TGFβRIIdn was determined by biotinylated TGFβR and streptavidin-PE staining. Histogram overlays of UTD, CAR D0181, and D0211 are shown on the right. (Figure 8C) Luciferase-based cytotoxicity assays were performed using the MSLN+ tumor lines NCI-H226, A431-MSLN, and MSLN-A431. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. (Figure 8D) CAR T cell culture supernatants were evaluated after overnight incubation with MSLN + NCI-H226 target cells at 10 different E:T ratios. Cytokine production of IFNγ and TNFα was analyzed by ELISA. Mean ± SEM of two technical replicates. Data represent one independent experiment from four separate donors. (Figure 8E) Kinetic killing assay testing the functionality of TGFβRIIdn-armed, boosted MSLN and ROR1 CAR T cells against the AsPc-1 tumor cell line in the presence or absence of TGFβ. [Figure 8E]In vitro characterization of the TGFβRIIdn-boosted MSLN CAR is shown. (Figure 8A) MSLN CAR D0181 consisted of the MSLN scFv binding domain, CD8 hinge and transmembrane domains, 41BB costimulatory domain, and CD3ζ activation domain. The boosted CAR D0211 was constructed from a mono-MSLN CAR, a 2A peptide linker, in-frame to the dominant-negative TGFβ receptor II (TGFβRIIdn). Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the MSLN CAR D0181 and boosted MSLN CAR D0211 constructs. (Figure 8B) CAR surface expression was assessed by flow cytometry using MSLN-His followed by anti-His-APC staining. Expression of TGFβRIIdn was determined by biotinylated TGFβR and streptavidin-PE staining. Histogram overlays of UTD, CAR D0181, and D0211 are shown on the right. (Figure 8C) Luciferase-based cytotoxicity assays were performed using the MSLN+ tumor lines NCI-H226, A431-MSLN, and MSLN-A431. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. (Figure 8D) CAR T cell culture supernatants were evaluated after overnight incubation with MSLN + NCI-H226 target cells at 10 different E:T ratios. Cytokine production of IFNγ and TNFα was analyzed by ELISA. Mean ± SEM of two technical replicates. Data represent one independent experiment from four separate donors. (Figure 8E) Kinetic killing assay testing the functionality of TGFβRIIdn-armed, boosted MSLN and ROR1 CAR T cells against the AsPc-1 tumor cell line in the presence or absence of TGFβ. [Figure 9A]Expression and cytotoxicity of the ROR1 CAR with TGFβRIIdn are demonstrated in an overnight endpoint killing assay at a range of effector to target cell ratios. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the ROR1 CAR LTG2529 and the ROR1CAR D0228 construct armed with boosted TGFβRIIdn. CAR surface expression was assessed by flow cytometry using ROR1 Fc followed by anti-Fc-AF647 staining. The percentage of CAR expression is plotted in the panel (Figure 9A). The ROR1+ target lines, OVAR3 (Figure 9B), CAPAN-2 (Figure 9C), and NCI-H226 (Figure 9D), were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at various effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration function in Prism, was used for curve fitting. Data represent one independent experiment from one different donor. [Figure 9B]Expression and cytotoxicity of the ROR1 CAR with TGFβRIIdn are demonstrated in an overnight endpoint killing assay at a range of effector to target cell ratios. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the ROR1 CAR LTG2529 and the ROR1CAR D0228 construct armed with boosted TGFβRIIdn. CAR surface expression was assessed by flow cytometry using ROR1 Fc followed by anti-Fc-AF647 staining. The percentage of CAR expression is plotted in the panel (Figure 9A). The ROR1+ target lines, OVAR3 (Figure 9B), CAPAN-2 (Figure 9C), and NCI-H226 (Figure 9D), were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at various effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration function in Prism, was used for curve fitting. Data represent one independent experiment from one different donor. [Figure 9C]Expression and cytotoxicity of the ROR1 CAR with TGFβRIIdn are demonstrated in an overnight endpoint killing assay at a range of effector to target cell ratios. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the ROR1 CAR LTG2529 and the ROR1CAR D0228 construct armed with boosted TGFβRIIdn. CAR surface expression was assessed by flow cytometry using ROR1 Fc followed by anti-Fc-AF647 staining. The percentage of CAR expression is plotted in the panel (Figure 9A). The ROR1+ target lines, OVAR3 (Figure 9B), CAPAN-2 (Figure 9C), and NCI-H226 (Figure 9D), were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at various effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration function in Prism, was used for curve fitting. Data represent one independent experiment from one different donor. [Figure 9D]Expression and cytotoxicity of the ROR1 CAR with TGFβRIIdn are demonstrated in an overnight endpoint killing assay at a range of effector to target cell ratios. Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding the ROR1 CAR LTG2529 and the ROR1CAR D0228 construct armed with boosted TGFβRIIdn. CAR surface expression was assessed by flow cytometry using ROR1 Fc followed by anti-Fc-AF647 staining. The percentage of CAR expression is plotted in the panel (Figure 9A). The ROR1+ target lines, OVAR3 (Figure 9B), CAPAN-2 (Figure 9C), and NCI-H226 (Figure 9D), were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at various effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration function in Prism, was used for curve fitting. Data represent one independent experiment from one different donor. [Figure 10A]The structures of MSLN and ROR1 CARs with ECM booster and surface expression in human primary T cells are shown. (Figure 10A) The MSLN-targeting CAR consisted of a fully human MSLN scFv targeting domain, CD8 hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3ζ activation domain. The ROR1-targeting CAR consisted of a fully human ROR1 scFv9 targeting domain, IgG4 short hinge, CD8 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ activation domain. The booster CAR contained a monotargeting CAR followed by a 2A peptide in frame with the ECM molecule. The booster molecules selected were heparanase (HPSE), metalloproteinase (MMP2), and hyaluronidase PH-20. (Figure 10B) Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding the CAR construct. Transduced T cells were assayed for CAR surface expression by flow cytometry using ROR1 Fc or MSLN-His staining followed by anti-Fc-AF647 or anti-His APC, respectively. CD4 staining was included to identify CD4+ and CD8+ populations. CAR-positive percentages are listed above the plots. UTD - untransduced control. [Figure 10B]The structures of MSLN and ROR1 CARs with ECM booster and surface expression in human primary T cells are shown. (Figure 10A) The MSLN-targeting CAR consisted of a fully human MSLN scFv targeting domain, CD8 hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3ζ activation domain. The ROR1-targeting CAR consisted of a fully human ROR1 scFv9 targeting domain, IgG4 short hinge, CD8 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ activation domain. The booster CAR contained a monotargeting CAR followed by a 2A peptide in frame with the ECM molecule. The booster molecules selected were heparanase (HPSE), metalloproteinase (MMP2), and hyaluronidase PH-20. (Figure 10B) Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding the CAR construct. Transduced T cells were assayed for CAR surface expression by flow cytometry using ROR1 Fc or MSLN-His staining followed by anti-Fc-AF647 or anti-His APC, respectively. CD4 staining was included to identify CD4+ and CD8+ populations. CAR-positive percentages are listed above the plots. UTD - untransduced control. [Figure 11A] Figure 11 shows the in vitro cytotoxicity of MSLN and ROR1 CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines: (Figure 11A) MEC-1 ROR1Hi MSLNHi, (Figure 11C) NCI H226, and ROR1-MSLN- tumor lines: (Figure 11B) MEC-1, and (Figure 11D) HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 1.25:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SEM of three technical replicates. A representative experiment from one donor is shown in the panel. [Figure 11B] Figure 11 shows the in vitro cytotoxicity of MSLN and ROR1 CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines: (Figure 11A) MEC-1 ROR1Hi MSLNHi, (Figure 11C) NCI H226, and ROR1-MSLN- tumor lines: (Figure 11B) MEC-1, and (Figure 11D) HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 1.25:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SEM of three technical replicates. A representative experiment from one donor is shown in the panel. [Figure 11C] Figure 11 shows the in vitro cytotoxicity of MSLN and ROR1 CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines: (Figure 11A) MEC-1 ROR1Hi MSLNHi, (Figure 11C) NCI H226, and ROR1-MSLN- tumor lines: (Figure 11B) MEC-1, and (Figure 11D) HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 1.25:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SEM of three technical replicates. A representative experiment from one donor is shown in the panel. [Figure 11D]Figure 11 shows the in vitro cytotoxicity of MSLN and ROR1 CAR constructs. Luciferase-based cytotoxicity assays were performed using ROR1+ MSLN+ tumor lines: (Figure 11A) MEC-1 ROR1Hi MSLNHi, (Figure 11C) NCI H226, and ROR1-MSLN- tumor lines: (Figure 11B) MEC-1, and (Figure 11D) HL-60. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 1.25:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SEM of three technical replicates. A representative experiment from one donor is shown in the panel. [Figure 12A] HPSE expression in booster CARs and its ability to promote CAR T cell migration in vitro are shown. (Figure 12A) HPSE secreted by CAR D0344 and CAR D0347 was measured by ELISA, with monoCAR D0181, CAR D0290, and untransduced T cells (UTD) from the same donor included as controls. After overnight incubation, CAR T cell culture supernatants were evaluated. (Figure 12B) HPSE functionality was assessed by migration assay using transwells coated with 0, 2.5, or 5 mg / ml Cultrex. One million thawed CAR T cells were seeded into the pre-coated transwells. After 24 hours, the total CAR T cells migrated to the bottom chamber were quantified using Absolute counting beads on a flow cytometer. [Figure 12B]HPSE expression in booster CARs and its ability to promote CAR T cell migration in vitro are shown. (Figure 12A) HPSE secreted by CAR D0344 and CAR D0347 was measured by ELISA, with monoCAR D0181, CAR D0290, and untransduced T cells (UTD) from the same donor included as controls. After overnight incubation, CAR T cell culture supernatants were evaluated. (Figure 12B) HPSE functionality was assessed by migration assay using transwells coated with 0, 2.5, or 5 mg / ml Cultrex. One million thawed CAR T cells were seeded into the pre-coated transwells. After 24 hours, the total CAR T cells migrated to the bottom chamber were quantified using Absolute counting beads on a flow cytometer. [Figure 13A] Figure 13 shows the in vivo activity of CAR T constructs in the JeKo-1 xenograft model. NSG mice were implanted with 5x105 JeKo-1 cells stably transduced with luciferase via the tail vein on day 0. Tumor burden was determined using bioluminescence imaging. Mice with similar mean tumor burdens were randomly assigned to each group and injected with 5x106 CAR+ T cells or UTD per mouse on day 7. Tumor kinetics were measured on days 13, 20, 27, 34, 41, and 48. (Figure 13A) Bioluminescence images of representative mice are shown at the indicated time points. (Figure 13B) The time course of tumor growth based on whole-body bioluminescence (radiance) was quantified as photons per second per cm2 per steradian. TA - tumor alone; UTD - untransduced T cell control. N = 6 mice, mean ± SEM. [Figure 13B]Figure 13 shows the in vivo activity of CAR T constructs in the JeKo-1 xenograft model. NSG mice were implanted with 5x105 JeKo-1 cells stably transduced with luciferase via the tail vein on day 0. Tumor burden was determined using bioluminescence imaging. Mice with similar mean tumor burdens were randomly assigned to each group and injected with 5x106 CAR+ T cells or UTD per mouse on day 7. Tumor kinetics were measured on days 13, 20, 27, 34, 41, and 48. (Figure 13A) Bioluminescence images of representative mice are shown at the indicated time points. (Figure 13B) The time course of tumor growth based on whole-body bioluminescence (radiance) was quantified as photons per second per cm2 per steradian. TA - tumor alone; UTD - untransduced T cell control. N = 6 mice, mean ± SEM. [Figure 14] Figure 1 shows mouse weight changes during the JeKo-1 xenograft study. NSG mice bearing JeKo-1 mantle cell lymphoma were treated with 5x106 CART+ cells per mouse, and mouse weights were recorded three times per week. Weight change was calculated as a percentage change from the start of the study. Mean ± SEM. TA - tumor alone, UTD - untransduced T cell control. N = 6 mice per group. [Figure 15A] Figure 15 shows the in vivo activity of CAR T constructs in an OVCAR-3 xenograft model. NSG mice were injected intraperitoneally with 1 x 10 OVCAR-3 luciferase cells on day 0. Tumor burden was measured using bioluminescence imaging with an IVIS-S5 instrument. Mice with similar tumor burdens were randomly assigned to each group and treated with 5 x 10 CAR+ T cells or UTD per mouse on day 7. The kinetics of tumor development were measured on days 10, 17, 24, 31, 38, 45, and 52. (Figure 15A) Bioluminescence images of mice are shown at the indicated time points. (Figure 15B) The time course of tumor growth based on whole-body bioluminescence (radiance) was quantified and plotted as photons per second per cm per steradian. TA, tumor alone; UTD, untransduced T cell control. N = 4–5 mice, mean ± SEM. [Figure 15B] Figure 15 shows the in vivo activity of CAR T constructs in an OVCAR-3 xenograft model. NSG mice were injected intraperitoneally with 1 x 10 OVCAR-3 luciferase cells on day 0. Tumor burden was measured using bioluminescence imaging with an IVIS-S5 instrument. Mice with similar tumor burdens were randomly assigned to each group and treated with 5 x 10 CAR+ T cells or UTD per mouse on day 7. The kinetics of tumor development were measured on days 10, 17, 24, 31, 38, 45, and 52. (Figure 15A) Bioluminescence images of mice are shown at the indicated time points. (Figure 15B) The time course of tumor growth based on whole-body bioluminescence (radiance) was quantified and plotted as photons per second per cm per steradian. TA, tumor alone; UTD, untransduced T cell control. N = 4–5 mice, mean ± SEM. [Figure 16] Figure 1 shows mouse weight changes during the OVCAR-3 study. NSG mice bearing disseminated OVCAR-3 tumors were treated with 5 x 106 CAR T-positive (CAR T+) cells per mouse, and mouse weights were recorded three times per week. Weight change was calculated as a percentage change from the start of the study. Mean ± SEM. TA - tumor alone, UTD - untransduced T cell control. N = 4-5 mice per group. [Figure 17A]The structures of ROR1 and FolR1 CARs with ECM booster and surface expression in human primary T cells are shown. A) The ROR1-targeting CAR consisted of a fully human ROR1 scFv9 targeting domain, an IgG4 short hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain. The FolR1-targeting CAR consisted of a fully human Farle scFv targeting domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain under the control of a PGK or EF1α promoter. The booster CAR contained a mono-targeting CAR followed by a 2A peptide in frame with the ECM molecule. The booster molecules selected were matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), hyaluronidase (PH-20), and heparanase (HPSE). In the hyaluronidase-expressing ROR1 CAR set, PH-20 was expressed with or without a GPI anchor, or with a 7-amino acid residue, either natively or under the tPA signal peptide. B) Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding CAR constructs. Transduced T cells were assayed for CAR surface expression by flow cytometry using ROR1-Fc or FolR1-Fc staining followed by anti-Fc-AF647. CD4 staining was included to identify CD4+ and CD8+ populations. CAR-positive percentages are listed above the plots. UTD - untransduced control. [Figure 17B]The structures of ROR1 and FolR1 CARs with ECM booster and surface expression in human primary T cells are shown. A) The ROR1-targeting CAR consisted of a fully human ROR1 scFv9 targeting domain, an IgG4 short hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain. The FolR1-targeting CAR consisted of a fully human Farle scFv targeting domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain under the control of a PGK or EF1α promoter. The booster CAR contained a mono-targeting CAR followed by a 2A peptide in frame with the ECM molecule. The booster molecules selected were matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), hyaluronidase (PH-20), and heparanase (HPSE). In the hyaluronidase-expressing ROR1 CAR set, PH-20 was expressed with or without a GPI anchor, or with a 7-amino acid residue, either natively or under the tPA signal peptide. B) Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with lentiviral vectors encoding CAR constructs. Transduced T cells were assayed for CAR surface expression by flow cytometry using ROR1-Fc or FolR1-Fc staining followed by anti-Fc-AF647. CD4 staining was included to identify CD4+ and CD8+ populations. CAR-positive percentages are listed above the plots. UTD - untransduced control. [Figure 18A]Figure 18A shows the in vitro cytotoxicity and cytokine release of ROR1 and FolR1 CAR constructs. A, B) Luciferase-based cytotoxicity assays were performed using antigen-specific tumor lines. ROR1 CARs were tested against the ROR1+ lines NCI-H226 and MEC-1 ROR1Hi, with MEC-1 used as a negative control line expressing basal levels of ROR1. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios: 10:1, 5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. (Figure 18A) Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in each panel. (Figure 18B) Data represent one independent experiment from one donor. Mean ± SD of three technical replicates. (Figure 18C) The FolR1 CAR was tested against the FolR1+ line OVCAR3, with HL-60 used as a negative control for nonspecific killing. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 10:1, 2.5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in the panel. All target lines were stably transduced with firefly luciferase. (Figure 18D, Figure 18E) Cytokine production of IFNγ and TNFα was analyzed by ELISA. (Figure 18D) Culture supernatants of CAR-T cells were assessed after overnight incubation with ROR1+ NCI-H226 target cells at E:T ratios of 10:1, 5:1, or 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. (Figure 18E) CAR-T cell culture supernatants were assessed after overnight incubation with FolR1+ OVCAR3 target cells at E:T ratios of 10:1, 2.5:1, and 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. [Figure 18B]Figure 18A shows the in vitro cytotoxicity and cytokine release of ROR1 and FolR1 CAR constructs. A, B) Luciferase-based cytotoxicity assays were performed using antigen-specific tumor lines. ROR1 CARs were tested against the ROR1+ lines NCI-H226 and MEC-1 ROR1Hi, with MEC-1 used as a negative control line expressing basal levels of ROR1. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios: 10:1, 5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. (Figure 18A) Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in each panel. (Figure 18B) Data represent one independent experiment from one donor. Mean ± SD of three technical replicates. (Figure 18C) The FolR1 CAR was tested against the FolR1+ line OVCAR3, with HL-60 used as a negative control for nonspecific killing. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 10:1, 2.5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in the panel. All target lines were stably transduced with firefly luciferase. (Figure 18D, Figure 18E) Cytokine production of IFNγ and TNFα was analyzed by ELISA. (Figure 18D) Culture supernatants of CAR-T cells were assessed after overnight incubation with ROR1+ NCI-H226 target cells at E:T ratios of 10:1, 5:1, or 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. (Figure 18E) CAR-T cell culture supernatants were assessed after overnight incubation with FolR1+ OVCAR3 target cells at E:T ratios of 10:1, 2.5:1, and 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. [Figure 18C]Figure 18A shows the in vitro cytotoxicity and cytokine release of ROR1 and FolR1 CAR constructs. A, B) Luciferase-based cytotoxicity assays were performed using antigen-specific tumor lines. ROR1 CARs were tested against the ROR1+ lines NCI-H226 and MEC-1 ROR1Hi, with MEC-1 used as a negative control line expressing basal levels of ROR1. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios: 10:1, 5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. (Figure 18A) Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in each panel. (Figure 18B) Data represent one independent experiment from one donor. Mean ± SD of three technical replicates. (Figure 18C) The FolR1 CAR was tested against the FolR1+ line OVCAR3, with HL-60 used as a negative control for nonspecific killing. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 10:1, 2.5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in the panel. All target lines were stably transduced with firefly luciferase. (Figure 18D, Figure 18E) Cytokine production of IFNγ and TNFα was analyzed by ELISA. (Figure 18D) Culture supernatants of CAR-T cells were assessed after overnight incubation with ROR1+ NCI-H226 target cells at E:T ratios of 10:1, 5:1, or 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. (Figure 18E) CAR-T cell culture supernatants were assessed after overnight incubation with FolR1+ OVCAR3 target cells at E:T ratios of 10:1, 2.5:1, and 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. [Figure 18D]Figure 18A shows the in vitro cytotoxicity and cytokine release of ROR1 and FolR1 CAR constructs. A, B) Luciferase-based cytotoxicity assays were performed using antigen-specific tumor lines. ROR1 CARs were tested against the ROR1+ lines NCI-H226 and MEC-1 ROR1Hi, with MEC-1 used as a negative control line expressing basal levels of ROR1. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios: 10:1, 5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. (Figure 18A) Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in each panel. (Figure 18B) Data represent one independent experiment from one donor. Mean ± SD of three technical replicates. (Figure 18C) The FolR1 CAR was tested against the FolR1+ line OVCAR3, with HL-60 used as a negative control for nonspecific killing. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 10:1, 2.5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in the panel. All target lines were stably transduced with firefly luciferase. (Figure 18D, Figure 18E) Cytokine production of IFNγ and TNFα was analyzed by ELISA. (Figure 18D) Culture supernatants of CAR-T cells were assessed after overnight incubation with ROR1+ NCI-H226 target cells at E:T ratios of 10:1, 5:1, or 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. (Figure 18E) CAR-T cell culture supernatants were assessed after overnight incubation with FolR1+ OVCAR3 target cells at E:T ratios of 10:1, 2.5:1, and 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. [Figure 18E]Figure 18A shows the in vitro cytotoxicity and cytokine release of ROR1 and FolR1 CAR constructs. A, B) Luciferase-based cytotoxicity assays were performed using antigen-specific tumor lines. ROR1 CARs were tested against the ROR1+ lines NCI-H226 and MEC-1 ROR1Hi, with MEC-1 used as a negative control line expressing basal levels of ROR1. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios: 10:1, 5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. (Figure 18A) Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in each panel. (Figure 18B) Data represent one independent experiment from one donor. Mean ± SD of three technical replicates. (Figure 18C) The FolR1 CAR was tested against the FolR1+ line OVCAR3, with HL-60 used as a negative control for nonspecific killing. CAR-T cells and tumor cells were cocultured overnight at the indicated effector-to-target (E:T) ratios of 10:1, 2.5:1, or 1.25:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from three different donors. Mean ± SD of three technical replicates. A representative experiment from one donor is shown in the panel. All target lines were stably transduced with firefly luciferase. (Figure 18D, Figure 18E) Cytokine production of IFNγ and TNFα was analyzed by ELISA. (Figure 18D) Culture supernatants of CAR-T cells were assessed after overnight incubation with ROR1+ NCI-H226 target cells at E:T ratios of 10:1, 5:1, or 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. (Figure 18E) CAR-T cell culture supernatants were assessed after overnight incubation with FolR1+ OVCAR3 target cells at E:T ratios of 10:1, 2.5:1, and 1.25:1. Mean ± SD of three technical replicates. Data represent three independent experiments from three separate donors. [Figure 19A]The expression of the enzymes in booster CARs and their ability to promote CAR-T cell migration in vitro are shown. (Figure 19A) Left: The concentration of MMP-9 secreted by ROR1-coexpressing MMP-9 (D0373). Untransduced and CAR D0290 were also measured by MMP-9 ELISA. Data represent one independent experiment from two different donors tested. Right: HPSE by CARs D0368 and D0369 was measured by ELISA. CAR D0351 and untransduced T cells (UTD) from the same donor were included as controls. CAR-T cell culture supernatants were evaluated from the final day of CAR-T generation. (Figure 19B) The functionality of MMP-2 and MMP-9 was evaluated by migration assay using transwells coated with 0 or 5 mg / ml Cultrex™. 0.5 million thawed CAR-T cells were seeded onto pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Absolute counting beads on a flow cytometer. (Figure 19C) The functionality of HPSE and PH-20 was evaluated in a migration assay using transwells coated with 0 or 5 mg / ml Cultrex™ (Figure 19C) or hyaluronan (Figure 19D), respectively. 0.5 million thawed CAR-T cells were seeded into the pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Cultrex™-coated Absolute counting beads on a flow cytometer. [Figure 19B]The expression of the enzymes in booster CARs and their ability to promote CAR-T cell migration in vitro are shown. (Figure 19A) Left: The concentration of MMP-9 secreted by ROR1-coexpressing MMP-9 (D0373). Untransduced and CAR D0290 were also measured by MMP-9 ELISA. Data represent one independent experiment from two different donors tested. Right: HPSE by CARs D0368 and D0369 was measured by ELISA. CAR D0351 and untransduced T cells (UTD) from the same donor were included as controls. CAR-T cell culture supernatants were evaluated from the final day of CAR-T generation. (Figure 19B) The functionality of MMP-2 and MMP-9 was evaluated by migration assay using transwells coated with 0 or 5 mg / ml Cultrex™. 0.5 million thawed CAR-T cells were seeded onto pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Absolute counting beads on a flow cytometer. (Figure 19C) The functionality of HPSE and PH-20 was evaluated in a migration assay using transwells coated with 0 or 5 mg / ml Cultrex™ (Figure 19C) or hyaluronan (Figure 19D), respectively. 0.5 million thawed CAR-T cells were seeded into the pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Cultrex™-coated Absolute counting beads on a flow cytometer. [Figure 19C]The expression of the enzymes in booster CARs and their ability to promote CAR-T cell migration in vitro are shown. (Figure 19A) Left: The concentration of MMP-9 secreted by ROR1-coexpressing MMP-9 (D0373). Untransduced and CAR D0290 were also measured by MMP-9 ELISA. Data represent one independent experiment from two different donors tested. Right: HPSE by CARs D0368 and D0369 was measured by ELISA. CAR D0351 and untransduced T cells (UTD) from the same donor were included as controls. CAR-T cell culture supernatants were evaluated from the final day of CAR-T generation. (Figure 19B) The functionality of MMP-2 and MMP-9 was evaluated by migration assay using transwells coated with 0 or 5 mg / ml Cultrex™. 0.5 million thawed CAR-T cells were seeded onto pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Absolute counting beads on a flow cytometer. (Figure 19C) The functionality of HPSE and PH-20 was evaluated in a migration assay using transwells coated with 0 or 5 mg / ml Cultrex™ (Figure 19C) or hyaluronan (Figure 19D), respectively. 0.5 million thawed CAR-T cells were seeded into the pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Cultrex™-coated Absolute counting beads on a flow cytometer. [Figure 19D]The expression of the enzymes in booster CARs and their ability to promote CAR-T cell migration in vitro are shown. (Figure 19A) Left: The concentration of MMP-9 secreted by ROR1-coexpressing MMP-9 (D0373). Untransduced and CAR D0290 were also measured by MMP-9 ELISA. Data represent one independent experiment from two different donors tested. Right: HPSE by CARs D0368 and D0369 was measured by ELISA. CAR D0351 and untransduced T cells (UTD) from the same donor were included as controls. CAR-T cell culture supernatants were evaluated from the final day of CAR-T generation. (Figure 19B) The functionality of MMP-2 and MMP-9 was evaluated by migration assay using transwells coated with 0 or 5 mg / ml Cultrex™. 0.5 million thawed CAR-T cells were seeded onto pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Absolute counting beads on a flow cytometer. (Figure 19C) The functionality of HPSE and PH-20 was evaluated in a migration assay using transwells coated with 0 or 5 mg / ml Cultrex™ (Figure 19C) or hyaluronan (Figure 19D), respectively. 0.5 million thawed CAR-T cells were seeded into the pre-coated transwells. After 24 hours, the total CAR-T cells that had migrated to the bottom chamber were quantified using Cultrex™-coated Absolute counting beads on a flow cytometer. [Figure 20A]Figure 20A shows the in vivo activity of FolR1 CAR-T cells co-expressing HPSE or PH-20 in an OVCAR3 xenograft model. NSG mice were implanted intraperitoneally with 1 x 10 OVCAR3 cells stably transduced with luciferase. Tumor burden was determined using bioluminescence imaging with an IVIS-S5 instrument. Mice with similar mean tumor burdens were randomly assigned to each group and injected with 5 x 10 CAR+ T cells or UTD per mouse on day 8. Tumor kinetics were measured on days 11, 18, 25, 32, and 39. (Figure 20A) Bioluminescence images of representative mice are shown at the indicated time points. (Figure 20B) The time course of tumor growth based on whole-body bioluminescence (radiance, photons / second / cm2 / sr) was quantified and plotted as indicated. TA - tumor alone; UTD - untransduced T cell control. N = 4 mice, mean ± SD. (Figure 20C) Mouse weight change during the OVCAR3 xenograft study. OVCAR3-bearing mice were treated with CAR-T cells and body weight was recorded three times per week. Weight change was calculated as a percentage change from the start of the study. Mean ± SEM. TA - tumor alone, UTD - untransduced T cell control. N = 4 mice / group. [Figure 20B]Figure 20A shows the in vivo activity of FolR1 CAR-T cells co-expressing HPSE or PH-20 in an OVCAR3 xenograft model. NSG mice were implanted intraperitoneally with 1 x 10 OVCAR3 cells stably transduced with luciferase. Tumor burden was determined using bioluminescence imaging with an IVIS-S5 instrument. Mice with similar mean tumor burdens were randomly assigned to each group and injected with 5 x 10 CAR+ T cells or UTD per mouse on day 8. Tumor kinetics were measured on days 11, 18, 25, 32, and 39. (Figure 20A) Bioluminescence images of representative mice are shown at the indicated time points. (Figure 20B) The time course of tumor growth based on whole-body bioluminescence (radiance, photons / second / cm2 / sr) was quantified and plotted as indicated. TA - tumor alone; UTD - untransduced T cell control. N = 4 mice, mean ± SD. (Figure 20C) Mouse weight change during the OVCAR3 xenograft study. OVCAR3-bearing mice were treated with CAR-T cells and body weight was recorded three times per week. Weight change was calculated as a percentage change from the start of the study. Mean ± SEM. TA - tumor alone, UTD - untransduced T cell control. N = 4 mice / group. [Figure 20C]Figure 20A shows the in vivo activity of FolR1 CAR-T cells co-expressing HPSE or PH-20 in an OVCAR3 xenograft model. NSG mice were implanted intraperitoneally with 1 x 10 OVCAR3 cells stably transduced with luciferase. Tumor burden was determined using bioluminescence imaging with an IVIS-S5 instrument. Mice with similar mean tumor burdens were randomly assigned to each group and injected with 5 x 10 CAR+ T cells or UTD per mouse on day 8. Tumor kinetics were measured on days 11, 18, 25, 32, and 39. (Figure 20A) Bioluminescence images of representative mice are shown at the indicated time points. (Figure 20B) The time course of tumor growth based on whole-body bioluminescence (radiance, photons / second / cm2 / sr) was quantified and plotted as indicated. TA - tumor alone; UTD - untransduced T cell control. N = 4 mice, mean ± SD. (Figure 20C) Mouse weight change during the OVCAR3 xenograft study. OVCAR3-bearing mice were treated with CAR-T cells and body weight was recorded three times per week. Weight change was calculated as a percentage change from the start of the study. Mean ± SEM. TA - tumor alone, UTD - untransduced T cell control. N = 4 mice / group. [Figure 21A] Figure 21A shows the in vivo characterization of Farle CAR-T cells boosted with the ECM enzymes HPSE or PH-20. (Figure 21A) CAR-T infiltration, CAR expression (percent and gMFI), and CD4:CD8 ratios were measured in the bone marrow (top) and spleen (middle) at the time of study death. All samples were normalized by volume and Absolute counting beads. Spleen weight was measured, and no significant differences were found between treatment groups. (Figure 21B) Memory phenotypes of CAR-T cells in the bone marrow (top) and spleen (bottom). Naive, central memory, effector memory, and effector cells were measured for CAR-T+ cells (left), CAR+CD4+ (middle), and CAR+CD8+ (right). Mean ± SD. Statistical differences were calculated using one-way ANOVA in Prism software. For B, statistical differences in effector populations between different treatment groups were measured. TA, tumor alone; UTD, untransduced T cell control. N = 4 mice / group. [Figure 21B] Figure 21A shows the in vivo characterization of Farle CAR-T cells boosted with the ECM enzymes HPSE or PH-20. (Figure 21A) CAR-T infiltration, CAR expression (percent and gMFI), and CD4:CD8 ratios were measured in the bone marrow (top) and spleen (middle) at the time of study death. All samples were normalized by volume and Absolute counting beads. Spleen weight was measured, and no significant differences were found between treatment groups. (Figure 21B) Memory phenotypes of CAR-T cells in the bone marrow (top) and spleen (bottom). Naive, central memory, effector memory, and effector cells were measured for CAR-T+ cells (left), CAR+CD4+ (middle), and CAR+CD8+ (right). Mean ± SD. Statistical differences were calculated using one-way ANOVA in Prism software. For B, statistical differences in effector populations between different treatment groups were measured. TA, tumor alone; UTD, untransduced T cell control. N = 4 mice / group. [Figure 22A] The structure and surface expression of ROR1 and CD276 CARs on transduced primary T cells are shown. (Figure 22A) ROR1 or CD276 CARs contained the ROR1 or CD276 scFv binding domain, IgG4 or CD8 hinge domain, CD8 transmembrane domain, 41BB costimulatory domain, and CD3ζ activation domain. (Figure 22B) Representative flow plots of CAR expression in transduced T cells. CAR and CAR / CCR T cells were stained with ROR1-Fc followed by anti-Fc AF647 to detect ROR1 CAR and CD276-His to detect CD276 CCR. (Figure 22C) Average CAR expression in T cells from three healthy donors. Error bars represent mean ± SEM. [Figure 22B]The structure and surface expression of ROR1 and CD276 CARs on transduced primary T cells are shown. (Figure 22A) ROR1 or CD276 CARs contained the ROR1 or CD276 scFv binding domain, IgG4 or CD8 hinge domain, CD8 transmembrane domain, 41BB costimulatory domain, and CD3ζ activation domain. (Figure 22B) Representative flow plots of CAR expression in transduced T cells. CAR and CAR / CCR T cells were stained with ROR1-Fc followed by anti-Fc AF647 to detect ROR1 CAR and CD276-His to detect CD276 CCR. (Figure 22C) Average CAR expression in T cells from three healthy donors. Error bars represent mean ± SEM. [Figure 22C] The structure and surface expression of ROR1 and CD276 CARs on transduced primary T cells are shown. (Figure 22A) ROR1 or CD276 CARs contained the ROR1 or CD276 scFv binding domain, IgG4 or CD8 hinge domain, CD8 transmembrane domain, 41BB costimulatory domain, and CD3ζ activation domain. (Figure 22B) Representative flow plots of CAR expression in transduced T cells. CAR and CAR / CCR T cells were stained with ROR1-Fc followed by anti-Fc AF647 to detect ROR1 CAR and CD276-His to detect CD276 CCR. (Figure 22C) Average CAR expression in T cells from three healthy donors. Error bars represent mean ± SEM. [Figure 23A]Figure 23 shows the cytotoxicity of ROR1 or CD276 CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using the ROR1+ CD276+ tumor lines (Figure 23A) OVCAR3; (Figure 23B) AsPC-1; and (Figure 23C) NCI-H226. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three in T cells from different donors. Error bars represent the mean ± SEM. [Figure 23B] Figure 23 shows the cytotoxicity of ROR1 or CD276 CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using the ROR1+ CD276+ tumor lines (Figure 23A) OVCAR3; (Figure 23B) AsPC-1; and (Figure 23C) NCI-H226. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three in T cells from different donors. Error bars represent the mean ± SEM. [Figure 23C]Figure 23 shows the cytotoxicity of ROR1 or CD276 CAR constructs in vitro. Luciferase-based cytotoxicity assays were performed using the ROR1+ CD276+ tumor lines (Figure 23A) OVCAR3; (Figure 23B) AsPC-1; and (Figure 23C) NCI-H226. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cocultured overnight at 10 effector-to-target (E:T) ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three in T cells from different donors. Error bars represent the mean ± SEM. [Figure 24A] The structure and surface expression of CD276 CCR-boosted ROR1 CAR on primary human T cells are shown. (Figure 24A) The CD276 CCR-boosted ROR1 CAR contains the ROR1 CAR linked in-frame to the CD276 CCR via a P2A ribosomal skip element. (Figure 24B) Transduced primary T cells were gated based on forward and side scatter, doublet exclusion, and viability dye negativity. Surface CAR expression of the ROR1-targeting domain or CD276-targeting domain of each binder was detected by co-staining with ROR1-Fc and CD276-His, followed by anti-Fc and anti-His FL conjugates. Representative flow plots of co-expression of ROR1 CAR and CD276 CCR are shown. (Figure 24C) Co-expression of CAR and CCR on the T cell surface was quantified. The average values of T cells transduced from three healthy donors are shown, and error bars indicate ±SEM. [Figure 24B]The structure and surface expression of CD276 CCR-boosted ROR1 CAR on primary human T cells are shown. (Figure 24A) The CD276 CCR-boosted ROR1 CAR contains the ROR1 CAR linked in-frame to the CD276 CCR via a P2A ribosomal skip element. (Figure 24B) Transduced primary T cells were gated based on forward and side scatter, doublet exclusion, and viability dye negativity. Surface CAR expression of the ROR1-targeting domain or CD276-targeting domain of each binder was detected by co-staining with ROR1-Fc and CD276-His, followed by anti-Fc and anti-His FL conjugates. Representative flow plots of co-expression of ROR1 CAR and CD276 CCR are shown. (Figure 24C) Co-expression of CAR and CCR on the T cell surface was quantified. The average values of T cells transduced from three healthy donors are shown, and error bars indicate ±SEM. [Figure 24C] The structure and surface expression of CD276 CCR-boosted ROR1 CAR on primary human T cells are shown. (Figure 24A) The CD276 CCR-boosted ROR1 CAR contains the ROR1 CAR linked in-frame to the CD276 CCR via a P2A ribosomal skip element. (Figure 24B) Transduced primary T cells were gated based on forward and side scatter, doublet exclusion, and viability dye negativity. Surface CAR expression of the ROR1-targeting domain or CD276-targeting domain of each binder was detected by co-staining with ROR1-Fc and CD276-His, followed by anti-Fc and anti-His FL conjugates. Representative flow plots of co-expression of ROR1 CAR and CD276 CCR are shown. (Figure 24C) Co-expression of CAR and CCR on the T cell surface was quantified. The average values of T cells transduced from three healthy donors are shown, and error bars indicate ±SEM. [Figure 25A]Figure 25 shows the cytotoxicity of the ROR1 CAR alone without the CD276 CCR construct in vitro. Luciferase-based cytotoxicity assays were performed using (Figure 25A) the ROR1+ CD276+ tumor line OVCAR3; (Figure 25B) the ROR1- CD276- tumor line RS4;11; and single-target-positive cell lines (Figure 25C) ROR1+ CD276- RS4;11-ROR1; (Figure 25D) ROR1- CD276+ RS4;11-CD276. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at a series of 10 effector-to-target ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three performed with T cells from three different donors. Error bars = mean ± SEM [Figure 25B] Figure 25 shows the cytotoxicity of the ROR1 CAR alone without the CD276 CCR construct in vitro. Luciferase-based cytotoxicity assays were performed using (Figure 25A) the ROR1+ CD276+ tumor line OVCAR3; (Figure 25B) the ROR1- CD276- tumor line RS4;11; and single-target-positive cell lines (Figure 25C) ROR1+ CD276- RS4;11-ROR1; (Figure 25D) ROR1- CD276+ RS4;11-CD276. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at a series of 10 effector-to-target ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three performed with T cells from three different donors. Error bars = mean ± SEM [Figure 25C]Figure 25 shows the cytotoxicity of the ROR1 CAR alone without the CD276 CCR construct in vitro. Luciferase-based cytotoxicity assays were performed using (Figure 25A) the ROR1+ CD276+ tumor line OVCAR3; (Figure 25B) the ROR1- CD276- tumor line RS4;11; and single-target-positive cell lines (Figure 25C) ROR1+ CD276- RS4;11-ROR1; (Figure 25D) ROR1- CD276+ RS4;11-CD276. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at a series of 10 effector-to-target ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three performed with T cells from three different donors. Error bars = mean ± SEM [Figure 25D] Figure 25 shows the cytotoxicity of the ROR1 CAR alone without the CD276 CCR construct in vitro. Luciferase-based cytotoxicity assays were performed using (Figure 25A) the ROR1+ CD276+ tumor line OVCAR3; (Figure 25B) the ROR1- CD276- tumor line RS4;11; and single-target-positive cell lines (Figure 25C) ROR1+ CD276- RS4;11-ROR1; (Figure 25D) ROR1- CD276+ RS4;11-CD276. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at a series of 10 effector-to-target ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of CAR expression. A nonlinear EC50 shift, x, is the logarithmic concentration, was used for curve fitting. Data represent one independent experiment out of three performed with T cells from three different donors. Error bars = mean ± SEM [Figure 26A]Figure 26 shows the relative potency of ROR1 CAR / CD276 CCR constructs in vitro. CAR T cells and ROR1+ tumor cells were co-cultured overnight at 10 effector-to-target ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of ROR1 CAR expression. Relative potency compared to ROR1 CAR LTG2529 was calculated using the nonlinear EC50 shift function in GraphPad Prism, where x is the logarithmic concentration. The relative potency of each construct targeting ROR1+ in tumor lines: (Figure 26A) OVCAR-3, (Figure 26B) RS4;11-ROR1, is plotted as a bar graph. Data represent the mean ± SEM of independent experiments using T cells from three different donors. [Figure 26B] Figure 26 shows the relative potency of ROR1 CAR / CD276 CCR constructs in vitro. CAR T cells and ROR1+ tumor cells were co-cultured overnight at 10 effector-to-target ratios. The percentage of specific target lysis was assessed by luminometry and normalized to the percentage of ROR1 CAR expression. Relative potency compared to ROR1 CAR LTG2529 was calculated using the nonlinear EC50 shift function in GraphPad Prism, where x is the logarithmic concentration. The relative potency of each construct targeting ROR1+ in tumor lines: (Figure 26A) OVCAR-3, (Figure 26B) RS4;11-ROR1, is plotted as a bar graph. Data represent the mean ± SEM of independent experiments using T cells from three different donors. [Figure 27A]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27B]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27C]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27D]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27E]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27F]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27G]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27H]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27I]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27J]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 27K]The novel anti-ROR1 LTG2529 (with scFV9 binder) exhibits higher expression and cytokine secretion than LTG2527 (with the control R12 binder), while exhibiting comparable cytotoxic potency in vitro and being effective against hematologic tumors in vivo. (Figure 27A) Schematic of the CAR construct. (Figure 27B) Left: Example flow plot of the percentage of CAR+ T cells; Center: Percentage of CAR+ T cells (n = 3 donors); Right: CAR density (n = 3 donors); All on day 8 of transduction. (Figure 27C) Quantification of ROR1 molecules per cell in different hematologic cell lines. Experiments were performed in duplicate using anti-ROR1 Ab from BD Biosciences; separate experiments were also performed in duplicate using anti-ROR1 Abs from Miltenyi Biotec and R&D systems, with similar results. (Figure 27D) In vitro cytotoxic activity of CAR-T when co-cultured with the MCL cell line Jeko-1 for 18 hours; left: representative killing curve; right: relative potency of LTG2529 vs. LTG2527 (n=3 donors). (Figure 27E) Quantification by ELISA of cytokines secreted in 18-hour co-culture of CAR T with the Jeko-1 cell line; representative data from 3 donors is shown. (Figures 27F-K): NSG mice were implanted with Jeko-1 cells on day -6 (iv, 0.5e6 cells / mouse; 6 mice / group), then staged on day -1 and administered CAR T cells on day 0 (iv, 3e6 CAR+T cells / mouse) (Figure 27F). Tumor progression was quantified by bioluminescence imaging (Figures 27G, 27H), body weight was monitored (Figure 27I), and blood was sampled at the indicated time points and tumor cells (Figure 27J) or T cells (Figure 27K) were quantified by flow cytometry. Note: *: p<0.05; **: p<0.01; n / s: not significant. [Figure 28A]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28B]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28C]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28D]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28E]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28F]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28G]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28H]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 28I]These results demonstrate that LTG2529, but not LTG2527, was effective in inhibiting solid tumor progression in vivo in the OVCAR-3 ovarian cancer xenograft model, despite comparable in vitro cytotoxic activity (higher cytokine production). (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; experiments were performed in duplicate using an anti-ROR1 Ab from BD Biosciences; separate experiments using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems were also performed in duplicate with similar results. (B) Representative killing curves of 18-hour cocultures of CAR-T against various solid tumor cell lines, showing the relative cytotoxic potency of LTG2529 versus LTG2527 on the right (n = 3 donors). (C) Quantification of cytokines secreted by ELISA in 18-hour cocultures of OVCAR-3 cell lines and CAR-T. Representative data are shown; three independent experiments were performed with three donors with similar results. (D-I): CAR-T efficacy in an in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (5 mice / group) were implanted (ip) with the OVCAR-3 cell line (10e6 cells / mouse) on day -7 and subsequently staged on day -1; CAR-T (5e6 CAR+ T cells / mouse) was administered (iv) on day 0 (D); tumor progression was quantified by bioluminescence imaging (E, F); body weight was monitored (G); and blood was sampled at the indicated time points to quantify CAR+ T cells in both CD8 and CD4 subpopulations (H) and memory T cells (I). [Figure 29A]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29B]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29C]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29D]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29E]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29F]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29G]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29H]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29I]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29J]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 29K]This shows that dominant-negative TGFbRII (DN) disrupted TGFb1 signaling in LTG2529-transduced T cells and reduced the inhibitory effect of TGFb1 on the cytotoxic activity of CAR-T against the pancreatic cancer cell line AsPC-1 in vitro. (Figure 29A) Schematic diagram of LTG2529 alone and LTG2529-armed DN (i.e., D0228) constructs. (Figure 29B) On day 8 after transduction, CD8+ and CD4+ T cells transduced with LTG2529 or D0228 were analyzed by flow cytometry for CAR expression (left: flow plot; center: graph from flow plot) and memory phenotype (right); three independent experiments were performed using three donors with similar results. (Fig. 29C) Expression of TGFbRII in T cells transduced with LTG2529 or D0228 was evaluated by flow cytometry; three independent experiments were performed using three donors with similar results. (Fig. 29D) CAR-T cells were synchronized by IL-2 starvation for 22 hours and then treated with TGFb1 (10 ng / mL) for 0.5 or 2 hours; cells were then stained with pSmad2 / 3 and subjected to flow analysis; upper panel: flow plot; lower panel: graph from the plot in the upper panel; data are representative of three independent experiments performed with three donors. (Fig. 29E) Expression of ROR1 in the AsPC-1 cell line was evaluated by flow cytometry. (Figure 29F) AsPC-1 were co-cultured with CAR-T without or with TGFb1 (1 ng / mL or 10 ng / mL); tumor cell lysis was measured by xCELLigence; left: % cell lysis; center: time to 50% tumor cell death (KT50); right: relative cytotoxicity of CAR-T treated with TGFb1 versus untreated; two independent experiments using two donors were performed in triplicate with similar results. (Figure 29G) Cytokine production from the experiments in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results.(Figure 29H, Figure 29I): Production of active or latent TGFb1 by various solid tumor cell lines (Figure 29H) or AsPC-1 ectopically overexpressing TGF1 (Figure 29I) was assessed by ELISA; data are representative of two independent experiments with similar results. (Figure 29J) AsPC-1 overexpressing TGFb1 (AsPC-1 / TGFb) or AsPC-1 ctrl were cocultured with CAR-T cells, and the percentage of tumor cell cytolysis is shown. (Figure 29K) Cytokine production from the experiment in (Figure 29E) was quantified by ELISA; two independent experiments using two donors were performed in triplicate with similar results. Note: *: p<0.05; **: P<0.01; ***: P<0.001. [Figure 30A]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30B]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30C]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30D]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30E]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30F]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30G]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30H]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30I]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 30J]In a pancreatic cancer xenograft model using AsPC-1, which produces low levels of TGFb1, TGFbRIIDN showed a higher frequency of CAR+ T cells (Figures 30A-30D). CAR-T efficacy in an in vivo pancreatic cancer AsPC-1 xenograft model: NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 cells (1e6 cells / mouse) on day -17, followed by staging and CAR-T injection (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 30A). Tumor volume was measured (Figure 30B) (left: tumor volume from mice across all groups; right: tumor volume from mice treated with armed and disarmed CARs starting 10 days after T cell administration). Body weight was monitored (Figure 30C). Blood was sampled from mice, and the CD8 subpopulation of CAR+ T cells was quantified (Figure 30D). (Figure 30E-J): At day 73 after T cell administration, mice (4 from the disarmed CAR-treated group and 3 from the armed CAR-treated group; note: 1 mouse from the armed group was euthanized 60 days after T cell infusion due to excessive weight loss) were re-challenged with AsPC-1 cells (1e6 cells / mouse in the left flank; as the first challenge was in the right flank) (Figure 30E); tumor volume (Figure 30F) and viability (Figure 30G) in both flanks were monitored; blood from mice was sampled at the indicated time points to quantify T cell memory phenotype (Figure 30H), percentage of CAR+ cells (Figure 30I); T cells isolated from spleen and bone marrow at termination were also analyzed for CAR+ T cell content by flow cytometry (Figure 30J). [Figure 31A]Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified. [Figure 31B] Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified. [Figure 31C]Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified. [Figure 31D] Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified. [Figure 31E]Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified. [Figure 31F] Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified. [Figure 31G]Figure 31A shows the attenuation of the inhibitory effect of TGFb by TGFbRIIDN-armed ROR1 CAR T cells in a pancreatic cancer xenograft model using TGFb-overexpressing AsPC-1. NSG mice (5 mice / group) were subcutaneously implanted with AsPC-1 / TGFb cells (1e6 cells / mouse) on day -15, followed by staged and CAR-T infusion (iv, 5e6 CAR+ T cells / mouse) on day 0 (Figure 31A); tumor volume was monitored (Figure 31B); blood was sampled from mice on days 5 and 15 after T cell infusion, and cytokines, including TGFb1 (left), IFNg (center), and GM-CSF (right), were quantified (Figure 31C); T cells isolated from blood at the indicated time points were quantified for total cell number (Figure 31D) and CAR+ content in both CD8 and CD4 subpopulations (Figure 31E). At termination (day 49 post-T cell infusion), T cells were collected from blood, spleen, and bone marrow, and the CAR+ content (Figure 31F) and memory phenotype (Figure 31G) in both CD4 and CD8 subpopulations were quantified.

[0058] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. For example, the term "antigen" includes single or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" without excluding other elements. The phrase "and / or" means "and" or "or." Furthermore, it should be understood that 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 illustrative purposes unless otherwise specified. Although many methods and materials similar or equivalent to those described herein can be used, particularly preferred methods and materials are described below. In the event of any conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

[0059] The term "about" when referring to a measurable value such as an amount, temporal duration, or the like, is meant to encompass variations of ±20%, ±10%, or more preferably ±5%, or ±1%, or even more preferably ±0.1% from the stated value, such as variations appropriate for practicing the disclosed methods.

[0060] Unless otherwise specified, technical terms in this specification are used according to conventional usage.The definition of general terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press in 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. in 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc. in 1995; and other similar references.

[0061] The present invention provides a novel anti-effector partial antibody or its antigen-binding domain, and a chimeric antigen receptor (CAR) containing such an effector partial antigen-binding domain, as well as a host cell (e.g., T cell) expressing the receptor and a nucleic acid molecule encoding the receptor.The CAR may consist of a single molecule expressed on the surface of an effector cell, or may consist of a CAR composed of an effector cell-expressed signal transduction module and a soluble targeting module (when the soluble targeting module is combined with the cell-expressed signal transduction module, a complete functional CAR is formed).The CAR exhibits high surface expression on transduced T cells, and has high cytolytic activity and in vivo expansion and persistence of transduced T cells.Methods of using the disclosed CAR, host cell, and nucleic acid molecule are also provided, for example, for treating cancer in a subject.

[0062] In its broadest aspect, provided herein are novel chimeric antigen receptors (CARs) comprising a CAR construct with a main effector moiety followed by one or more 2A sequences, in frame to one or more additional "booster" elements for improved function, including enhanced tumor penetration, to improve the therapeutic efficacy of CAR-T cells in solid tumors, hematological tumors, autoimmune diseases, genetic diseases, or other relevant indications. In yet another broad aspect, provided herein are novel chimeric antigen receptors (CARs) comprising boosted CARs, in which functionally co-expressed boosted CAR elements are expressed with high transduction efficiency from a single multicistronic vector, thereby simplifying CAR production and release and reducing the cost for market introduction. In one embodiment, the boosted CAR composition comprises one or more of the following features: i) high surface expression on the transduced T cells; ii) multitargeting to overcome antigen escape; iii) one or more armoured elements to overcome immunosuppression in the TME; iv) one or more cytokine stimulating elements to promote cytokine-autonomous T cell stimulation resulting in enhanced anti-tumor cytotoxicity, expansion, memory formation, cytokine secretion, persistence; v) one or more digestive enzymes to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and enable tumor penetration of the CAR T; vi) one or more pro-inflammatory immune activators; and vii) one or more on-switches or off-switches to control expression of the CAR; or any combination thereof, wherein the boosted CAR achieves a high degree of cytolytic activity and persistence to promote in vivo expansion of the transduced T cells, and persistence of patient-specific anti-tumor T cells resulting in tumor stabilisation, cancer reduction, elimination, remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0063] In yet another broad aspect, the novel chimeric antigen receptors (CARs) provided herein can comprise single-targeting, tandem-targeting, or multi-targeting CAR constructs (including those in DuoCAR format), or any combination thereof.

[0064] In certain embodiments, the novel boosted CAR is under the control of one or more constitutive, tissue-specific, or inducible promoters, or any combination thereof.

[0065] In certain embodiments, the novel boosted CAR may comprise one or more pro-inflammatory immune activators.

[0066] In certain embodiments, the one or more pro-inflammatory immune activators may include boosters that can turn a "cold" immune environment "hot," such as neutrophil-activating protein (NAP) from bacteria such as Helicobacter pylori, bacterial lipopolysaccharide (LPS) components, or polyinosinic-polycytidylic acid (poly(I:C)), which can induce or enhance an exogenous bystander response against solid tumors, or soluble inflammatory factors such as FLT3 ligand, or oncolytic viruses, or TNF family cytokines, including CD40 ligand (CD40L), tumor necrosis factor (TNF), and receptor activator of nuclear factor-κB (RANKL) / TRANCE. In one embodiment, when used as a booster for CAR T cell therapy, such elements may reduce or ablate tumor growth and / or increase survival regardless of the target antigen, tumor type, and host haplotype. Such boosters act by supporting dendritic cell maturation and bystander responses, potentially resulting in epitope spreading and infiltration of CD8+ cells targeting tumor-associated antigens other than the CAR T target antigen.

[0067] In certain embodiments, the one or more switches include a tag, a kill switch, an on switch, an off switch, and / or an adapter switch, or any combination thereof.

[0068] In certain embodiments, the novel boosted CAR switch may be a tag (CD19, CD34, CD22, EGFR), or a kill switch (iCAS9), or an [ON] switch, or an [OFF] switch, or an adapter switch, or any combination thereof.

[0069] In certain embodiments, novel chimeric antigen receptors (CARs) are provided that are single, tandem, multi-targeting, and DuoCAR (with or without one or more booster elements) and are used to transduce effector cells for the treatment of solid and hematological tumors and other diseases via the following target antigens (e.g., but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3, and the like). TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD 38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM, folate binding protein, folate receptor Folate receptor alpha (FOLR1), folate receptor beta (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, pl85, IL-2 receptor, interleukin-1 receptor accessory protein (IL1RAP), EGFRvIII (de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, ανβ3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, p53 non-mutant, NY-ESO-1, MelanA / MART 1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B l, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE, MAGEA3, CYP1B 1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES 1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL1, MUC1, MUC16 / CA125, MAGE A4, MAGE C2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endoglin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPRC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15, or any combination thereof, or fragment thereof, is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any of the foregoing, or a combination thereof).

[0070] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2, and / or anti-CD276, and / or anti-GPC2, and / or anti-FGFR2, and / or anti-PSMA, and / or anti-MUC1, and / or anti-MUC16, and / or anti-IL13Ralpha antibody, or any combination thereof, or fragment thereof, is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single chain Fv (ScFv).

[0071] In one embodiment, anti-GD2, anti-GD3, anti-GM2, anti-Ley, anti-polysialic acid, anti-fucosyl GMl, anti-GM3, anti-Tn, anti-STn, anti-sLe (animal), anti-GloboH, anti-CD5, anti-CD7, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CD37, anti-CD30, Anti-CD33, anti-CD38, anti-CD123, anti-CD45, anti-CAMPATH-1, anti-BCMA, anti-CS-1, anti-PD-L1, anti-CD276 / B7-H3, anti-B7-H4, anti-B7-DC, anti-HLA-DR carcinoembryonic antigen (CEA), anti-TAG-72, anti-EpCAM, anti-folate-binding protein Protein, anti-folate receptor alpha (FOLR1), anti-folate receptor beta (FOLR2), anti-A33, anti-G250, anti-prostate-specific membrane antigen (PSMA), anti-ferritin, anti-CA-125, anti-CA19-9, anti-CD44v6, anti-epidermal growth factor, anti-pl85, anti-IL-2 receptor, anti-interleukin-1 receptor accessory protein (IL1RAP), anti-EGFRvIII (de2-7), anti-fibroblast activation protein, anti-tenascin, anti-metalloproteinase, anti-endosialin, anti-vascular endothelial growth factor, anti-ανβ3, anti-WT1, anti-LMP2, anti-HPV E6, anti-HPV E7, anti-Her-2 / neu, anti-p53 non-mutant, anti-NY-ESO-1, anti-MelanA / MART 1, anti-Ras mutant, anti-gp100, anti-FGFR1, anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-GPC1, anti-GPC2, anti-GPC3, anti-p53 mutant, anti-PR1, anti-bcr-abl, anti-tyrosinase, anti-survivin, anti-PSA, anti-hTERT, anti-sarcoma translocation breakpoint fusion protein, anti-EphA2, anti-PAP, anti-ML-IAP, anti-AFP, anti-ERG, anti-NA17, anti-PAX3, anti-ALK, anti-androgen receptor, anti-cyclin B, anti-MYCN, anti-RhoC, anti-TRP-2, anti-mesothelin, anti-PSCA, anti-MAGE A1, anti-MAGE A3, anti-CYP1B 1, anti-PLAV1, anti-BORIS, anti-ETV6-AML, anti-NY-BR-1, anti-RGS5, anti-SART3, anti-carbonic anhydrase IX, anti-PAX5, anti-OY-TES 1, anti-sperm protein 17, anti-LCK, anti-HMWMAA, anti-AKAP-4, anti-SSX2, anti-XAGE 1, anti-B7H3, anti-Legumain, anti-Tie3. Anti-PAGE4, anti-VEGFR2, anti-MAD-CT-1, anti-PDGFR-B, anti-MAD-CT-2, anti-TRAIL1, anti-MUC1, anti-MUC16 / CA125, anti-MAGE A4, anti-MAGE C2, anti-GAGE, anti-EGFR, anti-EGFR1, anti-EGFR2 / Her2, anti-CMET, anti-HER3, anti-CA6, anti-NAPI2B, anti-TROP2, anti-TEM1, anti-TEM7, anti-TEM8 , anti-FAP, anti-LAP, anti-CLDN6, anti-CLDN8, anti-CLDN16, anti-CLDN18.2, anti-RON, anti-LY6E, anti-DLL3, anti-PTK7, anti-UPK1B, anti-STRA6, anti-TMP RSS3, anti-TMRRSS4, anti-TMEM238, anti-Clorfl86, anti-LIV1, anti-ROR1, anti-ROR2, anti-Fos-related antigen 1, anti-VEGFR1, anti-endoglin, anti-CD90, Anti-CD326, anti-CD70, anti-SSEA4, anti-CD318, anti-CLA, anti-TSPAN8, anti-GPRC5D, anti-EpCAM, anti-Thy1, anti-IL13Ra2, anti-BDCA1, anti-BDCA2, Isolated polynucleotides are provided that encode anti-BDCA3, anti-GD2, anti-PSMA, anti-FAP, anti-CLL1, anti-SLAMF7 / CS1, anti-CD147, anti-DPPA5, anti-GRP78, anti-CD66c, VISTA, LRRC5, LRRC15 antibodies, or any combination thereof, or fragments thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any of the foregoing, or any combination thereof.

[0072] In one embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded extracellular ROR1 and / or MSLN antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to ROR1 and / or MSLN.

[0073] In another embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded extracellular ROR1 and / or MSLN antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to ROR1 and / or MSLN.

[0074] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the encoded extracellular ROR1 and / or MSLN antigen-binding domain comprises an ScFv.

[0075] In yet another broad aspect, one or more of the above-identified novel boost chimeric antigen receptors (CARs) provided above with respect to SEQ ID NOs: 151-256 may comprise single-targeting, tandem-targeting, or multi-targeting CAR constructs (including those in DuoCAR format), or any combination thereof.

[0076] For each of the various aspects and embodiments of single, tandem, multitargeting, DuoCAR, CAR constructs (with or without one or more booster elements) specifically contemplated herein, the nucleotide sequence encoding the functional CAR (with or without one or more booster elements) comprises the nucleotide sequence of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 245, 247, 249, 251, 253, 255, or any combination thereof.

[0077] For each of the various aspects and embodiments of single, tandem, multitargeting, DuoCAR, CAR constructs (with or without one or more booster elements) specifically contemplated herein, each vector encodes a functional CAR (with or without one or more booster elements) comprising the amino acid sequence of SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, 256, or any combination thereof.

[0078] For each of the various aspects and embodiments, an isolated polynucleotide is provided encoding a fully human anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149.

[0079] For each of the various aspects and embodiments, provided herein are novel single, tandem, DuoCAR, or multitargeting CAR molecules (with or without one or more booster elements) comprising at least one extracellular antigen-binding domain comprising an anti-ROR1 and / or anti-MSLN antigen-binding domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain.

[0080] For each of the various aspects and embodiments, provided herein are novel single, tandem, DuoCAR, or multitargeting CAR molecules (with or without one or more booster elements) comprising at least one extracellular antigen-binding domain comprising an anti-ROR1 and / or anti-MSLN antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain.

[0081] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149.

[0082] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 and / or anti-MSLN anti-ROR1 and / or anti-MSLN and / or anti-FolR1, and / or anti-HER2 / ERBB2, and / or anti-GPC3, and / or anti-FGFR4, and / or anti-GD2 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150.

[0083] In one aspect, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeted chimeric antigen receptor (CAR) (with or without one or more boosting elements) comprising, from N-terminus to C-terminus, at least one ROR1 and / or MSLN antigen-binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149; at least one transmembrane domain, and at least one intracellular signaling domain.

[0084] In one aspect, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeted chimeric antigen receptor (CAR) (with or without one or more boosting elements) comprising, from N-terminus to C-terminus, at least one ROR1 and / or MSLN antigen-binding domain encoded by a nucleotide sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one transmembrane domain, and at least one intracellular signaling domain.

[0085] In one embodiment, the targeting domain of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and contains an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149, connected to an additional binding tag or epitope, while the effector cell expression component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is connected to a tag or epitope expressed on a soluble single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) module. contains a binding domain specifically directed to bind to an epitope, e.g., specific binding to the cell-binding component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) on the soluble component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) forms a fully functional single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) structure.

[0086] In another embodiment, the targeting domain of the single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and contains an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149, and an additional ScFv, while the effector cell expression component of the single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is an additional ScF expressed on a soluble single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) module. Specific binding of a tag or epitope specifically reactive with v, e.g., on the soluble component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), to the cell-binding component of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) forms a fully functional single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) structure.

[0087] In yet another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the encoded single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) extracellular ROR1 and / or MSLN antigen-binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to ROR1 and / or MSLN.

[0088] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 and / or MSLN antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0089] In another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the encoded ROR1 and / or MSLN extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0090] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) comprising at least one ROR1 and / or MSLN antigen binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 143, 145, 147, and 149, wherein the single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) encodes at least one of CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, GD2, GD3, GM2, Ley, polysialic acid, fucosyl GMl, GM3, Tn, STn, sLe (animal), GloboH, CD5, CD7, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD 38, CD123, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, CD276 / B7-H3, B7-H4, B7-DC, HLA-DR carcinoembryonic antigen (CEA), TAG-72, EpCAM, folate binding protein, folate receptor Folate receptor alpha (FOLR1), folate receptor beta (FOLR2), A33, G250, prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, CD44v6, epidermal growth factor, pl85, IL-2 receptor, interleukin-1 receptor accessory protein (IL1RAP), EGFRvIII(de2-7), fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, ανβ3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, non-mutant p53, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, FGFR1, FGFR2, FGFR3, FGFR4, GPC1, GPC2, GPC3, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE A1, MAGE A3, CYP1B1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, TRAIL1, MUC1, MUC16 / CA125, MAGE A4, MAGEC2, GAGE, EGFR, EGFR1, EGFR2 / Her2, CMET, HER3, CA6, NAPI2B, TROP2, TEM1, TEM7, TEM8, FAP, LAP, CLDN3, CLDN6, CLDN8, CLDN16, CLDN18.2, RON, LY6E, DLL3, PTK7, UPK1B, STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorfl86, LIV1, ROR1, ROR2, Fos-related antigen 1, VEGFR1, endoglin, CD90, CD326, CD70, SSEA4, CD318, CLA, TSPAN8, GPRC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, S and further encoding an extracellular antigen-binding domain that targets an antigen including, but not limited to, LAMF7 / CS1, CD147, DPPA5, GRP78, CD66c, VISTA, LRRC5, LRRC15, or any combination thereof, or a fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any of the foregoing, or any combination thereof.

[0091] In certain embodiments, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), further comprising an encoded extracellular antigen binding domain selected from the group consisting of an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-BCMA ScFv antigen binding domain, an anti-CD5 ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PSMA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ... The antigen-binding domain of the ScFv includes an anti-GD-2 ScFv, an anti-NY-ESO-1 TCR ScFv, an anti-MAGE A3 TCR ScFv, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0092] In one aspect, the single, tandem, DuoCAR, or multi-targeting CAR provided herein (with or without one or more boosting elements) further comprises a linker or spacer domain.

[0093] In one embodiment, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the extracellular ROR1 and / or MSLN antigen-binding domain, the intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

[0094] In one embodiment, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded linker domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19, or CD28 and is linked to a transmembrane domain.

[0095] In another embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, Fc epsilon R, or a combination thereof.

[0096] In yet another embodiment, an isolated nucleic acid molecule encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0097] In one embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded intracellular signaling domain is positioned C-terminal to the CD3 zeta intracellular domain.

[0098] In another embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0099] In another embodiment, an immunotherapeutic composition is provided, wherein at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), PD-1, GITR, CTLA-4, or any combination thereof.

[0100] In one embodiment, an isolated nucleic acid molecule is provided encoding a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) further containing a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43.

[0101] In yet another embodiment, an isolated nucleic acid molecule is provided that encodes a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements), wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

[0102] In one aspect, a single, tandem, DuoCAR, or multitargeting chimeric antigen receptor (CAR) (with or without one or more boosting elements) is provided that comprises, from N-terminus to C-terminus, at least one ROR1 and / or MSLN antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0103] In one embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular ROR1 and / or MSLN antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to the antigen, or at least one heavy-chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0104] In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0105] In some embodiments, single, tandem, DuoCAR, or multitargeting CARs (with or without one or more boosting elements) are provided, which single, tandem, DuoCAR, or multitargeting CARs (with or without one or more boosting elements) can further comprise one or more of the following: anti-CD19, anti-CD20, anti-CD22, anti-CD33, anti-CD38, anti-CD123 (IL3RA), anti-CD138, anti-GPC2, anti-GPC3, anti-FGFR4, anti-c-Met, anti-PSMA, anti-glycolipid F77, anti-EGFRvIII, anti-GD-2, anti-NY-ESO-1 TCR, anti-MAGE A3 TCR, anti-GD2, anti-GD3, anti-GM2, anti-Ley, anti-polysialic acid, anti-fucosyl GMl, anti-GM3, anti-Tn, anti-STn, anti-sLe (animal), anti-GloboH, anti-CD5, anti-CD7, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CD37, anti-CD30, anti-CD 33, anti-CD38, anti-CD123, anti-CD45, anti-CAMPATH-1, anti-BCMA, anti-CS-1, anti-PD-L1, anti-CD276 / B7-H3, anti-B7-H4, anti-B7-DC, anti-HLA-DR carcinoembryonic antigen (CEA), anti-TAG-72, anti-EpCAM, anti-folate binding protein Antibodies against fibroblasts, anti-folate receptor alpha (FOLR1), anti-folate receptor beta (FOLR2), anti-A33, anti-G250, anti-prostate-specific membrane antigen (PSMA), anti-ferritin, anti-CA-125, anti-CA19-9, anti-CD44v6, anti-epidermal growth factor, anti-pl85, anti-IL-2 receptor, anti-interleukin-1 receptor accessory protein (IL1RAP), anti-EGFRvIII (de2-7), anti-fibroblast activation protein, anti-tenascin, anti-metalloproteinase, anti-endosialin, anti-vascular endothelial growth factor, anti-ανβ3, anti-WT1, anti-LMP2, anti-HPV E6, anti-HPV E7, anti-Her-2 / neu, anti-p53 non-mutant, anti-NY-ESO-1, and anti-MelanA / MART1. Anti-Ras mutation, anti-gp100, anti-GPRC5D, anti-FGFR1, anti-FGFR2, anti-FGFR3, anti-FGFR4, anti-GPC1, anti-GPC2, anti-GPC3, anti-p53 mutation, anti-PR1, anti-bcr-abl, anti-チロシナーゼ, Anti-September, anti-PSA, anti-hTERT, anti-sarcoma ブレークポイントfusion polypeptide, anti-EphA2, anti-PAP, Anti-ML-IAP, anti-AFP, anti-ERG, anti-NA17, anti-PAX3, anti-ALK, anti-アンドロゲン receptor, anti-サイクリンB l, anti-MYCN, anti-RhoC, anti-TRP-2, anti-Mesoterin, anti-PSCA, anti-MAGE A1, anti-MAGE A3, anti-CYP1B 1, anti-PLAV1, anti-BORIS, anti-ETV6-AML, anti-NY-BR-1, anti-RGS5, anti-SART3, anti-carbonyl dehydratase IX, anti-PAX5, anti-OY-TES 1, anti-sperm tanpak 17, anti-LCK, anti-HMWMAA, anti-AKAP-4, anti-SSX2, anti-XAGE 1, anti-B7H3, anti-Legumain, anti-Tie 3, anti-PAGE4, anti-VEGFR2, anti-MAD-CT-1, anti-PDGFR-B, anti-MAD-CT-2, anti-TRAIL1, anti-MUC1, anti-MUC16 / CA125, anti-MAGE A4, anti-MAGEAnti-C2, anti-GAGE, anti-EGFR, anti-EGFR1, anti-EGFR2 / Her2, anti-CMET, anti-HER3, anti-CA6, anti-NAPI2B, anti-TROP2, anti-TEM1, anti-TEM7, anti-TEM8, anti-FAP, Anti-LAP, anti-CLDN6, anti-CLDN8, anti-CLDN16, anti-CLDN18.2, anti-RON, anti-LY6E, anti-DLL3, anti-PTK7, anti-UPK1B, anti-STRA6, anti-TMPRSS3, anti-TMRRSS 4, anti-TMEM238, anti-Clorfl86, anti-LIV1, anti-ROR1, anti-ROR2, anti-Fos-related antigen 1, anti-VEGFR1, anti-endoglin, anti-CD90, anti-CD326, anti-CD70, anti-SSEA4 , anti-CD318, anti-CLA, anti-TSPAN8, GPRC5D, EpCAM, Thy1, IL13Ra2, BDCA1, BDCA2, BDCA3, GD2, PSMA, FAP, CLL1, SLAMF7 / The antibody or fragment thereof encodes an extracellular antigen-binding domain comprising a CS1, anti-CD147, anti-DPPA5, anti-GRP78, anti-CD66c, anti-VISTA, anti-LRRC5, anti-LRRC15 antibody, or any combination thereof, or a fragment thereof, wherein the antibody or fragment thereof comprises a Fab fragment, a F(ab')2 fragment, an Fv fragment, a nanobody, a VHH, a ligand peptide, and a single-chain Fv (ScFv), or a fragment of any of the foregoing, or a molecule that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any of the foregoing, or any combination thereof, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or a fragment selected from the group consisting of:

[0106] In one embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular antigen binding domain is selected from the group consisting of an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, an anti-GD-2 ScFv antigen binding domain, an anti-NY-ESo-1 TCR Further comprising an ScFv antigen binding domain, an anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0107] In one embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular antigen binding domain is alternatively an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123(IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipidF77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, an anti-GD-2 ScFv antigen binding domain, an anti-NY-ESo-1 TCR The ScFv antigen-binding domain comprises an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0108] In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular antigen-binding domain is an immunoglobulin variable heavy chain only (VH) anti-CD19 antigen-binding domain, anti-CD20 VH antigen-binding domain, anti-CD22 VH antigen-binding domain, anti-CD33 VH antigen-binding domain, anti-CD38 VH antigen-binding domain, anti-CD123 (IL3RA) VH antigen-binding domain, anti-CD138 VH antigen-binding domain, anti-GPC2 VH antigen-binding domain, anti-GPC3 VH antigen-binding domain, anti-FGFR4 VH antigen-binding domain, anti-c-Met VH antigen-binding domain, anti-PMSA VH antigen-binding domain, anti-glycolipid F77 VH antigen-binding domain, anti-EGFRvIII VH antigen-binding domain, anti-GD-2 VH antigen-binding domain, anti-NY-ESO-1 TCR VH antigen-binding domain, anti-MAGE A3 TCR ... It further comprises a VH antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0109] In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular antigen-binding domain is alternatively an immunoglobulin variable heavy chain only (VH) anti-CD19 antigen-binding domain, anti-CD20 VH antigen-binding domain, anti-CD22 VH antigen-binding domain, anti-CD33 VH antigen-binding domain, anti-CD38 VH antigen-binding domain, anti-CD123 (IL3RA) VH antigen-binding domain, anti-CD138 VH antigen-binding domain, anti-GPC2 VH antigen-binding domain, anti-GPC3 VH antigen-binding domain, anti-FGFR4 VH antigen-binding domain, anti-c-Met VH antigen-binding domain, anti-PMSA VH antigen-binding domain, anti-glycolipid F77 VH antigen-binding domain, anti-EGFRvIII VH antigen-binding domain, anti-GD-2 VH antigen-binding domain, anti-NY-ESO-1 TCR VH antigen-binding domain, anti-MAGE A3 TCR ... VH antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0110] In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular antigen-binding domain is an anti-CD19 P antigen-binding domain, an anti-CD20 P antigen-binding domain, an anti-CD22 P antigen-binding domain, an anti-CD33 P antigen-binding domain, an anti-CD38 P antigen-binding domain, an anti-CD123 (IL3RA) P antigen-binding domain, an anti-CD138 P antigen-binding domain, an anti-BCMA (CD269) P antigen-binding domain, an anti-GPC2 P antigen-binding domain, an anti-GPC3 P antigen-binding domain, an anti-FGFR4 P antigen-binding domain, an anti-c-Met P antigen-binding domain, an anti-PMSA P antigen-binding domain, an anti-glycolipid F77 P antigen-binding domain, an anti-EGFRvIII P antigen-binding domain, an anti-GD-2 P antigen-binding domain, an anti-NY-ESO-1 TCR P antigen-binding domain, an anti-MAGE A3 TCR P antigen-binding domain, an anti-CD19 P antigen-binding domain, an anti-CD20 P antigen-binding domain, an anti-CD22 P antigen-binding domain, an anti-CD33 P antigen-binding domain, an anti-CD38 P antigen-binding domain, an anti-CD123 (IL3RA) P antigen-binding domain, an anti-CD138 P antigen-binding domain, an anti-BCMA (CD269) P antigen-binding domain, an anti-GPC2 P antigen-binding domain, an anti-GPC3 P antigen-binding domain, an anti-FGFR4 P antigen-binding domain, an anti-c-Met P antigen-binding domain, an anti-PMSA P antigen-binding domain, an anti-glycolipid F77 P antigen-binding domain, an anti-EGFRvIII P antigen-binding domain, an anti-GD-2 P antigen-binding domain, an anti-NY-ESO-1 TCR P antigen The CAR further comprises a protein or peptide (P) sequence capable of specifically binding to the target antigen, which may be derived from a natural or synthetic sequence comprising a P antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof. In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0111] In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the extracellular antigen-binding domain is alternatively an anti-CD19 P antigen-binding domain, an anti-CD20 P antigen-binding domain, an anti-CD22 P antigen-binding domain, an anti-CD33 P antigen-binding domain, an anti-CD38 P antigen-binding domain, an anti-CD123 (IL3RA) P antigen-binding domain, an anti-CD138 P antigen-binding domain, an anti-BCMA (CD269) P antigen-binding domain, an anti-GPC2 P antigen-binding domain, an anti-GPC3 P antigen-binding domain, an anti-FGFR4 P antigen-binding domain, an anti-c-Met P antigen-binding domain, an anti-PMSA P antigen-binding domain, an anti-glycolipid F77 P antigen-binding domain, an anti-EGFRvIII P antigen-binding domain, an anti-GD-2 P antigen-binding domain, an anti-NY-ESO-1 TCR P antigen-binding domain, an anti-MAGE A3 TCR P antigen-binding domain, an anti-CD19 P antigen-binding domain, an anti-CD20 P antigen-binding domain, an anti-CD22 P antigen-binding domain, an anti-CD33 P antigen-binding domain, an anti-CD38 P antigen-binding domain, an anti-CD123 (IL3RA) P antigen-binding domain, an anti-CD138 P antigen-binding domain, an anti-BCMA (CD269) P antigen-binding domain, an anti-GPC2 P antigen-binding domain, an anti-GPC3 P antigen-binding domain, an anti-FGFR4 P antigen-binding domain, an anti-c-Met P antigen-binding domain, an anti-PMSA P antigen-binding domain, an anti-glycolipid F77 P antigen-binding domain, an anti-EGFRvIII P antigen-binding domain, an anti-GD-2 P antigen-binding domain, an anti-NY-ESO-1 TCR The P antigen-binding domain comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof. In another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain. In yet another embodiment, a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0112] In one embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 151. In one embodiment, the nucleic acid sequence encodes a boosted CAR comprising the amino acid sequence of SEQ ID NO: 152.

[0113] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 153. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 154.

[0114] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 155. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 156.

[0115] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 157. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 158.

[0116] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 159. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 160.

[0117] In another embodiment, the nucleic acid sequence encoding the boosted CAR comprises the nucleic acid sequence of SEQ ID NO: 161. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 162.

[0118] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 163. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 164.

[0119] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 165. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 166.

[0120] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 167. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 168.

[0121] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 179. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 180. In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 181. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 182.

[0122] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 183. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 184.

[0123] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 185. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 186.

[0124] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 187. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 188. In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 189. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 190.

[0125] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 191. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 192.

[0126] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 193. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 194.

[0127] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 195. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 196.

[0128] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 197. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 198.

[0129] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 226. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 225.

[0130] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 228. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 227.

[0131] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 230. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 229.

[0132] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 232. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 231.

[0133] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 234. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 233.

[0134] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 236. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 235.

[0135] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 238. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 237.

[0136] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 240. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 239.

[0137] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 242. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 241.

[0138] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 244. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 243.

[0139] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 245. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 246.

[0140] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 247. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 248.

[0141] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 249. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 250.

[0142] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 251. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 252.

[0143] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 253. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 254.

[0144] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 255. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 256.

[0145] In one embodiment, the single, tandem, DuoCAR, or multitargeting CARs disclosed herein (with or without one or more boosting elements) are modified to express or contain a detectable marker for use in diagnosing, monitoring, and / or predicting treatment outcomes, such as progression-free survival, of cancer patients, or for monitoring the progress of such treatment.

[0146] In one embodiment, the nucleic acid molecule encoding the disclosed single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) can be contained in a vector, such as a viral vector. The vector can be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0147] In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0148] In yet another embodiment, vectors expressing single, tandem, DuoCAR, or multitargeted CARs (with or without one or more boosting elements) can be further modified to include one or more activating elements to control the expression of single, tandem, DuoCAR, or multitargeted CAR T cells (with or without one or more boosting elements) or to eliminate single, tandem, DuoCAR, or multitargeted CAR T cells (with or without one or more boosting elements) by a suicide switch. Suicide switches may include, for example, signaling cascades that induce apoptosis or drugs that induce cell death. In a preferred embodiment, vectors expressing single, tandem, DuoCAR, or multitargeted CARs (with or without one or more boosting elements) can be further modified to express enzymes such as thymidine kinase (TK) or cytosine deaminase (CD).

[0149] In another aspect, a host cell is also provided that comprises a nucleic acid molecule encoding a single, tandem, DuoCAR, or multi-targeting CAR (with or without one or more boosting elements). In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.

[0150] In yet another aspect, a pharmaceutical composition is provided comprising an antitumor-effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multitargeting chimeric antigen receptor (CAR) construct, wherein the CAR comprises at least one extracellular antigen-binding domain comprising an MSLN and / or ROR1 antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain, and the T cells are T cells of a human with cancer. The cancer is, inter alia, a blood cancer such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0151] In yet another aspect, a pharmaceutical composition comprises an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeted, boosted chimeric antigen receptor (CAR) construct, wherein the boosted CAR comprises at least one extracellular antigen binding domain comprising an MSLN and / or ROR1 antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain, followed by one or more 2A sequences, in combination with one or more arming molecules, one or more cellular signaling domains.

[0010] A pharmaceutical composition is provided, comprising: an antitumor-effective amount of a population of human T cells comprising in-frame extracellular matrix enzymes, one or more chemokine receptors, one or more stromal targeting molecules, one or more tumor microenvironment (TME) digestion elements, one or more switch tag elements, one or more chemoattractant receptors, one or more chemotactic molecule-secreted factors, one or more switches, and / or one or more cytokines, or any combination thereof; and a pharmaceutically acceptable excipient, wherein the boosted CAR is used to genetically modify one or more human T cell lymphocyte populations, and the T cells are T cells of a human with cancer. The cancer may be, inter alia, a blood cancer such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0152] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR (with or without one or more booster elements) contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0153] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer comprises adult cancers, including oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or combinations thereof.

[0154] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective amount of a population of human T cells from a human with cancer, wherein the cancer is a refractory cancer that does not respond to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, multiple myeloma (MM) with minimal residual disease (MRD), smoldering multiple myeloma (SMM), monoclonal gammopathy of undetermined significance (MGUS), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL) including mantle cell lymphoma (MCL), and Hodgkin's lymphoma (HL). Included are adult and pediatric hematologic malignancies, including chronic myeloid leukemia (CML), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other blood cancers and solid tumors, or any combination thereof.

[0155] In another aspect, a method of generating T cells containing single, tandem, DuoCAR, or multi-targeting CAR constructs (hereinafter "CAR-T cells") (with or without one or more booster elements) is provided. The method includes transducing T cells with a vector or nucleic acid molecule encoding the disclosed CARs that specifically bind to MSLN and / or ROR1, thereby generating CAR-T cells.

[0156] In yet another aspect, a method of generating a population of RNA-engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed single, tandem, DuoCAR, or multi-targeted CAR (with or without one or more booster elements) into cells of a subject, thereby generating single, tandem, DuoCAR, or multi-targeted CAR cells (with or without one or more booster elements).

[0157] In yet another aspect, a method is provided for diagnosing a disease, disorder, or condition associated with expression of MLSN and / or ROR1 on a cell, the method comprising: a) contacting the cell with a human anti-MLSN and / or ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; and b) detecting the presence of MSLN and / or ROR1, wherein the presence of MSLN and / or ROR1 diagnoses the disease, disorder, or condition associated with expression of MSLN and / or ROR1.

[0158] In one embodiment, the disease, disorder, or condition associated with expression of MSLN and / or ROR1 is cancer, including hematopoietic cancer, myelodysplastic syndrome pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myelogenous leukemia), adult B-cell malignancies including non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-lineage ALL (acute lymphocytic leukemia)), multiple myeloma lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0159] In another embodiment, a method for diagnosing, prognosing, or determining the risk of an MSLN- and / or ROR1-associated disease in a mammal is provided, the method comprising: a) detecting expression of MSLN and / or ROR1 in a sample from a mammal, the method comprising: a) contacting the sample with a human anti-MSLN and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; and b) detecting the presence of MSLN and / or ROR1, wherein the presence of MSLN and / or ROR1 diagnoses an MSLN- and / or ROR1-associated disease in the mammal.

[0160] In another embodiment, a method of inhibiting MSLN and / or ROR1-dependent T cell inhibition is provided, the method comprising contacting a cell with a human anti-MSLN and / or ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150. In one embodiment, the cell is selected from the group consisting of an MSLN- and / or ROR1-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0161] In another embodiment, a method is provided for blocking T cell inhibition mediated by MSLN- and / or ROR1-expressing cells and altering the tumor microenvironment to inhibit tumor growth in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising an isolated anti-MSLN and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150. In one embodiment, the cell is selected from the group consisting of an MSLN- and / or ROR1-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0162] In another embodiment, a method is provided for inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising an isolated anti-MSLN and / or anti-ROR1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150. In one embodiment, the antibody or fragment thereof inhibits interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a tumor cell expressing MSLN and / or ROR1, a tumor-associated macrophage, and any combination thereof.

[0163] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed single, tandem, or multi-targeting CARs (with or without one or more booster elements).

[0164] In another embodiment, a method of treating or preventing cancer in a mammal is provided, the method comprising administering to the mammal one or more of the disclosed single, tandem, or multitargeting CARs (with or without one or more booster elements) in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed single, tandem, or multitargeting CARs (with or without one or more booster elements) that specifically bind to MSLN and / or ROR1 and / or one or more of the aforementioned antigens under conditions sufficient to form an immune complex of the antigen-binding domain on the single, tandem, or multitargeting CAR (with or without one or more booster elements) and the extracellular domain of MSLN and / or ROR1 and / or one or more of the aforementioned antigens in the subject.

[0165] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeting chimeric antigen receptor (CAR) (with or without one or more booster elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more booster elements) comprises at least one MSLN and / or ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NOs: 144, 146, 148, and 150, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with cancer.

[0166] In yet another embodiment, there is provided a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeting chimeric antigen receptor (CAR) (with or without one or more booster elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more booster elements) comprises at least one MSLN and / or ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NOs: 144, 146, 148, and 150, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with cancer. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0167] In yet another embodiment, a method is provided for treating a mammal having an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multitargeting chimeric antigen receptor (CAR) (with or without one or more booster elements), wherein the single, tandem, or multitargeting CAR (with or without one or more booster elements) comprises at least one extracellular MSLN and / or ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NOs: 144, 146, 148, and 150, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject having the autoimmune, alloimmune, or autoaggressive disease, disorder, or condition.

[0168] In yet another embodiment, a method for treating an autoimmune, alloimmune, or autoaggressive disease in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multitargeting chimeric antigen receptor (CAR) (with or without one or more booster elements), wherein the single, tandem, or multitargeting CAR (with or without one or more booster elements) comprises at least one MSLN and / or ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NOs: 144, 146, 148, and 150, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0169] In yet another embodiment, a method is provided for treating a mammal having an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeting chimeric antigen receptor (CAR) (with or without one or more booster elements), the single, tandem, or multi-targeting CAR (with or without one or more booster elements) being selected from the group consisting of SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 23 , 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the T cell is a T cell of a subject with an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition.

[0170] In yet another embodiment, there is provided a method for treating an autoimmune disease, an alloimmune disease, or an autoaggressive disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a single, tandem, or multi-targeting chimeric antigen receptor (CAR) (with or without one or more booster elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more booster elements) is selected from the group consisting of SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 2 at least one MSLN and / or ROR1 antigen-binding domain comprising the amino acid sequence of: 4, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256, or any combination thereof; at least one linker or spacer domain; at least one transmembrane domain; and at least one intracellular signaling domain, wherein the T cell is a T cell of a subject with an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0171] For each of the various aspects and embodiments of the methods for treating an autoimmune, alloimmune, or autoaggressive disease in a subject in need thereof, the nucleotide sequences encoding any of the single, tandem, multitargeted, DuoCARs, CAR constructs (with or without one or more booster elements) specifically contemplated above and / or below, the aforementioned functional CARs (with or without one or more booster elements) referenced above and / or below, can be used to treat an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition.

[0172] For each of the various aspects and embodiments of the methods for treating an autoimmune, alloimmune, or autoaggressive disease in a subject in need thereof, the amino acid sequences encoding the single, tandem, multitargeted, DuoCARs, CAR constructs (with or without one or more booster elements) specifically contemplated above and / or below, any of the aforementioned functional CARs (with or without one or more booster elements) referenced above and / or below, can be used to treat an autoimmune, alloimmune, or autoaggressive disease, disorder, or condition.

[0173] For various aspects and embodiments of the methods for treating an autoimmune disease, alloimmune disease, or autoaggressive disease described herein, illustrative non-limiting examples of autoimmune diseases include chronic graft-versus-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, Goodpasture's disease, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, pemphigus vulgaris, Graves' disease, autoimmune hemolytic anemia, hemophilia A, primary Sjogren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, Evan's syndrome, IgM-mediated neuropathy, thyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired hemoglobinemia, and the like. Examples of alloimmune diseases include ductal edema, chronic urticaria, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasia, while illustrative, non-limiting examples of alloimmune diseases include allosensitization (see, e.g., Blazar et al., 2015, Am. J. Transplant., Vol. 15(4):931-41), or sensitization to foreign antigens such as may occur with hematopoietic or solid organ transplantation, xenosensitization from blood transfusions, pregnancy with fetal allosensitization, alloimmune thrombocytopenia of the newborn, hemolytic disease of the newborn, enzyme or protein replacement therapy, blood products, and replacement of inherited or acquired deficiency disorders treated with gene therapy.

[0034] Antigen-binding domains specific for ligands on B cells, plasma cells, or plasmablasts are useful in the methods of treating autoimmune, alloimmune, or autoaggressive diseases described herein. For example, the CAR construct may contain, but is not limited to, an antigen binding domain specific to CD19, CD20, CD22, CD138, BCMA, CD319, CD10, CD24, CD27, CD38, or CD45R. In addition, the CAR construct may contain, but is not limited to, an antigen binding domain specific to an autoimmune-specific antigen. Autoimmune-specific antigens include, for example, antigens that cause systemic lupus erythematosus (SLE), Graves' disease, celiac disease, type 1 diabetes mellitus, rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, polymyositis (PM), dermatomyositis (DM), mucous membrane pemphigus vulgaris, and myasthenia gravis.See, e.g., Ellebrecht et al., 2016, Science, 353:179-84.

[0174] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a single, tandem, or multi-targeting CAR (with or without one or more booster elements), wherein the single, tandem, or multi-targeting CAR (with or without one or more booster elements) comprises at least one MSLN and / or ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 144, 146, 148, and 150, or any combination thereof; at least one transmembrane domain; and at least one intracellular signaling domain, wherein the persistent population of engineered T cells, or a progeny population of T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0175] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0176] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens that can be treated or prevented or ameliorated using one or more of the single, tandem, or multi-targeting CARs (with or without one or more booster elements) disclosed herein is In yet another aspect, there is provided a kit for generating the chimeric antigen receptor T cells described above, or for preventing, treating, or ameliorating any of the cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in a subject described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, and instructions for use of the kit.

[0177] In one aspect of the present invention, immunotherapy compositions are provided comprising single, tandem, DuoCAR, or multitargeted CARs (with or without one or more booster elements) that can be used to transduce autologous lymphocytes to generate active patient-specific anti-tumor lymphocyte cell populations that can be directly infused back into the patient to promote in vivo expansion, persistence of patient-specific anti-tumor T cells that result in tumor stabilization, cancer reduction, elimination, remission, or prevention or amelioration of cancer recurrence, or a combination thereof, in a patient-specific manner.

[0178] In yet another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen-binding domain comprising an anti-ROR1 and / or anti-MSLN antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 146, 148, and 150; at least one linker domain; at least one transmembrane domain; and at least one intracellular signaling domain; and one or more arming molecules (e.g., TGFβRIIdn, truncated PD-1 (decoy), PD-1 dominant negative (PD-1dn), synthetic PD-1 activating receptor, truncated CTLA-4, truncated Tim-3, truncated TIGIT, TIGIT neutralizing antibody, TIGIT intrabody, TIGITshRNA), one or more extracellular matrix enzymes (ECM), one or more chemokine receptors (CXCL8, CCL2), one or more stromal targeting molecules (FAP, LRRC15, CD276 / B7-H3, TEM7, TEM8, TEM1), one or more TME digestion elements (heparanase (HPSE), MMPs (MMP-1, MMP-2, MMP-9, MMP-12, MMP-13) and hyaluronidase 1, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4, PH-20, and hyaluronoglucosaminidase pseudogene 1 (HYALP1), tissue inhibitor of metalloproteinases (TIMPs) (TIMP-1, TIMP-2, TIMP-3, TIMP-4), hyaluronidase), one or more switches (tag, kill switch, on switch, off switch, adaptor switch, Pharmaceutical compositions are provided that comprise at least one boosting element comprising a truncated EGF receptor, truncated CD19, truncated CD20, a CD20 mimotope, truncated CD34, truncated LNGF receptor), a chimeric costimulatory receptor (CCR), and / or one or more cytokines (membrane-bound or soluble IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ (each of the foregoing with or without an FC (antibody fragment crystallizable) element)), or a combination of a membrane-bound receptor and a tethered cytokine ligand (mbIL15, mbIL7, mbIL-21), a natural system-derived ligand (TLR ligand, LPS, bacterial product), or any combination thereof, and the T cell is a T cell of a human with cancer. The cancer is, inter alia, a blood cancer such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0179] In one embodiment, a pharmaceutical composition is provided in which at least one transmembrane domain of a single, tandem, DuoCAR, or multitargeting CAR (with or without one or more boosting elements) contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0180] It will be understood that single, tandem, DuoCAR, or multitargeting CARs (with or without one or more booster elements), host cells, nucleic acids, and methods are useful beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

[0181] In one aspect of the above-identified invention, the DuoCAR (with or without one or more boosters) disclosed herein comprises at least two vectors, each vector encoding a functional CAR (with or without one or more boosters), whereby the combination of the vectors results in the expression of two or more non-identical binding domains, wherein the binding domains encoded by each vector are covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0182] In certain embodiments of the boosted CAR of the present invention, an immunotherapy composition is provided, comprising one or more isolated nucleic acid molecules encoding at least two vectors, each vector encoding a functional DuoCAR (with or without one or more booster elements), whereby the combination of vectors results in the expression of two or more non-identical binding domains, each vector-encoded binding domain being covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and which can be used to transduce autologous lymphocytes to generate an active patient-specific anti-tumor lymphocyte cell population that can be directly infused back into the patient to promote in vivo expansion, persistence of patient-specific anti-tumor T cells, or a combination thereof, in a patient-specific manner, resulting in tumor stabilization, cancer reduction, elimination, remission, or prevention or amelioration of cancer recurrence. Also provided herein are novel adoptive immunotherapy compositions comprising such two or more vector-transduced lymphocytes, and methods for their use in patient-specific combination immunotherapy that can be used to treat cancer and other diseases and conditions.

[0183] Thus, in one aspect, provided herein are lentiviral vectors expressing Duo chimeric antigen receptors (DuoCARs) (with or without one or more booster elements), and nucleic acid molecules encoding the lentiviral vectors expressing DuoCARs (with or without one or more booster elements).Methods of using the disclosed lentiviral vectors expressing DuoCARs (with or without one or more booster elements), host cells, and nucleic acid molecules are also provided, for example, to treat cancer in a subject.

[0184] In one aspect, an immunotherapeutic composition is provided, the immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least two vectors (DuoCAR) (with or without one or more booster elements), each vector encoding a functional CAR (with or without one or more booster elements), wherein at least one binding domain in one of the vectors is non-identical, such that the combination of the vectors results in expression of two or more non-identical binding domains, and wherein the binding domain encoded by each vector is covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0185] In one embodiment, an immunotherapeutic composition is provided, the immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least three vectors (TrioCARs) (with or without one or more booster elements), each vector encoding a functional CAR (with or without one or more booster elements), whereby the combination of vectors results in expression of two or more non-identical binding domains, each vector-encoded binding domain covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0186] In one embodiment, an immunotherapeutic composition is provided, the immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least four vectors (QuatroCARs) (with or without one or more booster elements), each vector encoding a functional CAR (with or without one or more booster elements), the combination of vectors resulting in expression of two or more non-identical binding domains, each vector-encoded binding domain covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs.

[0187] In yet another embodiment, an immunotherapeutic composition is provided, the immunotherapeutic composition comprising one or more isolated nucleic acid molecules encoding at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 vectors (e.g., "nCARs") (with or without one or more booster elements), each vector encoding a functional CAR (with or without one or more booster elements), whereby the combination of the vectors results in expression of two or more non-identical binding domains, each vector-encoded binding domain being covalently linked to a transmembrane domain and one or more non-identical intracellular signaling motifs, and each unique member of the nCAR set, when assembled into a CAR product, constitutes a unique CAR composition, referred to herein as an "nCAR" (with or without one or more booster elements) (e.g., DuoCAR, TrioCAR, QuatroCAR, PentaCAR, HexaCAR, HeptaCAR, OctaCAR, NonaCAR, and DecaCAR, etc.).

[0188] In another embodiment, DuoCAR (with or without one or more boosters) is used to enhance the immune response against tumors mediated by therapeutic T cell populations. The immune response is enhanced in multiple ways.

[0189] First, DuoCARs enable multitargeting of tumor cells, reducing the risk of tumor antigen escape and allowing for efficient elimination of antigen-heterogeneous tumors. This feature is particularly important when targeting solid tumors, which often exhibit antigenic heterogeneity and antigen deficiency. Table 1 below illustrates a CAR with the dual targeting capability of the solid tumor antigens mesothelin and ROR1.

[0190] Additionally, the DuoCAR format allows for the introduction of multiple costimulatory domains into the CAR structure, providing a stronger overall stimulation for CAR T cell effector function, differentiation, memory formation, and persistence. For example, the same CAR T cells can benefit from CD28 stimulation, necessary for potent CAR T cell activation, expansion, and cytokine production, as well as 4-1BB stimulation to extend CAR T cell survival and persistence in patients. Each DuoCAR chain can be a second- or third-generation DuoCAR and incorporate one or two costimulatory domains. By providing a third T cell activation sequence on a separate vector CAR construct (with or without one or more boosters), we can recapture the benefits of expressing two or more targeting domains, improved costimulation, and booster payloads, without the disadvantage of reduced CAR expression on the T cell surface at the CAR% level.

[0191] In a second aspect, the DuoCAR of the present invention (with or without one or more boosters) can target cell types other than the tumor that mediate immunosuppressive effects. For example, if immunosuppressive cells expressing one of the target antigens are present in the tumor lesion and also inhibit anti-tumor immunity by producing IL-10 or other mediators, a second advantage to using a tumor-specific T cell population expressing DuoCAR (with or without one or more boosters) is that the immunosuppressive cell population is also removed.

[0192] For example, if immunosuppressive B cells are present within solid tumor lesions, they can be eliminated by using B cell-specific DuoCARs (e.g., CD19-specific DuoCARs with or without one or more boosters). If immunosuppressive fibroblast-like cells are present, they can be eliminated by stroma-specific DuoCARs (with or without one or more boosters) (e.g., by targeting fibroblast activation protein alpha (FAP)). If malformed blood vessels are the cause of the lack of an effective immune response, DuoCARs specific to these types of blood vessels or lymphatic vessels (e.g., anti-VEGFR) may also impr...

Claims

1. 1. An isolated nucleic acid molecule encoding a boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR), comprising at least one extracellular antigen binding domain comprising a ROR1, MSLN, FOLR1, and / or CD276 / B7-H3 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain operably linked to one or more booster elements, wherein the boosted single, tandem, multitargeted, or DuoCAR 1. An isolated nucleic acid molecule, wherein the CAR is encoded by a nucleotide sequence comprising SEQ ID NO: 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 245, 247, 249, 251, 253, or 255, or any combination thereof.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multitargeted, or DuoCAR CAR further comprises one or more booster elements including: i) an arming element to overcome immunosuppression in the tumor microenvironment (TME); ii) a cytokine stimulating element to promote autonomous T cell stimulation by cytokines; iii) a digestive enzyme element to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and allow CAR T tumor penetration; iv) a neutrophil-activating protein (NAP); or v) an on-switch element or an off-switch element to control expression of the CAR; or any combination thereof, wherein the boosted single, tandem, multitargeted, or DuoCAR CAR exhibits one or more properties in a patient-specific manner.

3. 2. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multi-targeted, or DuoCAR CAR further comprises one or more booster elements comprising an arming element comprising TGFβRIIdn, truncated PD-1, PD-ldn, a synthetic PD-1 activating receptor, truncated CTLA-4, truncated Tim-3, truncated TIGIT, or any combination thereof, to overcome immunosuppression in the tumor microenvironment (TME) in a patient-specific manner.

4. 10. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multi-targeting, or DuoCAR CAR further comprises one or more booster elements comprising a cytokine stimulatory element comprising membrane-bound IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21 TNFα, IFNγ, or any combination thereof, to promote cytokine-autonomous T cell stimulation in a patient-specific manner.

5. 2. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multi-targeted, or DuoCAR CAR further comprises one or more booster elements comprising digestive enzyme elements including heparinase / HPSE, MMP-1, MMP-2, MMP-9, MMP-12, MMP-13, and hyaluronidase 1, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4, PH-20, and HYALP1, or any combination thereof, to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and enable tumor penetration of the CAR T in a patient-specific manner.

6. 2. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multi-targeted, HPSE, MMP-2, MMP-9 and PH-20 or DuoCAR CAR further comprises one or more booster elements comprising an on-switch element or an off-switch element comprising a truncated EGF receptor, a truncated TGFβ receptor (TGFβRIIdn), a truncated CD19, a truncated CD20, a CD20 mimotope, a truncated CD34, a truncated LNGF receptor, a chimeric costimulatory receptor (CCR), or any combination thereof, to control expression of the CAR in a patient-specific manner.

7. 2. The isolated nucleic acid molecule of claim 1, wherein the boosted single, tandem, multitargeted, or DuoCAR CAR exhibits one or more properties in a patient-specific manner, the properties further comprising: i) multitargeting to overcome antigen escape; ii) high surface expression on the transduced T cells; or iii) high cytolysis and in vivo expansion of transduced T cells resulting in tumor stabilization, reduction, elimination, remission of cancer or autoimmune disease, alloimmune disease, or autoaggressive disease, or prevention or amelioration of recurrence of cancer or autoimmune disease, alloimmune disease, or autoaggressive disease, or a combination thereof, and persistence to promote in vivo expansion, persistence of patient-specific anti-tumor T cells.

8. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one ROR1 and / or MSLN antigen-binding domain, at least one intracellular signaling domain, or both, is connected to the transmembrane domain by a linker or spacer domain.

9. 9. The isolated nucleic acid molecule of claim 8, wherein the encoded linker or spacer domain is derived from the extracellular domain of IgG1, IgG2, IgG3 or IgG4, CD8, TNFRSF19 or CD28 and is linked to a transmembrane domain.

10. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular ROR1 and / or MSLN antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

11. 11. The isolated nucleic acid molecule of claim 10, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 13, which encodes the leader amino acid sequence of SEQ ID NO: 14, or SEQ ID NO: 39, which encodes the leader amino acid sequence of SEQ ID NO: 40, or SEQ ID NO: 41, which encodes the leader amino acid sequence of SEQ ID NO: 42, or SEQ ID NO: 43, which encodes the leader amino acid sequence of SEQ ID NO:

44.

12. 2. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

13. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular antigen-binding domain comprises a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73, 75, or 77, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

14. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

15. 15. The isolated nucleic acid molecule of claim 14, wherein the encoded at least one intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

16. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, a chimeric costimulatory receptor (CCR), or any combination thereof.

17. 17. The isolated nucleic acid molecule of claim 16, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137), PD-1, GITR, and CTLA-4, or any combination thereof.

18. 10. A boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.

19. at least one extracellular antigen-binding domain comprising a ROR1, MSLN, FOLR1, and / or CD276 antigen-binding domain, and wherein the boosted single, tandem, multi-targeting, or DuoCAR is selected from the group consisting of SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 20. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 18, comprising an amino acid sequence of 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain.

20. 20. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 19, wherein said transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

21. 21. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 20, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO:27, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:

28.

22. at least one extracellular antigen-binding domain comprising a ROR1, MSLN, FOLR1, and / or CD276 antigen-binding domain, and said at least one intracellular signaling domain, or both, is connected to said transmembrane domain by a linker or spacer domain; and said boosted single, tandem, multi-targeting, or DuoCAR is selected from the group consisting of:

20. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 19, comprising an amino acid sequence of 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 98, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain.

23. 23. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 22, wherein the linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, IgG1, IgG2, IgG3, IgG4, or CD28 and is linked to a transmembrane domain.

24. 20. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 19, wherein said at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

25. 25. The boosted single, tandem, multitargeted, or DuoCAR CAR of claim 24, wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137), PD-1, GITR, and CTLA-4, or a combination thereof.

26. A vector comprising the nucleic acid molecule of claim 1.

27. 27. The vector of claim 26, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

28. 27. The vector of claim 26, further comprising a promoter.

29. 29. The vector of claim 28, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

30. A cell comprising the vector of claim 26.

31. 31. The cell of claim 30, wherein the cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a regulatory T cell (Treg), an inducible regulatory T (iTreg) cell, a B cell, a dendritic cell (DC), a gamma delta T cell, a macrophage, a stem cell, or an induced pluripotent stem (iPS) cell.

32. T cells are CD8 ＋ T cells, or CD4 ＋ The cell of claim 30, which is a T cell.

33. The cell of claim 30 , wherein the cell is a human cell.

34. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a boosted single, tandem, multi-targeted, or DuoCAR chimeric antigen receptor (CAR), wherein the boosted single, tandem, multi-targeted, or DuoCAR CAR comprises the amino acid sequence of SEQ ID NO: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256. A pharmaceutical composition comprising at least one extracellular antigen-binding domain comprising a ROR1, MSLN, FOLR1, and / or CD276 antigen-binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cell is a T cell of a human having cancer, an autoimmune disease, an alloimmune disease, an autoaggressive disease, or any combination thereof.

35. 35. The pharmaceutical composition of claim 34, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

36. 35. The pharmaceutical composition of claim 34, wherein the T cells are T cells of a human with a hematological cancer.

37. 37. The pharmaceutical composition of claim 36, wherein the hematological cancer is leukemia or lymphoma.

38. 38. The pharmaceutical composition of claim 37, wherein the leukemia is acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic T-cell leukemia (T-ALL), or acute lymphoblastic B-cell leukemia (B-ALL).

39. 38. The pharmaceutical composition of claim 37, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma.

40. 37. The pharmaceutical composition of claim 36, wherein the hematological cancer is multiple myeloma.

41. 37. The pharmaceutical composition of claim 36, wherein the autoimmune disease, alloimmune disease, or autoaggressive disease is rheumatoid arthritis, lupus, celiac disease, Sjogren's syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, post-streptococcal autoimmune disease, anti-neuronal antibody-mediated neuropsychiatric disorder, immune-mediated extrapyramidal movement disorder, Sydenham's chorea, autoimmune hemolytic disease, pulmonary fibrosis, systemic sclerosis, and fibrotic disease.

42. 35. The pharmaceutical composition of claim 34, wherein the human cancer comprises adult cancers including oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

43. 27. A method of producing a cell, the method comprising transducing a T cell with the vector of claim 26.

44. 10. A method for producing a population of RNA-engineered cells, comprising introducing in vitro transcribed or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule of claim 1.

45. 31. A method of conferring anti-tumor immunity in a mammal, comprising administering to said mammal an effective amount of the cells of claim 30.

46. 20. A method of treating or preventing cancer in a mammal, comprising administering to the mammal the boosted single, tandem, multitargeted, or DuoCAR CAR of claim 19 in an amount effective to treat or prevent cancer, an autoimmune disease, an alloimmune disease, an autoaggressive disease, or any combination thereof in the mammal.

47. 1. A method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR), wherein the boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR) comprises a nucleic acid sequence encoding a boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR). 250, 252, 254, or 256; and wherein the CAR comprises at least one extracellular antigen-binding domain comprising a ROR1, MSLN, FOLR1, and / or CD276 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256; at least one linker or spacer domain; at least one transmembrane domain; and at least one intracellular signaling domain; and wherein the T cells are T cells of the subject with cancer.

48. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR), wherein the boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR) comprises a nucleic acid sequence encoding a boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor (CAR). 250, 252, 254, or 256; and wherein the CAR comprises at least one extracellular antigen-binding domain comprising a ROR1, MSLN, FOLR1, and / or CD276 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 246, 248, 250, 252, 254, or 256; at least one linker or spacer domain; at least one transmembrane domain; and at least one intracellular signaling domain; and wherein the T cells are T cells of the subject with cancer.

49. 49. The method of claim 47 or 48, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

50. 10. A method for generating a boosted single, tandem, multi-targeted, or DuoCAR chimeric antigen receptor-expressing cell, comprising introducing the isolated nucleic acid of claim 1 into the cell.

51. 51. The method for generating boosted single, tandem, multitargeted, or DuoCAR chimeric antigen receptor-expressing cells of claim 50, wherein the cells are T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, dendritic cells (DCs), gamma delta T cells, macrophages, stem cells, or induced pluripotent stem (iPS) cells, or a cell population containing T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, dendritic cells (DCs), gamma delta T cells, macrophages, stem cells, or induced pluripotent stem (iPS) cells.

Images