Compositions and methods for treating cancer by anti-ROR1 immunotherapy

Abstract

To provide new B cell malignancy targets such as ROR1.SOLUTION: Chimeric antigen receptors containing ROR1 antigen binding domains are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the chimeric antigen receptors, are also disclosed. Methods of treating or preventing cancer in a subject, and methods of making chimeric antigen receptor T cells are also disclosed.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 581,284, filed on November 3, 2017. The entire disclosure of the U.S. Provisional Patent Application is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format, and the entire sequence listing is incorporated herein by reference. The file name of this ASCII copy (created on October 25, 2018) is "SequenceListing.txt", and the size is 90.0 kilobytes.

[0003] Description of Research or Development Sponsored by the Federal Government This invention was made by the realization 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 This application relates to the field of cancer, in particular, to the ROR1 antigen - binding domain, a chimeric antigen receptor (CAR) containing this ROR1 antigen - binding domain, and methods of using the same.

Background Art

[0005] Background Cancer is one of the most lethal threats to human health. In the United States alone, nearly 1.3 million people are newly diagnosed with cancer each year, making it the second leading cause of death after cardiovascular disease, and accounting for one in four deaths. Most of these deaths are caused by solid tumors. Although medical treatments for some specific cancers have advanced significantly, the 5-year survival rate for all cancers combined has only improved by about 10% over the past 20 years. Treating cancer is extremely difficult because the metastasis and growth of cancer or malignant tumors are rapid and uncontrollable.

[0006] In the treatment of solid and liquid tumors, many therapeutic requirements are not met. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an embryonic protein that is highly expressed in many types of cancer, including chronic lymphocytic leukemia (CLL), breast cancer, glioblastoma, lung adenocarcinoma, and sarcomas (Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and fibrosarcoma), but is generally absent 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 transmembrane glycoprotein consisting of 937 amino acids (signal peptide 1-29) with a molecular weight of 104 kDa (up to 120 kDa depending on glycosylation). It includes a protein and two variants, in intracellular and secreted forms, smaller than it (GeneBank NP_005003, Masiakowski, P. and Carroll, R.D., 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 indicates that targeting ROR1 may enable the development of new cancer treatments for many liquid cancers such as chronic lymphocytic leukemia (CLL) and 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] Generally, it does not exist in adult tissues, but according to at least one report, ROR1 expression has been found in the parathyroid gland, pancreatic islets, and the 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 provides a basis for 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 the signaling of Wnt and EGFR (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). Due to its role in contributing to the tumor phenotype, it may play an important function in tumor initiation or progression, and thus is shown to be a driver protein.

[0008] Previous approaches in cancer treatment include surgery, radiotherapy, chemotherapy, and bone marrow transplantation for hematomas. However, current primary treatments indicate that further improvement is needed. Such improvement is required for new immunotherapy strategies. Current ongoing pre - clinical and clinical trial studies targeting the ROR1 antigen are constructed using multiple modalities. T lymphocytes expressing ROR1 - specific CAR 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 116:4532-41.). The lack of toxicity in non-human primates has led to the confidence that studies in humans can be initiated (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 treatment for B-lineage leukemia is thought to involve consolidation therapy following remission induction with high-dose chemotherapy or radiotherapy, and may be characterized by stem cell transplantation and further chemotherapy as needed (see cancer.gov on the World Wide Web). Such treatments are highly toxic and carry a risk of complications such as relapse, secondary malignancies, or GVHD, and thus better alternative treatments are being explored. 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 access date: September 20, 2017.), and ROR1-specific antibodies for breast cancer within the scope of 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 access date: September 20, 2017).

[0009] Chimeric antigen receptors (CARs) are hybrid molecules consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a hinge / 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 generally engineered to mimic the single-chain fragment variable (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs such as receptor ligands (e.g., IL-13 engineered to bind to the IL-13 receptor expressed in tumors), full-length immunoreceptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D) are also incorporated by design. Other cell targets for CAR expression (such as 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 highly active T-cell population to transduce with a CAR vector and finding optimal culture and expansion techniques requires considerable further effort to elucidate the structure of the CAR protein itself in detail at the molecular level.

[0010] The CAR binding motif may 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 to a position far from the T cell membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the surface membrane of tumor cells (such as disialoganglioside GD2; Orentas et al., this observation is unpublished). All of the signaling motifs used in CARs to date include the CD3-zeta chain. This is because this core motif is an important signal for T cell activation. The first reported second-generation CARs were characterized by the CD28 signaling domain and the CD28 transmembrane sequence. This motif was used in third-generation CARs that further contain the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the emergence of new technologies, it is no longer necessary for the CAR itself to encode the activation of T cells by beads conjugated with anti-CD3 and anti-CD28 antibodies, as well as the presence of the classical "signal 2" from CD28. Third-generation vectors using bead activation were found not to outperform second-generation vectors in in vitro assays, and furthermore did not provide a clear benefit over second-generation vectors in a leukemia mouse model (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 has been demonstrated by the clinical success of CD19-specific CARs in second-generation 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 modalities (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 persistence signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions for culturing CAR T cell populations, such as including cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther. 2015;22(2):72-78).

[0011] Currently, the challenges in more broadly and effectively applying CAR therapy to cancer are related to the fact that there are few promising targets. The generation of binders that bind to cell surface antigens can now be easily achieved, but the discovery of cell surface antigens that are tumor-specific and do not affect normal tissues remains very difficult. As a method that can confer stronger target cell specificity to CAR-expressing T cells than before, there is a method of using a combination of multiple CAR approaches. In one system, the CD3-zeta signal unit and the CD28 signal unit are divided into each of two different CAR constructs expressed in the same cell, and in another system, two CARs are expressed in the same T cell, but since one of them has a lower affinity, it is necessary to first bind the other CAR to maximize the activity of the latter CAR (Lanitis E et al. Cancer Immu (nol Res. 2013; 1(1): 43-53; Kloss CC et al. Nat Biotechnol. 2013; 31(1): 71-5). The second issue in creating a CAR based on a single ScFv as an immunotherapeutic agent is that tumor cells are not homogeneous. At least one group has developed a treatment method for glioblastoma using CAR, and this method targets multiple antigens (HER2, IL-13Ra, EphA2) simultaneously in the effector cell population and attempts to avoid the growth of the population without the target antigen (Hegde M et al. Mol Ther. 2013; 21(11): 2087-101).

[0012] T cell-based immunotherapy has become a new cutting-edge area in synthetic biology. Multiple promoters and gene products have been conceived for the purpose of inducing these highly potent cells into the tumor microenvironment, and T cells can mediate effective tumor death by avoiding negative control signals in the tumor microenvironment. The method of removing unwanted T cells by dimerization of an inducible caspase 9 construct (drug-induced dimerization) using a dimerization-inducing chemical such as AP1903 presents one strategy that can pharmacologically initiate a powerful switch to control the T cell population (Di Stasi A et al. N Engl J Med. 2011; 365(18): 1673-83). Furthermore, the method of creating an effector T cell population that does not respond to the negative regulatory action of transforming growth factor β by expressing a decoy receptor indicates the extent to which effector T cells can be manipulated to obtain optimal antitumor activity (Foster AE et al. J Immunother. 2008; 31(5): 500-5). Therefore, although CAR seems to be able to trigger T cell activation in a manner similar to the endogenous T cell receptor, at present, the limited proliferation of CAR+ T cells in vivo, the disappearance of these cells soon after infusion, and the poor clinical activity are major obstacles, and the clinical application of this technology has not advanced. This may be partly due to the fact that some of the CAR sequences used are of mouse origin.

Summary of the Invention

Problems to be Solved by the Invention

[0013] Whether patients who have received either antibody or CAR-T therapy need to subsequently undergo HSCT to sustain the response is still an area of active debate. High responses have been reported in CD19 CAR-T trials, but at least 20% of patients have ended unsuccessfully in a short period (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). As the best result reported at the 12-month time point after CAR19 treatment, 55% of the patients who could receive the T cell product at the University of Pennsylvania showed RFS, and 79% showed OS (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). Since the expected long-term response was less than 50%, there is still a great clinical need for new B cell malignancy targets such as ROR1.

Means for Solving the Problems

[0014] The present invention addresses the above-described need by providing CAR compositions and treatment methods that can be used for the treatment of cancer and other diseases and / or conditions. In particular, the present invention disclosed and described herein provides a CAR that can be used for the treatment of diseases, disorders, or conditions associated with dysregulated expression of ROR1, and this CAR contains a ROR1 antigen-binding domain with high surface expression on transduced T cells, and the degree of cell lysis of ROR1-expressing cells is high, and the transduced T cells proliferate and persist in vivo. ysis of ROR1-expressing cells is high, and the transduced T cells proliferate and persist in vivo.

[0015] Summary Provided herein are novel anti-ROR1 antibodies, or antigen-binding domains thereof, and chimeric antigen receptors (CARs) containing such ROR1 antigen-binding domains, and host cells (e.g., T cells) expressing such receptors, and nucleic acid molecules encoding such receptors. The CAR has high surface expression on transduced T cells, high cytotoxicity, and the transduced T cells proliferate and persist in vivo. Further provided are methods of using the disclosed CARs, host cells, and nucleic acid molecules, e.g., for treating cancer in a subject.

[0016] Thus, in one aspect, provided is an isolated polynucleotide encoding a human anti-ROR1 antibody or a fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and 7.

[0017] In one embodiment, provided is an isolated polynucleotide encoding a fully human anti-ROR1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, an F(ab’)2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

[0018] In one embodiment, provided is an isolated polynucleotide encoding a fully human anti-ROR1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 8.

[0019] In one aspect, provided is an isolated nucleic acid molecule encoding a chimeric antigen receptor (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 NO: 1 and 7, from N-terminus to C-terminus, at least one transmembrane domain, and at least one intracellular signaling domain.

[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 encoded extracellular ROR1 antigen-binding domain of the 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 bound 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 located downstream of a sequence encoding a leader or signal peptide.

[0025] In yet another embodiment, an isolated nucleic acid molecule 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 NO: 1 and 7 is provided, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen comprising, 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, there is provided an isolated nucleic acid molecule encoding a CAR, 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, an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

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

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

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

[0031] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises the 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 co-stimulatory 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 co-stimulatory 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 combinations thereof.

[0035] In one embodiment, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) further containing a leader sequence or a signal peptide is provided, 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, a chimeric antigen receptor (CAR) comprising, from the N-terminus to the C-terminus, at least one ROR1 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain is provided herein.

[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 the at least one transmembrane domain comprises the 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, where 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, where the extracellular antigen-binding domain comprises 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 an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0042] In another embodiment, a CAR is provided, where at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0043] In yet another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory 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 comprises the nucleic acid sequence of SEQ ID NO: 3 (LTG 1941 LP-ScFV4-CD8H / CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 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-CD3 zeta CAR amino acid sequence (Figure 2A)).

[0045] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5 (LTG 2528 LP-ScFv4-IgG4H / CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2B)). 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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 including the CAR nucleic acid sequence (Figure 2D)). 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-CD3 zeta CAR amino acid sequence (Figure 2D)).

[0048] In one aspect, the CARs disclosed herein are modified to express or contain a detectable marker for use in the diagnosis, monitoring, and / or prediction of treatment outcomes such as progression-free survival of cancer patients or for monitoring the progress of such treatments.

[0049] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector such as a viral vector. The vector is 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 vector expressing the CAR may be further modified to include one or more operable elements for controlling the expression of CAR T cells or for removing CAR-T cells by a suicide switch. The suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0052] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is further provided. 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 comprising an anti-tumor effective amount of a population of human T cells is provided, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises 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 human T cells having cancer. The cancer includes, inter alia, blood cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma), or multiple myeloma, or combinations thereof.

[0054] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of a CAR contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a 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 combinations thereof.

[0055] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer includes adult cancers such as oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, interhepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung, and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, genital system (cervix, corpus uteri, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous systems, or any combination thereof.

[0056] In yet another embodiment, a pharmaceutical composition is provided that contains a population of human T cells from a human having cancer in an anti-tumor effective amount, wherein the cancer is a refractory cancer that does not respond to one or more chemotherapeutic agents. The cancer is hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (B-line including ALL (acute lymphoblastic 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 aspect, a method for generating CAR-containing T cells (hereinafter referred to as "CAR-T cells") is provided. This method includes the step of transducing a vector or nucleic acid molecule encoding a CAR (disclosed) that specifically binds to ROR1 into T cells, thereby generating CAR-T cells.

[0058] In yet another aspect, a method for generating a population of RNA-engineered cells is provided, which includes the step of introducing in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding the disclosed CAR into the 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 the expression of ROR1 in a cell is provided, which includes: a) contacting the cell with a human anti-ROR1 antibody or a fragment thereof, wherein the antibody or the fragment thereof includes 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 that it is a disease, disorder, or condition associated with the expression of ROR1 when ROR1 is present.

[0060] In one embodiment, the disease, disorder, or condition associated with the expression of ROR1 is a cancer including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adult B-cell malignancies including non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (including B-line ALL (acute lymphoblastic 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, prognosticating, or determining the risk of an ROR1-related disease in a mammal is provided, which comprises the step of detecting the expression of ROR1 in a sample derived from the mammal, the step comprising: a) contacting the sample with a human anti-ROR1 antibody or a fragment thereof, the antibody or fragment thereof comprising 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 that the mammal has an ROR1-related disease when ROR1 is present.

[0062] In another embodiment, a method for inhibiting ROR1-dependent T cell inhibition is provided, which comprises the step of contacting a cell with a human anti-ROR1 antibody or a fragment thereof, the antibody or fragment thereof comprising 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 ROR1-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0063] In another embodiment, a method for blocking T cell inhibition mediated by ROR1-expressing cells and changing the tumor microenvironment to inhibit tumor growth in a mammal is provided, which comprises administering to the mammal, in an effective amount, a composition comprising an isolated anti-ROR1 antibody or a fragment thereof, the antibody or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 8. In one embodiment, the cell is selected from the group consisting of ROR1-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0064] In another embodiment, the immunity of an anti-tumor or anti-cancer immune response in a mammal Methods are provided for inhibiting, suppressing, or preventing, which include administering to a mammal, in an effective amount, 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 the interaction between a first cell and a T cell, and the first cell is selected from the group consisting of ROR1-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0065] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, which includes administering to a mammal, in a therapeutically effective amount, T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0066] In another embodiment, a method for treating or preventing cancer in a mammal is provided, which includes administering to the mammal, in an amount effective for treating or preventing cancer in the mammal, one or more of the disclosed CARs. The above method includes administering to the subject, in a therapeutically effective amount, host cells expressing a CAR (disclosed) that specifically binds to one or more of ROR1 and / or the above-described antigens under conditions sufficient to form an immune complex consisting of the antigen-binding domain of the CAR, the extracellular domain of ROR1, and / or one or more of the above-described antigens in the subject.

[0067] In yet another embodiment, a method for treating a mammal having a disease, disorder, or condition associated with increased expression of a tumor antigen is provided, the method including administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises 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, at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer.

[0068] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, which comprises administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and 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 linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer. In some embodiments of the methods described above, the at least one transmembrane domain comprises the 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 for generating a population of persistently genetically engineered T cells in a human diagnosed with cancer is provided. In one embodiment, the method comprises administering to the human T cells genetically engineered to express a 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 transmembrane domain, and at least one intracellular signaling domain, and the population of persistently genetically engineered T cells, or the 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 comprise 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 cancer, disease, disorder, or condition associated with increased expression of a tumor antigen, as described above, can be treated, prevented, or alleviated using one or more of the CARs disclosed herein.

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

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

[0074]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0075] Detailed Description Definitions As used herein, the singular forms "a", "an", and "the" refer to both the singular and plural forms unless the context clearly dictates otherwise. For example, the term "an antigen" can include 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 phrase "and / or" means "and" or "or". Further, unless otherwise specified, 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 values provided for illustrative purposes. Many methods and materials similar or equivalent to those described herein can be used, but particularly preferred methods and materials are described below. In case of any conflict, the present specification (including explanations of terms) will control. In addition, 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] When the term "about" refers to measurable values such as amounts and durations, it means including a variable 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. This is because such variables are appropriate for the implementation of the disclosed methods.

[0077] Unless otherwise specified, scientific terms in this specification are used in their conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by 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 materials.

[0078] The present disclosure provides an ROR1 antibody or a fragment thereof, and a chimeric antigen receptor (CAR) having such an ROR1 antigen-binding domain. Improving the functional activity of the CAR is directly related to improving the functional activity of CAR-expressing T cells. As a result of making one or more of such modifications, the CAR exhibits both cytokine-induced cytolysis and cell surface expression in transduced T cells at high levels, and the proliferation of T cells in vivo and and the persistence of transduced CAR-expressing T cells are at high levels.

[0079] The unique ability to combine functional parts derived from different protein domains is an innovative feature of chimeric antigen receptors (CARs). The choice of which of these protein domains to select is an important design feature, just like the manner in which they bind specifically. Individual design domains are essential components that can be used in any heterologous CAR platform for the purpose of manipulating lymphocyte function. For example, the selection of the extracellular binding domain can render a CAR that is otherwise ineffective effective.

[0080] The non-variable framework components of the sequence of an immunoglobulin-derived protein used to create the extracellular antigen-binding domain of a CAR may be completely neutral or may be such that they self-associate and cause metabolic exhaustion of T cells, significantly reducing the effectiveness of the therapeutic T cells expressing this CAR. This phenomenon occurs independently of the antigen-binding function of this CAR domain. Furthermore, the selection of the intracellular signaling domain can also govern the activity and durability of the population of therapeutic lymphocytes used in immunotherapy. Here, the ability to bind a target antigen and to transmit an activation signal to T cells through each of the extracellular and intracellular domains described above are important CAR design aspects, but it has also become clear that the choice of the source of the extracellular antigen-binding fragment can have a significant effect on the efficacy of the CAR and thus potentially play a decisive role in the function and clinical utility of the CAR.

[0081] Surprisingly and unexpectedly, it has been found that using 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 cell clearance in the host) (see clinical trial using a mouse-derived SS1 ScFv sequence with funding from the University of Pennsylvania, NCT02159716), can determine the functional activity of CAR-expressing T cells.

[0082] In light of this discovery, multiple ROR1 binders from a human scFv expression library were developed. Since these fully human ROR1 CARs are not of murine origin, they are 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, injecting patients after expressing these "fully human" CARs in T cells may lead to higher therapeutic efficacy. Such CAR binders derived from human sequences can be used for the treatment of cancers, leukemias, and lymphomas (including but not limited to B - CLL, ovarian cancer, triple - negative breast cancer, lung adenocarcinoma, and glioblastoma) that express the ROR1 antigen in humans (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, E.H. et al., Cell Biochem Funct, 34:149 - 157).

[0083] The CARs disclosed herein are expressed at high levels in cells. The cells expressing this CAR have a high growth rate in vivo, produce large amounts of cytokines, and have high cytotoxic activity against cells having the ROR1 antigen to which the CAR binds on their surface. As a result of using the human extracellular ROR1 antigen - binding domain, CARs with improved in vivo function were generated, while at the same time, the induction of anti - CAR immunity in the host immune response and the death of the CAR T cell population were avoided. CARs expressing the fully human extracellular ROR1 ScFv antigen - binding domain exhibit excellent activity and / or properties, which include i) CAR T's Prevention of poor persistence and poor function (seen in the mouse-derived binding sequences), ii) lack of delivery of the CAR to a specific region (i.e., intrapleural) for efficacy, and iii) the ability to enable the design of CAR T cells based on both high and low affinity ROR1 binders. The latter property allows researchers to better regulate the efficacy vs. toxicity and / or tissue specificity of CAR T products. This is because ROR1 is more highly expressed in tumors than in normal tissues, so the lower affinity binder may have higher specificity for tumors than for normal tissues, thereby preventing on-target off tumor toxicity and bystander cell killing.

[0084] Next, the CARs of the present invention will be described in detail. This description includes descriptions of its extracellular ROR1 antigen-binding domain, transmembrane domain, and intracellular domain, as well as further descriptions of CARs, antibodies, and their antigen-binding fragments, conjugates, nucleotides, expression, vectors, and host cells, treatment methods using the disclosed CARs, compositions, and kits.

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

[0086] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (ScFv)) that is linked to a T-cell signaling domain via a transmembrane domain. The characteristics of CAR include the ability to redirect the specificity and reactivity of T cells to a selected target in a manner not restricted by MHC and to utilize the antigen-binding properties of monoclonal antibodies. Since it can recognize antigens without MHC restriction, T cells expressing CAR have the ability to recognize antigens independently of antigen processing and, as a result, can avoid the major mechanisms of tumor escape. Also, when expressed in T cells, CAR advantageously does 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 CAR may include, for example, a T-cell receptor signaling domain, a T-cell costimulatory signaling domain, or both. The T-cell receptor signaling domain refers to a portion of CAR that includes an intracellular domain of a T-cell receptor, such as (but not limited to) the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of CAR that includes an intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for lymphocytes to efficiently respond to an antigen.

[0088] 1. Extracellular domain In one embodiment, CAR includes a target-specific binding element, also referred to as an antigen-binding domain or site. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can function as ligands for the antigen-binding domain in CAR include those associated with viral infection, bacterial infection, and parasitic infection, autoimmune diseases, and cancer cells.

[0089] In one embodiment, the CAR can be designed to target a desired tumor antigen by designing a desired antigen-binding domain that specifically binds to an antigen on the tumor cell. Tumor antigens are proteins produced by tumor cells that manifest an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain may 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 carcinoma 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 merely by way of example. The listing is not intended to be limiting, and other examples will be readily recognized by those skilled in the art.

[0090] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. 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 prostate 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 cancer gene HER-2 / Neu / ErbB-2. Further, as another group of target antigens, there are cancer fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins correspond to truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, CD22, BCMA, ROR1, and CD37 are also candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) are used as targets for passive immunotherapy using monoclonal antibodies, but have not achieved sufficient success.

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

[0092] The type of tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur on other cells of the body. TAA is not unique to tumor cells; instead, it is expressed on normal cells under conditions where immune tolerance to this antigen is not induced. The expression of this antigen in tumors can occur under conditions that allow the immune system to respond to this antigen. TAA can be an antigen that is expressed on normal cells during fetal development when the immune system is not mature and cannot respond to antigens, or TAA can be an antigen that is normally present at very low levels on normal cells but is expressed at a significantly higher level on tumor cells.

[0093] Examples of TSA or TAA include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed cancer genes and mutant tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7, but are not limited to these. Other large protein-based antigens include 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, p15, p16, 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 antigens 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, the 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 to the amino acid sequence of SEQ ID NO: 2.

[0097] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular ROR1 antigen-binding domain ScV9 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 to the amino acid sequence of SEQ ID NO: 8.

[0098] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular ROR1-controlled 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-controlled 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 to the amino acid sequence of SEQ ID NO: 14.

[0099] In various embodiments of the ROR1-specific CARs disclosed herein, a schematic scheme is depicted in FIG. 1, which, from the N-terminus to the C-terminus, includes 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, where the boldface letters represent the cloning sites for 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-CD3 zeta amino acid sequence (shown in FIG. 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1941 LP-ScFv4-CD8H / CD8TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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-CD3 zeta 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2528 LP-ScFv4-CD8H / CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1942 LP-ScFv9-CD8H / CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2529 LP-ScFv9-IgG4H / CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1943 LP-control ScFv-CD8H / CD8TM-41BB-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2527 LP - control ScFv - IgG4H / CD8TM - 41BB - CD3 zeta amino acid sequence (shown in Figure 2F)].

[0112] Surface expression of anti - ROR1 CAR incorporating a single - chain variable fragment (ScFv) sequence reactive with the ROR1 antigen is shown in Example 2 below, and an overview is shown 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 a recombinant ROR1 - Fc peptide followed by an anti - human Fc F(ab’)2 fragment conjugated to AF647 and detected in the APC channel (see Figure 3). The ScFv - based anti - ROR1 CAR constructs LTG1941, LTG1942 - LTG1943 were highly expressed in human primary T cells (shown by the gated population) compared to non - transduced T cell controls (ungated cell population). Representative results from one donor are shown.

[0113] As shown in Example 2 and FIG. 4, a lentiviral vector (LV) expressing the following CARs was prepared and tested for anti-leukemia activity, and high cytolytic activity of the ROR1 CAR was shown. All CARs used in the experiments contained the described 4-1BB / CD3 zeta chain signaling motif and specific anti-ROR1 binding motif / domain. Leukemia target strains 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 had no specific lytic activity against Reh (see FIG. 4). The Jeko-lysing abilities of anti-ROR1 CARs LTG1941 and LTG1942 were different, indicating that their biological activities were also different. From these results, the efficiency and specificity of the prepared CAR constructs were shown.

[0114] Next, the cytokine secretion ability of anti-ROR1 CAR T cells was evaluated. Tumor cells were incubated overnight with CAR T cells or control T cells at an effector-target ratio of 10:1, and the culture supernatants were analyzed for IFN gamma, TNF alpha, and IL-2 by ELISA (see FIG. 5). Notably, CAR T expression Cells LTG1942 and LTG1943 produced high levels of IFN gamma, and LTG1941 produced IFN-gamma in moderate amounts in response to Jeko, but not in the case of the A431 leukemia cell line. Similar results were also seen for the expression of IL-2 and TNF-alpha. Negative controls (untransduced T cells, UN, or T cells transduced with control LTG1398 LV) did not show measurable cytokine induction. Importantly, what the results seen in cytotoxicity and cytokine function clearly show is that LTG1942 has a similar ability to LTG1943, which is a CAR with a control ScFv, but the cytokine production of LTG1941 is significantly lower than this, and the ability to lyse the Jeko leukemia cell line is also significantly lower. Nevertheless, what the A431 lysis ability of LTG1941 shows is that there may be another preferred option if the activity or high toxicity of LTG1942 becomes apparent in clinical studies of CAR-T.

[0115] Although not intended to be limited to any particular mechanism of action, reasons for the improved therapeutic function associated with exemplary CARs according to the present invention include, for example, a) signal transduction became more efficient because the lateral movement in the cell membrane was improved, b) the ability to interact with transmembrane signal transduction cascades related to T cell activation was improved because of the excellent position in the cell membrane microdomain (such as lipid rafts), c) the position in the cell membrane was excellent because there was a preferential movement away from inhibitory or downregulatory interactions, for example, the distance from phosphatases such as CD45 was relatively far or the interaction with the phosphatase was relatively small, and d) the assembly into the T cell receptor signal transduction complex (i.e., immune synapse) was excellent, or any combination of these is considered, but not limited to these.

[0116] So far, 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 the heavy chain-only binding domain or a subset thereof, and thus may include an ROR1 antigen-binding domain 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, when ROR1 is the desired antigen to be targeted, an antibody against ROR1 may be used as the antigen-binding domain incorporated into the CAR.

[0118] In an 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, where the nucleic acid sequence of the anti-CD33 ScFv includes the sequence of SEQ ID NO: 34. In one embodiment, the anti-CD33 ScFv includes 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 includes the amino acid sequence of SEQ ID NO: 35.

[0119] In an 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, where the nucleic acid sequence of the anti-mesothelin ScFv includes the sequence of SEQ ID NO: 36. In one embodiment, the anti-mesothelin ScFv includes 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 includes the amino acid sequence of SEQ ID NO: 37.

[0120] In an exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD19 It is assumed that. Preferably, the antigen-binding domain in the CAR is an anti-CD19 ScFv, where the nucleic acid sequence of the anti-mesothelin ScFv contains the sequence of SEQ ID NO: 32. In one embodiment, the anti-mesothelin ScFv contains 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 contains the amino acid sequence of SEQ ID NO: 33.

[0121] In one aspect of the present invention, for example, retroviridae (such as human immunodeficiency viruses such as HIV-1 and HIV-LP), picornaviridae (such as poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), 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 (such as herpes simplex virus type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus), poxviridae (such as variola virus, vaccinia virus, and poxvirus), or hepatitis C virus, or antigens (not limited thereto) derived from any combination of these are provided with CARs that can bind to antigens other than TSA or TAA.

[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. In particular, for example, Helicobacter pylori, Legionella pneumophilia, Mycobacterium species (such as Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis pneumophilia), Mycobacterium species (such as Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis CARs are provided that can bind to antigens derived from infectious bacteria such as species of meningitides, Listeria, 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 the transmembrane domain, the 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 a natural or synthetic source. When the source is natural, this domain may be derived from any membrane-bound or transmembrane protein.

[0125] Transmembrane regions particularly useful in the CARs described herein may be derived from the alpha, beta, or zeta chains 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 (i.e., comprising at least their transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it may predominantly contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2 to 10 amino acids in length, may form the linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain. A doublet of glycine and serine 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 domains described above.

[0127] In some examples, the transmembrane domain can be selected or amino acid substituted so that the domain avoids binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize the interaction between the domain and other receptor complex components.

[0128] In one embodiment, the transmembrane domain in the CAR according to the present 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 is the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence in which at least 1, 2, or 3 modifications (e.g., substitutions) are added to a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 22, provided that the modifications (e.g., substitutions) are 20, 10, or 5 or less.

[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 binds to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0132] 3. Spacer (hinge, H) domain In a CAR, a spacer domain may be disposed 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 couple the transmembrane domain to the extracellular domain and / or couple the transmembrane domain to the intracellular domain. The spacer domain contains 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 include a spacer element, and when the spacer element is present, the linker is enlarged by the spacer element, increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art and include those listed 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, 5,521,284, 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 U.S. Patent Publications 20110212088 and 20110070248 (which are hereby incorporated by reference in their entirety).

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

[0135] As this spacer domain, all or part of amino acid numbers 137 to 206 (SEQ ID NO: 25) of the hinge region of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acid numbers 135 to 195 of CD8 beta (GenBank: AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: NP__000607.1), or amino acid numbers 137 to 152 of CD28 (NCBI RefSeq: NP__006130.1) can be used. Also, as this spacer domain, a part of the constant region of the H chain or L chain of an antibody can also be used. Furthermore, this spacer domain may be an artificially synthesized sequence.

[0136] Also, the spacer domain may consist of elements of an immunoglobulin (Ig) constant domain, including a sequence that binds to an immunoglobulin domain containing an immunoglobulin protein, such as one derived from IgG4. The spacer or hinge domain is present at the C-terminus of the scFv ROR1 binding domain and extends to the CAR transmembrane domain. In one embodiment, the IgG4 hinge (H) domain contains the nucleic acid sequence of SEQ ID NO: 38. In one embodiment, the CD8 hinge domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 39. In another embodiment, the CD8 hinge domain contains the amino acid sequence of SEQ ID NO: 39, or a sequence that is 95 - 99% identical thereto.

[0137] In some examples, the IgG4 constant region functions as a hinge and binds to the transmembrane domain of CD8. In one embodiment, the IgG4 H domain binds to the CD8 transmembrane domain and together contains the nucleic acid sequence of SEQ ID NO: 40. In one embodiment, the IgG4 H domain bound to the CD8 transmembrane domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41. In another embodiment, the IgG4 H bound to the CD8 transmembrane domain contains the amino acid sequence of SEQ ID NO: 41, or a sequence that is 95 - 99% identical thereto.

[0138] Furthermore, in the CAR, a signal peptide sequence may be attached to the N-terminus. This signal peptide sequence is present at the N-terminus of many secreted and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above as the intracellular domain 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 the CAR is responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to the specialized functions of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the protein portion that transmits effector function signals and directs the cell to perform specialized functions. 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, this truncated portion may be used in place of the full chain as long as it can transmit effector function signals. Therefore, the meaning of the term "intracellular signal transduction domain" includes any truncated portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[0140] Preferred examples of intracellular signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that cooperate to initiate signaling after binding of the antigen and the receptor, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional ability.

[0141] Signals emitted only through the TCR are known to be insufficient to fully activate T cells, and a second or co-stimulatory signal is further required. Thus, T cell activation involves two distinct types of cytoplasmic signaling sequences: one that initiates antigen-dependent first activation via the TCR (the first cytoplasmic signaling sequence) and one that acts in an antigen-independent manner to provide a second or co-stimulatory signal (the second cytoplasmic signaling sequence).

[0142] The first cytoplasmic signaling sequence regulates the first activation of the TCR complex in either a stimulatory or inhibitory manner. The first cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor activation tyrosine 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 acid numbers 51 - 164 of CD3 zeta (NCBI RefSeq:NP__932170.1), amino acid numbers 45 - 86 of Fc epsilon RI gamma (NCBI RefSeq:NP__004097.1), amino acid numbers 201 - 244 of Fc epsilon RI beta (NCBI RefSeq:NP__000130.1), amino acid numbers 139 - 182 of CD3 gamma (NCBI RefSeq:NP__000064.1), amino acid numbers 128 - 171 of CD3 delta (NCBI RefSeq:NP__000723.1), amino acid numbers 153 - 207 of CD3 epsilon (NCBI RefSeq:NP__000724.1), amino acid numbers 402 - 495 of CD5 (NCBI RefSeq:NP__055022.2), amino acid numbers 707 - 847 of 0022 (NCBI RefSeq:NP__001762.2), amino acid numbers 166 - 226 of CD79a (NCBI RefSeq:NP__001774.1), amino acid numbers 182 - 229 of CD79b (NCBI RefSeq:NP__000617.1), and peptides having the sequence of amino acid numbers 177 - 252 of CD66d (NCBI RefSeq:NP__001806.2), and variants having the same function as these peptides, including, but not limited to, these. Amino acid numbers based on the NCBI RefSeq ID or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor of each protein (including signal peptide sequences, 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 itself include a CD3-zeta signaling domain or may be combined with any other desirable cytoplasmic domain useful in the context of the CAR. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands necessary for lymphocytes to efficiently respond to antigens. Examples of such co-stimulatory 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, among others. Specific examples of such co-stimulatory molecules include peptides having the sequences of amino acids 236 to 351 of CD2 (NCBI RefSeq:NP__001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq:NP__000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq:NP__055022.2), amino acids 207 to 235 of CD8 alpha (NCBI RefSeq:NP__001759.3), amino acids 196 to 210 of CD83 (GenBank:AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq:NP__006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBI RefSeq:NP__001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq:NP__003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq:NP__036224.1), as well as variants having the same function as these peptides, but are not limited thereto. Thus, the present disclosure has so far been exemplified mainly using 4-1BB as a co-stimulatory signaling element, but other co-stimulatory elements are also within the scope of the present disclosure. are cell surface molecules other than antigen receptors or their ligands. Examples of such co-stimulatory 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, among others. Specific examples of such co-stimulatory molecules include peptides having the sequences of amino acids 236 to 351 of CD2 (NCBI RefSeq:NP__001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq:NP__000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq:NP__055022.2), amino acids 207 to 235 of CD8 alpha (NCBI RefSeq:NP__001759.3), amino acids 196 to 210 of CD83 (GenBank:AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq:NP__006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBI RefSeq:NP__001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq:NP__003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq:NP__036224.1), as well as variants having the same function as these peptides, but are not limited thereto. Thus, the present disclosure has so far been exemplified mainly using 4-1BB as a co-stimulatory signaling element, but other co-stimulatory elements are also within the scope of the present disclosure.

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

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

[0147] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the nucleic acid sequence of SEQ ID NO: 26, and the signaling domain of CD3-zeta includes 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 include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the nucleic acid sequence of SEQ ID NO: 26, and the signaling domain of CD3-zeta includes 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 include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 27, and the signaling domain of CD3-zeta includes 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 include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB includes the amino acid sequence of SEQ ID NO: 27, and CD3-zeta the signaling domain of which includes the amino acid sequence of SEQ ID NO: 29 and the variant amino acid sequence of SEQ ID NO: 31.

[0150] 5. Further description of the CAR The functional portions of the CARs disclosed herein are also clearly included within the scope of the present invention. As used in reference to a CAR, the term "functional portion" refers to any portion or fragment of one or more of the CARs disclosed herein, which portion or fragment retains 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, to the same extent as, or to a greater extent than the parent CAR. With respect to a parent CAR, a functional portion can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

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

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

[0153] Functional variants may include, for example, those in which at least one conservative amino acid substitution is made to the amino acid sequence of the parental CAR. Alternatively or additionally, functional variants may include those in which at least one non-conservative amino acid substitution is made to the amino acid sequence of the parental CAR. In this case, it is preferred that this non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. This 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 parental CAR.

[0154] Amino acid substitutions in the 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 certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include substituting an acidic / negatively charged polar amino acid (e.g., Asp or Glu) with another acidic / negatively charged polar amino acid, substituting a nonpolar side chain-containing amino acid (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another nonpolar 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, Gln, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, substituting a beta-branched side chain-containing amino acid (e.g., Ile, Thr, and Val) with another beta-branched side chain-containing amino acid, substituting an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid and the like may be possible.

[0155] The CAR may essentially consist of one or more of the designated amino acid sequences described herein, such that the biological activity of the functional variant is not substantially altered by other components (e.g., other amino acids).

[0156] A CAR (including functional parts and functional variants) may be of any length, i.e., may contain any number of amino acids, as long as the CAR (or its functional part or functional variant) retains a biological activity such as, for example, the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent a disease in a mammal. For example, the CAR may be about 50 to about 5000 amino acids in length, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids in length.

[0157] A CAR (including functional parts and functional variants according to the present invention) may contain synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-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, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0158] CAR (including functional moieties and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridging), or converted to an acid addition salt, and / or optionally may be dimerized or polymerized or conjugated.

[0159] CAR (including its functional moieties and functional variants) can be obtained by methods well known in the art. CAR may be made by any suitable polypeptide or protein production method. Suitable methods for newly synthesizing 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. Also, polypeptides and proteins may be produced recombinantly using the nucleic acids described herein and standard recombinant methods. For example, Sambrook et al., Molecular Cloning: A Laboratory Manua See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 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. Further, some of the 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 (including functional portions and functional variants thereof) described herein may be commercially synthesized by a company. In this regard, the 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 the 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, "T cell expressing a CAR" or "CAR T cell" means a T cell that expresses a CAR and has antigen specificity, e.g., as determined by the antibody-derived targeting domain of the CAR.

[0161] As used herein, "antigen-binding domain" may include an antibody and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and includes diverse 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 well known in the art that retain binding affinity for an antigen, as well as variants and fragments thereof.

[0162] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up this population are identical except for natural mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigen epitope. The modifier "monoclonal" indicates the property that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted to mean that the antibody must be produced by any particular method. In some examples, monoclonal antibodies are produced by a single clone of B lymphocytes or by cells transfected with nucleic acids encoding the light and heavy chain variable regions of the antibody (or antigen-binding fragment thereof) or their progeny. In some examples, monoclonal antibodies are isolated from a subject. Monoclonal antibodies 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, see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition Cold Spring Harbor Publications, New York (2013).

[0163] Typically, an immunoglobulin has heavy (H) and light (L) chains that are linked to each other by disulfide bonds. Immunoglobulin genes include 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 main classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule (IgM, IgD, IgG, IgA, and IgE).

[0164] The heavy and light chains each have a constant region (or constant domain) and a variable region (or containing a variable domain (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting of only heavy chains are functional and stable without a light chain (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 those of Fv, ScFv, dsFv, or Fab.

[0165] The variable regions of the light and heavy chains contain "framework" regions and three hypervariable regions (also called "complementary determining regions" or "CDRs") that interrupt it (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department 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] The CDRs are primarily responsible for binding to the antigen epitope. The boundaries of the amino acid sequence of a given CDR are defined by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health), Bethesda, MD, 1991; “Kabat” numbering scheme), 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,” It can be readily determined using any of a plurality of well-known schemes, including the scheme described by Dev. Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus towards the C-terminus), and further typically are identified by the chain in which the CDR is located. Thus, VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody containing it, and VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody containing 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] An “antigen-binding fragment” is a portion of a full-length antibody and various combinations of such portions that retain the ability to specifically recognize cognate antigens. 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 made by modification of the whole antibody or newly synthesized using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0168] A single-chain antibody (ScFv) is a genetically engineered molecule containing a VH domain and a VL domain of one or more antibodies linked by a suitable polypeptide linker to form a gene-fused single-chain molecule (see, for example, 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 the ScFv typically does not determine the ScFv. Thus, ScFvs having both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0169] In dsFv, the variable chains of the heavy and light chains have disulfide bonds introduced by mutation to stabilize the binding of both chains. Diabodies are also included, which are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to connect the two domains into one chain is used, so the two domains are connected to the complementary domains of another chain, forming two antigen-binding sites (see, for example, 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 mouse 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] Antibodies that do not occur naturally can be constructed using solid-phase peptide synthesis, or produced recombinantly, or obtained, for example, by screening a combinatorial library consisting of variable heavy and variable light chains as described by Huse et al., Science 246:1275-1281 (1989) (incorporated herein by reference). These methods, as well as other methods for making, 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 an antagonistic assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in an antagonistic assay. Antibody antagonistic assays are well known, and exemplary antagonistic assays are provided herein.

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

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

[0175] A "fully human antibody" or "human antibody" is an antibody that contains sequences from the human genome (or derived therefrom) and does not contain sequences from another species. In some embodiments, a human antibody comprises CDRs, framework regions, and (if present) an Fc region from the human genome. Human antibodies can be identified and isolated, for example, by using antibody production techniques based on human genome-derived sequences, such as by phage display or the use of genetically engineered animals (e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st Edition New York: Cold Spring Harbor Laboratory Press, 2004. Print.; see Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0176] An antibody may have one or more binding sites. When there is more than one binding site, these binding sites may be the same as each other or different. For example, a natural 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 antibody ability to bind to any functional part of a CAR are well known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assay (see, for example, Janeway et al. below, U.S. Patent Application Publication No. 2002 / 0197266 Al, and U.S. Patent No. 7,338,929).

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

[0179] C. Conjugates A CAR, a T cell expressing the CAR, a monoclonal antibody, or an antigen-binding fragment thereof, specific for one or more of the antigens disclosed herein, may be conjugated to an agent such as an effector molecule or a detectable marker using any of a number of means well known to those of skill in the art. Either covalent or non-covalent means may be used. The conjugate includes, but is not limited to, a molecule in which an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein is covalently conjugated to an effector molecule or a detectable marker. It will be understood by those of skill in the art that a variety of effector molecules and detectable markers can be used, including, but not limited to, chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C, 3 H, and 35 radiation agents such as S, as well as other labels, target sites, and ligands, etc.

[0180] The choice of a specific effector molecule or detectable marker depends on the specific 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 specific 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 (-NH2), or sulfhydryl (-SH) groups, which can be utilized in reactions with suitable functional groups on the antibody, resulting in the attachment of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may be associated with the attachment of any of a number of well-known linker molecules such as those available from the Pierce Chemical Company (Rockford, IL). A linker may be any molecule used to couple an antibody or antigen-binding fragment to an effector molecule or detectable marker. A 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 of skill 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 linker may be attached to the constituent amino acids via its side chain (e.g., to cysteine via a disulfide bond) or to the amino and carboxy groups of the alpha carbon of the terminal amino acids.

[0182] In some embodiments, the linker may include a spacer element, and when the spacer element is present, the spacer element causes the linker to be larger, increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art and include those listed 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, 5,521,284, 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, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (each of which is incorporated herein 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 another embodiment, 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 within the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker may be, for example, a peptide linker cleaved by an intracellular protease, or a protease enzyme including, but not limited to, a lysosomal protease or an endosomal protease. Some In an embodiment, the peptide linker is at least two amino acids in length, or at least three amino acids in length. However, the linker can be four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acids in length, e.g., one to two amino acids, one to three amino acids, two to five amino acids, three to ten amino acids, three to fifteen amino acids, one to five amino acids, one to ten amino acids, one to fifteen amino acids in length. The proteases may include cathepsin B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics See 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin-B can be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is hereby incorporated by reference herein. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citruline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345. This document describes the synthesis of doxorubicin with a valine-citruline linker).

[0184] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, such pH-sensitive linkers hydrolyze under acidic conditions. For example, acid-labile linkers that can hydrolyze within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amide, orthoesters, acetals, or ketals, etc.) can be used. (See, e.g., U.S. Pat. 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 unstable at pH 5.5 or less than 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bonded to the therapeutic agent via an acylhydrazone linkage (see, e.g., U.S. Pat. No. 5,622,929, etc.)).

[0185] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are well known in the art and include, 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, for example, 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. 68:92809290, 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 a 3'-N-amide analog (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 U.S. Publication No. 2005 / 0238649, which is hereby incorporated by reference in its entirety).

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

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

[0190] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art, can be isolated from natural sources according to well-known methods, can be 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 mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from Maytenus serrata, a shrub native to East Africa (U.S. Patent No. 3,896,111). Subsequently, it was further discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent 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, 4,313,946, 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 hereby incorporated by reference herein. Conjugates containing maytansinoids, methods for their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163, and European Patent No. EP0425235 B1, the disclosures of which are hereby expressly incorporated by reference herein.

[0191] Additional toxins can be used with the CAR, T cells expressing the CAR, an antibody, or an antigen-binding portion thereof. Examples of toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A-F. Such toxins are well known in the art and many of them are readily available from commercial suppliers (e.g., Sigma Chemical Company, St. Louis, MO). Intended toxins also include variants of such toxins (see, e.g., U.S. Pat. Nos. 5,079,163 and 4,689,401). 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 does not have a mechanism for specifically entering cells and thus needs to be conjugated to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein in order to efficiently enter cells.

[0192] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins has reduced or eliminated nonspecific toxicity due to mutations. A mutant known as CRM107 has sufficient enzymatic activity but significantly reduced nonspecific toxicity and has been well known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Pat. No. 5,792,458 and U.S. Pat. No. 5,208,021.

[0193]

[0194] ​Ricin is the lectin RCA60 obtained from Ricinus communis (Castor bean). See, for example, U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) has two forms, which are designated RCA 60 and RCA 120 because their molecular weights are approximately 65 kD and approximately 120 kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for the inactivation of protein synthesis and cell death. The B chain binds ricin to cell surface galactose residues and facilitates the 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 binding to target molecules (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins 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 first isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes DNA double-strand breaks and induces apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug has been in clinical trials and is at the toxic site of immunotoxins (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0196] Abrin contains a toxic lectin obtained from Abrus precatorius. Its toxic components, abrin a, b, c, and d, have a molecular weight of about 63 to 67 kD and are composed of two polypeptide chains A and B linked by disulfide bonds. Chain A inhibits protein synthesis, and chain B (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, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof that are specific for one or more of the antigens disclosed herein may also be conjugated to a detectable marker, such as a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging diagnostic techniques (computerized tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound diagnosis, fiber optic examination, and laparoscopic examination, etc.). Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. CARs, T cells expressing CARs, 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 a CAR, T cell expressing a CAR, antibody, or antigen-binding portion thereof is conjugated to a detectable enzyme, it can be detected by adding a further reagent that produces a reaction product distinguishable from that used for the enzyme. For example, in the presence of the agent horseradish peroxidase, a colored reaction product is obtained by addition of hydrogen peroxide and diaminobenzidine, which can be detected visually. CARs, T cells expressing CARs, antibodies, or antigen-binding portions thereof may also be conjugated to biotin and may be detected by indirectly measuring the binding of avidin or streptavidin.Notably, avidin itself may be bound to an enzyme or a fluorescent label.

[0198] The CAR, the T cell expressing the CAR, the antibody, or an antigen-binding portion thereof may be bound to a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. The antibody may also be bound to lanthanides (such as europium and dysprosium) and manganese. The antibody or antigen-binding fragment may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a binding site for a secondary antibody, a metal-binding domain, an epitope tag, etc.).

[0199] The CAR, the T cell expressing the CAR, the antibody, or an antigen-binding portion thereof may also be bound to a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For example, the radiolabel may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, luminescence spectroscopy, or other diagnostic techniques. Further, the radiolabel may be used as a toxin in the treatment of treating tumors in a subject, for example, treating neuroblastoma. Examples of labels for polypeptides are 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 radioisotopes or radiolabeled nucleotides including, but not limited to, I and the like.

[0200] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, a radiolabel may be detected using a photographic film or a scintillation counter, and a fluorescent marker may be detected by detecting emitted light using a light detector. Enzyme labels typically involve providing a substrate to the enzyme and Detected by detecting the reaction product generated therefrom, and the colorimetric label is detected simply by visualizing the colored label.

[0201] D. Nucleotides, Expression, Vectors, and Host Cells According to one embodiment of the present invention, there is further provided a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein. The nucleic acid according to the present invention may include 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 have modified codons. Without being bound by any theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but is more readily translated by a tRNA that is more readily available within the cell, and thus, the translation efficiency may be increased. Optimization of the nucleotide sequence may also be one that reduces secondary mRNA structures that may interfere with translation, and thus, the translation efficiency may be increased.

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

[0204] As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally may be single-stranded or double-stranded, may be obtained from synthetic or 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 found between nucleotides of unmodified oligonucleotides), meaning a polymer of DNA or RNA. In some embodiments, the nucleic acid contains no insertions, deletions, inversions, and / or substitutions whatsoever. However, as described herein, in some instances, it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0205] A recombinant nucleic acid may have a sequence that does not exist naturally or may have a sequence in which two regions that are distant in the sequence are artificially combined. This artificial combination is often achieved by chemical synthesis or, more generally, by artificially manipulating distant nucleic acid regions by genetic engineering techniques such as those described in the Sambrook et al. literature mentioned above. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, the Sambrook et al. literature and the Ausubel et al. literature mentioned above. For example, nucleic acids may be chemically synthesized using natural nucleotides or nucleotides modified in various ways (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the double-stranded structure 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 adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 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, methyl ester of uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine, including but not limited to these. Alternatively, one or more of the nucleic acids according to the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0206] The nucleic acid may comprise any of the above-described CARs or any isolated or purified nucleotide sequence encoding a functional portion or functional 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 complementary to the nucleotide sequence of any of the nucleic acids described herein, or a nucleotide sequence 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 hybridize under highly stringent conditions. "Highly stringent conditions" means that the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein), and the amount thereof is detectably more than non-specific hybridization. Highly stringent conditions include conditions that can distinguish a polynucleotide having a strictly complementary sequence or having only 2 to 3 scattered mismatches from a random sequence that accidentally has 2 to 3 small regions (for example, 3 to 10 bases) that match the nucleotide sequence. Such small complementary regions are more easily melted than full-length complementary regions of 14 to 17 bases or more in length, and can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions may include, for example, low salt and / or high temperature conditions such as about 0.02 to 0.1M NaCl or equivalent and a temperature of about 50 to 70 °C. Thus, highly stringent conditions allow for very low levels of mismatches, if any, between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any of the CARs of the present invention. It is generally understood that the conditions can be made more stringent by increasing the amount of formamide added.

[0209] Also provided are nucleic acids comprising a nucleotide sequence having at least about 70% or more identity, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with any of the nucleic acids described herein.

[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 the purposes described herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector and a host cell are contacted under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the host cell, the host cell is capable of expressing the mRNA, protein, polypeptide, or peptide. Such vectors are generally not found in nature.

[0211] However, some of such vectors may exist in nature. 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, may be synthetic or obtained in part from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain natural or non-natural inter-nucleotide linkages, or both types of linkages. Preferably, the non-natural or modified nucleotides or inter-nucleotide linkages 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 can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for purposes of propagation and growth, or for expression, or for both purposes (such as plasmids and viruses). 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 λΤΙΟ, λΤΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, for example, a retroviral vector or a 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 can be used in a clinic include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc. Non-clinical grade lentiviral vectors are also available and may be well known to those skilled in the art.

[0214] Multiple transfection techniques are generally well 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 co-precipitation (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 transfection (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid introduction using a high-velocity microparticle gun (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0216] In one embodiment, the recombinant expression vector may be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., supra, and Ausubel et al., supra. The circular or linear construct of the expression vector may be prepared to contain a replication mechanism that functions in a prokaryotic or eukaryotic host cell. The replication mechanism may be derived from, for example, ColE1, 2μ plasmid, λ, SV40, and bovine papillomavirus, among others.

[0217] The recombinant expression vector is appropriately specific for the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is introduced, and may contain regulatory sequences such as transcription codons, translation initiation codons, and stop codons, taking into account whether the vector is based on DNA or RNA. The recombinant expression vector may contain restriction sites for facilitating cloning.

[0218] The recombinant expression vector may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include, for example, biocide resistance such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Marker genes suitable 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 contain a natural or non-natural promoter operably linked to a nucleotide sequence encoding a CAR (including its functional parts and functional variants), or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a CAR. The selection of the promoter (e.g., strong, weak, inducible, tissue-specific, and developmental-specific, etc.) is within the scope of the ordinary knowledge of those skilled in the art. Similarly, the binding of the nucleotide sequence to the promoter is also within the scope of the ordinary knowledge of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, for example, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or the promoter found in the terminal repeat sequence of the murine stem cell virus.

[0220] The recombinant expression vector may be designed for either transient expression, stable expression, or both. Further, the recombinant expression vector may be prepared for constitutive expression or inducible expression.

[0221] Furthermore, the recombinant expression vector may be prepared to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene may confer sensitivity to an agent, such as a drug, on the cells expressing the gene, such that contact or exposure of the cells to the agent causes the death of the cells. Suicide genes are well known in the art (see, e.g., 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, the 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 the recombinant expression vector according to the present invention. The host cell may be a eukaryotic cell such as, for example, a plant, an animal, a fungus, or an alga, or may be a prokaryotic cell such as, for example, a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism such as, for example, a human). The 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 Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is amplification or replication of the recombinant expression vector, the host cell may be a prokaryotic cell such as, for example, DH5a cells. When the purpose is production of 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 developmental stage, 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 the purposes described herein, the T cells can be any T cells, which can be cultured T cells (e.g., primary T cells, etc.), or T cells from cultured T cell lines (e.g., Jurkat, SupTl, etc.), or T cells obtained from mammals. When obtained from mammals, the T cells can be obtained from a wide variety of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cells can be enriched or purified. The T cells can be human T cells. The T cells can be T cells isolated from humans. The T cells can be any type of T cells and can be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells such as 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, etc. The T cells can 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 cells are those having immune effector functions, such as, for example, NK cells and T-like cells generated from pluripotent stem cells.

[0225] Also provided in one embodiment is a population of cells comprising at least one host cell described herein. This population of cells can be a heterogeneous population that includes, in addition to host cells containing any of the described recombinant expression vectors, at least one other cell, such as a host cell that does not contain any of the recombinant expression vectors (e.g., T cells), or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells can be recombinant It may be a substantially homogeneous population, mainly comprising (e.g., consisting essentially of) host cells containing the expression vector. Also, the population may be a clonal cell population in which all cells of the population are clones of a single host cell containing the recombinant expression vector, and thus all cells of the population contain this recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells containing the recombinant expression vector described herein.

[0226] CAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding parts thereof) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the purity of the host cells is higher than that in the natural environment in vivo. Such host cells may be prepared, for example, by standard purification techniques. In some embodiments, the preparation of host cells is purified such that the host cells occupy at least about 50%, such as at least about 70%, of the total cell content of the preparation. For example, this purity may be at least about 50%, or may exceed about 60%, about 70%, or about 80%, or may be about 100%.

[0227] E. Treatment methods It is intended that the CARs disclosed herein may be used in methods for treating or preventing diseases in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, which comprises administering to the mammal a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or its antigen-binding part, and / or pharmaceutical composition in an amount effective for treating or preventing cancer in the mammal.

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

[0229] For the purposes of the methods in which a host cell or population of cells is administered, the cell may be a xenogeneic cell derived from the same species as the mammal, or an autologous cell thereof. Preferably, the cell may be an autologous cell of the mammal. As used herein, "xenogeneic" means any material that is derived from an animal that is of the same species as, but a different individual from, the individual into which the material is introduced. Two or more individuals are said to be xenogeneic to each other when the genes at one or more loci are not identical. In some embodiments, xenogeneic materials from individuals of the same species may be genetically different enough to interact antigenically with each other. As used herein, "autologous" means any material that is derived from the same individual as the individual into which 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 rodent mammals such as mice and hamsters and lagomorph mammals such as rabbits. The mammal may be of the order Carnivora, including the families Felidae (cats) and Canidae (dogs). The mammal may be of the order Artiodactyla, including the subfamily Bovinae (cattle) and swine (pigs), or of the order Perissodactyla, including the family Equidae (horses). The mammal may be of the order Primates, Ceboids, or Simoids (monkeys), or of the superfamily Hominoidea (humans and apes). Preferably, the mammal is a human.

[0231] Regarding the methods described above, cancer can be any cancer, including, but not limited to, acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., urothelial carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, oropharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0232] The terms "treatment" and "prevention" and their derivatives, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that would be recognized as beneficial or having a therapeutic effect by a person of ordinary skill in the art. In this regard, the methods can provide any amount or level of cancer treatment or prevention in a mammal.

[0233] Furthermore, the treatment or prevention provided by the methods can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented (e.g., cancer). Also, for the purposes described herein, "prevention" can include delaying the onset of a disease or its symptoms or conditions.

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

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

[0236] Regarding one embodiment of a method for detecting the presence of a proliferative disorder, such as cancer, in a mammal, a sample containing mammalian cells may be a sample containing whole cells, their lysates, or whole cell lysate fractions, such as nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions. When the sample contains whole cells, these cells may be any cells of the mammal, such as cells of any organ or tissue (including blood cells or endothelial cells).

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

[0238] Also, the detection of the complex may be performed by any of a plurality of methods well known in the art. For example, the CAR disclosed herein, the polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations described herein, or The antibody, or antigen-binding portion thereof, may be labeled with a detectable label such as, for example, a radioisotope, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles), as disclosed above.

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

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

[0241] Any method of administration, including local and systemic administration, may be used with the disclosed therapeutic agents. For example, topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administrations may be used. Specific modes of administration and dosing regimens may be selected by the attending clinician, taking into account the details of the case (e.g., the subject, the disease, the associated disease state, and whether the treatment is prophylactic). When more than one agent or composition is administered, more than one route 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. The method of administration includes 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, non-volatile 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 an area suspected of having a tendency to develop tumors. In some embodiments, sustained intratumoral (or near the 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 or intravitreally into the eye as an eye drop.

[0242] The disclosed therapeutic agent may be formulated in unit dosage forms suitable for administering an exact dosage each time. In addition, the disclosed therapeutic agent may be administered according to a schedule of single or multiple administrations. The schedule of multiple administrations may be such that more than one administration (e.g., 1 to 10 administrations) is carried out separately each time in the first series of treatments, and subsequently, if necessary, the remaining administrations may be carried out at time intervals to maintain or increase the action of the composition. The treatment may involve administering the compound once or multiple times a day (multi-daily doses) over a period of 2 to 3 days to several months, or even several years. Thus, the dosing regimen may be determined based at least in part on the specific requirements of the subject to be treated and may depend 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, such as from about 0.1 to about 10 mg / kg.

[0244] In a specific example, the subject is administered a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or a further agent according to a multiple daily dosing schedule, such as for a period of several weeks, several months, or several years, for at least 2 consecutive days and up to 10 consecutive days. In one example, the subject is administered a conjugate, an antibody, a composition, or a further agent for a period of at least 30 days, such as 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 to a subject surgery, radiation therapy, and / or chemotherapy, in combination with (e.g., sequentially, substantially simultaneously, or simultaneously) the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR. 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. Preparations and dosing schedules for additional agents may be used according to the manufacturer's instructions or based on the judgment of a skilled physician's experience. Also, preparations and dosing schedules for such chemotherapy are 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 cross-linkers, 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-angiogenic agent may be administered in combination with a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages for such agents are well known to those of skill in the art and may be determined by a skilled clinician.

[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 folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (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 including, but not limited to, other agents such as 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, vemurafinib, vandetanib, and tretinoin. The selection of such agents and the therapeutic dosages are well known to those of ordinary skill in the art and may be determined by a skilled clinician.

[0248] Combination therapies can be synergistic and can be shown to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the sum of the effects obtained when the same compounds are used separately. A synergistic effect can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit dosage preparation, (2) delivered as separate preparations, alternately or in parallel, or (3) when some other regimen is used. When delivered alternately, for example, by separate injection with separate syringes, a synergistic effect can occur when the compounds are administered or delivered sequentially. Generally, in the alternate case, the effective dosage of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the effective dosages of two or more active ingredients are administered together.

[0249] In one embodiment, an effective amount of an antibody, antigen-binding fragment, or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject having a tumor after anti-cancer treatment. After sufficient time has elapsed for the administered antibody, antigen-binding fragment, or conjugate to form an immune complex with the antigen expressed on the respective cancer cells, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, if the immune complex is increased compared to a control obtained prior to the treatment, it is indicated that the treatment is ineffective, and if the immune complex is decreased compared to a control obtained prior to the treatment, it is indicated that the treatment is effective.

[0250] F. Biopharmaceutical Composition A biopharmaceutical composition or biological composition (hereinafter, "composition") containing one or more of the disclosed CARs, or T cells expressing CARs, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein is provided herein for use in gene therapy, immunotherapy, and / or cell therapy. The composition 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 composition may be formulated for systemic (such as intravenous) or local (such as intratumoral) administration. In one example, the disclosed CARs, or T cells expressing CARs, antibodies, antigen-binding fragments, conjugates are formulated for parenteral administration such as intravenous administration. Compositions containing the CARs, or T cells expressing CARs, conjugates, antibodies, or antigen-binding fragments disclosed herein are useful, for example, in the treatment and detection of tumors (such as, but not limited to, neuroblastoma). In some examples, the composition is useful in the treatment or detection of cancer. Compositions containing the CARs, or T cells expressing CARs, conjugates, antibodies, or antigen-binding fragments disclosed herein are also useful, for example, in the detection of pathological angiogenesis.

[0251] This composition for administration may contain a solution in which a CAR, or a T cell expressing a CAR, conjugate, antibody, or antigen-binding fragment is dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. Such a solution is sterilized and generally free of undesirable substances. This composition may be sterilized by conventional and well-known sterilization techniques. This composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, toxicity modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as necessary to approximate physiological conditions. The concentration of the CAR, or the T cell expressing the CAR, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected according to the particular mode of administration selected and the requirements of the subject, mainly based on factors such as the volume of the fluid, viscosity, and body weight. The actual method of preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy is well known or will be apparent to those skilled in the art.

[0252] A typical composition for intravenous administration contains from about 0.01 to about 30 mg / kg per day per subject of an antibody or antigen-binding fragment or conjugate (or the corresponding dosage of a CAR, or a T cell expressing a CAR, conjugate containing an antibody or antigen-binding fragment). The actual method of preparing the composition for administration may be well known or apparent to those skilled in the art and is described in more detail in publications such as Remington’s Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0253] A CAR, or a T cell expressing a CAR, an antibody, an antigen-binding fragment, or a conjugate may be provided in lyophilized form and reconstituted with sterile water for administration, but may also be provided in solution dissolved in a sterile solution of a known concentration. The solution of the CAR, or a T cell expressing a CAR, an antibody, or an antigen-binding fragment, or a conjugate is then filled into an infusion bag containing 0.9% sodium chloride (USP) and, optionally, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art regarding the administration of antibodies or antigen-binding fragments and conjugates as drugs. For example, antibody drugs have been on the US market since the approval of Rituxan® in 1997. The CAR, or a T cell expressing a CAR, an antibody, its antigen-binding fragment, and a conjugate may be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher loading dose is administered, followed by a maintenance dose administered at a lower level. For example, an antibody or antigen-binding fragment is infused at an initial loading dose of 4 mg / kg (or the corresponding dose of a conjugate containing the antibody or antigen-binding fragment) over about 90 minutes, and if this initial dose is well tolerated, subsequently, a maintenance dose of 2 mg / kg once a week is infused over 30 minutes each time for 4 to 8 weeks.

[0254] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particle systems. A comprehensive overview of protein delivery systems can be found in Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain therapeutic proteins, such as cytotoxins or drugs, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules with a size of less than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since capillaries have a diameter of about 5 μm, only nanoparticles are administered intravenously. Microparticles typically have a diameter of about 100 μm and are administered subcutaneously or intramuscularly. For example, see 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 De livery, edited by A. Kydonieus, Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0255] The polymer may be used for ion-controlled release of the CAR, or T cells expressing the CAR, antibodies, or antigen-binding fragments, or conjugates, 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 polaxamer 407 exists as a mobile liquid with viscosity at low temperatures but forms a semi-solid gel at body temperature. This 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 aspect, liposomes are used for the controlled release of lipid-encapsulated drugs and for drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). In addition to these, a very large number of systems for the controlled delivery of therapeutic proteins are well known (U.S. Patent No. 5,055,303, U.S. Patent No. 5,188,837, U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028, U.S. Patent No. 4,957,735, U.S. Patent No. 5,019,369, U.S. Patent No. 5,055,303, U.S. Patent No. 5,514,670, U.S. Patent No. 5,413,797, U.S. Patent No. 5,268,164, U.S. Patent No. 5,004,697, U.S. Patent No. 4,902,505, U.S. Patent No. 5,506,206, U.S. Patent No. 5,271,961, U.S. Patent No. 5,254,342, and U.S. Patent No. 5,534,496).

[0256] G. Kit In one aspect, kits are further provided that use the CARs disclosed herein. For example, kits for treating tumors in a subject or for generating CAR T cells that express one or more of the CARs disclosed herein. Such kits may typically include the antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR may be included in the kit.

[0257] The kit may include a container and a label or package insert attached to 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 contains a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR. 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 to be used for treating a particular condition.

[0258] The label or package insert may typically further include, for example, an explanation of the use of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR in a method of treating or preventing a tumor or in a method of generating CAR T cells. The package insert typically contains the instructions customarily included in the commercial package of a therapeutic product It contains, and this manual contains information regarding indications, usage, dosage, administration, contraindications, and / or warnings related to the use of the therapeutic product. The content of the manual may be described in electronic form (such as a floppy disk or a compact disk) or visual form (such as a video file). The kit may further contain additional components to facilitate a specific use for which the kit is designed. Thus, for example, the kit may further contain a label detection means (such as an enzyme substrate for enzyme labeling, a filter set for fluorescence label detection, or an appropriate secondary label such as a secondary antibody). The kit may further contain buffers and other reagents customarily used in the implementation of a specific method. Such kits and appropriate contents are well-known to those skilled in the art.

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

Examples

[0260] Isolation of ROR1-Specific Binders from Phage Display and Yeast Displayed Full-Human ScFv Libraries Materials and Methods: a) Generation of Full-Human ScFc (ScFv with an Fc domain for analysis) Binders Against Human ROR1 A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library constructed from peripheral blood B cells of 50 healthy donors (with an approximate diversity of 10 unique specificities) 10)(Z.Y. Zhu and D.S. Dimitrov, data not published) were used to select ScFvs for recombinant human ROR1 protein. An amplified library of 10 12 individual ScFvs was incubated with ROR1 coated at 5 μg, 3 μg, and 1 μg, respectively, in a volume of 5 × 100 μl (equally distributed among five wells of a 96-well plate) for 2 hours each time at room temperature during the first, second, and third rounds of biopanning. After each incubation, the wells were washed 5 times after the first round and 10 times after subsequent rounds with phosphate-buffered saline containing 0.05% Tween20 (PBST) to remove non-specifically bound phages. The bound phages were mixed with TG1 competent cells at 37°C for 1 hour to amplify the phages from the infected cells for use in the next round of biopanning. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells, and each one was inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate. After the optical density at 600 nm (OD600) of this bacterial culture reached 0.5, helper phage M13K07 with a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) of kanamycin were added to the medium, and the plate was incubated at 30°C overnight in a shaker at 250 rpm. The phage supernatant was mixed with 3% skim milk (in PBS) at a volume ratio of 4:1 and used in an enzyme-linked immunosorbent assay (ELISA) to identify phage clones presenting ScFvs with high ROR1 binding affinity. The supernatant was incubated with 50 ng each of recombinant human ROR1 coated in each well of a 96-well plate for 2 hours at room temperature and washed 5 times with PBST (incubated overnight at 4°C, blocked with 3% skim milk (in PBS), and washed 3 times with PBS containing 0.05% Tween20). ROR1-binding phages were detected using horseradish peroxidase-labeled goat anti-M13 antibody. After incubation with this target antibody, non-specifically bound antibodies were removed by washing the wells, and 3,3’,5,5’-tetramethylbenzidine (TMB ) A matrix was added, and the absorbance of the solution was measured at 450 nm (A450). ROR1-binding clones with A450 exceeding 1.0 were selected for further characterization.

[0261] b) Expression and purification of the selected soluble ScFv The DNA sequences of VH and VL of the selected clones were determined, and ScFv with unique sequences encoded by the clones was expressed and purified as described below. HB2151 cells were transformed using the plasmid extracted from the clone. A single colony was selected from the plate containing the newly 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 OD at 600 nm of this culture reached 0.90, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was incubated at 30 °C overnight. 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 at 4 °C for 25 minutes, and the supernatant was used to purify ScFv using Ni-NTA resin according to the vendor'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 at 4°C overnight. Purified ScFv with His-tag and Flag-tag was serially diluted and added into the wells coated with the target protein. After washing, HRP-labeled anti-Flag antibody diluted at 1:3000 was added at room temperature for 1 hour. After washing, 3,3,5,5’-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes, then 1N H2SO4 was added to stop the reaction, and the O.D. at 450 nm was read to quantify the relative ROR1 binding ability of the ScFv.

[0263] Results: 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 results indicate that these two binders are 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 LTG1941, LTG1942, LTG2528, and LTG2529 human anti-ROR1 binders is outlined in Example 2 below.

Example

[0264] CAR expressing a fully human binding sequence against ROR1. Homo sapiens ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a well-studied cancer embryonic cell surface glycoprotein that is expressed in various solid tumors such as chronic lymphocytic leukemia (CLL), and some sarcomas, cancers, or 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). The results of this primary study are awaited. A phase 1 clinical trial using ROR1-specific CAR-T is also in the initial stage (funded by the Fred Hutchinson Cancer Research Institute, NCT02706392). The present applicants included published ROR1 binders as our controls (LTG1943, LTG2527) in order to evaluate our research based on certain criteria and to demonstrate the activity of our constructs (Hudecek et al., 2013, Clin Cancer Res 1 See 9:3153-3164). Considering the current progress of T cell-based therapies, including the fact that anti-CD19 CAR is commercially available, the development of cell-based immunotherapy featuring the CAR constructs presented herein for CLL and other malignancies expressing ROR1 is an innovative and novel approach for treating human diseases using binding sites derived from human sequences.

[0265] The novel anti-ROR1 CAR-T constructs described herein have 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, an ROR1 CAR was designed using an ROR1-binding sequence derived from an ScFv candidate identified by phage display and cloned under the control of the EF1a promoter into a lentiviral expression vector containing the selected structural and signaling domains for characterization and tested in vitro for transduction efficiency, killing function, and cytokine production in both model cell lines and primary human T cells. Table 1 shows an overview of the academic terms used. The CAR construct LTG1943 is a valid comparison as this sequence has been proposed for clinical use (see KTE-C19, Kite Pharma, and CTL019, Novartis).

[0266]

Table 1

[0267] Materials and Methods (a) Cell Lines All cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA), unless otherwise noted. The acute lymphoblastic leukemia cell line REH and the mantle cell lymphoma line Jeko-1 (ACC-553 DSMZ, Leibniz Institute DSMZ, Braunschwieg, Germany), as well as the chronic myelogenous leukemia line K562, were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY).

[0268] (b) Generation of Chimeric Antigen Receptor (CAR) Expression Vectors The ROR1 CAR construct was made by linking each scFv in the framework to either the hinge and transmembrane domains of CD8 (aa 141 - 182, UniProt accession number P0173 2) or the IgG4 hinge domain (aa 99 - 110, UniProt accession number P01861), and subsequently linking the CD8 transmembrane domain (aa 183 - 203, UniProt accession number P01732), the 4 - 1BB (CD137, aa 214 - 255, UniProt accession number Q07011) trans - activation domain, and the CD3 zeta signaling domain (CD247, aa 52 - 163, reference sequence number: NP_000725.1). To facilitate the 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 sequences of the CAR constructs 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 + cells were purified from whole blood or buffy coats (purchased from civilian suppliers with written donor consent) from healthy volunteers using immunomagnetic bead selection of cells according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured at a density of 0.3 - 2×10 6Cultured at [number of cells] / ml, activated using CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), and on the second day, transduced overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) using a lentiviral vector encoding the CAR construct. The medium was changed on the third day. The cultures were expanded in TexMACS medium supplemented with 200 IU / ml IL-2 and then 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 ratios (E:T) 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). The assay range was determined using target-only wells (maximum CPS) and target-only wells with 1% Tween-20 added (minimum CPS). The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). A cytokine release assay was performed on the supernatant collected after co-incubating effector and tumor cell lines at an E:T ratio of 10. Cytokines IFNγ and subsequently IL-2 were measured in triplicate by ELISA (Thermo Fischer, Waltham, MA).

[0271] Flow cytometry analysis For cell staining, 500,000 cells transduced with CAR T were recovered from the culture, washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and stained with ROR1-Fc peptide (R&D, Minneapolis, MN), followed by anti-Fc-AF647 conjugate to detect CAR surface expression (Jackson ImmunoResearch, West Grove, PA). Non-transduced cells were used as negative controls. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). The cells were washed twice, resuspended in 200 μl of staining buffer, and then quantitatively analyzed by flow cytometry. Flow cytometry analysis was performed using a MACSQuant(registered trademark) 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR). For flow cytometry analysis, a MACSQuant(registered trademark) 10 Analyzer (Miltenyi Biotec) was used, and data plots were generated using FlowJo software (Ashland, OR).

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

[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 supernatants encoding the CAR test constructs or controls and expanded until day 8-10 in culture. In flow cytometry analysis, all test CAR constructs demonstrated surface expression in transduced human T cells using recombinant ROR1-Fc fusion protein followed by anti-Fc APC (Figure 3).

[0274] [Table 2]

[0275] Anti-ROR1 chimeric antigen receptor-transduced T cells exhibit cytokine expression and cytolytic activity.

[0276] To evaluate the cytotoxic function of CAR T, CAR T cells were cultured on days 8–10 of culture using ROR1 + 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 (reference 9) and negative control T cells transduced with a lentiviral vector encoding GFP(1398) or non-transduced T cells (UTD) were included for comparison. All constructs shown (LTG1941-1943) showed dose-dependent ROR1-specific tumor killing. ROR1 + Maximum cytotoxicity against tumor lines (Jeko-1 ROR1 + 80%) at an E:T ratio of 40:1 in cells was demonstrated by the CAR construct LTG1942, which reduced tumor death The degree of killing was equivalent to that of the control CAR construct LTG1943, based on R12 anti-ROR1 scFv (reference 9). The CAR construct LTG1941 also showed ROR1 + Although relatively small, it showed cytotoxicity against tumor strains, and the specific lysis at an E:T ratio of 40:1 against Jeko-1 cells was up to 40%. In the A431 strain, which is relatively less sensitive to CAR T-mediated lysis, CAR LTG1941 was not effective, but CAR LTG1942 was equivalent to or more effective than the positive control CAR LTG1943.

[0277] Next, when induced by ROR1-positive and ROR1-negative tumor cell lines, ROR1 The concentrations of the inflammatory cytokines IFN-gamma, TNF-alpha, and IL-2 secreted by CAR T cells transduced with the CAR construct were measured (Figure 4). For the purpose of examining the basal levels of cytokine production, CAR T cells alone were included in each construct. T cells transduced with GFP (LTG1398) were included as a negative control. ROR1 + When induced by the Jeko-1 or A431 cell line, the levels of TNF-alpha, IFN-gamma, and IL-2 were strongly induced by the CAR T cell constructs LTG1942 and the positive control LTG1493, and to a lesser extent by CAR LTG1491. However, ROR1 -It was not induced in the case of Reh control stock. No cytokine induction was observed in the GFP or UTD groups of the negative control. In particular, the cytokine production level of LTG1941 was very low, indicating that the ability to activate T cells expressing this vector is different. This corresponded to the fact that in the comparison between LTG1941 and LTG1942, the cell lysis level against the Jeko cell line was lower, but not so against A431. This indicates, first of all, that simply identifying the binder is not sufficient to examine the true activity of the CAR containing this binder in the sequence. Importantly, such differences may be very important and there may be a possibility of regulating the activity of the CAR according to the desired target antigen density. The ability to lyse tumor targets is complexly involved in both the CAR expression level on the T cell surface and the target expression level of the tumor antigen on the surface of target tumor cells (Walker, A. et al., 2017, Mol Ther 25:2189 - 2201). Therefore, for example, if the expression of ROR1 on normal T cells induces CAR-T activity by LTG1492, a construct such as LTG1941 may be used to discriminate and target cancer against the low expression level on normal tissues. Furthermore, none of the constructs showed cytokine production above the baseline in the absence of tumor cell targets. This is an important proof that there is no such thing as autoactivation as previously reported for some CAR constructs (Long, A.H. et al., 2015, Nature Med 20:581 - 590). From the above, the CAR T constructs LTG1941, LTG1942, and LTG1943 were specific for the ROR1 tumor antigen expressed in the model tumor cell line.

[0278] Overall, CAR LTG1942 and LTG1941 showed tumor lysis activity and functional specificity manifested as cytokine synthesis in response to tumors. + Therefore, the constructs described in the present application are promising candidates for clinical use.

[0279] Each application and patent cited herein, as well as each document or prior art document cited in each application and patent (including each issued patent "application cited reference" in litigation), and each PCT application or patent and foreign application or patent corresponding to and / or claiming priority from any of the foregoing applications and patents, and each document cited or referenced in each application cited reference, are hereby expressly incorporated by reference into this specification, and these may be used in the practice of the present invention. More generally, the documents or prior art documents are cited in any of the text, the list of prior art documents before the claims, or the text itself, and each of the documents or prior art documents (including manufacturer's specifications, instructions, etc.) cited in each of the documents or prior art documents cited in this specification (hereinafter referred to as "prior art documents cited in this specification") are hereby expressly incorporated by reference into this specification. The above description of some specific embodiments provides sufficient information for others to easily modify or adapt the specific embodiments for various uses without departing from the general concept by applying the latest knowledge. Therefore, such adaptations and modifications should be understood to be within the meaning and scope of the equivalents of the disclosed embodiments. It is understood that the terms or technical terms used in this specification are for explanatory purposes and not for limiting purposes. In the drawings and the description, exemplary embodiments are disclosed and specific terms may be used, but these are used only for general and explanatory purposes only and not for limiting purposes, unless otherwise specified, and thus the scope of the claims is not limited thereby. Also, those skilled in the art will understand that the specific steps of the methods disclosed herein may be performed in a different order or the steps may be combined. Therefore, it is intended that the claims appended to the specific embodiments disclosed herein not be limited. Those skilled in the art can recognize many equivalents of the embodiments of the present invention described herein or can grasp them using only conventional experimental methods. Such equivalents are included in the following claims.

[0280] The above description of some specific embodiments provides sufficient information for others to easily modify or adapt the specific embodiments for various uses without departing from the general concept by applying the latest knowledge. Therefore, such adaptations and modifications should be understood to be within the meaning and scope of the equivalents of the disclosed embodiments. It is understood that the terms or technical terms used in this specification are for explanatory purposes and not for limiting purposes. In the drawings and the description, exemplary embodiments are disclosed and specific terms may be used, but these are used only for general and explanatory purposes only and not for limiting purposes, unless otherwise specified, and thus the scope of the claims is not limited thereby. Also, those skilled in the art will understand that the specific steps of the methods disclosed herein may be performed in a different order or the steps may be combined. Therefore, it is intended that the claims appended to the specific embodiments disclosed herein not be limited. Those skilled in the art can recognize many equivalents of the embodiments of the present invention described herein or can grasp them using only conventional experimental methods. Such equivalents are included in the following claims.

[0281] The 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 the 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 reference is made to the shown strand. In the accompanying Sequence Listing: SEQ ID NO:1 Nucleotide sequence of anti-ROR1 binder: ScFV4 CAGGTGCAGCTGCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATACCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACACCTGGGGGGTGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGAGGCGGATCCCTGCCTGTGCTGACTCAGCCCCCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGGAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTACTTCTGTCAGTCTTATGATAGCAGCAATCCCGTGGTATTCGGCGGAGGGACCCAGCTCACCGTTTTA Amino acid sequence of Array No. 2 anti-ROR1 binder: ScFV4 QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARHLGGDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSN SGNTATLTISGTQAMDEADYFCQSYDSSNPVVFGGGTQLTVL Array No. 3 ROR1-CAR DNA SEQ LTG1941 (LP-ScFV4-CD8H / CD8TM-41BB-CD3 zeta) Array number 4 ROR1-CAR AA SEQ LTG1941 (LP-ScFV4-CD8H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARHLGGDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISGTQAMDEADYFCQSYDSSNPVVFGGGTQLTVLAAATTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Array number 5 ROR1-CAR DNA SEQ LTG2528 (LP-ScFV4-IgG4H / CD8TM-41BB-CD3 zeta) Array No. 6 ROR1-CAR AA SEQ LTG2528 (LP-ScFV4-IgG4H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARHLGGDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAP VLVVYDDSDRPSGIPERFSGSNSGNTATLTISGTQAMDEADYFCQSYDSSNPVVFGGGTQLTVLAAAESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Array No. 7 Nucleotide sequence of anti-ROR1 binder: ScFV9 CAGGCGGCCCAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAGGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTTATAATGATGCTTTTGATATCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCAATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCATTGTGATCTATGAGGATGATCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACACCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGAGTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGAGCCCGGCAATGGGGTATTCGGCGGAGGGACCAAGGTCACCGTCCTA Sequence number 8: Amino acid sequence of anti-ROR1 binder: ScFV9 QAAQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNYAQRFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASYNDAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTIVIYEDDQRPSGVPDRFSGSIDTSSNSASLTISGLQSEDEADYYCQSYEPGNGVFGGGTKVTVL Sequence number 9 ROR1-CAR DNA SEQ LTG1942 (LP-ScFV9-CD8H / CD8TM-41BB-CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAGGCGGCCCAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAGGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTTATAATGATGCTTTTGATATCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCAATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACG GTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCATTGTGATCTATGAGGATGATCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACACCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGAGTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGAGCCCGGCAATGGGGTATTCGGCGGAGGGACCAAGGTCACCGTCCTAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Array No. 10 ROR1-CAR AA SEQ LTG1942 (LP-ScFV9-CD8H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQAAQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNYAQRFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASYNDAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTIVIYEDDQRPSGVPDRFSGSIDTSSNSASLTISGLQSEDEADYYCQSYEPGNGVFGGGTKVTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Array No. 11 ROR1-CAR DNA SEQ LTG2529 (LP-ScFV9-IgG4H / CD8TM-41BB-CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAGGCGGCCCAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAGGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTA Array No. 12 ROR1-CAR AA SEQ LTG2529 (LP-ScFV9-IgG4H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQAAQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNYAQRFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASYNDAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTIVIYEDDQRPSGVPDRFSGSIDTSSNSASLTISGLQSEDEADYYCQSYEPGNGVFGGGTKVTVLAAAESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Array No. 13 Nucleotide sequence of control anti-ROR1 binder: CAAGAACAGCTTGTAGAGTCCGGCGGTAGATTGGTGACACCGGGGGGGAGCCTTACCCTGTCTTGTAAGGCATCTGGGTTCGATTTCAGTGCGTATTATATGAGCTGGGTTCGGCAGGCGCCCGGGAAGGGGCTGGAATGGATAGCCACTATATACCCGTCATCCGGCAAGACTTACTACGCGACTTGGGTAAACGGGAGGTTTACGATAAGCTCAGATAACGCCCAAAACACGGTTGATCTCCAAATGAATAGCTTGACCGCCGCTGATAGGGCGACCTATTTCTGTGCGCGGGACTCTTACGCTGATGACGGGGCCCTCTTCAATATATGGGGACCGGGAACGCTCGTAACCATATC ATCTGGAGGAGGTGGGAGCGGAGGCGGAGGGTCAGGTGGGGGCGGGAGCGAACTCGTACTTACACAATCTCCAAGCGTAAGCGCAGCGTTGGGGAGTCCAGCAAAGATCACCTGCACTTTGTCAAGCGCCCACAAAACGGATACGATAGATTGGTATCAGCAACTCCAAGGTGAAGCGCCACGATATCTCATGCAGGTACAGAGCGACGGGAGTTATACTAAGAGGCCCGGGGTCCCAGACAGATTCAGTGGCAGCAGTTCAGGTGCCGACAGATACCTGATAATACCCTCAGTTCAAGCCGATGATGAAGCCGATTACTACTGTGGGGCTGACTACATAGGTGGGTATGTTTTCGGGGGCGGCACTCAATTGACAGTTACAGGG Accession No. 14 Amino acid sequence of control anti-ROR1 binder: QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG Accession No. 15 ROR1-CAR DNA SEQ control LTG1943 (LP-control ScFv-CD8H / CD8TM-41BB-CD3 zeta) CCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Sequence number 16 ROR1-CAR AA SEQ control LTG1943 (LP-control ScFv-CD8H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence number 17 ROR1-CAR DNA SEQ control LTG2527 (LP-control ScFv-IgG4H / CD8TM-41BB-CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAAGAACAGCTTGTAGAGTCCGGCGGTAGATTGGTGACACCGGGGGGGAGCCTTACCCTGTCTTGTAAGGCATCTGGGTTCGATTTCAGTGCGTATTATATGAGCTGGGTTCGGCAGGCGCCCGGGAAGGGGCTGGAATGGATAGCCACTATATACCCGTCATCCGGCAAGACTTACTACGCGACTTGGGTAAACGGGAGGTTTACGATAAGCTCAGATAACGCCCAAAACACGGTTGATCTCCAAATGAATAGCTTGACCGCCGCTGATAGGGCGACCTATTTCTGTGCGCGGGACTCTTACGCTGATGACGGGGCCCTCTTCAATATATGGGGACCGGGAACGCTCGTAACCATATCATCTGGAGGAGGTGGGAGCGGAGGCGGAGGGTCAGGTGGGGGCGGGAGCGAACTCGTACTTACACAATCTCCAAGCGTAAGCGCAGCGTTGGGGAGTCCAGCAAAGATCACCTGCACTTTGTCAAGCGCCCACAAAACGGATACGATAGATTGGTATCAGCAACTCCAAGGTGAAGCGCCACGATATCTCATGCAGGTACAGAGCGACGGGAGTTATACTAAGAGGCCCGGGGTCCCAGACAGATTCAGTGGCAGCAGTTCAGGTGCCGACAGATACCTGATAATACCCTCAGTTCAAGCCGATGATGAAGCCGATTACTACTGTGGGGCTGACTACATAGGTGGGTATGTTTTCGGGGGCGGCACTCAATTGACAGTTACAGGGGCGGCCGCAGAGTCAAAATACGGTCCTCCGTGCCCTCCGTGTCCGATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAA GAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Sequence number 18 ROR1-CAR AA SEQ control LTG2527 (LP-control ScFv-IgG4H / CD8TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGAAAESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Array No. 19 leader / signal peptide sequence (LP) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG Amino acid sequence of Array No. 20 leader / signal peptide sequence (LP) MLLLVTSLLLCELPHPAFLLIP Nucleotide sequence of Array No. 21 DNA CD8 transmembrane domain ATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGC Amino acid sequence of Array No. 22 CD8 transmembrane domain IWAPLAGTCGVLLLSLVITLYC Nucleotide sequence of Array No. 23 DNA CD8 hinge domain ACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTAC Amino acid sequence of Array No. 24 CD8 hinge domain TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY Amino acid sequence of hinge and transmembrane regions of amino acid numbers 137 - 206 of CD8 alpha (NCBI REFSEQ:NP__001759.3) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC Nucleotide sequence of Array No. 26 4 - 1BB signaling domain AAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATG CGAACTG Amino acid sequence of the signal transduction domain of 4-1BB, SEQ ID NO: 27 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Nucleotide sequence of the intracellular signal transduction domain of CD3-zeta, SEQ ID NO: 28 CGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of CD3-zeta, SEQ ID NO: 29 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of the intracellular signal transduction domain, variant, of CD3-zeta, SEQ ID NO: 30 CGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATAAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 31, CD3-zeta signal transduction domain, variant RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of SEQ ID NO: 32, ScFV CD19 (FMC63) GACATTCAGATGACTCAGACCACCTCTTCCTTGTCCGCGTCACTGGGAGACAGAGTGACCATCTCGTGTCGCGCAAGCCAGGATATCTCCAAGTACCTGAACTGGTACCAACAGAAGCCCGACGGGACTGTGAAGCTGCTGATCTACCACACCTCACGCCTGCACAGCGGAGTGCCAAGCAGATTCTCCGGCTCCGGCTCGGGAACCGATTACTCGCTTACCATTAGCAACCTCGAGCAGGAGGACATCGCTACCTACTTCTGCCAGCAAGGAAATACCCTGCCCTACACCTTCGGCGGAGGAACCAAATTGGAAATCACCGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCGAAGTGAAGCTCCAGGAGTCCGGCCCCGGCCTGGTGGCGCCGTCGCAATCACTCTCTGTGACCTGTACCGTGTCGGGAGTGTCCCTGCCTGATTACGGCGTGAGCTGGATTCGGCAGCCGCCGCGGAAGGGCCTGGAATGGCTGGGTGTCATCTGGGGATCCGAGACTACCTACTACAACTCGGCCCTGAAGTCCCGCCTGACTATCATCAAAGACAACTCGAAGTCCCAGGTCTTTCTGAAGATGAACTCCCTGCAAACTGACGACACCGCC ATCTATTACTGTGCTAAGCACTACTACTACGGTGGAAGCTATGCTATGGACTACTGGGGGCAAGGCACTTCGGTGACTGTGTCAAGC Amino acid sequence of SEQ ID NO: 33 ScFV CD19 (FMC63) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS Nucleotide sequence of SEQ ID NO: 34 anti-CD33 ScFV (LTG1936) CAGGTGCAGCTGGTGCAATCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATTCAGTTTTCCCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTAGTTGGAGATGGCTACAATACGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCGATATTGTGATGACCCACACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAAGACCTATTTGTATTGGTACCTGCAGAAGCCAGGCCAGCCTCCACAGCTCCTGATCTATGGAGCTTCCAACCGGTTCTCTGGAGTGCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA SEQ ID NO: 35 Amino acid sequence of anti-CD33 ScFV (LTG1936) QVQLVQSGAEVKKPGESLRISCKGSGFSFPTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLVGDGYNTGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTHTPLSLSVTPGQPASISCKSSQSLLHSNGKTYLYWYLQKPGQPPQLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPITFGQGTRLEIK Sequence number 36. Nucleotide sequence of anti-methotrexate ScFV (LTG1904) GAGGTCCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATTTATCGTCAGTGGCTGGACCCTTTAACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGCGGTGGC GGATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGCGGAGGCACCCAGCTGACCGTCCTCGGT SEQ ID NO: 37 Amino acid sequence of anti-methotrexate ScFv (LTG1904) EVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLSSVAGPFNYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTQLTVLG 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 conjugated to CD8 TM (transmembrane) GAGTCAAAATACGGTCCTCCGTGCCCTCCGTGTCCGATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGC Amino acid sequence of the hinge domain of IgG4H bound to Array No. 41 CD8 TM (transmembrane) ESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYC

Claims

1. An isolated chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain comprising an scFv ROR1 antigen-binding domain having the amino acid sequence of SEQ ID NO: 8, a transmembrane domain comprising a CD8 transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of 4-1BB and a CD3 zeta intracellular domain.

2. The isolated CAR according to claim 1, wherein the at least one extracellular antigen-binding domain comprising the scFv ROR1 antigen-binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:

7.

3. The isolated CAR according to claim 2, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

22.

4. The isolated CAR according to claim 2, wherein the at least one extracellular antigen-binding domain and the intracellular signaling domain, or both, are linked to the transmembrane domain by a linker or spacer domain.

5. The isolated CAR according to claim 4, wherein the linker or spacer domain is obtained from the extracellular domain of IgG4, CD8, or CD28 and is linked to the transmembrane domain.

6. The isolated CAR according to claim 1, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.

7. A method for producing a cell, comprising the step of transducing in vitro a T cell with a vector comprising a nucleic acid molecule encoding a CAR comprising at least one extracellular antigen-binding domain comprising an scFv ROR1 antigen-binding domain having the amino acid sequence of SEQ ID NO: 8, a transmembrane domain comprising a CD8 transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of 4-1BB and a CD3 zeta intracellular domain.

8. The method according to claim 7, wherein at least one extracellular antigen-binding domain comprising the scFv ROR1 antigen-binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:

7.

9. The method according to claim 7, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

22.

10. The method according to claim 7, wherein the at least one extracellular antigen-binding domain and the intracellular signaling domain, or both, are linked to the transmembrane domain by a linker or spacer domain.

11. The method according to claim 10, wherein the linker or spacer domain is obtained from the extracellular domain of IgG4, CD8, or CD28 and is bound to the transmembrane domain.

12. The method according to claim 7, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.

13. A method for generating a population of RNA-engineered cells, comprising the step of introducing in vitro transcribed RNA or synthetic RNA into cells in vitro, wherein the RNA comprises a nucleic acid molecule encoding a CAR comprising at least one extracellular antigen-binding domain comprising the scFv ROR1 antigen-binding domain having the amino acid sequence of SEQ ID NO: 8, a transmembrane domain comprising a CD8 transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of 4-1BB and a CD3 zeta intracellular domain.

14. The method according to claim 13, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

22.

15. The method according to claim 13, wherein the at least one extracellular antigen-binding domain and the intracellular signaling domain, or both, are coupled to the transmembrane domain by a linker or spacer domain. **Claim 16** The method according to claim 15, wherein the linker or spacer domain is obtained from the extracellular domain of IgG4, CD8, or CD28 and is coupled to the transmembrane domain. **Claim 17** The method according to claim 13, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12. **Claim 18** An isolated CAR comprising the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.

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