Compositions and methods for treating cancer by Anti-ROR1 immunotherapy

Abstract

To provide chimeric antigen receptors comprising ROR1 antigen binding domain, and nucleic acid, recombinant expression vector, host cell, antigen binding fragment, and pharmaceutical composition related to the chimeric antigen receptors, as well as methods for treating or preventing cancer in subjects and methods for preparing chimeric antigen receptor T cells.SOLUTION: Provided herein is an isolated nucleic acid molecule encoding a chimeric antigen receptor comprising: at least one extracellular antigen binding domain comprising an ROR1 antigen binding domain encoded by a specific nucleotide sequence; at least one transmembrane domain; and at least one intracellular signaling domain.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority pursuant to 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 581,284, filed November 3, 2017, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy (created October 25, 2018) has the filename "SequenceListing.txt" and is 90.0 kilobytes in size.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made through the fulfillment of a Cooperative Research and Development Agreement between the National Institutes of Health and an agency of the U.S. Department of Health and Human Services. The U.S. Government has certain rights in this invention.

[0004] Field of the Disclosure The present application relates to the field of cancer, in particular to a ROR1 antigen-binding domain, a chimeric antigen receptor (CAR) containing this ROR1 antigen-binding domain, and methods of use thereof. [Background technology]

[0005] background Cancer is one of the most deadly threats to human health. With nearly 1.3 million new cases each year in the United States alone, it is the second leading cause of death after cardiovascular disease, accounting for one in four deaths. Most of these deaths are caused by solid tumors. While significant advances have been made in the medical treatment of some specific cancers, the five-year survival rate for all cancers combined 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.

[0006] There are numerous unmet therapeutic needs in the treatment of solid and liquid tumors. ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an embryonic protein that is highly expressed in many types of cancer, including CLL, breast cancer, glioblastoma, lung adenocarcinoma, and sarcomas (Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, and fibrosarcoma), but is not commonly present in normal tissues (Suping Zhang et al., 2012, The Onco-Embryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers. Am J Pathol, 181:1903-1910; Ashwini Balakrishnan et al., 2017, Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues. Clin Cancer Res 23:3061-3071; Borcherding, Nicholas et al., 2017, ROR1, an Embryonic Protein with an Emerging Role in Cancer Biology. Protein & Cell 5.7(2014):496-502). ROR1 has three splice variants, including a 104 kDa (up to 120 kDa depending on glycosylation) transmembrane glycoprotein consisting of 937 amino acids (signal peptides 1-29). The protein contains two smaller variants, an intracellular form and a secreted form (GeneBank NP_005003, Masiakowski, P. and Carroll, RD, 1992, A Novel Family of Cell Surface Receptors with Tyrosine Kinase-like Domain, J Biol Chem 36:26181-26190.) The presence of ROR1 on the surface of transformed cells suggests that targeting ROR1 may enable the development of novel cancer treatments for many liquid cancers, such as chronic lymphocytic leukemia (CLL), as well as other solid tumors (Borcherding, N., Kusner, D. et al., 2014, ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell, 5:496-502).

[0007] Although not generally present in adult tissues, at least one report has found ROR1 expression in the parathyroid gland, pancreatic islets, and regions of the esophagus, stomach, and duodenum (Ashwini Balakrishnan et al., 2017, Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues., Clin Cancer Res 23:3061-3071), which warrants caution in the clinical application of anti-cancer therapies targeting ROR1. The ROR1 receptor contains a cytosolic protein kinase domain, which, according to some reports, is involved in Wnt and EGFR signaling (Borcherding, N., Kusner, D. et al., 2014, ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell, 5:496-502). In tumors, ROR1 induces epithelial-mesenchymal transition (EMT), promotes tumor growth, invasion, and metastasis formation, and can affect apoptosis resistance (Yamaguchi, Tomoya et al., 2012, "NKX2-1 / TITF1 / TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma." Cancer Cell 21.3:348-361; Borcherding, N., Kusner, D. et al., 2014, ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell, 5:496-502). Its role in contributing to the tumor phenotype indicates that it may play an important function in tumor initiation or progression, and thus is a driver protein.

[0008] Previous approaches to cancer treatment include surgery, radiation therapy, chemotherapy, and bone marrow transplantation for hematomas. However, current first-line treatments demonstrate the need for further improvement. Such improvements are required for novel immunotherapeutic strategies. Ongoing preclinical and clinical trial studies targeting the ROR1 antigen are being conducted using multiple modalities. T lymphocytes expressing ROR1-specific CARs have been tested in both mouse and non-human primate systems (Huang X, Park H, Greene J, Pao J, Mulvey E, Zhou SX, et al., 2015, IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High-Risk Sarcomas. PLoS ONE 10(7):e0133152; Hudecek M, Schmitt TM, Baskar S, Lupo-Stanghellini MT, Nishida T, Yamamoto TN, Bleakley M, Turtle CJ, Chang WC, Greisman HA, Wood B, Maloney DG, Jensen MC,Rader C,Riddell SR,2010,The B-cell tumor-associated antigen ROR1 can be targ eted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood 116:4532-4541.) The lack of toxicity in non-human primates provides confidence that human studies can be undertaken (Berger, C. et al., 2015, Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer Immunol Res 3:2016-216.) Furthermore, both unmodified ROR1 antibodies and ROR1 antibodies conjugated to immunotoxins have been proposed for therapeutic use (Yang, Jiahui et al., 2011, "Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.” PloS One 6.6:e21018; Baskar, Sivasubramanian et al., 2012, “Targeting malignant B cells with an immunotoxin against ROR1.” MAbs, 4:3, 349-361.) The current standard of care for B-lineage leukemias is thought to involve induction treatment with high-dose chemotherapy or radiation therapy followed by consolidation therapy, which may be characterized by stem cell transplantation and further chemotherapy if necessary (see cancer.gov on the World Wide Web). Because these treatments are toxic and carry the risk of complications such as relapse, secondary malignancies, or graft-versus-host disease, better alternatives are being sought. Current open clinical trials include ROR1-targeted T cells for hematologic malignancies (Genetically Modified T-Cell Therapy in Treating Patients with Advanced ROR1+ Malignancies, NCT02706392, Funding: Fred Hutchinson Cancer Research Center, ClinicalTrials.gov accessed September 20, 2017), and a ROR1-specific antibody for breast cancer within chemotherapy (Study of Circumtuzumab and Paclitaxel for Metastatic or Locally Advanced, Unresectable Breast Cancer, NCT02776917, Funding: Barbara Parker, MD, University of California, San Diego, ClinicalTrials.gov accessed September 20, 2017).

[0009] Chimeric antigen receptors (CARs) are hybrid molecules consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a binding / 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 modeled after a single-chain fragment variable fragment (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs, such as receptor ligands (i.e., IL-13 was engineered to bind to the IL-13 receptor expressed in tumors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D), have also been incorporated into CAR designs. Other cell targets for expressing CARs (e.g., NK 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). Identifying the most active T cell populations to transduce with CAR vectors and optimizing their culture and expansion techniques will be crucial. Considerable further effort must be expended to find the CAR protein and to elucidate the molecular structure of the CAR protein itself in detail.

[0010] The binding motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed as a long, flexible linker. Structural motifs, such as those derived from the IgG constant domain, can be used to extend the ScFv binding domain far from the T cell membrane surface. This may be important for some tumor targets (e.g., disialoganglioside GD2; Orentas et al., this observation unpublished) whose binding domains are particularly close to the tumor cell surface membrane. All signaling motifs used in CARs to date include the CD3-zeta chain, as this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was used in third-generation CARs, which also contained the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the advent of new technologies, it is no longer necessary for the CAR itself to encode T cell activation by beads coupled to anti-CD3 and anti-CD28 antibodies, as well as the presence of the classic "signal 2" from CD28. Third-generation vectors using bead activation have not been shown to be superior to second-generation vectors in in vitro assays, and furthermore, they offer no clear advantage 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 evidenced by the clinical success of second-generation CD19-specific CARs in the CD28 / CD3-zeta (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD137 / CD3-zeta signaling modes (Porter DL et al. N Engl J Med. 2011;365(8):725-33). In addition to CD137, other members of the tumor necrosis factor receptor superfamily, 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, such as the inclusion of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther.2015;22(2):72-78).

[0011] Currently, the challenge to applying CAR therapy more broadly and effectively against cancer relates to the scarcity of promising targets. While engineering binders that bind to cell surface antigens is now easily achievable, finding cell surface antigens that are tumor-specific and spare normal tissues remains extremely challenging. A potential strategy to confer stronger target cell specificity to CAR-expressing T cells is to combine multiple CAR approaches. In one system, the CD3-zeta and CD28 signaling units are split into two separate CAR constructs expressed in the same cell. In another system, two CARs are expressed in the same T cell, but one has a lower affinity, so the other CAR must be bound first to maximize its activity (Lanitis E et al. Cancer Immunol. Nol Res. 2013;1(1):43-53; Kloss CC et al. Nat Biotechnol. 2013;31(1):71-5). A second challenge in generating a single ScFv-based CAR as an immunotherapeutic agent is the heterogeneity of tumor cells. At least one group has developed a CAR-based treatment for glioblastoma in which effector cell populations simultaneously target multiple antigens (HER2, IL-13Ra, and EphA2) in an attempt to avoid the proliferation of non-targeted populations (Hegde M et al. Mol Ther. 2013;21(11):2087-101).

[0012] T cell-based immunotherapy has become a new frontier in synthetic biology. Multiple promoters and gene products have been designed to target these highly potent cells to the tumor microenvironment, where they can circumvent negative regulatory signals and mediate effective tumor killing. Eliminating unwanted T cells through drug-induced dimerization of inducible caspase-9 constructs using dimerization-inducing chemicals such as AP1903 offers one way to pharmacologically trigger a powerful switch that can control T cell populations (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Furthermore, generating effector T cell populations that are resistant to the negative regulatory effects of transforming growth factor-β by expressing a decoy receptor demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, although CARs appear to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, clinical application of this technology is currently hindered by the limited in vivo expansion of CAR+ T cells, their rapid loss after infusion, and poor clinical activity, which may be due in part to the murine origin of some of the CAR sequences used. Summary of the Invention [Problem to be solved by the invention]

[0013] Whether patients receiving either antibody or CAR-T therapy should undergo subsequent HSCT to sustain their response remains an area of ​​active debate. Although high responses have been reported in CD19 CAR-T trials, at least 20% of patients experience short-term failure (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). At 12 months after CAR19 treatment, the best reported outcome was a 55% RFS and 79% OS for patients receiving T-cell products at the University of Pennsylvania (Maude SL, Teachey DT, Rheingold SR, Shaw PA, Aplenc R, Barrett DM, Barker CS, Callahan C, Frey NV, Farzana N, Lacey SF, Zheng A, Levine B, Melenhorst JJ, Motley L, Prter DL, June CH, Grupp SA, 2016, J Clin Oncol 34, no. 15_suppl (May 2016) 3011-3011). With anticipated long-term responses below 50%, there remains a great need for new B-cell malignancy targets, such as ROR1, in the clinic. [Means for solving the problem]

[0014] The present invention addresses the above-mentioned needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the invention disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with dysregulated expression of ROR1, which contain a ROR1 antigen-binding domain that has high surface expression on transduced T cells and inhibits cytolysis of ROR1-expressing cells. There is a high degree of resolution and the transduced T cells expand and persist in vivo.

[0015] overview Provided herein are novel anti-ROR1 antibodies, or their antigen-binding domains, and chimeric antigen receptors (CARs) containing such ROR1 antigen-binding domains, as well as host cells (e.g., T cells) expressing the receptors and nucleic acid molecules encoding the receptors. The CARs exhibit high surface expression on transduced T cells, a high degree of cytolysis, and in vivo proliferation and persistence of the transduced T cells. Additionally, methods of using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject, are provided.

[0016] Thus, in one aspect, an isolated polynucleotide encoding a human anti-ROR1 antibody or fragment thereof comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 7 is provided.

[0017] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 antibody 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, and a single-chain Fv (ScFv).

[0018] In one embodiment, an isolated polynucleotide encoding a fully human anti-ROR1 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: 2 and 8.

[0019] In one embodiment, an isolated nucleic acid molecule is provided encoding a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one ROR1 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 7.

[0020] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular ROR1 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to ROR1.

[0021] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular ROR1 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to ROR1.

[0022] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular ROR1 antigen-binding domain of the encoded CAR further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to ROR1.

[0023] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0024] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular antigen-binding domain of ROR1 is preceded by a sequence encoding a leader or signal peptide.

[0025] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one ROR1 antigen binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 7, wherein the CAR further encodes an extracellular antigen binding domain that targets an antigen including (but not limited to) CD19, CD20, CD22, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0026] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the further encoded extracellular antigen binding domain is an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-mesothelin 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-BCMA (CD269) 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-TSLPR 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, or an anti-EGFRvIII ScFv antigen binding domain. 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 that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0027] In one embodiment, the CAR provided herein further comprises a linker or spacer domain.

[0028] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular ROR1 antigen-binding domain, the intracellular signaling domain, or both, is connected to the transmembrane domain by a linker (L), hinge (H), or spacer domain.

[0029] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is linked to the transmembrane domain.

[0030] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR 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, CD83, CD86, CD134, CD137, and CD154, or a combination thereof.

[0031] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0032] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is located N-terminal to the CD3 zeta intracellular domain.

[0033] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0034] In a further embodiment, an isolated nucleic acid molecule encoding a CAR is provided, 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, and 4-1BB (CD137), or a combination thereof.

[0035] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided that further contains a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide (LP) comprises the nucleotide sequence of SEQ ID NO:19.

[0036] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:20.

[0037] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one ROR1 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0038] In one embodiment, a CAR is provided, wherein the extracellular ROR1 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.

[0039] In another embodiment, a CAR 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, CD154, TNFRSF19, or a combination thereof.

[0040] In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen-binding domain comprising CD19, CD20, CD22, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0041] In one embodiment, a CAR is provided, wherein the extracellular antigen-binding domain is an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-CD22 ScFv antigen-binding domain, an anti-mesothelin 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-BCMA (CD269) 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-TSLPR 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 , anti-EGFRvIII ScFv antigen binding domain, anti-GD-2 ScFv antigen binding domain, anti-NY-ESO-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0042] In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0043] In yet another embodiment, a CAR 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.

[0044] In one embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 3 (LTG 1941 LP-ScFv4-CD8H / CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG. 2A). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4 (LTG 1941 LP-ScFv4-CD8H / CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG. 2A)).

[0045] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 5 (LTG 2528 LP-ScFv4-IgG4H / CD8TM-41BB-CD3 Zeta In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 6 (LTG 2528 LP-ScFv4-1-IgG4H / CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2B)).

[0046] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9 (LTG1942 LP-ScFv9-CD8H / CD8TM-41BB-CD3zeta CAR nucleotide sequence (Figure 2C)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 10 (LTG1942 LP-ScFv9-CD8H / CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2C)).

[0047] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 11 (LTG2529 LP-ScFv9-IgG4H / CD8TM-41BB-CD3 Zeta In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 12 (LTG2529 LP-ScFv9-IgG4H / CD8 TM-41BB-CD3zeta CAR amino acid sequence (Figure 2D)).

[0048] In one embodiment, a CAR disclosed herein is 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.

[0049] In one embodiment, a nucleic acid molecule encoding a disclosed CAR can be contained in a vector, such as a viral vector, which 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.

[0050] In certain embodiments, the vector further comprises a promoter, which is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0051] In yet another embodiment, the CAR-expressing vector may be further modified to include one or more operable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. This suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the CAR-expressing vector may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0052] In another aspect, a host cell is further provided that comprises a nucleic acid molecule encoding a CAR. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.

[0053] 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 chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a human ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, 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. Cancers include, inter alia, hematological cancers 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.

[0054] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR 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, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0055] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer is selected from the group consisting of oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), gastrointestinal cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct), and rectal cancer. duct, gallbladder, pancreas), respiratory tract (larynx, lung, and bronchus), bone and joint cancer, soft tissue cancer, adult cancers including skin cancer (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), as well as cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicles, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous system, or any combination thereof.

[0056] 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 is unresponsive to one or more chemotherapeutic agents. The cancer may be hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), adult B-cell malignancies including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (B-cell malignancies), and the like. ALL (including acute lymphocytic leukemia), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other blood cancers and solid tumors, or any combination thereof.

[0057] In another embodiment, a method of producing CAR-containing T cells (hereinafter "CAR-T cells") is provided, which method comprises transducing T cells with a vector or nucleic acid molecule encoding a CAR (as disclosed) that specifically binds to ROR1, thereby producing CAR-T cells.

[0058] In yet another embodiment, a method for 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 CAR into cells of a subject, thereby generating CAR-expressing cells.

[0059] In yet another aspect, a method for diagnosing a disease, disorder, or condition associated with expression of ROR1 in a cell is provided, comprising: a) contacting the cell with a human 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 NO: 2 or 8; and b) detecting the presence of ROR1, and diagnosing the disease, disorder, or condition associated with expression of ROR1 if ROR1 is present.

[0060] In one embodiment, the disease, disorder, or condition associated with expression of ROR1 is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), 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 blood cancers and solid tumors, or any combination thereof.

[0061] In another embodiment, a method for diagnosing or prognosing or assessing the risk of a ROR1-associated disease in a mammal is provided, comprising the steps of: detecting expression of ROR1 in a sample derived from the mammal, the steps comprising: a) contacting the sample with a human 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 NO: 2 or 8; and b) detecting the presence of ROR1, and diagnosing the mammal as having a ROR1-associated disease if ROR1 is present.

[0062] In another embodiment, a method of inhibiting ROR1-dependent T cell inhibition is provided, comprising contacting a cell with a human 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 NO: 2 or 8. In one embodiment, the cell is selected from the group consisting of a ROR1-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0063] In another embodiment, a method is provided for blocking T cell inhibition mediated by ROR1-expressing cells and altering the tumor microenvironment to inhibit tumor growth in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated 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: 2 and 8. In one embodiment, the cell is selected from the group consisting of a ROR1-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0064] In another embodiment, the immunogens of an anti-tumor or anti-cancer immune response in a mammal are Methods for inhibiting, suppressing, or preventing suppression are provided, comprising administering to a mammal an effective amount of a composition comprising an isolated 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: 2 and 8. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a ROR1-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0065] 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 a disclosed CAR.

[0066] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs 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 a CAR (disclosed) that specifically binds to ROR1 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex in the subject consisting of the antigen-binding domain of the CAR, the extracellular domain of ROR1, and / or one or more of the above-mentioned antigens.

[0067] 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 an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular ROR1 antigen-binding domain comprising the amino acid sequences of SEQ ID NOs: 2 and 8, 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 cells are T cells of a subject with cancer.

[0068] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, 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 cells are T cells of a subject with cancer. In some embodiments of the above-described method, the at least one transmembrane domain comprises a transmembrane of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0069] 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 CAR, wherein the CAR comprises at least one ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the persistent population of engineered T cells, or a population of progeny of the 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.

[0070] In one embodiment, the progeny T cells in the human include memory T cells. In another embodiment, the T cells are autologous T cells.

[0071] In all aspects and embodiments of the methods described herein, any of the cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens described above can be treated or prevented or ameliorated using one or more of the CARs disclosed herein.

[0072] In yet another aspect, a kit is provided for generating the above-described chimeric antigen receptor T cells, or for preventing, treating, or ameliorating any of the above-described cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in a subject, comprising a container containing any one of the above-disclosed nucleic acid molecules, vectors, host cells, or compositions, or any combination thereof, or instructions for use of the kit.

[0073] It is understood that the above-described CARs, host cells, nucleic acids, and methods are useful beyond the scope of the specific aspects and embodiments described in detail herein. The features and advantages of the present disclosure described above will become more apparent from the following detailed description, which is provided with reference to the accompanying drawings. [Brief explanation of the drawings]

[0074] [Figure 1] Schematic diagram of the general domain structure of a CAR with the sequence of the novel extracellular ROR1 antigen-binding domain. The chimeric antigen receptor is composed of an extracellular ROR1-binding ScFv domain, a spacer or hinge domain (derived from IgG4 or CD8), a transmembrane domain, an intracellular signaling CD137 costimulatory domain, and a CD3 zeta signaling domain. [Figure 2A]Figure 2A shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing the sequence of a novel human extracellular ROR1 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide (SP or LP, leader peptide), a human anti-ROR1 binder single-chain variable fragment (ScFv), an extracellular linker (or hinge, H), a transmembrane domain (TM), a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. The nucleic acid sequence (SEQ ID NO: 3) of CAR LTG1941 (LP-ScFv4-CD8H / CD8TM-41BB-CD3 zeta) and the lentiviral vector expressing the encoded amino acid sequence (SEQ ID NO: 4) are shown. [Figure 2B] Figure 2B shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing the sequence of the novel human extracellular ROR1 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide (SP or LP, leader peptide), a human anti-ROR1 binder single-chain variable fragment (ScFv), an extracellular linker (or hinge, H), a transmembrane domain (TM), a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. The nucleic acid sequence (SEQ ID NO: 5) of CAR LTG2528 (LP-ScFv4-IgG4H / CD8TM-41BB-CD3 zeta) and the lentiviral vector expressing its encoded amino acid sequence (SEQ ID NO: 6) are shown. [Figure 2C]Figure 2C shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing the sequence of the novel human extracellular ROR1 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide (SP or LP, leader peptide), a human anti-ROR1 binder single-chain variable fragment (ScFv), an extracellular linker (or hinge, H), a transmembrane domain (TM), a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. The nucleotide sequence (SEQ ID NO: 9) of CAR LTG1942 (LP-ScFv9-CD8H / CD8TM-41BB-CD3 zeta) and the lentiviral vector expressing its encoded amino acid sequence (SEQ ID NO: 10) are shown. [Figure 2D] Figure 2D shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing the sequence of the novel human extracellular ROR1 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide (SP or LP, leader peptide), a human anti-ROR1 binder single-chain variable fragment (ScFv), an extracellular linker (or hinge, H), a transmembrane domain (TM), a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. The lentiviral vector expressing the CAR LTG2529 (LP-ScFv9-IgG4H / CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 11) and its encoded amino acid sequence (SEQ ID NO: 12) is shown. [Figure 2E]Figure 2E shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing the sequence of the novel human extracellular ROR1 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide (SP or LP, leader peptide), a human anti-ROR1 binder single-chain variable fragment (ScFv), an extracellular linker (or hinge, H), a transmembrane domain (TM), a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. The lentiviral vector expressing the CAR LTG 1943 (LP-control ScFv-CD8H / CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 15) and its encoded amino acid sequence (SEQ ID NO: 16) is shown. [Figure 2F] Figure 2F shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing the sequence of the novel human extracellular ROR1 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide (SP or LP, leader peptide), a human anti-ROR1 binder single-chain variable fragment (ScFv), an extracellular linker (or hinge, H), a transmembrane domain (TM), a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. The lentiviral vector expressing CAR LTG2527: (LP-control ScFv-IgG4H / CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 17) and the encoded amino acid sequence (SEQ ID NO: 18) is shown. [Figure 3]Figure 1 shows anti-ROR1 CAR surface expression in primary human T cells. CAR T cells redirected to the ROR1 tumor antigen using the ScFv domain were generated by lentiviral transduction of the CAR expression construct. CAR T detection was performed by flow cytometry. T cells were washed twice in cold PBS-EDTA buffer and stained with ROR1-Fc peptide followed by fluorescently labeled anti-human-Fc polyclonal F(ab)'2 fragment. Cells were gated based on forward and side scatter, singlet discrimination, and 7AAD negativity, allowing analysis of only live cells. Data were acquired using a MACSQuant 10 flow cytometer in the APC channel. Analyzed samples are listed on the left axis: UTD, untransduced negative control cells, GFP-LV control transduction, LTG1941 (ScFv4), LTG1942 (ScFv9), and LTG1943 (control-ScFv). The vertical dotted line designates the gate for CAR expression and the percentage of CAR expression in each population is listed, CAR %MFI. [Figure 4] Figure 1 shows anti-ROR1 CAR T cells incorporating ScFv binders (LTG1941, LTG1942, and LTG1943) mediate cytolysis of ROR1-positive tumors in vitro. CAR T cells expressing anti-ROR1 constructs were incubated overnight with ROR1-positive cell lines (Jeko-Luc and A431-Luc) or ROR1-negative lines (Reh-luc) stably transduced with firefly luciferase at the effector-target ratio (E:T) indicated on the x-axis. CAR T cytotoxic activity was assessed by luciferase activity measurement as described in Materials and Methods. UTD—untransduced T cell negative control; 1538—LTG1538 FMC63 murine anti-CD19 CAR positive control. [Figure 5]Figure 1 shows high levels of cytokine production by ROR1-specific CAR T cells when co-cultured with ROR1-positive leukemia lines (Jeko, gray, or A432, light gray) or when T cells were incubated with a non-expressing line (Reh) or alone (gray, last row). The assay was performed overnight at an E:T ratio of 10:1, and then supernatants were analyzed for cytokine concentrations by ELISA. N=2 technical replicates + / - SD. Negative controls: UT - untransduced T cells, LTG1941, LTG1942, LTG1943, anti-ROR1 transduced T cells. LTG1398, GFP-LV transduced control T cells, are indicated on the x-axis. DETAILED DESCRIPTION OF THE INVENTION

[0075] 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 contradicts. For example, the term "an antigen" includes one or more 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 term "and / or" means "and" or "or." Furthermore, unless otherwise specified, it is understood that any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes. 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. Additionally, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. To facilitate identification of various embodiments, explanations of terms are provided below.

[0076] The term "about," when referring to measurable possible values ​​such as amounts and durations, is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for the practice of the disclosed methods.

[0077] Unless otherwise specified, scientific terms herein are used in their conventional manner. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science, 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, 1995; and other similar reference works.

[0078] The present disclosure provides ROR1 antibodies or fragments thereof, and chimeric antigen receptors (CARs) having such ROR1 antigen-binding domains. Improving the functional activity of CARs directly correlates with improving the functional activity of CAR-expressing T cells. As a result of one or more of these modifications, CARs exhibit high levels of both cytokine-induced cytolysis and cell surface expression in transduced T cells, and promote T cell proliferation and proliferation in vivo. and high levels of persistence of transduced CAR-expressing T cells.

[0079] The unique ability to combine functional moieties from different protein domains is an innovative feature of chimeric antigen receptors (CARs). The choice of these protein domains, as well as the specific binding mode, are key design features. Individual engineered domains are essential components that can be used in any heterogeneous CAR platform to manipulate lymphocyte function. For example, selection of the extracellular binding domain can render an otherwise ineffective CAR effective.

[0080] The non-variable framework components of the immunoglobulin-derived protein sequence used to generate the extracellular antigen-binding domain of a CAR can be completely neutral, or they can be self-binding and drive T cells into a metabolically exhausted state, significantly reducing the efficacy of therapeutic T cells expressing the CAR. This phenomenon occurs independently of the antigen-binding function of the CAR domain. Furthermore, the selection of the intracellular signaling domain can also govern the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability to bind target antigens and transmit activation signals to T cells via the above-described extracellular and intracellular domains are important CAR design aspects, it has become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on CAR potency and therefore may play a crucial role in CAR function and clinical utility.

[0081] Surprisingly and unexpectedly, it was found that the use of a fully human antigen-binding domain in a CAR, rather than a mouse-derived antigen-binding fragment (which tends to induce an anti-mouse immune response and CAR T elimination in the host) (see: University of Pennsylvania-funded clinical trial using a mouse-derived SS1 ScFv sequence, NCT02159716), can determine the functional activity of CAR-expressing T cells.

[0082] In light of this discovery, several ROR1 binders from human scFv expression libraries have been developed. These fully human ROR1 CARs are no longer derived from mice and are therefore less likely to induce allergic or rejection reactions in patients (see Maus MV, Haas AR, Beatty GL, Albeda SM, Levine BL, Liu X, Zhao Y, Kalos M, June CH, 2013, Cancer Immunology Research, 1:26-31). As a result, these "fully human" CARs may be more therapeutically effective when expressed in T cells and then infused into patients. Such human sequence-derived CAR binders may be used to treat cancers, leukemias, and lymphomas that express the ROR1 antigen in humans, including but not limited to B-CLL, ovarian cancer, triple-negative breast cancer, lung adenocarcinoma, and glioblastoma (Balakrishnan, A. et al., 2016, Clin Cancer Res, 23:3061-3071; and Baskar, S. et al., 2008, Clin Cancer Res 14:396-404; and Jung, EH et al., Cell Biochem Funct, 34:149-157).

[0083] The CARs disclosed herein are expressed at high levels in cells. Cells expressing these CARs have high proliferation rates in vivo, produce large amounts of cytokines, and exhibit high cytotoxicity against cells bearing the ROR1 antigen to which the CAR binds. The use of a human extracellular ROR1 antigen-binding domain results in the creation of CARs with improved in vivo function, while avoiding the induction of anti-CAR immunity and the extinction of the CAR T cell population in the host immune response. CARs expressing the fully human extracellular ROR1 ScFv antigen-binding domain exhibit superior activity and / or properties, including: i) enhanced CAR T cell proliferation; These include ii) the prevention of poor persistence and poor function (as seen with murine-derived binding sequences); ii) the lack of targeting of the CAR to specific regions (i.e., intrapleural) for efficacy; and iii) the ability to engineer CAR T cells based on both high and low ROR1 affinity binders. The latter property allows researchers to better tune the efficacy versus toxicity and / or tissue specificity of CAR T products, since ROR1 is more highly expressed in tumors than in normal tissues, so lower affinity binders may have higher specificity for tumors than normal tissues, thereby preventing on-target off-tumor toxicity and bystander cell killing.

[0084] The CARs of the present invention are now described in detail, including a description of their extracellular ROR1 antigen-binding domain, transmembrane domain, and intracellular domain, as well as further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, treatment methods, compositions, and kits using the disclosed CARs.

[0085] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein comprise at least one ROR1 antigen-binding domain capable of binding to ROR1, at least one transmembrane domain, and at least one intracellular domain.

[0086] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. Characteristics of CARs include the ability to redirect T cell specificity and reactivity to selected targets in an MHC-independent manner, leveraging the antigen-binding properties of monoclonal antibodies. Because of their ability to recognize antigens without MHC restriction, CAR-expressing T cells have the ability to recognize antigens independently of antigen processing, thereby circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0087] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens.

[0088] 1. Extracellular domain In one embodiment, CAR comprises target-specific binding element, also referred to as antigen binding domain or site.The selection of domain depends on the type and number of ligands that define the surface of target cells.For example, antigen binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cells related to specific disease state.Therefore, examples of cell surface markers that can act as the ligand of antigen binding domain in CAR include those related to viral infection, bacterial infection, and parasitic infection, autoimmune disease, and cancer cell.

[0089] In one embodiment, a CAR can be designed to target a tumor antigen of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on the tumor cell. Antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding domain can depend on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD20, CD22, ROR1, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD19. The tumor antigens disclosed herein are included by way of example only, and the list is not intended to be limiting, as other examples will be readily apparent to those skilled in the art.

[0090] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express multiple proteins that can serve as target antigens for immune attack. Such molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules include those belonging to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins represent truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, CD22, BCMA, ROR1, and CD37, are also potential target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but without much success.

[0091] In a preferred embodiment, the tumor antigen is ROR1, and tumors associated with ROR1 expression include lung mesothelioma, ovarian and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein ROR1.

[0092] A type of tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells; instead, they are also expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Expression of the antigen in a tumor may occur under conditions that allow the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development, when the immune system is not mature and cannot respond to the antigen, or a TAA may be an antigen that is normally present at very low levels on normal cells but is expressed at significantly higher levels on tumor cells.

[0093] Examples of TSAs or TAAs include, but are not limited to, differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and the like; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include, but are not limited to, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0094] In one embodiment, the antigen binding domain portion of the CAR targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, and MAGE A3 TCR.

[0095] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular ROR1 antigen.

[0096] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular ROR1-binding domain scFv4 comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen-binding domain scFv4 comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0097] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular ROR1 antigen-binding domain ScFv9 comprises the nucleotide sequence of SEQ ID NO: 7, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen-binding domain ScFv9 comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0098] In one preferred embodiment, an isolated nucleic acid molecule encoding an extracellular ROR1 control ScFv antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 13, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 control ScFv antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0099] In various embodiments of the ROR1-specific CAR disclosed herein, a general scheme is set forth in FIG. 1, which includes, from N- to C-terminus, a signal or leader peptide, an anti-ROR1 ScFv, an extracellular linker or hinge (H) domain, a transmembrane (TM) domain, 4-1BB, and CD3 zeta, with bold letters indicating linker domains. This represents the cloning site of the linking domain.

[0100] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4 [LTG1941 LP-ScFv4-CD8H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2A)].

[0101] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1941 LP-ScFv4-CD8H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2A)].

[0102] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 14 [LTG2528 LP-ScFv4-IgG4H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2B)].

[0103] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR with the amino acid sequence of SEQ ID NO: 6, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2528 LP-ScFv4-CD8H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2B)].

[0104] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 10 [LTG1942 LP-ScFv9-CD8H / CD8TM-41BB-CD3zeta CAR amino acid sequence (shown in Figure 2C)].

[0105] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 10, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1942 LP-ScFv9-CD8H / CD8TM-41BB-CD3zeta CAR amino acid sequence (shown in Figure 2C)].

[0106] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 11 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 12 [LTG2529 LP-ScFv9-IgG4H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2D)].

[0107] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 11, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 12, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2529 LP-ScFv9-IgG4H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2D)].

[0108] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 16 [LTG1943 LP-control ScFv-CD8H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2E)].

[0109] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 16, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1943 LP-control ScFv-CD8H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2E)].

[0110] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 18 [LTG2527 LP-Control ScFv-IgG4H / CD8TM-41BB-CD3 Zeta amino acid sequence (shown in Figure 2F)].

[0111] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 18, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2527 LP-control ScFv-IgG4H / CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2F)].

[0112] The surface expression of anti-ROR1 CARs incorporating single-chain variable fragment (ScFv) sequences reactive to the ROR1 antigen is shown in Example 2 below, and a summary is provided in Table 2. The expression level of each ScFv-containing CAR was determined by flow cytometry analysis of LV-transduced T cells from healthy donors using recombinant ROR1-Fc peptide followed by anti-human Fc F(ab')2 fragments conjugated to AF647, detected in the APC channel (see Figure 3). The ScFv-based anti-ROR1 CAR constructs LTG1941, LTG1942, and LTG1943 were highly expressed in human primary T cells (indicated by the gated population) compared with the non-transduced T cell control (ungated cell population). Representative results from one donor are shown.

[0113] As shown in Example 2 and Figure 4, lentiviral vectors (LVs) expressing the following CARs were constructed and tested for anti-leukemia activity, demonstrating the high cytolytic activity of the ROR1 CAR. All CARs used in the experiment contain the described 4-1BB / CD3 zeta chain signaling motif and specific anti-ROR1 binding motif / domain. Leukemia target lines with ROR1 surface expression were used: Jeko and A431; and ROR1-negative Reh. The ScFv-based anti-ROR1 CAR constructs LTG1941, LTG1942, and LTG1943 could efficiently lyse A431, but they did not have specific lytic activity against Reh (see Figure 4). The anti-ROR1 CARs LTG1941 and LTG1942 possessed different abilities to lyse Jeko, indicating different biological activities. These results demonstrate the efficiency and specificity of the constructed CAR constructs.

[0114] The cytokine secretion capacity of anti-ROR1 CAR T cells was then assessed. Tumor cells were incubated overnight with CAR T cells or control T cells at an effector-target ratio of 10:1, and culture supernatants were analyzed for IFN-gamma, TNF-alpha, and IL-2 by ELISA (see Figure 5). Notably, CAR T expression Cells LTG1942 and LTG1943 produced high levels of IFN-gamma, while LTG1941 produced moderate amounts of IFN-gamma in response to Jeko but not A431 leukemia cell line. Similar results were observed for IL-2 and TNF-alpha expression. Negative controls (untransduced T cells, UN, or T cells transduced with control LTG1398 LV) showed no measurable cytokine induction. Importantly, the results for cytolytic and cytokine function clearly demonstrate that LTG1942 has similar potency to the control ScFv-carrying CAR, LTG1943, while LTG1941 produced significantly less cytokine and was significantly less able to lyse the Jeko leukemia cell line. Nevertheless, the ability of LTG1941 to dissolve A431 suggests that there are alternatives that may be preferred if LTG1942 proves inactive or highly toxic in CAR-T clinical studies.

[0115] While not intending to be limited to any particular mechanism of action, reasons for improved therapeutic function associated with exemplary CARs of the present invention may include, but are not limited to, for example, a) more efficient signaling due to improved lateral movement in the plasma membrane, b) better location in plasma membrane microdomains (such as lipid rafts) that allow for improved interaction with transmembrane signaling cascades associated with T cell activation, c) better location in the plasma membrane due to preferential movement away from reducing or down-regulating interactions, e.g., greater distance from or less interaction with phosphatases such as CD45, and d) better assembly into the T cell receptor signaling complex (i.e., immune synapse), or any combination thereof.

[0116] To this point, the present disclosure has been illustrated using an exemplary extracellular ROR1 ScFv antigen-binding domain, however, other nucleotide and / or amino acid variants in the ROR1 variable ScFv antigen-binding domain can also be used to derive heavy chain single binding domains or subsets thereof, and thus may comprise ROR1 antigen-binding domains for use in the CARs described herein.

[0117] Depending on the desired antigen to be targeted, the CAR may be further engineered to include an appropriate antigen-binding domain specific for the desired antigen target. For example, if ROR1 is the desired antigen to be targeted, an antibody to ROR1 may be used as the antigen-binding domain to be incorporated into the CAR.

[0118] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD33. Preferably, the antigen-binding domain in the CAR is an anti-CD33 ScFv, wherein the nucleic acid sequence of the anti-CD33 ScFv comprises the sequence of SEQ ID NO: 34. In one embodiment, the anti-CD33 ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 35. In another embodiment, the anti-CD33 ScFv portion of the CAR comprises the amino acid sequence of SEQ ID NO: 35.

[0119] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets mesothelin. Preferably, the antigen-binding domain in the CAR is an anti-mesothelin ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the sequence of SEQ ID NO: 36. In one embodiment, the anti-mesothelin ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 37. In another embodiment, the anti-mesothelin ScFv portion of the CAR comprises the amino acid sequence of SEQ ID NO: 37.

[0120] In one exemplary embodiment, the antigen binding domain portion of the CAR further targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the sequence of SEQ ID NO: 32. In one embodiment, the anti-mesothelin ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 33. In another embodiment, the anti-CD19 ScFv portion of the CAR comprises the amino acid sequence of SEQ ID NO: 33.

[0121] In one embodiment of the present invention, CARs are provided that can bind to non-TSAs or non-TAAs, including, but not limited to, antigens derived from Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enteroviruses, human coxsackieviruses, rhinoviruses, and echoviruses), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., herpes simplex virus types 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes viruses), Poxviridae (e.g., smallpox virus, vaccinia virus, and poxviruses), or hepatitis C virus, or any combination thereof.

[0122] In another aspect of the present invention, CARs are provided that can bind to antigens derived from bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella, among others, e.g., Helicobacter pylori, Legionella pneumophila, and the like. pneumophilia), mycobacterial species (e.g., Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis The present invention provides CARs capable of binding to antigens derived from infectious bacteria such as species of Lactobacillus meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof.

[0123] 2. Transmembrane domain With respect to transmembrane domains, a CAR comprises one or more transmembrane domains fused to the extracellular ROR1 antigen-binding domain of the CAR.

[0124] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0125] Particularly useful transmembrane regions in the CARs described herein can be derived from (i.e., comprise at least the transmembrane region 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, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. Alternatively, the transmembrane domain can be synthetic and, in this case, can comprise primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine can be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine and serine doublet provides a particularly suitable linker.

[0126] In one embodiment, a transmembrane domain originally associated with one of the domains in the CAR is used in addition to the transmembrane domain described above.

[0127] In some instances, the transmembrane domain can be selected or amino acid substituted to prevent the domain from binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interactions of the domain with other receptor complex components.

[0128] In one embodiment, the transmembrane domain in a CAR of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 21. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22.

[0129] In one embodiment, the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 22 with at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions).

[0130] In some examples, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 23. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 24 or a sequence having 95-99% identity thereto.

[0131] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0132] 3. Spacer (hinge, H) domain In CARs, a spacer domain may be located between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer domain refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0133] In some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,2 84, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, which are incorporated by reference in their entireties.

[0134] The spacer domain preferably has a sequence that promotes binding between the CAR and the antigen and increases signal transduction into the cell. Examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine at potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0135] The spacer domain can be the entire or a portion of amino acids 137-206 (SEQ ID NO: 25) of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). A portion of the constant region of an antibody heavy or light chain can also be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0136] The spacer domain may also consist of elements of an immunoglobulin (Ig) constant domain, including sequences that link immunoglobulin domains comprising immunoglobulin proteins, such as those derived from IgG4. The spacer or hinge domain is present C-terminal to the scFv ROR1-binding domain and extends into the CAR transmembrane domain. In one embodiment, the IgG4 hinge (H) domain comprises the nucleic acid sequence of SEQ ID NO: 38. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 39. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 39, or a sequence 95-99% identical thereto.

[0137] In some examples, the IgG4 constant region acts as a hinge (H) and is attached to the CD8 transmembrane domain. In one embodiment, the IgG4H domain is attached to the CD8 transmembrane domain and together therewith comprises the nucleic acid sequence of SEQ ID NO: 40. In one embodiment, the IgG4H domain attached to the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41. In another embodiment, the IgG4H attached to the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 41, or a sequence 95-99% identical thereto.

[0138] Furthermore, a signal peptide sequence may be attached to the N-terminus of the CAR. This signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above as intracellular domains have a signal peptide sequence, this signal peptide can be used as the signal peptide for the CAR. In one embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 20.

[0139] 4. Intracellular domain The cytoplasmic domain or intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, it may be used instead of the complete chain, as long as the truncated portion is capable of transmitting the effector function signal. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. The meaning of "transduction domain" includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0140] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that cooperate to initiate signal transduction following binding of an antigen to the receptor, as well as any derivatives or variants of these sequences, and any synthetic sequence with the same functional capability.

[0141] It is known that signals emitted through the TCR alone are insufficient to fully activate T cells, and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct types of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (first cytoplasmic signaling sequence), and one that acts in an antigen-independent manner to provide a secondary or costimulatory signal (second cytoplasmic signaling sequence).

[0142] The first cytoplasmic signaling sequence regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. A first cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM.

[0143] Examples of ITAMs containing a first cytoplasmic signaling sequence that are particularly useful in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP__932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP__004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP__000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP__000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP__000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP__000724.1), and CD5 (NCBI RefSeq: NP__000724.1). The amino acids 402 to 495 of CD79a (NCBI RefSeq:NP__055022.2), 707 to 847 of CD79a (NCBI RefSeq:NP__001762.2), 166 to 226 of CD79a (NCBI RefSeq:NP__001774.1), 182 to 229 of CD79b (NCBI RefSeq:NP__000617.1), and CD66d (NCBI RefSeq:NP__000617.1) were also included. RefSeq:NP_001806.2), as well as variants having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information in NCBI RefSeq ID or GenBank described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0144] In a preferred embodiment, the intracellular domain of the CAR may be designed to contain a CD3-zeta signaling domain by itself, or may be combined with any other desired cytoplasmic domain useful in the context of a CAR. For example, the intracellular domain of a CAR may contain a CD3-zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. Specific examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP__001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP__000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP__055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP__006130.1), and CD137 (4-1BB, NCBI The costimulatory signaling elements of the present disclosure include, but are not limited to, peptides having amino acids 214-255 of CD134 (OX40, NCBI RefSeq:NP_001552.2), amino acids 241-277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acids 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants having the same function as these peptides. Thus, although the present disclosure has thus far been exemplified primarily using 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the present disclosure.

[0145] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form this linkage. A glycine and serine doublet provides a particularly suitable linker.

[0146] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0147] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence of SEQ ID NO: 26, and the signaling domain of CD3-zeta comprises the nucleic acid sequence of SEQ ID NO: 28 and the variant nucleic acid sequence of SEQ ID NO: 30. In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence of SEQ ID NO: 26, and the signaling domain of CD3-zeta comprises the nucleic acid sequence of SEQ ID NO: 28 and the variant nucleic acid sequence of SEQ ID NO: 30.

[0148] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 27, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 29 and a variant nucleic acid encoding the amino acid sequence of SEQ ID NO: 31.

[0149] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 27 and the signaling domain of CD3-zeta The signaling domain of comprises the amino acid sequence of SEQ ID NO:29 and the variant amino acid sequence of SEQ ID NO:31.

[0150] 5. Further explanation of CAR Functional portions of the CARs disclosed herein are also expressly included within the scope of the present invention. The term "functional portion," when used in reference to a CAR, refers to any one or more portions or fragments of the CARs disclosed herein, which retain the biological activity of the CAR (parent CAR). Functional portions include, for example, CAR portions that retain the ability to recognize target cells or detect, treat, or prevent disease to a similar extent as the parent CAR, to the same extent as the parent CAR, or to a greater extent than the parent CAR. With respect to the parent CAR, the functional portion can, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0151] A functional portion may contain additional amino acids at the amino or carboxy terminus, or both, of the portion that are not found in the amino acid sequence of the parent CAR. Desirably, these additional amino acids do not interfere with the biological function of the functional portion, such as, for example, target cell recognition, cancer detection, cancer treatment, or prevention. More desirably, these additional amino acids improve such biological activity over the biological activity of the parent CAR.

[0152] Functional variants of the CARs disclosed herein are included within the scope of this disclosure. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with the parent CAR, and this functional variant retains the biological activity of the CAR from which it is derived. Functional variants include, for example, variants of the CARs (parent CARs) described herein that retain the ability to recognize target cells to a similar extent to, or to a greater extent than, the parent CAR. With respect to the parent CAR, functional variants may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more amino acid sequence identity with the parent CAR.

[0153] A functional variant may, for example, comprise at least one conservative amino acid substitution in the amino acid sequence of the parent CAR. Alternatively, or in addition, a functional variant may comprise at least one non-conservative amino acid substitution in the amino acid sequence of the parent CAR. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may improve the biological activity of the functional variant, such that the biological activity of the functional variant is superior to that of the parent CAR.

[0154] The amino acid substitutions in CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include substituting an acidic / charged polar amino acid (e.g., Asp or Glu) with another acidic / charged polar amino acid, substituting a non-polar side chain-containing amino acid (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another non-polar side chain-containing amino acid, substituting a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, substituting a polar side chain-containing uncharged amino acid (e.g., Asn, Gin, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, substituting a beta-branched side chain-containing amino acid (e.g., He, Thr, and Val) with another beta-branched side chain-containing amino acid, and substituting an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid. It may be a substitution or the like.

[0155] A CAR can consist essentially of one or more of the specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0156] CARs (including functional portions and functional variants) can be of any length, i.e., contain any number of amino acids, so long as the CAR (or functional portion or variant thereof) retains biological activity, such as, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal. For example, a CAR can be from about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more amino acids in length.

[0157] CARs (including functional portions and functional variants according to the invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid. acid), a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0158] CARs (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized or conjugated.

[0159] CAR (including its functional parts and functional variants) can be obtained by methods well known in the art. CAR may be produced by any suitable polypeptide or protein production method. Suitable methods for de novo synthesis of polypeptides and proteins are described in prior art documents such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins may also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 1, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional portions and functional variants thereof) may be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) may be commercially synthesized by companies. In this regard, CARs may be synthetic, recombinant, isolated, and / or purified.

[0160] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding domain or portion thereof, that specifically binds to one or more of the antigens disclosed herein. As used herein, a "T cell expressing a CAR" or "CAR T cell" refers to a T cell that expresses a CAR and has antigen specificity, e.g., determined by the antibody-derived targeting domain of the CAR.

[0161] As used herein, an "antigen-binding domain" may include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Examples of antibodies include, but are not limited to, intact immunoglobulins and variants and fragments thereof well known in the art that retain binding affinity for an antigen.

[0162] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for possible natural mutations, which may be present in trace amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the property of the antibody being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some instances, a monoclonal antibody is produced by a single clone of B lymphocytes or by cells transfected with nucleic acid encoding the light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or their progeny. In some instances, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are well known and are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, 2nd Edition. Cold Spring Harbor Publications, New York (2013).

[0163] Typically, immunoglobulins have heavy (H) chains and light (L) chains linked together by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0164] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or The heavy and light chain variable domains comprise a heavy chain variable region (VH) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind to an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH," or "VH," refer to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment, such as Fv, ScFv, dsFv, or Fab. References to "VL," or "VL," refer to the variable domain of an antibody light chain, including that of an Fv, ScFv, dsFv or Fab.

[0165] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity-determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Vol. 1, No. 1, pp. 111-114, 2002). (See, for example, the "International Framework for Antibody Development" (International Standards Board of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved among species. The framework regions of an antibody, i.e., the framework regions of the constituent light and heavy chains, together position and align the CDRs in three-dimensional space.

[0166] CDRs are primarily responsible for binding to an antigenic epitope. The amino acid sequence boundaries of a given CDR are determined by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, USA). Al-Lazikani et al. (JMB 273,927-948,1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N- to C-terminus) and are further typically identified by the chain in which the CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody that contains it, and a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody that contains it. Light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3.

[0167] "Antigen-binding fragments" are portions of full-length antibodies, and various combinations of such portions, that retain the ability to specifically recognize their cognate antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments generated by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0168] Single-chain antibodies (ScFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies joined by a suitable polypeptide linker into a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains within an ScFv is typically not determinative of the ScFv. Thus, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0169] In dsFvs, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the bond between the two chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but are connected to complementary domains on another chain using a linker that is too short to connect the two domains into a single chain, thereby forming two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0170] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0171] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, or can be made recombinantly, or can be obtained by screening combinatorial libraries composed of variable heavy and light chains, for example, as described by Huse et al., Science 246:1275-1281 (1989), incorporated herein by reference. These methods, as well as other methods for producing, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies, are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0172] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are well known, and exemplary competition assays are provided herein.

[0173] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment that provides the CDRs is called the "donor," and the framework regions The human antibody or antigen-binding fragment that provides the CDR is referred to as the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant regions may be absent, but if present, may be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90% (e.g., about 95% or more) identical. Thus, all parts of the humanized antibody or antigen-binding fragment (possibly except for the CDRs) are substantially identical to the corresponding parts of a natural human antibody sequence.

[0174] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and the CDRs and / or framework regions from another human antibody.

[0175] A "fully human antibody" or "human antibody" is an antibody that contains sequences from (or derived from) the human genome and no sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated by using antibody production techniques based on sequences derived from the human genome, for example, by phage display or the use of transgenic animals (see, e.g., Barbas et al., Phage Display). (See display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0176] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For example, a native immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0177] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are well known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929, below).

[0178] Additionally, the CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof may be modified to contain a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., a gold particle).

[0179] C.conjugates CARs, CAR-expressing T cells, or monoclonal antibodies, or antigen-binding fragments thereof, specific for one or more of the antigens disclosed herein may be conjugated to agents such as effector molecules or detectable markers using any of a number of means well known to those skilled in the art. Both covalent and non-covalent means may be used. Conjugates include, but are not limited to, molecules in which an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein is covalently linked to an effector molecule or detectable marker. Those skilled in the art will be familiar with the use of conjugates that are specifically linked to chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C. 3 H, and 35 Radiation such as S It will be understood that a wide variety of effector molecules and detectable markers can be used, including, but not limited to, inhibitors, other labels, targeting moieties, and ligands.

[0180] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin used to cause the death of a particular target cell (such as a tumor cell).

[0181] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on an antibody, resulting in the attachment of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of several well-known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). The linker can be any molecule used to connect an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker can form covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may be attached to constituent amino acids through their side chains (e.g., to cysteine ​​via a disulfide bond) or to the amino and carboxy groups of the alpha carbon of the terminal amino acid.

[0182] In some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,28 Nos. 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated by reference in its entirety.

[0183] In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by degradation of the antibody. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In embodiments, the peptide linker is at least 2 amino acids in length, or at least 3 amino acids in length. However, the linker may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids in length. Proteases may include cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics). 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin-B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0184] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, such pH-sensitive linkers are hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, or ketals, etc.) that can be hydrolyzed in lysosomes can be used. (See, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions (such as in blood), but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

[0185] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0186] In yet another specific embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(1) 0):1299-1304), or the 3'-N-amide analogue (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0187] In yet another embodiment, the linker is not cleavable, and the effector molecule or detectable marker is released by degradation of the antibody (see US Publication No. 2005 / 0238649, incorporated herein by reference in its entirety).

[0188] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating a conjugate containing the desired linker with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of effector molecule or detectable marker free in the plasma. A variety of exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0189] In some embodiments, conjugates of a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof, with one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a dolastatin, an auristatin, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0190] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to well-known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogs can be prepared synthetically according to well-known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, certain microorganisms were further discovered to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, and 4, Nos. 313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for their preparation, and therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and 6,441,163, and European Patent No. EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0191] Additional toxins can be used in conjunction with the CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Examples of toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxin. Toxins include toxins A-F. Such toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of such toxins (see, e.g., U.S. Pat. Nos. 5,079,163 and 4,689,401).

[0192] Saporin is a toxin derived from Saponaria officinalis that inhibits protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin lacks a mechanism for specific intracellular entry and therefore must bind to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein for efficient entry into cells.

[0193] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins has been mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0194] Ricin is the lectin RCA60 obtained from Ricinus communis (Castor bean). For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, with molecular weights of approximately 65 kD and 120 kD, respectively, and hence RCA. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds lysine to cell surface galactose residues, facilitating transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0195] Ribonucleases have also been used as immunotoxins by conjugating them to target molecules (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribonucleases, such as α-sarcin and restrictocin, are described, for example, in Rathore et al., Gene 190:31-5, 1997, and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin was originally isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes DNA double-strand breaks, leading to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). The drug is the toxic moiety of immunotoxins in clinical trials (see, e.g., Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0196] Abrin includes toxic lectins obtained from Abrus precatorius. The toxic components, abrins a, b, c, and d, have molecular weights of approximately 63-67 kD and consist of two disulfide-bonded polypeptide chains, A and B. The A chain inhibits protein synthesis, while the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978). ).

[0197] CARs, CAR-expressing T cells, monoclonal antibodies, and antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can also be conjugated to a detectable marker, such as a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (such as computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy). Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioactive isotypes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, and lanthanide illuminators. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also useful. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, may also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof are conjugated to a detectable enzyme, they can be detected by adding additional reagents that produce a distinguishable reaction product when used with the enzyme. For example, in the presence of the agent horseradish peroxidase, addition of hydrogen peroxide and diaminobenzidine yields a colored reaction product that can be detected visually. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding.Of note, the avidin itself may be conjugated to an enzyme or fluorescent label.

[0198] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may also be conjugated with lanthanides (such as europium and dysprosium) and manganese. Antibodies or antigen-binding fragments may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a secondary antibody binding site, a metal binding domain, an epitope tag, etc.).

[0199] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may also be conjugated with radiolabeled amino acids. Radiolabels may be used for both diagnostic and therapeutic purposes. For example, radiolabels may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabels may be used in therapy as toxins to treat tumors in subjects, such as neuroblastoma. Examples of labels for polypeptides include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 These include, but are not limited to, radioisotopes such as I or radionucleotides.

[0200] Means of detecting such detectable markers are well known to those of skill in the art. Thus, for example, radioactive labels may be detected using photographic film or scintillation counters, fluorescent markers may be detected by detecting emitted light using a photodetector. Enzyme labels typically involve providing the enzyme with a substrate and reacting with the enzyme on the substrate. Colorimetric labels are detected by simply visualizing the colored label, whereas colorimetric labels are detected by simply visualizing the colored label.

[0201] D. Nucleotides, Expression, Vectors, and Host Cells

[0013] Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof described herein (including functional portions and functional variants thereof). The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0202] In some embodiments, the nucleotide sequence may be codon-modified.Without being bound by any theory, it is believed that the codon optimization of the nucleotide sequence can increase the translation efficiency of mRNA transcripts.The codon optimization of the nucleotide sequence may involve replacing natural codons with other codons that code for the same amino acid but can be translated by tRNAs that are more easily utilized in cells, thus increasing translation efficiency.The optimization of the nucleotide sequence may also reduce secondary mRNA structures that may interfere with translation, thus increasing translation efficiency.

[0203] In one embodiment of the invention, a nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid may comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0204] As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, may be synthetic or obtained from natural sources (e.g., by isolation and / or purification), may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages (such as phosphoramidate or phosphorothioate linkages instead of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides). In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be preferred for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0205] Recombinant nucleic acids may have sequences that do not occur in nature or sequences that are an artificial combination of two separate regions of a sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificially manipulating separate nucleic acid regions using genetic engineering techniques, such as those described in Sambrook et al., supra. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the duplex formed by hybridization. Examples of modified nucleotides that can be used in nucleic acid production are 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino Methyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methylaminomethyl-2-thiouracil ... Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), uracil-5-carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention may be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0206] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any of the above-described CARs, or a functional portion or variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence degenerate to any of the above-described sequences, or a combination of degenerate sequences.

[0207] One embodiment further provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0208] Nucleotide sequences that hybridize under stringent conditions may also hybridize under highly stringent conditions. "Highly stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount detectably greater than nonspecific hybridization. Highly stringent conditions include conditions under which polynucleotides with exactly complementary sequences or those with only a few scattered mismatches can be distinguished from random sequences that coincidentally contain a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more in length, and can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions include, for example, conditions under which a polynucleotide with an exactly complementary sequence or one with only a few scattered mismatches can be distinguished from a random sequence that coincidentally contains a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more in length, and can be easily distinguished by highly stringent hybridization. These conditions may include low salt and / or high temperature conditions, such as NaCl or its equivalent at a temperature of about 50-70°C. Such highly stringent conditions tolerate very little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0209] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical.

[0210] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids described above. For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and that is capable of causing a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with a host cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide in the host cell. Such vectors generally do not occur in nature.

[0211] However, some of these vectors may be naturally occurring. The recombinant expression vector may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or derived in part from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain natural or non-natural internucleotide bonds, or both types of bonds. Preferably, the non-natural or altered nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0212] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those (such as plasmids and viruses) designed for propagation and propagation, or for expression, or both. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0213] Bacteriophage vectors such as λυTIO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, for example, LENTIVECTOR® gene transfer technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc. Non-clinical versions of lentiviral vectors are also available and will be known to those skilled in the art.

[0214] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

[0215] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid transfer using high-velocity particle bombardment (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0216] In one embodiment, recombinant expression vectors may be prepared using standard recombinant DNA techniques, e.g., as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear constructs of expression vectors may be prepared to contain replication mechanisms that function in prokaryotic or eukaryotic host cells. Replication mechanisms may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, and bovine papilloma virus.

[0217] Recombinant expression vectors may contain regulatory sequences, such as transcription and translation initiation and termination codons, that are appropriate for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector is to be introduced, and taking into account whether the vector is DNA or RNA based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0218] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0219] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding the CAR (including functional portions and functional variants thereof) or a nucleotide sequence complementary to or hybridizing to the CAR-encoding nucleotide sequence. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific, developmental-specific, etc.) is within the ordinary skill of those in the art. Similarly, the association of a nucleotide sequence with a promoter is also within the ordinary skill of those in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0220] Recombinant expression vectors may be designed for either transient expression, stable expression, or both, and may be engineered for constitutive or inducible expression.

[0221] Additionally, recombinant expression vectors may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell in which it is expressed. A suicide gene can be engineered to cause death in a cell in which it is expressed, for example by administering a drug. The suicide gene may be a gene that confers sensitivity to an agent, such as a drug, causing the death of the cell when the cell comes into contact with or is exposed to the agent. Suicide genes are well known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0222] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. A host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. A host cell may be a cultured cell or a primary cell (i.e., isolated directly from an organism, such as a human). A host cell may be an adherent cell or a suspension cell (i.e., a cell that grows in suspension). Suitable host cells are well known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is to amplify or replicate a recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. When the purpose is to produce a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, may be derived from any type of tissue, and may be at any stage of development, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0223] For purposes described herein, a T cell may be any T cell, including cultured T cells (e.g., primary T cells), T cells from a cultured T cell line (e.g., Jurkat, SupTl, etc.), or T cells obtained from a mammal. If obtained from a mammal, T cells may be obtained from a wide variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may be enriched or purified. T cells may be human T cells. T cells may be T cells isolated from a human. T cells may be any type of T cell and at any developmental stage, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm, and naive T cells. T cells may be CD8+ T cells or CD4+ T cells.

[0224] In one embodiment, the CARs described herein can be used in suitable cells that are not T cells, such as those that have immune effector functions, such as NK cells and T-like cells developed from pluripotent stem cells.

[0225] One embodiment also provides a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population that includes, in addition to host cells that contain any of the described recombinant expression vectors, at least one other cell, e.g., a host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a heterogeneous population that includes host cells that contain any of the described recombinant expression vectors, as well as at least one other cell, e.g., a host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a heterogeneous population that includes at least one host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), a host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. The population may be a substantially homogeneous population that primarily comprises (e.g., consists essentially of) host cells that contain the expression vector. The population may also be a clonal cell population, in which all cells in the population are clones of a single host cell that contains the recombinant expression vector, and therefore all cells in the population contain this recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population that comprises host cells that contain a recombinant expression vector described herein.

[0226] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the host cells are more pure than they are in their natural environment in the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, or may be greater than about 60%, about 70%, or about 80%, or may be about 100%.

[0227] E. Treatment Method It is contemplated that the CARs disclosed herein may be used in methods for treating or preventing disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.

[0228] One embodiment further comprises lymphodepleting the mammal prior to administering a CAR disclosed herein. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0229] For purposes of this method, in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells may be autologous to the mammal. As used herein, allogeneic refers to any material derived from an animal of the same species as the individual into which the material is introduced, but from a different individual. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci for those individuals are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically different to be able to interact antigenically. As used herein, "autologous" refers to any material derived from the same individual into whom the material will later be reintroduced.

[0230] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, Rodentia mammals, such as mice and hamsters, and Logomorpha mammals, such as rabbits. The mammal may be of the Order Carnivora, which includes Felidae (cats) and Canidae (dogs). The mammal may be of the Order Artiodactyla, which includes Bovinae (cattle) and Porcinae (pigs), or Persodactyla, which includes Equidae (horses). The mammal may be of the Order Primates, Ceboids, or Simoids (monkeys), or Apes (humans and apes). Preferably, the mammal is a human.

[0231] For the methods described above, the cancer may be selected from acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, The cancer may be any cancer, including any of laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0232] The terms "treatment" and "prevention," and derivatives thereof, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as potentially beneficial or therapeutic. In this regard, the method can provide any amount or level of cancer treatment or prevention in a mammal.

[0233] Furthermore, the treatment or prevention provided by the present methods may include treatment or prevention of one or more conditions or symptoms of the disease (e.g., cancer) being treated or prevented. For purposes herein, "prevention" may also encompass delaying the onset of the disease or its symptoms or conditions.

[0234] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody, and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0235] The sample may be obtained by any suitable method, such as, for example, biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may involve the collection of tissue and / or cells from the individual for subjecting the removed tissue and / or cells to an experimental procedure. The experimental procedure may include an experiment to determine whether the individual has and / or is suffering from a particular condition or disease state. The condition or disease may be, for example, cancer.

[0236] For one embodiment of the method for detecting the presence of a proliferative disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a whole cell lysate fraction, e.g., a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction. When the sample contains whole cells, the cells can be any cells of the mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0237] The contacting described above may occur in vitro or in vivo in a mammal. Preferably, the contacting occurs in vitro.

[0238] Additionally, detection of the complex may be performed by any of several methods known in the art, such as detecting a CAR disclosed herein, a polypeptide described herein, a protein, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, or the like. The antibody, or antigen-binding portion thereof, may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., a gold particle), for example, as disclosed above.

[0239] The method for testing the target cell recognition ability and antigen specificity of CAR is well known in the art.For example, Clay et al., J.Immunol,163:507-513(1999) teaches the method for measuring the release of cytokines (for example, interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)).In addition, the function of CAR can be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J.Immunol,174:4415-4423(2005).

[0240] Another embodiment provides the use of a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody, or antigen-binding portion thereof, and / or pharmaceutical composition of the invention to treat or prevent a proliferative disorder, such as cancer, in a mammal, which may be any of the cancers described herein.

[0241] Any administration method, including local and systemic administration, may be used for the disclosed therapeutic agents. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration may be used. The specific mode of administration and dosing regimen may be selected by the attending clinician, taking into consideration the specifics of the case (e.g., the subject, the disease, any associated disease states, and whether the treatment is prophylactic). When more than one agent or composition is administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally, and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic and pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue area after tumor removal or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops or intravitreously to the eye.

[0242] The disclosed therapeutic agents may be formulated in unit dosage forms suitable for administering precise dosage amounts one at a time. Additionally, the disclosed therapeutic agents may be administered in a single-dose or multiple-dose schedule. A multiple-dose schedule may involve an initial series of treatment in which more than one dose (e.g., 1-10 doses) may be administered separately, followed by subsequent doses at intervals, as needed, to maintain or enhance the effect of the composition. Treatment may involve administering the compound once daily or multiple times daily (multi-daily doses) for a period ranging from two to three days to several months or even years. Accordingly, the dosage regimen may be determined, at least in part, based on the specific requirements of the subject being treated and may be dependent on the judgment of the administering physician.

[0243] Typical dosages of the antibody or conjugate may range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0244] In a specific example, a subject is administered a therapeutic composition comprising one or more of a conjugate, antibody, composition, CAR, CAR T cell, or additional agent on a multiple daily dosing schedule, such as at least 2 consecutive days and at least 10 consecutive days, for a period of, e.g., weeks, months, or years. In one example, a subject is administered a conjugate, antibody, composition, or additional agent for a period of at least 30 days, for example, for a period of at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0245] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy, and / or chemotherapy in combination (e.g., sequentially, substantially simultaneously, or simultaneously) with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells. Methods and therapeutic dosages for such agents and treatments are well known to those of skill in the art and may be determined by a skilled clinician. Preparation and administration schedules for additional agents may be used according to manufacturer's instructions or according to the experienced judgment of a skilled physician. Preparation and administration schedules for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

[0246] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Examples of additional therapeutic agents that can be used in combination therapy include, but are not limited to, microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenesis agent may be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. The methods and therapeutic dosages for such agents are well known to those skilled in the art and can be determined by skilled clinicians.

[0247] Additional chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; , etoposide, and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antineoplastic antibiotics such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and and other agents such as, but not limited to, alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are well known to those skilled in the art and can be determined by a skilled clinician.

[0248] Combination therapy can produce synergistic effects and can be proven to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the combined effect achieved when the same compounds are used separately. Synergistic effects can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit-dose preparation, (2) delivered alternately or in parallel as separate preparations, or (3) when some other regimen is used. In the case of alternate delivery, synergistic effects can occur when the compounds are administered or delivered sequentially, for example, by separate injections in separate syringes. Generally, in the case of alternate administration, an effective dosage of each active ingredient is administered sequentially, i.e., consecutively, while in combination therapy, effective dosages of two or more active ingredients are administered together.

[0249] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more antigens disclosed herein is administered to a tumor-bearing subject after anti-cancer treatment. After a sufficient time has passed, the administered antibody or antigen-binding fragment or conjugate forms an immune complex with the antigen expressed on each cancer cell, and the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control obtained before the treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control obtained before the treatment indicates that the treatment is effective.

[0250] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biological compositions (hereinafter "compositions") comprising one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells that specifically bind to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier), for use in gene therapy, immunotherapy, and / or cell therapy. The compositions may be prepared in unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome. The compositions may be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments are useful, for example, for the treatment and detection of tumors (e.g., but not limited to, neuroblastoma). In some instances, the compositions are useful for treating or detecting cancer. Compositions comprising the CARs disclosed herein, or T cells, conjugates, antibodies, or antigen-binding fragments expressing the CARs, are also useful for detecting, for example, pathological angiogenesis.

[0251] The composition for administration may comprise a solution of the CAR, or T cells, conjugates, antibodies, or antigen-binding fragments expressing the CAR, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. Such solutions are sterile and generally free of undesirable matter. The compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and adjuvants, as needed to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of CAR, or CAR-expressing T cells, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected depending primarily on fluid volume, viscosity, body weight, and the like, depending on the particular mode of administration selected and the requirements of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are well known or will be apparent to those skilled in the art.

[0252] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing compositions for administration will be well known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0253] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be provided in lyophilized form and administered by reconstitution with sterile water, or may be provided dissolved in a sterile solution of known concentration. The CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugate solutions are then loaded into infusion bags containing 0.9% sodium chloride (USP) and, in some cases, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art in administering antibody, antigen-binding fragment, and conjugate drugs; for example, antibody drugs have been available on the U.S. market since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and conjugates thereof, may be administered by slow infusion rather than intravenous push or bolus. In one example, a higher loading dose is administered, followed by a lower maintenance dose. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or the corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) may be infused over approximately 90 minutes, and if this initial dose is well tolerated, then weekly maintenance doses of 2 mg / kg may be infused over 30 minutes for 4-8 weeks.

[0254] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particulate systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries are about 5 μm in diameter, only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery. Livery, A. Kydonieus (ed.), Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0255] Polymers may be used for ion-controlled release of the CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugate compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are well known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous, mobile liquid at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the preparation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous other systems for controlled delivery of therapeutic proteins are known (U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,957,735, U.S. Pat. No. 5,019,369, U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,514,670, U.S. Pat. No. 5,413,797, U.S. Pat. No. 5,268,164, U.S. Pat. No. 5,004,697, U.S. Pat. No. 4,902,505, U.S. Pat. No. 5,506,206, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,254,342, and U.S. Pat. No. 5,534,496).

[0256] G.Kit In one embodiment, further provided is a kit that uses the CAR disclosed herein.For example, a kit for treating tumors in a subject or for generating CAR T cells that express one or more of the CARs disclosed herein.Such kits can typically include the antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cells that express CAR disclosed herein.More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cells that express CAR can be included in the kit.

[0257] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used for treating a particular condition.

[0258] The label or package insert may typically further include instructions for using the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells, for example, in methods for treating or preventing tumors or in methods for generating CAR T cells. The package insert will typically be similar to the instructions customarily included in commercial packaging for therapeutic products. The kit may further include instructions, which include information about the indications, usage, dosage, administration, contraindications, and / or warnings associated with the use of the therapeutic product. The contents of the instructions may be written in electronic format (e.g., floppy disk or compact disk) or visual format (e.g., video file). The kit may further include additional components to facilitate the particular use for which the kit is designed. Thus, for example, the kit may further include label detection means (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, or an appropriate secondary label such as a secondary antibody). The kit may further include buffers and other reagents routinely used in the practice of a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0259] Example The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. On the contrary, it is clearly understood that reliance must be placed on various other embodiments, modifications, and equivalents, which may occur to those skilled in the art after reading the description herein without departing from the spirit of the invention and / or the scope of the appended claims. [Example]

[0260] Isolation of ROR1-specific binders from phage- and yeast-displayed entirely human ScFv libraries material and method: a) Generation of fully human ScFc (ScFv with Fc domain for analysis) binders to human ROR1 A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library (approximately 10 unique specificities as diversity) constructed from peripheral blood B cells of 50 healthy donors was generated. 10) (ZYZhu and D.S.Dimitrov, unpublished data) was used to select ScFv for recombinant human ROR1 protein. 12 The amplified ScFv library was incubated with ROR1 coated at 5 μg, 3 μg, and 1 μg in 5 × 100 μl volumes (equally distributed among five wells of a 96-well plate) for 2 hours at room temperature during the first, second, and third rounds of biopanning. After each incubation, the wells were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST) five times after the first round and ten times after each subsequent round to remove nonspecifically bound phage. The bound phage were mixed with TG1 competent cells for 1 hour at 37°C, and the phage were amplified from the infected cells and used in the next round of biopanning. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells and individually inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate. After the bacterial culture reached an optical density at 600 nm (OD600) of 0.5, helper phage M13K07 at a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) of kanamycin were added to the medium, and the plate was further incubated overnight at 30°C in a shaker at 250 rpm. The phage supernatant was mixed with 3% nonfat milk in PBS at a volume ratio of 4:1 and used in an enzyme-linked immunosorbent assay (ELISA) to identify phage clones displaying ScFvs with high ROR1 binding affinity. The supernatant was incubated with 50 ng of recombinant human ROR1 coated onto each well of a 96-well plate at room temperature for 2 hours and washed five times with PBST (after overnight incubation at 4°C, blocking was performed with 3% nonfat milk in PBS and three washes with PBS containing 0.05% Tween 20). ROR1-binding phages were detected using horseradish peroxidase-conjugated goat anti-M13 antibody. After incubation with this target antibody, the wells were washed to remove nonspecifically bound antibody and then incubated with 3,3',5,5'-tetramethylbenzidine (TMB). ) Substrate was added and the absorbance of the solution was measured at 450 nm (A450). ROR1-binding clones with A450 greater than 1.0 were selected for further characterization.

[0261] b) Expression and purification of selected soluble ScFvs The DNA sequences of the VH and VL of selected clones were determined, and the ScFvs encoded by the clones, each with a unique sequence, were expressed and purified as described below. Plasmids extracted from the clones were used to transform HB2151 cells. A single colony was picked from a plate containing freshly transformed cells and inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture reached an OD at 600 nm of 0.90, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was further incubated overnight at 30°C. The bacterial pellet was collected by centrifugation at 8,000 × g for 20 minutes and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation at room temperature for 30 minutes with rotation at 50 rpm, the resuspended pellet was centrifuged at 25,000 × g for 25 minutes at 4°C, and the supernatant was used to purify the ScFv using Ni-NTA resin according to the manufacturer's protocol (Qiagen).

[0262] c) ELISA Binding Assay 50 μl of recombinant human ROR1 diluted to 2 μg / ml in PBS was coated onto a 96-well plate overnight at 4°C. Serial dilutions of purified ScFvs bearing His and Flag tags were added to the target protein-coated wells. After washing, a 1:3000 dilution of HRP-labeled anti-Flag antibody was added for 1 hour at room temperature. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated for 10 minutes at room temperature. The reaction was stopped by adding 1N H2SO4, and the OD at 450 nm was read to quantify the relative ROR1-binding ability of the ScFvs.

[0263] result: Two ScFv clones specific for recombinant human ROR1 were identified and classified as human anti-ROR1 ScFv binders, ScFv4 and ScFv9. The very low binding activity indicates that these two binders represent completely unique and important new ROR1 binding sites that can be incorporated into CAR and immunoglobulin therapeutic constructs. The generation of chimeric antigen receptors expressing the LTG1941, LTG1942, LTG2528, and LTG2529 human anti-ROR1 binders is outlined in Example 2 below. [Example]

[0264] CAR expressing anti-ROR1 fully human binding sequence. Homo sapiens ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a well-studied cancer cell surface glycoprotein expressed in various solid tumors, including chronic lymphocytic leukemia (CLL) and some sarcomas, carcinomas, and adenocarcinomas of the lung. A phase 1 study of the anti-ROR1 antibody UC-961 (cirtuzumab) for relapsed or refractory chronic lymphocytic leukemia is currently underway (funded by Thomas Kipps, NCT02222688). Results of this first-in-class study are awaited. A phase 1 clinical trial using ROR1-specific CAR-T cells is also in its early stages (funded by Fred Hutchinson Cancer Institute, NCT02706392). We included published ROR1 binders as our controls (LTG1943, LTG2527) to benchmark our study and demonstrate the activity of our constructs (Hudecek et al., 2013, Clin Cancer Res 1 9:3153-3164). Given the current advances in T cell-based therapies, including the recent commercial availability of anti-CD19 CARs, the development of cell-based immunotherapies featuring the CAR constructs presented herein for ROR1-expressing CLL and other malignancies represents an innovative and novel approach to treating human disease using binding sites derived from human sequences.

[0265] The novel anti-ROR1 CAR-T constructs described herein exhibit high levels of cell surface expression in primary human T cells, specificity for ROR1-positive tumor cells, and potential cytotoxicity and cytokine function. As in Example 1, ROR1 CARs were designed using ROR1-binding sequences derived from ScFv candidates identified by phage display. For characterization, ROR1 CARs were cloned into lentiviral expression vectors containing selected structural and signaling domains under the control of the EF1a promoter and tested in vitro for transduction efficiency, killing function, and cytokine production in both model cell lines and primary human T cells. Table 1 provides an overview of the nomenclature used. The CAR construct LTG1943 is a relevant comparison, as this sequence has been proposed for clinical use (see KTE-C19, Kite Pharma, and CTL019, Novartis).

[0266] [Table 1]

[0267] material and method (a) Cell line All cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA) unless otherwise stated. The acute lymphocytic leukemia cell line REH and mantle cell lymphoma line Jeko-1 (ACC-553 DSMZ, Leibniz Institute DSMZ, Braunschwieg, Germany), and the chronic myeloid leukemia line K562 were cultured in 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Rapids, UT). The cells were cultured in RPMI-1640 medium supplemented with erythrocytes (Pan-1640, University of California, San Diego, NY).

[0268] (b) Construction of chimeric antigen receptor (CAR) expression vectors The ROR1 CAR construct was constructed by inserting each scFv in frame between the hinge and transmembrane domains of CD8 (aa 141182–191, UniProt no. P0173 CAR constructs were generated by combining either the CD8 transmembrane domain (aa 183-203, UniProt SEQ ID NO: P01732), the 4-1BB (CD137, aa 214-255, UniProt SEQ ID NO: Q07011) transactivation domain, and the CD3 zeta signaling domain (CD247, aa 52-163, Reference SEQ ID NO: NP_000725.1) with either the IgG4 hinge domain (aa 99-110, UniProt SEQ ID NO: P01861), followed by the CD8 transmembrane domain (aa 183-203, UniProt SEQ ID NO: P01732), the 4-1BB (CD137, aa 214-255, UniProt SEQ ID NO: Q07011) transactivation domain, and the CD3 zeta signaling domain (CD247, aa 52-163, Reference SEQ ID NO: NP_000725.1). To facilitate transport of the CAR to the T cell membrane, a leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit was included in all constructs. The CAR construct sequences were codon-optimized and cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology, Gaithersburg, MD).

[0269] Purification and transduction of primary T cells CD4 + and CD8 + Human primary T cells from healthy volunteers were purified from whole blood or buffy coats (purchased from a commercial supplier, with written donor consent) using immunomagnetic bead selection of cells according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured at densities of 0.3–2 × 10 in TexMACS medium supplemented with 200 IU / ml IL-2. 6Cells were cultured at 1000 / ml, activated with CD3 / CD28 MACS® GMP T Cell TransAct Reagent (Miltenyi Biotec), and transduced overnight with a lentiviral vector encoding a CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 2, with the medium changed on day 3. Cultures were propagated in TexMACS medium supplemented with 200 IU / ml IL-2 and harvested on days 8–12.

[0270] (d) Immune effector assays (CTL and cytokines) To examine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various effector-to-target (E:T) ratios and incubated overnight. SteadyGlo reagent (Promega, Madison, WI) was added to each well, and the resulting luminescence was analyzed using an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells with 1% Tween-20 (minimum CPS) were used to determine the assay range. The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Cytokine release assays were performed on supernatants collected after co-incubation of effector and tumor cell lines at an E:T ratio of 10. The cytokines IFNg followed by IL-2 were measured in triplicate by ELISA (Thermo Fischer, Waltham, MA).

[0271] Flow cytometry analysis For cell staining, 0.5 million CAR T-transduced cells were harvested from culture and washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with ROR1-Fc peptide (R&D, Minneapolis, MN) followed by anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). Non-transduced cells were used as a negative control. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice, resuspended in 200 μl of staining buffer, and then quantitatively analyzed by flow cytometry. MACSQuant® Flow cytometry analysis was performed using a Target 10 Analyzer (Miltenyi Biotec) and data plots were generated using FlowJo software (Ashland, OR).

[0272] result: A fully human CAR T construct targeting the ROR1 tumor antigen was designed by combining in-frame the sequences of a leader peptide derived from GMCSFR, an anti-human ROR1 ScFv, a CD8 or IgG4 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z activation domain. A schematic diagram of the CAR T construct and a list of the designed constructs and each ScFv target domain are provided (Figure 1 and Table 1). Untransduced T cells (UTD) grown under the same conditions were included as a control.

[0273] All test and control CAR constructs were cloned into an LV backbone expression vector under the control of the human Ef1-alpha promoter and used to generate lentiviral vector particles by transfection into 293 cells using a standard four-plasmid system. Activated human primary T cells were transduced with LV supernatant encoding the CAR test constructs or controls and expanded for 8–10 days in culture. Flow cytometry analysis using recombinant ROR1-Fc fusion protein followed by anti-Fc APC demonstrated surface expression of all test CAR constructs in transduced human T cells (Figure 3).

[0274] [Table 2]

[0275] T cells transduced with anti-ROR1 chimeric antigen receptors exhibit cytokine expression and cytolytic activity.

[0276] To assess the cytotoxic function of CAR T, CAR T cells were transfected with ROR1 T cells on days 8–10 of culture. + Mantle cell lymphoma Jeko-1, ROR1 + Epidermoid carcinoma A431, or ROR1 - The constructs were combined with Reh leukemia cells at E:T ratios of 40:1, 20:1, or 10:1 (Figure 3). A positive control CAR construct, LTG1943, based on the anti-ROR1 scFv R12 (see reference 9), and negative control T cells transduced with a lentiviral vector encoding GFP (1398) or untransduced T cells (UTD) were included for comparison. All constructs shown (LTG1941-1943) demonstrated dose-dependent ROR1-specific tumor killing. + Maximum cytotoxicity against tumor lines (Jeko-1 ROR1 + In cells, 80% (at an E:T ratio of 40:1) was demonstrated by the CAR construct LTG1942, and its tumor death The degree of inhibition was comparable to that of the control CAR construct LTG1943, based on the R12 anti-ROR1 scFv (reference 9). CAR construct LTG1941 also inhibited ROR1. + They showed relatively little cytotoxicity against tumor lines, with a maximum specific lysis of 40% against Jeko-1 cells at an E:T ratio of 40:1. In the A431 line, which is relatively less susceptible to CAR T-mediated lysis, CAR LTG1941 was ineffective, whereas CAR LTG1942 was as effective as or more effective than the positive control CAR LTG1943.

[0277] ROR1 was then expressed when induced by ROR1-positive and ROR1-negative tumor cell lines. The concentrations of the inflammatory cytokines IFN-gamma, TNF-alpha, and IL-2 secreted by CAR T cells transduced with the CAR constructs were measured (Figure 4). CAR T cells alone were included for each construct to examine basal levels of cytokine production. GFP (LTG1398)-transduced T cells were included as a negative control. + When challenged with Jeko-1 or A431 strains, TNF-alpha, IFN-gamma, and IL-2 levels were strongly induced by the CAR T cell construct LTG1942 and the positive control LTG1493, and to a lesser extent by the CAR LTG1491, but not by ROR1. -No cytokines were induced in the Reh control cell line. No cytokines were induced in the negative control GFP or UTD groups. Notably, the cytokine production level of LTG1941 was very low, indicating a difference in the ability to activate T cells expressing this vector. This corresponds to the lower cytolysis levels of LTG1941 vs. LTG1942 against the Jeko cell line, but not against A431. This indicates that, first of all, simply identifying a binder is not sufficient to determine the true activity of a CAR containing this binder in its sequence. Importantly, these differences may be crucial and allow for the modulation of CAR activity depending on the desired target antigen density. The ability to lyse tumor targets is intricately related to both the CAR expression level on the T cell surface and the target expression level of tumor antigens on the surface of target tumor cells (Walker, A. et al., 2017, Mol Ther 25:2189-2201). Therefore, if ROR1 expression on normal T cells, such as by LTG1492, induces CAR-T activity, constructs such as LTG1941 may be used to differentiate and target cancers despite low expression levels on normal tissues. Furthermore, none of the constructs demonstrated cytokine production above baseline in the absence of tumor cell targets, an important demonstration of the lack of autoactivation, as previously reported for some CAR constructs (Long, AH et al., 2015, Nature Med 20:581-590). Thus, the CAR-T constructs LTG1941, LTG1942, and LTG1943 were specific for the ROR1 tumor antigen expressed in model tumor cell lines.

[0278] Taken together, CARs LTG1942 and LTG1941 have oncolytic activity and ROR1 + The constructs described herein exhibited functional specificity, manifested as cytokine synthesis in response to tumors, and are therefore promising candidates for clinical use.

[0279] Each application and patent cited in the text, and each literature or prior art document cited therein (including each issued patent in litigation "application cited document"), and each PCT application or patent and foreign application or patent corresponding to and / or claiming priority to any of the applications and patents, and each literature cited or referenced in each application cited document, are expressly incorporated herein by reference and may be used in the practice of the invention. More generally, literature or prior art documents are cited either in the text, in a prior art list before the claims, or in the text itself, and each of the literature or prior art documents is expressly incorporated herein by reference. ("Incorporated Prior Art Documents"), and each document or prior art document cited within each Incorporated Prior Art Document (including manufacturer's specifications, instructions, etc.) is expressly incorporated herein by reference.

[0280] The above description of several specific embodiments provides sufficient information to enable others, by application of current knowledge, to easily modify or adapt the specific embodiments for various uses without departing from the general concept; therefore, such adaptations and modifications should be understood to be within the meaning and range of equivalents of the disclosed embodiments. It is understood that the words or terminology used herein are for purposes of description and not limitation. In the drawings and description, exemplary embodiments are disclosed, and specific terms may be used; however, these, unless otherwise indicated, are used generically and for purposes of description only and not of limitation, and therefore should not be construed as limiting the scope of the claims. Furthermore, those skilled in the art will understand that certain steps of the methods disclosed herein may be performed in a different order or steps may be combined. Therefore, it is not intended that the claims appended hereto be limited to the specific embodiments disclosed herein. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the inventive embodiments described herein. Such equivalents are encompassed by the following claims.

[0281] Sequences according to the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strand is included when referring to the displayed strand. In the accompanying sequence listing: SEQ ID NO: 1: Nucleotide sequence of anti-ROR1 binder: ScFV4 CAGGTGCAGCTGCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGTCGAGTCACCATACCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACACCTGGGGGGTGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGAG GTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGAGGCGGATCCCTGCCTGTGCTGACTCAGCCCCCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGT CTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTACTTCTGTCAGTCTTATGATAGCAGCAATCCCGTGGTATTCGGCGGAGGGACCCAGCTCACCGTTTTA SEQ ID NO: 2: Amino acid sequence of anti-ROR1 binder: ScFV4 QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARHLGGDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSN SGNTATLTISGTQAMDEADYFCQSYDSSNPVVFGGGTQLTVL SEQ ID NO: 3 ROR1-CAR DNA SEQ LTG1941 (LP-ScFV4-CD8H / CD8TM-41BB-CD3 Zeta) SEQ ID NO: 4 ROR1-CAR AA SEQ LTG1941 (LP-ScFV4-CD8H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARHLGGDAFDIWGQGTTVTV SSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISGTQAMDEADYFCQSYDSSNPVVFGGGTQLTVLAAATTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 5 ROR1-CAR DNA SEQ LTG2528 (LP-ScFV4-IgG4H / CD8TM-41BB-CD3 Zeta) SEQ ID NO: 6 ROR1-CAR AA SEQ LTG2528 (LP-ScFV4-IgG4H / CD8TM-41BB-CD3 Zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARHLGGDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAP VLVVYDDSDRPSGIPERFSGSNSGNTATLTISGTQAMDEADYFCQSYDSSNPVVFGGGTQLTVLAAAESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 7 Nucleotide sequence of anti-ROR1 binder: ScFV9 CAGGCGGCCCAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAGGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTTATAATGATGCTTTTGATATCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCAATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCATTGTGATCTATGAGGATGATCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACACCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGAGTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGAGCCCGGCAATGGGGTATTCGGCGGAGGGACCAAGGTCACCGTCCTA Sequence number 8 Amino acid sequence of anti-ROR1 binder: ScFV9 QAAQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNYAQRFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASYNDFDIWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTIVIYEDDQRPSGVPDRFSGSIDTSSNASLTISGLQSEDEADYCQSYEPGNGVFGGGTKVTVL SEQ ID NO:9 ROR1-CAR DNA SEQ LTG1942(LP-ScFV9-CD8H / CD8TM-41BB-CD3ゼータ) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAGGCGGCCCAGGTACAGCTGCAGCAGTCAGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAAACAGTGGTGGCACAAACTATGCAC AGAGGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTTATAATGATGCTTTTGATATCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGATCCAATTTTATGCTGACTCAGCCCACTCTGTGTCGGGATCTCCGGGGAAGACG GTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCATTGTGATCTATGAGGATGATCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACACCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGAGTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGAGCCCGGCAATGGGGTATTCGGCGGAGGGACCAAGGTCACCGTCCTAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 10 ROR1-CAR AA SEQ LTG1942 (LP-ScFV9-CD8H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQAAQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNYAQRFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAS YNDAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTIVIYEDDQRPSGVPDRFSGSIDTSSNSASLTISGLQSEDEADYY CQSYEPGNGVFGGGTKVTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 11 ROR1-CAR DNA SEQ LTG2529 (LP-ScFV9-IgG4H / CD8TM-41BB-CD3 Zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAGGCGGCCCAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAGGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTA SEQ ID NO: 12 ROR1-CAR AA SEQ LTG2529 (LP-ScFV9-IgG4H / CD8TM-41BB-CD3 Zeta) MLLLVTSLLLCELPHPAFLLIPQAAQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNYAQRFQGRVTMTRDTSISTAYMELSRLRSD DTAVYYCASYNDAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTIVIYEDDQRPSGVPDRFSGSIDTSSNS ASLTISGLQSEDEADYYCQSYEPGNGVFGGGTKVTVLAAAESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 13 Nucleotide sequence of a control anti-ROR1 binder: CAAGAACAGCTTGTAGAGTCCGGCGGTAGATTGGTGACACCGGGGGGGAGCCTTACCCTGTCTTGTAAGGCATCTGGGTTCGATTTCAGTGCGTATTATATGAGCTGGGTTCGGCAGGCGCCCGGGAAGGGGCTGGAATGGATAGCCACTATATACCCGTCATCCGGCAAGACTTACTA CGCGACTTGGGTAAACGGGAGGTTTACGATAAGCTCAGATAACGCCCAAAACACGGTTGATCTCCAAATGAATAGCTTGACCGCCGCTGATAGGGCGACCTATTTCTGTGCGCGGGACTCTTACGCTGATGACGGGGCCCTCTTCAATATATGGGGACCGGGAACGCTCGTAACCATATC ATCTGGAGGAGGTGGGAGCGGAGGCGGAGGGTCAGGTGGGGGCGGGAGCGAACTCGTACTTACACAATCTCCAAGCGTAAGCGCAGCGTTGGGGAGTCCAGCAAAGATCACCTGCACTTTGTCAAGCGCCCACAAAACGGATACGATAGATTGGTATCAGCAACTCCAAGGTGAAGCGCCACGATATCTCAT GCAGGTACAGAGCGACGGGAGTTATACTAAGAGGCCCGGGGTCCCAGACAGATTCAGTGGCAGCAGTTCAGGTGCCGACAGATACCTGATAATACCCTAGTTCAAGCCGATGATGAAGCCGATTACTACTGTGGGCTGACTACATAGGTGGGTATGTTTTCGGGGGCGGCACTCAATTGACAGTTACAGGG SEQ ID NO: 14. Amino acid sequence of a control anti-ROR1 binder: QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGG GSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG SEQ ID NO: 15 ROR1-CAR DNA SEQ Control LTG1943 (LP-Control ScFv-CD8H / CD8TM-41BB-CD3 Zeta) CCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAA AGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 16 ROR1-CAR AA SEQ Control LTG1943 (LP-Control ScFv-CD8H / CD8TM-41BB-CD3 Zeta) MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYA DDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADY YCGADYIGGYVFGGGTQLTVTGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 17 ROR1-CAR DNA SEQ Control LTG2527 (LP-Control ScFv-IgG4H / CD8TM-41BB-CD3 Zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAAGAACAGCTTGTAGAGTCCGGCGGTAGATTGGTGACACCGGGGGGGAGCCTTACCCTGTCTTGTAAGGCATCTGGGTTCGATTTCAGTGCGTATTATATGAGCTGGGTTCGGCAGGCGCCCGGGAAGGGGCTGGAATGGATAGCCACTATATACCCGTCATCCGGCAAGACTTACTACGCGACTTGGGTAAACGGGAGGTTTACGATAAGCTCAGATAACGCCCAAAACACGGTTGATCTCCAAATGAATAGCTTGACCGCCGCTGATAGGGCGACCTATTTCTGTGCGCGGGACTCTTACGCTGATGACGGGGCCCTCTTCAATATATGGGGACCGGGAACGCTCGTAACCATATCATCTGGAGGAGGTGGGAGCGGAGGCGGAGGGTCAGGTGGGGGCGGGAGCGAACTCGTACTTACACAATCTCCAAGCGTAAGCGCAGCGTTGGGGAGTCCAGCAAAGATCACCTGCACTTTGTCAAGCGCCCACAAAACGGATACGATAGATTGGTATCAGCAACTCCAAGGTGAAGCGCCACGATATCTCATGCAGGTACAGAGCGACGGGAGTTATACTAAGAGGCCCGGGGTCCCAGACAGATTCAGTGGCAGCAGTTCAGGTGCCGACAGATACCTGATAATACCCTCAGTTCAAGCCGATGATGAAGCCGATTACTACTGTGGGGCTGACTACATAGGTGGGTATGTTTTCGGGGGCGGCACTCAATTGACAGTTACAGGGGCGGCCGCAGAGTCAAAATACGGTCCTCCGTGCCCTCCGTGTCCGATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAA GAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGG AAACCACGGCGGAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGCGGAAGCCTACTCAGAAATCGGGAATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO:18 ROR1-CAR AA SEQ CONTROLLTG2527(LP-CONTROLScFv-IgG4H / CD8TM-41BB-CD3ゼータ) MLLLVTSLLLCELPHPAPFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGAR DRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGAAAESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 19 Nucleotide sequence of leader / signal peptide sequence (LP) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG SEQ ID NO: 20: Amino acid sequence of leader / signal peptide sequence (LP) MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 21 DNA CD8 transmembrane domain nucleotide sequence ATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGC SEQ ID NO: 22: Amino acid sequence of CD8 transmembrane domain IWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 23 DNA CD8 hinge domain nucleotide sequence ACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTAC SEQ ID NO: 24: Amino acid sequence of CD8 hinge domain TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY SEQ ID NO: 25: Amino acid sequence of the hinge and transmembrane region of CD8 alpha from amino acids 137 to 206 (NCBI REFSEQ: NP__001759.3) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 26 Nucleotide sequence of the signaling domain of 4-1BB AAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATG CGAACTG SEQ ID NO: 27: Amino acid sequence of the signaling domain of 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 28 Nucleotide sequence of the intracellular signaling domain of CD3-zeta CGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAA GGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 29: Amino acid sequence of CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 30 Nucleotide sequence of the intracellular signaling domain of CD3-zeta, variant CGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATAAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAACCACGGCGGAAAAACCCTCAGGAA GGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 31: Amino acid sequence of CD3-zeta signaling domain, variant RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 32 Nucleotide sequence of ScFV CD19 (FMC63) GACATTCAGATGACTCAGACCACCTCTTCCTTGTCCGCGTCACTGGGAGACAGAGTGACCATCTCGTGTCGCGCAAGCCAGGATATCTCCAAGTACCTGAACTGGTACCAACAGAAGCCCGACGGGACTGTGAAGCTGCTGATCTACCACACCTCACGCCTGCACAGCGGAGTGCCAAGCAGATTCTCCGGCTCCGGCTCGGGAACCGATTACTCGCTTACCATTAGCAACCTCGAGCAGGAGGACATCGCTACCTACTTCTGCCAGCAAGGAAATACCCTGCCCTACACCTTCGGCGGAGGAACCAAATTGGAAATCACCGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCGAAGTGAAGCTCCAGGAGTCCGGCCCCGGCCTGGTGGCGCCGTCGCAATCACTCTCTGTGACCTGTACCGTGTCGGGAGTGTCCCTGCCTGATTACGGCGTGAGCTGGATTCGGCAGCCGCCGCGGAAGGGCCTGGAATGGCTGGGTGTCATCTGGGGATCCGAGACTACCTACTACAACTCGGCCCTGAAGTCCCGCCTGACTATCATCAAAGACAACTCGAAGTCCCAGGTCTTTCTGAAGATGAACTCCCTGCAAACTGACGACACCGCC ATCTATTACTGTGCTAAGCACTACTACTACGGTGGAAGCTATGCTATGGACTACTGGGGGCAAGGCACTTCGGTGACTGTGTCAAGC Amino acid sequence of SEQ ID NO: 33 ScFV CD19 (FMC63) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG SEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQ ID NO: 34 Nucleotide sequence of anti-CD33 ScFV (LTG1936) CAGGTGCAGCTGGTGCAATCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATTCAGTTTTCCCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTAGTTGGAGATGGCTACAATACGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCGATATTGTGATGACCCACACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAAGACCTATTTGTATTGGTACCTGCAGAAGCCAGGCCAGCCTCCACAGCTCCTGATCTATGGAGCTTCCAACCGGTTCTCTGGAGTGCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA Sequence number 35 Amino acid sequence of anti-CD33 ScFV (LTG1936) QVQLVQSGAEVKKPGESLRISCKGSGFSFPTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLVGDGYNTGAFDIWGQGTMVTVSSGG GGSGGGGSGGGGSDIVMTHTPLSVTPGQPASISCKSSQSLLHSNGKTYLYWYLQKPGQPPQLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCMQSIQLPITFGQGTRLEIK SEQ ID NO: 36 Nucleotide sequence of anti-mesothelin ScFV (LTG1904) GAGGTCCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCC GATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATTTATCGTCAGTGGCTGGACCCTTTAACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGCGGTGGC GGATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGC CCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGCGGAGGCACCCAGCTGACCGTCCTCGGT SEQ ID NO: 37 Amino acid sequence of anti-mesothelin ScFV (LTG1904) EVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLSSVAGPFNYWGQGTLVTVSSGG GGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTQLTVLG SEQ ID NO: 38 Nucleotide sequence of IgG4H (hinge) GAGTCAAAATACGGTCCTCCGTGCCCTCCGTGTCCG SEQ ID NO: 39 Amino acid sequence of IgG4H (hinge) ESKYGPPCPPCP SEQ ID NO: 40 Nucleotide sequence of the hinge domain of IgG4H bound to CD8 TM (transmembrane) GAGTCAAAATACGGTCCTCCGTGCCCTCCGTGTCCGATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGC SEQ ID NO: 41 Amino acid sequence of the hinge domain of IgG4H bound to CD8 TM (transmembrane) ESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYC

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain comprising a ROR1 antigen-binding domain encoded by a nucleotide sequence comprising SEQ ID NO:1 or SEQ ID NO:7, at least one transmembrane domain, and at least one intracellular signaling domain.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one ROR1 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to ROR1.

3. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one ROR1 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to ROR1.

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

5. 5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of IgG4, CD8, or CD28 and is linked to a transmembrane domain.

6. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular ROR1 antigen-binding domain is located after a leader nucleotide sequence encoding a leader peptide.

7. 7. The isolated nucleic acid molecule of claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 19, which encodes the leader amino acid sequence of SEQ ID NO:

20.

8. 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.

9. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular ROR1 antigen-binding domain comprises a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, or 11, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

10. 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.

11. 11. The isolated nucleic acid molecule of claim 10, wherein the encoded at least one intracellular signaling domain is located N-terminal to the CD3 zeta intracellular domain.

12. 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, or any combination thereof.

13. The encoded at least one costimulatory domain may be OX40, CD70, CD27 , CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

14. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.

15. The CAR of claim 14, comprising at least one extracellular antigen-binding domain comprising a ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, at least one transmembrane domain, and at least one intracellular signaling domain.

16. The CAR of claim 15, wherein the ROR1 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to ROR1.

17. The CAR of claim 15, wherein the ROR1 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to ROR1.

18. 16. The CAR of claim 15, 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, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

19. 19. The CAR of claim 18, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

22.

20. The CAR of claim 15, wherein at least one extracellular antigen-binding domain comprising the ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, and the at least one intracellular signaling domain, or both, are linked to the transmembrane domain by a linker or spacer domain.

21. The CAR of claim 20, wherein the linker or spacer domain is derived from the extracellular domain of IgG4, CD8, or CD28 and is linked to the transmembrane domain.

22. The CAR of claim 15, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

23. The CAR of claim 22, 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, and 4-1BB (CD137), or a combination thereof.

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

25. The vector may 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.

25. The vector of claim 24, selected from the group consisting of:

26. 25. The vector of claim 24, further comprising a promoter.

27. 27. The vector of claim 26, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

28. A cell comprising the vector of claim 24.

29. 29. The cell of claim 28, wherein the cell is a T cell.

30. 30. The cell of claim 29, wherein the T cell is a CD8+ T cell.

31. 29. The cell of claim 28, wherein the cell is a human cell.

32. A method for producing cells, comprising the step of transducing a T cell with the vector of claim 24.

33. 10. A method for generating a population of RNA-engineered cells, comprising the step of introducing in vitro transcribed or synthesized RNA into cells, said RNA comprising the nucleic acid molecule of claim 1.

34. 30. A method of providing anti-tumor immunity to a mammal, comprising administering to said mammal an effective amount of the cells of claim 28.

35. A method for treating or preventing cancer in a mammal, comprising a step of administering to the mammal the CAR of claim 15 in an amount effective for treating or preventing cancer in the mammal.

36. 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 chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, including a ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a human with cancer.

37. 37. The pharmaceutical composition of claim 36, 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.

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

39. 39. The pharmaceutical composition of claim 38, wherein the hematological cancer is leukemia or lymphoma.

40. 40. The pharmaceutical composition of claim 39, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML).

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

42. 39. The pharmaceutical composition of claim 38, wherein the hematological cancer is multiple myeloma.

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

44. 1. A method 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 an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, the T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, the T cells being T cells of the subject with cancer.

45. 1. A method of treating cancer in a subject in need thereof, the method comprising: administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, the T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a ROR1 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of the subject with cancer.

46. 46. ​​The method of claim 44 or 45, 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.

47. 10. A process for producing a chimeric antigen receptor-expressing cell, the process comprising introducing the isolated nucleic acid of claim 1 into a cell.

48. 48. The process for producing a chimeric antigen receptor-expressing cell of claim 47, wherein the cell is a T cell or a cell population containing a T cell.

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