Compositions and methods for treating cancer with Anti-CD19 immunotherapy

Abstract

The invention relates to compositions and methods for the treatment of cancer with anti-CD19 immunotherapy. Chimeric antigen receptors comprising a human CD19 antigen binding domain are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments and pharmaceutical compositions related to the chimeric antigen receptors are also disclosed. Also disclosed are methods of treating or preventing cancer in a subject, as well as methods of preparing chimeric antigen receptor T cells.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880074006.X and invention name “Compositions and methods for treating cancer with anti-CD19 immunotherapy”. The original application is the PCT international application PCT / US2018 / 051167 filed on September 14, 2018, which entered the Chinese national phase on May 14, 2020.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. Section 119(e) to U.S. Provisional Patent Application No. 62 / 559,297, filed on September 15, 2017, the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on September 13, 2018 is named Sequence.Listing.txt and is 93.7 kilobytes in size.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made in performance of a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the Department of Health and Human Services. The U.S. Government has certain rights in this invention. Technical Field

[0008] The present application relates to the field of cancer, and in particular to a CD19 antigen binding domain and a chimeric antigen receptor (CAR) comprising such a CD19 antigen binding domain and methods of using the same. Background Art

[0009] Cancer is one of the most deadly threats to human health. In the U.S. alone, cancer affects nearly 1.3 million new patients each year, and it is the second leading cause of death after cardiovascular disease, causing about a quarter of deaths. Solid tumors are the cause of most of these deaths. Although significant progress has been made in the medical treatment of some cancers, the overall 5-year survival rate of all cancers has only increased by about 10% in the past 20 years. Cancer or malignant tumors metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult.

[0010] CD19 is a transmembrane cell surface glycoprotein receptor of 85 to 95kDa.CD19 is a member of the immunoglobulin (Ig) superfamily of proteins, and comprises two extracellular Ig-like domains, a transmembrane domain and an intracellular signaling domain (Tedder TF, Isaacs, CM, 1989, JImmunol 143: 712-171).CD19 modifies B cell receptor signaling, reduces the triggering threshold of B cell receptor to antigen (Carter, RH, and Fearon, DT, 1992, Science, 256: 105-107), and cooperates with CD81 and CD21 to regulate this essential B cell signaling complex (Bradbury, LE, Kansas GS, Levy S, Evans RL, Tedder TF, 1992, JImmunol, 149: 2841-50). During B cell ontogeny, CD19 is able to signal independently of antigen receptors at the pro-B, pre-pre-B cell, pre-B, and early B cell stages, and is associated with the Src family of protein tyrosine kinases, is tyrosine phosphorylated, and induces both intracellular calcium mobilization and inositol phospholipid signaling (Uckun FM, Burkhardt AL, Jarvis L, Jun X, Stealy B, Dibirdik I, Myers DE, Tuel-Ahlgren L, Bolen JB, 1983, J Biol Chem 268: 21172-84). A key point relevant to the treatment of B cell malignancies is that CD19 is expressed in a tightly regulated manner on normal B cells, restricted to early B cell precursors, mature B cells at the stage of IgH gene rearrangement, but not on hematopoietic stem cells or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood 63: 1424-1433).

[0011] The current standard of care for B-lineage leukemias may consist of remission induction therapy with high-dose chemotherapy or radiation, followed by consolidation, and may feature stem cell transplantation and additional chemotherapy courses as needed (see the World Wide Web at cancer.gov). The high toxicity associated with these treatments and the risk of complications (e.g., relapse, secondary malignancies, or GVHD) have prompted the search for better treatment alternatives. Expression of CD19 on both adult and pediatric (pre-B-ALL) B-cell malignancies has led to both antibody-directed and chimeric antigen receptor (CAR)-T cell-based therapies exploiting this target (Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA, Lanier BJ, Morgan RA, Rosenberg SA, 2010, Blood 116:4099-102; Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL, 2015, Lancet 385:517-28).

[0012] Many new approaches have been developed to treat B-cell leukemias and lymphomas, including bispecific antibodies that link an anti-CD19 binding motif to a T-cell binding motif (i.e., blinatumomab, Suitable for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). To date, many binding parts of CD19 used in CAR constructs have utilized domains derived from mouse antibodies. Many of these products are currently under consideration for approval, including those developed by Novartis and Kite Pharmaceuticals. In April 2017, Novartis announced that CTL019 (tisagenlecleucel) had received FDA breakthrough designation for the treatment of adult patients with refractory or relapsed (r / r) DLBCL (diffuse large B cell lymphoma) who have failed two or more previous treatments, adding this designation to that for r / r B-cell acute lymphoblastic leukemia (ALL). These indications are based on the Phase II JULIET study (NCT02445248) and the ELIANA study (NCT02435849), respectively. The JULIET trial showed an overall response rate (ORR) of 45%, with 37% complete response (CR) and 8% partial response (PR) at three months. In the ELIANA study, 82% of patients infused with the product achieved CR or CR with incomplete count recovery, and the relapse-free survival rate was 60% at 6 months. The CAR-T product (KTE-C19, axicabtagene ciloleucel) from Kite Pharmaceuticals was granted breakthrough designation for diffuse large B-cell lymphoma (DLBLC), transformed follicular lymphoma (TFL) and primary mediastinal B-cell lymphoma (PMBCL). In the Kite ZUMA-3 Phase II trial of KTE-C19 in r / r ALL, 73% CR (at 2 months or longer) was reported. All information is from company press releases. Regardless of whether antibodies are utilized for CAR-T therapy, there is still a significant number of patients who are not helped by these treatments, and there is considerable room for improved treatment approaches.

[0013] Chimeric antigen receptors (CARs) are hybrid molecules comprising three basic units: (1) an extracellular antigen binding motif, (2) a junction / 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 a CAR typically emulates the minimal binding domain of an immunoglobulin (Ig) molecule, a single chain variable fragment (ScFv). Alternative antigen binding motifs have also been engineered, such as receptor ligands (i.e., IL-13 has been engineered to bind to an IL-13 receptor expressed by a tumor), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or γ-δ 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). Much work remains to be done to define the most active T cell populations for transduction with CAR vectors, determine optimal culture and expansion techniques, and define the molecular details of the CAR protein structure itself.

[0014] The connecting motif of CAR can be a relatively stable domain (such as the constant domain of IgG) or a flexible linker designed to be extended. Structural motifs (such as those derived from IgG constant domains) can be used to extend the ScFv binding domain away from the surface of the T cell plasma membrane. This can be important for some tumor targets in which the binding domain is particularly close to the surface membrane of tumor cells (for example, for disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motif for CAR always includes the CD3-ζ chain because the core motif is a key signal for T cell activation. The first reported second-generation CAR is characterized by a CD28 signaling domain and a CD28 transmembrane sequence. This motif is also used in a third-generation CAR containing a CD137 (4-1BB) signaling motif (Zhao Y et al JImmunol.2009; 183 (9): 5563–74). With the advent of new technologies, T cells are activated with beads connected to anti-CD3 and anti-CD28 antibodies, and the presence of the typical "signal 2" from CD28 no longer needs to be encoded by the CAR itself. By using bead activation, it was found that the third-generation vector was not superior to the second-generation vector in in vitro assays, and it did not provide significant benefits relative to the second-generation vector in a mouse model of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, YuanCM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, MackallCL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting Bcell precursor acute lymphoblastic leukemia, Blood. 2013; 121 (7): 1165–74; Kochenderfer JN et al. Blood. 2012; 119 (12): 2709–20). This is confirmed by the clinical success of second-generation CD28 / CD3-ζ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD19-specific CARs with CD137 / CD3-ζ signaling formats (Porter DL et al. N Engl J Med. 2011; 365(8):725–33).In addition to CD137, other tumor necrosis factor receptor superfamily members (such as OX40) can also provide important sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15 (18): 5852–60). Equally important are the culture conditions for culturing CAR T cell populations, such as the presence of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther. 2015; 22 (2): 72-78).

[0015] The challenge in the wider and effective adaptation of CAR therapy currently used for cancer is related to the lack of powerful targets. Establishing a binder of cell surface antigens is now easily achievable, but it is still a daunting challenge to find cell surface antigens that are specific to tumors while not harming normal tissues. A potential way to give CAR-expressing T cells greater target cell specificity is to use a combined CAR approach. In one system, CD3-ζ and CD28 signaling units are separated between two different CAR constructs expressed in the same cell; in another system, two CARs are expressed in the same T cell, but one has a lower affinity and therefore requires another CAR to first engage (engage) to make the second fully active (Lanitis E et al. Cancer Immunol Res. 2013; 1 (1): 43–53; Kloss CC et al. Nat Biotechnol. 2013; 31 (1): 71–5). The second challenge in producing a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma in which the effector cell population simultaneously targets multiple antigens (HER2, IL-13Ra, EphA2), hoping to avoid the consequences of a target antigen-negative population (Hegde M et al. Mol Ther. 2013; 21(11): 2087–101).

[0016] T cell-based immunotherapy has emerged as a new frontier in synthetic biology; a variety of promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can not only evade negative regulatory signals but also mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of inducible caspase 9 constructs with chemical-based dimers (e.g., AP1903) suggests a way in which a powerful switch that can control T cell populations can be turned on pharmacologically (Di Stasi A et al. N Engl J Med. 2011; 365(18): 1673–83). Generation of effector T cell populations immune to the negative regulatory effects of transforming growth factor β through expression of decoy receptors further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008; 31(5): 500–5). Therefore, although it appears that CAR can trigger T cell activation in a manner similar to endogenous T cell receptors, the main obstacle to the clinical application of this technology so far is limited by the in vivo expansion of CAR+ T cells, the rapid disappearance of cells after infusion, and disappointing clinical activity. This can be partly attributed to the mouse origin of some of the CAR sequences used.

[0017] For critically ill patients receiving this treatment, the use of blinotumomab (bispecific anti-CD19 and anti-CD3 antibodies) has shown impressive results. However, the durable remission rate is less than 40%, and at most only 50% of responders can be saved by hematopoietic stem cell transplant (HSCT) (see Gore et al., 2014, NCT01471782 and Von Stackelberg, et al., 2014, NCT01471782, summarized in Benjamin, JE, Stein AS, 2016, Therapeutic Advances in Hematology 7: 142-156). The requirement for patients who have received bispecific antibodies or CAR-T therapy to subsequently undergo HSCT to maintain a durable response is still an area of ​​active debate. Although high responses have been reported for CD19 CAR-T trials, some even greater than 90%, this figure may be closer to 70% if the trials are recast as “intent-to-treat” trials (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). The best results reported 12 months after CAR19 treatment showed an RFS of 55% and an OS of 79% in patients who were able to receive T-cell products at the University of Pennsylvania (Maude SL, Teachey DT, Rheingold SR, Shaw PA, Aplenc R, Barrett DM, Barker CS, Callahan C, Frey NV, Farzana N, Lacey SF, Zheng A, Levine B, Melenhorst JJ, Motley L, Prter DL, June CH, Grupp SA, 2016, J Clin Oncol 34, no15_suppl (May 2016) 3011-3011).

[0018] Therefore, there is an urgent and long-felt need in the art to find new compositions and methods for treating B-ALL and other CD19-expressing B-cell malignancies using approaches that can exhibit specific and potent anti-tumor effects without the aforementioned drawbacks.

[0019] The present invention solves these needs by providing CAR compositions and treatment methods that can be used to treat cancer and other diseases and / or conditions. In particular, the present invention as disclosed and described herein provides the following CAR: It can be used to treat diseases, disorders or conditions associated with dysregulated CD19 expression, and the CAR comprises a CD19 antigen binding domain that exhibits high surface expression on transduced T cells, exhibits a high degree of cell lysis of CD19-expressing cells, and wherein the transduced T cells show in vivo expansion and persistence. Summary of the invention

[0020] Provided herein are new anti-CD19 antibodies or their antigen binding domains and chimeric antigen receptors (CARs) comprising such CD19 antigen binding domains, and host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. The CARs show high surface expression on transduced T cells, and have a high degree of cell lysis and transduced T cell amplification and persistence in vivo. Also provided are methods using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject.

[0021] Thus, in one aspect, an isolated polynucleotide encoding a human anti-CD 19 antibody or a fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15.

[0022] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD19 antibody or a fragment thereof is provided, wherein the antibody or fragment thereof comprises a Fab fragment, a F(ab') 2 Fragments, Fv fragments and single-chain Fv (ScFv) fragments.

[0023] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD19 antibody or a fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16.

[0024] In one aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, wherein the chimeric antigen receptor comprises from N-terminus to C-terminus at least one CD19 antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the CD19 antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15.

[0025] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD19.

[0026] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19.

[0027] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD19 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) that binds to CD19.

[0028] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD19 antigen binding domain is connected to the transmembrane domain via a linker domain.

[0029] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD19 extracellular antigen binding domain is preceded by a sequence encoding a leader or signal peptide.

[0030] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the CAR comprises at least one CD19 antigen binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15, and wherein the CAR further encodes an extracellular antigen binding domain targeting an antigen including but not limited to CD20, CD22, ROR1, 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.

[0031] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the additional encoded extracellular antigen binding domain comprises an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 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-PMSAS ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain. ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2 ScFv antigen binding domain, anti-NY-ESO-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0032] In one aspect, a CAR provided herein further comprises a linker or spacer domain.

[0033] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular CD19 antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0034] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is connected to the transmembrane domain.

[0035] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the α, β or ζ chain of a T cell receptor; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD83; CD86; CD134; CD137 and CD154; or a combination thereof.

[0036] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0037] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is arranged at the N-terminal side relative to the CD3 zeta intracellular domain.

[0038] 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.

[0039] In other embodiments, 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 a combination thereof.

[0040] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, which further comprises a leader sequence or a signal peptide, wherein the leader or signal peptide nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 17.

[0041] In another embodiment, an isolated nucleic acid molecule encoding CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:18.

[0042] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD19 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0043] In one embodiment, a CAR is provided, wherein the extracellular CD19 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.

[0044] In another embodiment, a CAR is provided, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the α, β or ζ chain of a T cell receptor; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; TNFRSF19; or a combination thereof.

[0045] In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen binding domain comprising CD20, CD22, ROR1, 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.

[0046] In one embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 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-PMSAScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESO-1TCR ScFv antigen binding domain, anti-MAGE A3 TCRScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0047] In another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0048] In another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory domain, wherein the co-stimulatory domain comprises 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.

[0049] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (LTG2050LP-M19217-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2A )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 20 (LTG 2050LP-M19217-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2A ))'s CAR.

[0050] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 21 (LTG2065LP-M19217-1-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2B )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 22 (LTG 2065LP-M19217-1-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2B ))'s CAR.

[0051] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 23 (LTG2066 LP-M19217-2-CD8 TM-41BB-CD3ζCAR nucleotide sequence ( Figure 2C )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 24 (LTG 2066LP-M19217-2-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2C ))'s CAR.

[0052] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 25 (LTG2067LP-M19217-7-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2D)). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 26 (LTG 2067LP-M19217-7-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2D ))'s CAR.

[0053] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 27 (LTG2068LP-M19217-23-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2E )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 28 (LTG2068 LP-M19217-23-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2E ))'s CAR.

[0054] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 29 (LTG2069LP-M19217-29-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2F )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 30 (LTG 2069LP-M19217-29-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2F ))'s CAR.

[0055] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 31 (LTG2070LP-M19217-38-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2G )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 32 (LTG 2070LP-M19217-38-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2G ))'s CAR.

[0056] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 33 (LTG2071LP-M19217-40-CD8 TM-41BB-CD3ζCAR nucleic acid sequence ( Figure 2H )). In one embodiment, the nucleic acid sequence encodes an amino acid sequence comprising SEQ ID NO: 34 (LTG 2071LP-M19217-40-CD8 TM-41BB-CD3ζCAR amino acid sequence ( Figure 2H ))'s CAR.

[0057] In one aspect, the CAR disclosed herein is modified to express or comprise a detectable marker for diagnosis, monitoring, and / or prediction of treatment outcome (e.g., progression-free survival of a cancer patient) or for monitoring the progress of such treatment.

[0058] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector (e.g., 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 lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

[0059] In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0060] In another embodiment, the vector expressing CAR may also be modified to include one or more control elements for CAR T cell expression or for eliminating CAR-T cells by suicide switch. The suicide switch may include, for example, a drug that induces apoptosis-inducing signaling cascade or induces cell death. In a preferred embodiment, the vector expressing CAR may also be modified to express an enzyme, such as thymidine kinase (thymidine kinase, TK) or cytosine deaminase (cytosinedeaminase, CD).

[0061] In another aspect, a host cell comprising a nucleic acid molecule encoding CAR is also 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.

[0062] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a human CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16, wherein the T cell is a T cell of a person suffering from cancer. The cancer particularly includes hematological cancer, such as leukemia (e.g. chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML), lymphoma (e.g. mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0063] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of: the α, β or ζ chain of a T cell receptor; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; mesothelin; CD33; CD37; CD64; CD80; CD83; CD86; CD134; CD137; CD154; TNFRSF19; or a combination thereof.

[0064] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer comprises adult cancer, including oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), as well as breast cancer, reproductive system cancer (cervix, uterine body, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system cancer (bladder, kidney and renal pelvis, ureter), eye cancer and orbit cancer, endocrine system cancer (thyroid), as well as brain cancer and other nervous system cancers, or any combination thereof.

[0065] In another embodiment, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population of a human with cancer is provided, wherein the cancer is a refractory cancer that is not responsive to one or more chemotherapeutic agents. The cancers include hematopoietic cancers, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, 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)), childhood B-cell malignancies (including B-lineage ALL (acute lymphoblastic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0066] In another aspect, a method for preparing a T cell (hereinafter "CAR-T cell") comprising a CAR is provided. The method includes transducing a T cell with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds to CD19, thereby preparing a CAR-T cell.

[0067] In another aspect, a method for producing an RNA-engineered cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA encoding a nucleic acid molecule of the disclosed CAR into cells of a subject, thereby producing cells expressing the CAR.

[0068] In another aspect, a method for diagnosing a disease, disorder or condition associated with CD19 expression on a cell is provided, comprising a) contacting the cell with a human anti-CD19 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16; and b) detecting the presence of CD19, wherein the presence of CD19 is diagnostic of a disease, disorder or condition associated with CD19 expression.

[0069] In one embodiment, the disease, disorder or condition associated with CD19 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), childhood B-cell malignancies (including B-lineage ALL (acute lymphoblastic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0070] In another embodiment, a method for diagnosing, prognosing or determining the risk of a disease associated with CD19 in a mammal is provided, which comprises detecting the expression of CD19 in a sample derived from the mammal, comprising: a) contacting the sample with a human anti-CD19 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16; and b) detecting the presence of CD19, wherein the presence of CD19 is diagnostic of a disease associated with CD19 in the mammal.

[0071] In another embodiment, a method of inhibiting CD19-dependent T cell suppression is provided, comprising contacting a cell with a human anti-CD19 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. In one embodiment, the cell is selected from a tumor cell expressing CD19, a tumor-associated macrophage, and any combination thereof.

[0072] In another embodiment, a method of blocking T cell suppression mediated by cells expressing CD19 and altering the tumor microenvironment to inhibit tumor growth in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD19 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, 4, 6, 8, 10, 12, 14 and 16. In one embodiment, the cell is selected from a tumor cell expressing CD19, a tumor associated macrophage, and any combination thereof.

[0073] In another embodiment, a method of inhibiting, preventing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD19 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a tumor cell expressing CD19, a tumor-associated macrophage, and any combination thereof.

[0074] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0075] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering one or more disclosed CARs to a mammal in an amount effective to treat or prevent cancer in a mammal. The method includes administering to a subject a therapeutically effective amount of a host cell expressing a disclosed CAR specifically binding to CD19 and / or one or more of the aforementioned antigens, the conditions being sufficient to form an immune complex in the subject with the antigen binding domain on the CAR and the extracellular domain of CD19 and / or one or more of the aforementioned antigens.

[0076] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD19 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the extracellular CD19 antigen binding domain comprises an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16, or any combination thereof, and wherein the T cells are T cells of a subject suffering from cancer.

[0077] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one CD19 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the CD19 antigen binding domain comprises an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16, or any combination thereof, wherein the T cells are T cells of a subject suffering from cancer. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the alpha, beta or zeta chain of a T cell receptor; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD19; CD22; mesothelin; CD33; CD37; CD64; CD80; CD83; CD86; CD134; CD137; CD154; TNFRSF16; TNFRSF19; or a combination thereof.

[0078] In another embodiment, a method for producing a persistent genetically modified T cell population in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express a CAR, wherein the CAR comprises at least one CD19 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the CD19 antigen binding domain comprises an amino acid sequence of SEQ ID NO.2, 4, 6, 8, 10, 12, 14 or 16, or any combination thereof, wherein after administration, the persistent genetically modified T cell population or the progeny population of the T cell persists in the human body 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.

[0079] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0080] In all aspects and embodiments of the methods described herein, one or more CARs disclosed herein can be used to treat, prevent or ameliorate any of the aforementioned cancers, diseases, disorders or conditions associated with elevated expression of tumor antigens.

[0081] In another aspect, a kit is provided for preparing a chimeric antigen receptor T cell as described above or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression in a subject as described above, comprising a container and instructions for using the kit, wherein the container comprises any one or any combination of the nucleic acid molecules, vectors, host cells or compositions disclosed above.

[0082] It should be understood that CAR, host cells, nucleic acids and methods are also applicable outside of the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which is carried out with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 A schematic diagram of the general domain structure of a CAR with a novel extracellular CD19 antigen binding domain sequence is shown. The chimeric antigen receptor is composed of an extracellular CD19 binding ScFv domain, a CD8 spacer and transmembrane domain, an intracellular signaling CD137 co-stimulatory domain, and a CD3 z signaling domain.

[0084] Figures 2A to 2H Several nucleic acid and amino acid sequences of chimeric antigen receptors (CARs) comprising novel human extracellular CD19 antigen binding domain sequences are shown. The general scheme of CAR comprises a signal peptide, a human anti-CD19 binder single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 ζ signaling domain from the N-terminus to the C-terminus.

[0085] Figure 2A The lentiviral vector expressing the CARLTG2050 (19217ScFv-CD8 TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 19) and the encoded amino acid sequence (SEQ ID NO: 20) is shown.

[0086] Figure 2BThe lentiviral vector expressing the CARLTG2065 (M19217-1 ScFv-CD8 TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 21) and the encoded amino acid sequence (SEQ ID NO: 22) is shown.

[0087] Figure 2C The lentiviral vector expressing the CARLTG2066 (M19217-2 ScFv CD8 TM-41BB-CD3ζ) nucleotide sequence (SEQ ID NO: 23) and the encoded amino acid sequence (SEQ ID NO: 24) is shown.

[0088] Figure 2D The lentiviral vector expressing the CARLTG2067 (M19217-7 ScFv CD8 TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 25) and the encoded amino acid sequence (SEQ ID NO: 26) is shown.

[0089] Figure 2E The lentiviral vector expressing the CARLTG2068 (M19217-23 ScFv CD8TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 27) and the encoded amino acid sequence (SEQ ID NO: 28) is shown.

[0090] Figure 2F Shown is a lentiviral vector expressing the CAR LTG2069 (M19217-29 ScFv CD8TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 29) and the encoded amino acid sequence (SEQ ID NO: 30).

[0091] Figure 2G The lentiviral vector expressing the CARLTG2070 (M19217-38 ScFv CD8TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 31) and the encoded amino acid sequence (SEQ ID NO: 32) is shown.

[0092] Figure 2H The lentiviral vector expressing the CARLTG2071 (M19217-40 ScFv CD8TM-41BB-CD3ζ) nucleic acid sequence (SEQ ID NO: 33) and the encoded amino acid sequence (SEQ ID NO: 34) is shown.

[0093] Figure 3Anti-CD19 CAR surface expression in primary human T cells is shown.CAR T cells redirected to CD19 tumor antigens using ScFv domains (as listed in each group) are produced by lentiviral transduction with CAR expression constructs.CART detection is performed by flow cytometry.T cells are washed twice in cold PBS-EDTA buffer and stained with CD19-Fc peptide followed by anti-Fc-AF647 reagent.At least 20,000 cells are obtained for each analysis.Based on forward scatter and side scatter, single peak discrimination and 7AAD negative, cells are gated so that only live cells are analyzed.Data are acquired in the APC channel on a MACSQuant 10 flow cytometer.UTD, untransduced negative control cells.The markers in each group identify the population expressing CAR and are listed as the percentage of the total transduced T cells analyzed.

[0094] Figure 4 Anti-CD19 CAR T cells incorporated with ScFv conjugates (LTG2050, 2065-2071) are shown, and the ScFv conjugates mediate the cell lysis of CD19 positive tumors in vitro. CAR T cells expressing anti-CD19 constructs are incubated overnight with CD19 positive cell lines (Raji and Reh) or CD19 negative cell lines (K562 and 293T), and the cell lines are stably transduced with firefly luciferase, and the effector to target ratio is 5, 10, 20 and 40 (x-axis). Then, CART cytotoxic activity is assessed by luciferase activity measurement, as described in Materials and Methods. Each bar is the mean value of 3 technical repetitions and the error bar represents SD. UTD-untransduced T cell negative control, 1538-LTG1538 FMC63 mouse anti-CD19 CAR positive control.

[0095] Figure 5 Shown is the production of high cytokine levels of CD19-specific CART cells when co-cultured with CD19-positive leukemia cell lines (dark gray) or cultured T cells alone (gray). The assay was performed overnight at an E:T ratio of 10:1, and the cytokine concentration of the supernatant was then analyzed by ELISA. N = 2 technical repeats + / -SD. Negative control: UT-untransduced T cells, 1538-FMC63 mouse CD19 positive control transduced T cells. The number of LTGs for each LV used to transduce human T cells is listed on the x-axis. DETAILED DESCRIPTION

[0096] definition

[0097] Unless the context clearly indicates otherwise, nouns used in this article without quantifier modification refer to one or more. For example, the term "antigen" includes single or multiple antigens, and can be considered to be equivalent to the phrase "at least one antigen". The term "comprising" used herein means "comprising". Thus, "comprising antigens" means "comprising antigens" without excluding other elements. The phrase "and / or" means "and" or "or". It should also be understood that, unless otherwise indicated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular mass values ​​are approximate and are provided for descriptive purposes. Although many methods and materials similar or equivalent to the methods and materials described herein can be used, specific suitable methods and materials are described below. In the event of a conflict, this specification (including the explanation of the term) will prevail. In addition, materials, methods and examples are only illustrative and are not intended to be limited. For the convenience of a review of various embodiments, the following term explanations are provided:

[0098] The term "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to encompass variations of +-.20%, or in some cases +-.10%, or in some cases +-.5%, or in some cases +-.1%, or in some cases +-.0.1% from the particular value, as such variations are suitable for performing the disclosed methods.

[0099] Unless otherwise indicated, the technical terms herein are used according to conventional usage. Definitions of commonly used 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 Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0100] The present disclosure provides CD19 antibodies or fragments thereof and chimeric antigen receptors (CARs) with such CD19 antigen binding domains. The enhancement of the functional activity of CAR is directly related to the enhancement of the functional activity of T cells expressing CAR. As a result of one or more of these modifications, CAR exhibits both high cytokine-induced cell lysis and cell surface expression on transduced T cells, as well as the persistence of the increased in vivo T cell expansion level and transduced CAR expressing T cells.

[0101] The unique ability to combine functional parts derived from different protein domains is a key innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains is a key design feature, as is the way in which its specificity is combined. Each design domain is an essential component that can be used between different CAR platforms to transform the function of lymphocytes. For example, the selection of extracellular binding domains can make otherwise ineffective CARs effective.

[0102] The constant framework components of the immunoglobulin-derived protein sequences used to establish the extracellular antigen binding domain of the CAR can be completely neutral or can self-associate and drive T cells to a state of metabolic exhaustion, thereby making the effect of the therapeutic T cells expressing the CAR much lower. This occurs independently of the antigen binding function of the CAR domain. In addition, the selection of intracellular signaling domains can also control the activity and persistence of therapeutic lymphocyte populations for immunotherapy. Although the ability to bind to target antigens and the ability to transmit activation signals to T cells through these extracellular and intracellular domains, respectively, are important aspects of CAR design, it has become apparent that the choice of the source of the extracellular antigen binding fragment can have a significant effect on the efficacy of the CAR, thereby limiting the function and clinical utility of the CAR.

[0103] Surprisingly and unexpectedly, it has now been discovered that the use of fully human antigen-binding domains in CARs, rather than using mouse-derived antigen-binding fragments that are prone to inducing anti-mouse immune responses and CAR T elimination in the host (see, UPenn-sponsored clinical trial using mouse-derived SS1 ScFv sequence, NCT02159716), can also determine the functional activity of CAR-expressing T cells.

[0104] According to this discovery, a series of CD19 binders from human scFv expression libraries have been developed. These fully human CD19 CARs are no longer mouse-derived and are therefore unlikely to induce allergic or rejection reactions through patients (see MausMV, Haas AR, Beatty GL, Albeda SM, Levine BL, Liu X, Zhao Y, Kalos M, June CH, 2013, Cancer Immunology Research, 1: 26-31). Therefore, when these "fully human" CARs are expressed in T cells and then injected into patients, they may be more therapeutically effective. These CAR binders derived from human sequences can be used to treat cancers, leukemias, and lymphomas of people expressing CD19 antigens, including but not limited to B-ALL, DLBCL, and FL.

[0105] CAR disclosed herein is expressed at high levels in cells. Cells expressing CAR have a high in vivo proliferation rate, produce a large amount of cytokines, and have high cytotoxic activity against cells with CD19 antigens bound to CAR on the surface. The use of human extracellular CD19 antigen binding domains leads to the production of CARs that work better in vivo, while avoiding inducing anti-CAR immunity and killing CAR T cell populations in the host immune response. CARs expressing fully human extracellular CD19 ScFv antigen binding domains exhibit excellent activity / characteristics, including: i) as seen with mouse-derived binding sequences, preventing poor CAR T persistence and function; ii) CARs lacking regional (i.e., intrapleural) delivery are effective; and iii) the ability to produce CAR T cell designs based on binders with both high affinity and low affinity for CD19. The latter property allows researchers to better tune the potency versus toxicity and / or tissue specificity of CAR T products, as lower affinity binders may have higher specificity for tumors compared to normal tissues due to higher expression of CD19 on tumors than normal tissues, which could prevent off-target tumor toxicity and bystander cell killing.

[0106] The following is a detailed description of the CAR of the present invention, including a description of its extracellular CD19 antigen binding domain, transmembrane domain and intracellular domain, as well as other descriptions of CAR, antibodies and antigen binding fragments thereof, conjugates, nucleotides, expression, vectors and host cells, treatment methods, compositions and kits using the disclosed CAR.

[0107] A. Chimeric Antigen Receptor (CAR)

[0108] The CAR disclosed herein comprises at least one CD19 antigen binding domain capable of binding to CD19, at least one transmembrane domain and at least one intracellular domain.

[0109] Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide comprising an antigen binding domain (e.g., single-chain variable fragment (ScFv)) of an antibody connected to a T cell signaling domain via a transmembrane domain. The characteristics of CAR include its ability to redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner, and the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0110] As disclosed herein, the intracellular T cell signaling domain of a CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a CAR that includes an intracellular domain of a T cell receptor, such as, but not limited to, an intracellular portion of a CD3ζ protein. A co-stimulatory signaling domain refers to a portion of a CAR that includes an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to an antigen.

[0111] 1. Extracellular domain

[0112] In one embodiment, CAR comprises a target-specific binding element, which is also referred to as an antigen binding domain or portion. The selection of the domain depends on the type and quantity of the surface ligands that define the target cell. For example, the antigen binding domain can be selected to identify the ligand that acts as a cell surface marker associated with a specific disease state on the target cell. Therefore, some examples of cell surface markers that can serve as ligands for the antigen binding domain in CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0113] In one embodiment, the CAR can be modified to target a tumor antigen by modifying a desired antigen binding domain that specifically binds to an antigen on a tumor cell. Tumor antigens are proteins produced by tumor cells that cause an immune response, especially a T cell-mediated immune response. The choice of antigen binding domain will depend on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, 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, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and CD19. The tumor antigens disclosed herein are included by way of example only. This list is not meant to be exclusive and other examples will be apparent to those skilled in the art.

[0114] In one embodiment, tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumor.Malignant tumor expresses multiple proteins that can be used as target antigens for immune attack.These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.Other target molecules belong to transformation-related molecule groups, such as oncogene HER-2 / Neu / ErbB-2.Another group of target antigens is cancer-embryonic antigen, such as carcinoembryonic antigen (CEA).In B cell lymphoma, tumor-specific idiotype immunoglobulin constitutes the real tumor-specific immunoglobulin antigen unique to individual tumor.B cell differentiation antigens (such as CD19, CD20, CD22, BCMA, ROR1 and CD37) are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies with limited success.

[0115] In a preferred embodiment, the tumor antigen is CD19, and tumors associated with CD19 expression include lung mesothelioma, ovarian cancer, and pancreatic cancer, which express high levels of the extracellular protein CD19, or any combination thereof.

[0116] Types of tumor antigens may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor may occur under conditions that enable the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or a TAA may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0117] Non-limiting examples of TSAs or TAAs include the following: 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 oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens generated by 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. 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, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, 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-associated protein, TAAL6, TAG72, TLP and TPS.

[0118] In one embodiment, the antigen binding domain portion of CAR targets an antigen, including but not limited to CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1TCR, MAGE A3 TCR, etc.

[0119] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD19 antigen.

[0120] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 VH-2 antigen binding domain 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 CD19 VH-2 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0121] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 VH-4 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 3, 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 CD19 VH-4 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0122] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 9 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 5, 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 CD19 ScFv 9 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0123] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 10 antigen binding domain 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 CD19 ScFv 10 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0124] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 12 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 9, 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 CD19 ScFv 12 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0125] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 15 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 11, 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 CD19 ScFv15 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0126] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 15 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 CD19 ScFv15 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0127] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 15 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 15, 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 CD19 ScFv15 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0128] In various embodiments of the CD19-specific CAR disclosed herein, the general scheme is Figure 1 , and includes, from N-terminus to C-terminus, a signal peptide or leader peptide, anti-CD19 ScFv, an extracellular linker, CD8 transmembrane, 4-1BB, CD3ζ, wherein the bold text represents the cloning site of the connecting domain.

[0129] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19, and encodes an amino acid sequence as shown in SEQ ID NO: 20 [LTG 2050LP-M19217-CD8 TM-41BB-CD3ζ amino acid sequence (as shown in Figure 2A CAR shown in ).

[0130] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 20 [LTG 2050LP-M19217-CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2A )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0131] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 21, and encodes an amino acid sequence as shown in SEQ ID NO: 22 [LTG 2065LP-M19217-1-CD8 TM-41BB-CD3ζ amino acid sequence (as shown in Figure 2B CAR shown).

[0132] In another embodiment, the nucleic acid sequence encoding CAR comprises a nucleic acid sequence of SEQ ID NO: 21, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a nucleic acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 22 [LTG 2065LP-M19217-1-CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2B )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0133] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 23, and encodes an amino acid sequence as shown in SEQ ID NO: 24 [LTG 2066LP-M19217-2-CD8 TM-41BB-CD3ζCAR amino acid sequence (as shown in Figure 2C CAR shown in ).

[0134] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 23, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 24 [LTG 2066LP-M19217-2-CD8 TM-41BB-CD3ζCAR amino acid sequence (as Figure 2C )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0135] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 25, and encodes an amino acid sequence as shown in SEQ ID NO: 26 [LTG2067LP-M19217-7-CD8 TM-41BB-CD3ζ amino acid sequence (as Figure 2D CAR shown in ).

[0136] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 25, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 26 [LTG2067LP-M19217-7-CD8 TM-41BB-CD3 amino acid sequence (such as Figure 2D )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0137] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 27, and encodes an amino acid sequence as shown in SEQ ID NO: 28 [LTG2068LP-M19217-23-CD8 TM-41BB-CD3ζ amino acid sequence (as shown in Figure 2E CAR shown in ).

[0138] In another embodiment, the nucleic acid sequence encoding CAR comprises a nucleic acid sequence of SEQ ID NO: 27, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 28 [LTG2068LP-M19217-23-CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2E )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0139] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 29, and encodes an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 [(LTG2069LP-M19217-29-CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2F CAR shown in ).

[0140] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 29, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 30 [LTG2069LP-M19217-29-CD8 TM-41BB-CD3ζ amino acid sequence (as Figure 2F )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0141] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 31, and encodes an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 32 [(LTG2070LP-M19217-38-CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2G CAR shown in ).

[0142] In another embodiment, the nucleic acid sequence encoding CAR comprises a nucleic acid sequence of SEQ ID NO: 31, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 32 [(LTG2070LP-M19217-38-CD8 TM-41BB-CD3 ζ amino acid sequence (as Figure 2G )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0143] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 33, and encodes an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 34 [(LTG2071LP-M19217-40-CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2H CAR shown in ).

[0144] In another embodiment, the nucleic acid sequence encoding CAR comprises a nucleic acid sequence of SEQ ID NO: 33, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes an amino acid sequence as shown in SEQ ID NO: 34 [(LTG2071LP-M19217-40-CD8 TM-41BB-CD3ζ amino acid sequence (as Figure 2H )] or a CAR having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0145] Surface expression of anti-CD19 CARs incorporating single chain variable fragment (ScFv) sequences reactive with the CD19 antigen is shown in Example 2 below and summarized in Table 2. The expression level of each ScFv- or VH-containing CAR was determined by flow cytometry analysis of LV-transduced T cells from healthy donors using recombinant CD19-Fc peptides followed by anti-human Fc F(ab')2 fragments conjugated to AF647 and detected in the APC channel (see Figure 3 ). ScFv-based anti-CD19 CAR constructs LTG2050, LTG2065-LTG2071 were highly expressed in human primary T cells (as shown in the gated population) compared to untransduced T cell controls (non-gated population). Representative results from one donor are shown.

[0146] As in Example 2 and Figure 4As shown in , when a lentiviral vector (LV) expressing the following CAR was constructed and tested for anti-leukemia activity, high cell lytic activity of CD19 CAR was shown. Each experimental CAR contains a 4-1BB / CD3-ζ chain signaling motif and a specific anti-CD19 binding motif / domain indicated therein. Leukemia target lines with different CD19 surface expression were used: Raji and Reh; and CD19-negative K562 and 293T. The ScFv-based anti-CD19 CAR constructs LTG2050 and LTG2065-2071 are able to efficiently lyse high CD19 tumor lines Raji and Reh, however, they have no specific lytic activity against K562 or 293T (see Figure 4 ). These results show the efficiency and specificity of the generated CAR constructs.

[0147] The ability of anti-CD19 CAR T cells to secrete cytokines was then evaluated. Tumor cells were co-incubated with CAR T cells or control T cells at an effector to target ratio of 10:1 overnight, and culture supernatants were analyzed by ELISA for IFNγ, TNFα, and IL-2 (see Figure 5 ). It is noteworthy that cells LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070 and LTG2071 expressing CAR T showed high IFNγ, TNFα and IL-2 levels, while the negative control (untransduced, UN) did not produce significant cytokine induction. Unexpectedly, CD19 CAR LTG2050 produced significantly lower induced cytokine levels for the tested tumor lines. CAR tested as a positive control, LTG1538 expressing mouse FMC63 CD19 conjugate (SEQ ID NO: 47), has lower activity than all conjugates except LTG2050. The high in vitro cytolytic function of LTG2050 with low cytokine production capacity indicates that multiple CAR T functional endpoints need to be tested on a construct-by-construct basis. Furthermore, the superiority of the LTG2065-2071 human CD19 binder over the murine FMC63 scFv has been clearly shown in cytokine production assays.

[0148] Without intending to be limited to any particular mechanism of action, it is believed that possible reasons for the enhanced therapeutic function associated with the exemplary CARs of the present invention include, for example, but not limited to, a) improved lateral movement within the plasma membrane allowing more efficient signal transduction, b) a preferential location within the plasma membrane microdomain (e.g., lipid rafts), and a greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) a preferential location within the plasma membrane by preferential movement away from inhibitory (dampening) or downregulatory interactions (e.g., less proximity to or interaction with phosphatases (e.g., CD45)), and d) preferential assembly into T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0149] Although the disclosure has been illustrated with exemplary extracellular human CD19 ScFv antigen binding domains, other nucleotides and / or amino acid variants within the CD19 variable ScFv antigen binding domains can be used to obtain only a heavy chain binding domain or a subset thereof, and thus include CD19 antigen binding domains for use in CAR described herein. In one embodiment, other nucleotides and / or amino acid variants within the CD19 variable ScFv antigen binding domains include, for example, variants labeled as FMC63 (SEQ ID NO: 46 and SEQ ID NO: 47 (nucleotide sequence and amino acid sequence, respectively) comprising CD19 variable ScFv antigen binding domains (see Example 1).

[0150] Depending on the desired antigen to be targeted, CAR can be additionally engineered to include a suitable antigen binding domain specific to the desired antigen target. For example, if CD19 is a desired antigen to be targeted, an antibody against CD19 can be used as an antigen binding domain incorporated into the CAR.

[0151] In an exemplary embodiment, the antigen binding domain portion of the CAR additionally targets CD33. Preferably, the antigen binding domain in the CAR is an anti-CD33 ScFv, wherein the nucleic acid sequence of the anti-CD33ScFv comprises the sequence shown in SEQ ID NO: 48. In one embodiment, the anti-CD33 ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 49. In another embodiment, the anti-CD33 ScFv portion of the CAR comprises the amino acid sequence shown in SEQ ID NO: 49.

[0152] In an exemplary embodiment, the antigen binding domain portion of the CAR additionally targets mesothelin. Preferably, the antigen binding domain in the CAR is an anti-mesothelin ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the nucleotide sequence shown in SEQ ID NO: 50. In one embodiment, the anti-mesothelin ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 51. In another embodiment, the anti-mesothelin ScFv portion of the CAR comprises the amino acid sequence shown in SEQ ID NO: 51.

[0153] In one aspect of the present invention, a CAR capable of binding to a non-TSA or non-TAA is provided, wherein the non-TSA or non-TAA includes, for example, but not limited to, an antigen derived from the following: Retroviridae (e.g., human immunodeficiency virus (e.g., HIV-1 and HIV-LP)), Picornaviridae (e.g., 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 [e.g., herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV) and herpes virus], poxviridae (e.g., smallpox virus, vaccinia virus and poxvirus) or hepatitis C virus, or any combination thereof.

[0154] In another aspect of the present invention, a CAR is provided that is capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, CARs are provided that can bind to antigens derived from the following infectious bacteria: for example, Helicobacter pyloris, Legionella pneumophilia, bacterial strains of the genus Mycobacteria sp. (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, or Mycobacterium gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, A Streptococcus), Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae or Clostridium tetani, or a combination thereof.

[0155] 2. Transmembrane domain

[0156] With respect to transmembrane domains, the CAR comprises one or more transmembrane domains fused to the extracellular CD19 antigen binding domain of the CAR.

[0157] The transmembrane domain may be derived from natural or synthetic sources. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0158] The transmembrane region with a specific use in the CAR described herein may be derived from the following (i.e., at least the following transmembrane region): α, β or ζ chain of the T cell receptor; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; mesothelin; CD33; CD37; CD64; CD80; CD83; CD86; CD134; CD137; CD154; TNFRSF16 or TNFRSF19. Alternatively, the transmembrane domain may be synthetic, in which case it will mainly contain hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) may form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine dimers provide particularly suitable connectors.

[0159] In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used in addition to the transmembrane domains described above.

[0160] In some cases, transmembrane domains may be selected or amino acid substitutions may be made to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0161] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 35. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 36. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 36.

[0162] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:36, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:36.

[0163] In some cases, the transmembrane domain of CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence of SEQ ID NO: 37. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 38. In another embodiment, the CD8 hinge domain comprises an amino acid sequence of SEQ ID NO: 38, or a sequence having 95% to 99% identity thereto.

[0164] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0165] 3. Spacer domain

[0166] In CAR, the spacer domain may be arranged between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide for connecting the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0167] In some embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill and include those listed in the following: 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, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0168] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signal transduction into the cell. Some examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0169] As the spacer domain, all or part of amino acids 137 to 206 of the hinge region of CD8.α. (SEQ ID NO: 39) (NCBI RefSeq: NP.sub.--001759.3), amino acids 135 to 195 of CD8.β. (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP.sub.--000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP.sub.--006130.1) can be used. In addition, as the spacer domain, a part of the constant region of the antibody H chain or L chain can be used. In addition, the spacer domain can be an artificially synthesized sequence.

[0170] In addition, in CAR, the signal peptide sequence can be connected to the N-terminus. The signal peptide sequence is present at the N-terminus of many secreted proteins and membrane proteins and is 15 to 30 amino acids in length. Since many protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 18.

[0171] 4. Intracellular domain

[0172] The cytoplasmic domain or additional intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces effector function signals and instructs cells to perform specialized functions. Although the entire intracellular signaling domain can usually be used, in many cases the entire chain does not need to be used. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used to replace the complete chain as long as it transduces the effector function signal. Therefore, the term intracellular signaling domain means any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0173] Some preferred examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same functional capabilities.

[0174] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, T cell activation can be considered to be mediated by two different kinds of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0175] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0176] Some examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful for the CARs disclosed herein include those derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3.ζ. (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of FcεRI.γ. (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of FcεRI.β. (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ. (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3.δ. (NCBI RefSeq: NP.sub.--000723.1), amino acids 153 to 207 of CD3.ε. (NCBI RefSeq: NP.sub.--000723.1), amino acids 207 to 211 of FcεRI.β. (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ. (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3.δ. (NCBI RefSeq: NP.sub.--000723.1), amino acids 153 to 207 of CD3.ε. (NCBI RefSeq: NP.sub.--000 RefSeq: NP.sub.--000724.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1), and amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), as well as variants thereof having the same functions as these peptides. The amino acid numbering based on the amino acid sequence information of NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor of each protein (including signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0177] In a preferred embodiment, the intracellular domain of CAR can be designed to include a CD3-ζ signaling domain alone or in combination with any other desired cytoplasmic domain that can be used in CAR. For example, the intracellular domain of CAR may include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the intracellular domain of the CAR that contains a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the efficient response of lymphocytes to antigens. Some 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 ligands that specifically bind to CD83, etc. Specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8.α (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--001759.3), amino acids 207 to 235 of CD83 (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--001759.3 ...96 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--001759.1), amino acids 181 to 220 RefSeq: NP.sub.--006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBIRefSeq: NP.sub.--001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and variants thereof having the same functions as these peptides. Therefore, although the disclosure herein is mainly illustrated by 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the disclosure.

[0178] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of CAR can be connected to each other in a random or specific order. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0179] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28 and 4-1BB.

[0180] 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-ζ, wherein the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO: 40 and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO: 42 and the variant nucleic acid sequence shown in SEQ ID NO: 44. 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-ζ, wherein the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO: 40 and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO: 42, or in another embodiment, includes the CD3-ζ variant nucleic acid sequence shown in SEQ ID NO: 44.

[0181] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:41 and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:43, or in another embodiment, comprises a CD3-ζ variant nucleic acid encoding the amino acid sequence of SEQ ID NO:45.

[0182] 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-ζ, wherein the signaling domain of 4-1BB includes the amino acid sequence shown in SEQ ID NO:41 and the signaling domain of CD3-ζ includes the amino acid sequence shown in SEQ ID NO:43 and the variant amino acid sequence shown in SEQ ID NO:45.

[0183] 5. Additional description of CAR

[0184] Also explicitly included within the scope of the present invention are the functional parts of CAR disclosed herein.When used with reference to CAR, the term "functional part" refers to any of the following parts or fragments of one or more CAR disclosed herein, and the part or fragment retains the biological activity of CAR (the part or fragment is a part thereof) (parent CAR). The functional part covers, for example, the following parts of CAR, which retain the ability to identify target cells or detect, treat or prevent diseases to a similar extent, to the same extent or to a higher extent compared to the parent CAR. With reference to parent CAR, the functional part may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0185] The functional part may include additional amino acids at the amino or carboxyl terminus or at both ends of the part, which are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological functions of the functional part, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance biological activity compared to the biological activity of the parent CAR.

[0186] Included within the scope of the present disclosure are functional variants of CAR disclosed herein. The term "functional variant" used herein refers to a CAR, polypeptide or protein having a significant or significant sequence identity or similarity to a parent CAR, and the functional variant retains the biological activity of CAR (the functional variant is its variant). Functional variants include, for example, those variants of CAR (parent CAR) described herein, which retain the ability to identify target cells to a similar extent, to the same extent or to a higher extent compared to the parent CAR. With reference to the parent CAR, the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or higher identity in terms of amino acid sequence with the parent CAR.

[0187] Functional variants may, for example, comprise the amino acid sequence of a parent CAR having at least one conservative amino acid substitution. As an alternative or supplement, functional variants may comprise the amino acid sequence of a parent CAR having at least one non-conservative amino acid substitution. In this case, non-conservative amino acid substitutions preferably do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is improved compared to the parent CAR.

[0188] The amino acid substitutions of CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with specific physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr and Val), an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

[0189] The CAR may consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0190] CAR (including functional parts and functional variants) can have any length, that is, can contain any number of amino acids, provided that CAR (or its functional part or functional variant) retains its biological activity, such as the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, CAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0191] CAR (including functional parts and functional variants of the present invention) may include synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, di ... Indole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and a-tert-butylglycine.

[0192] CAR (including functional parts and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, a disulfide bridge) or converted into an acid addition salt and / or optionally dimerized or multimerized, or conjugated.

[0193] CAR (including its functional part and functional variant) can be obtained by methods known in the art. CAR can be prepared by any suitable method for preparing polypeptides or proteins. Suitable methods for synthesizing polypeptides and proteins from scratch are described in, for example, the following references: 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. In addition, the nucleic acids described herein can be used to recombinantly produce polypeptides and proteins using standard recombinant methods. See, for example, 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. In addition, some CARs (including functional parts and functional variants thereof) can be separated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). The methods of separation and purification are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by a company. In this regard, CARs can be synthetic, recombinant, separated and / or purified.

[0194] B. Antibodies and Antigen-binding Fragments

[0195] One embodiment also provides a CAR that specifically binds to one or more antigens disclosed herein, a T cell expressing CAR, an antibody, or an antigen binding domain or portion thereof. "CAR-expressing T cell" or "CAR T cell" as used herein means a T cell expressing CAR and having an antigen specificity determined by, for example, an antibody-derived targeting domain of CAR.

[0196] As used herein, "antigen binding domain" may include antibodies and antigen binding fragments thereof. The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments thereof, as long as they exhibit the desired antigen binding activity. Non-limiting examples of antibodies include, for example, complete immunoglobulins and variants and fragments thereof known in the art that retain binding affinity to antigens.

[0197] "Monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies, and is not interpreted as requiring the production of antibodies by any particular method. In some instances, a monoclonal antibody is an antibody produced by a monoclone of a B lymphocyte or by a cell or its progeny in which a nucleic acid encoding a single antibody light chain variable region and a heavy chain variable region (or its antigen-binding fragment) has been transfected. In some instances, a monoclonal antibody is separated from an object. A monoclonal antibody may have a conservative amino acid substitution that has substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, see Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0198] In general, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain species (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0199] Each heavy and light chain is comprised of a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6 thed., WH Freeman and Co., page 91 (2007).). In some embodiments, the heavy chain and light chain variable regions are combined to specifically bind to an antigen. In other embodiments, only the heavy chain variable region is required. For example, naturally occurring camel antibodies consisting of only heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including, for example, the variable region of an antigen-binding fragment of Fv, ScFv, dsFv or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including the variable domain of Fv, ScFv, dsFv or Fab.

[0200] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework regions of the constituent light and heavy chains) is used to position and arrange the CDRs in three-dimensional space.

[0201] The CDRs are primarily responsible for binding to antigenic epitopes. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5 thEd. 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 Igsuperfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2 and CDR3 (from N-terminus to C-terminus), and are also usually identified by the chain in which the particular CDR is located. Thus, VH CDR3 is CDR3 from the heavy chain variable domain of the antibody in which it is present, and VL CDR1 is CDR1 from the light chain variable domain of the antibody in which it is present. The light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

[0202] "Antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize cognate antigens, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed by antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of whole antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press, 2010).

[0203] Single-chain antibodies (ScFv) are genetically engineered molecules comprising the VH and VL domains of one or more antibodies connected as a genetically fused single-chain molecule by an appropriate polypeptide linker (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, ID Rugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is generally not critical for ScFv. Therefore, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0204] In dsFv, the heavy and light variable chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90: 6444 6448, 1993; Poljak et al., Structure, 2: 1121 1123, 1994).

[0205] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0206] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced, or can be obtained, for example, by screening a combinatorial library consisting of a variable heavy chain and a variable light chain, as described in Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference. These and other methods for preparing, for example, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, single-chain antibodies, and bifunctional antibodies are well known to those skilled 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, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0207] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and vice versa, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0208] "Humanized" antibodies or antigen-binding fragments comprise a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDR is referred to as a "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as a "recipient." In one embodiment, all CDRs in a humanized immunoglobulin are from a donor immunoglobulin. The constant region need not be present, but if it is present, it may be substantially identical to a human immunoglobulin constant region, e.g., an identity of at least about 85% to 90%, e.g., about 95% or higher. Therefore, all parts of a humanized antibody or antigen-binding fragment (except possibly CDR) are substantially identical to corresponding parts of a natural human antibody sequence.

[0209] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies, which are generally 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.

[0210] "Fully human antibodies" or "human antibodies" are antibodies that contain sequences from (or derived from) the human genome and do not contain sequences from another species. In some embodiments, human antibodies contain CDRs, framework regions, and (if present) Fc regions from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Printing; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0211] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different from each other. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0212] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., supra, U.S. Patent Application Publication No. 2002 / 0197266A1 and U.S. Patent No. 7,338,929).

[0213] In addition, CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0214] C. Conjugates

[0215] CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof that are specific for one or more antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which effector molecules or detectable markers are covalently attached to antibodies or antigen-binding fragments that specifically bind to one or more antigens disclosed herein. Those skilled in the art will appreciate that a variety of effector molecules and detectable markers can be used, including but not limited to chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g. 125 I. 32 P. 14 C. 3 H and 35 S) and other labels, target moieties and ligands, etc.

[0216] The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin used to cause the death of a particular target cell (eg, a tumor cell).

[0217] The procedures used to attach effector molecules or detectable markers to antibodies or antigen-binding fragments vary depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups that can be used to react with appropriate functional groups on antibodies to bind effector molecules or detectable markers; for example, carboxylic acid groups (COOH), free amine groups (-NH 2 ) or sulfhydryl (-SH) groups. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment of any of a variety of known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker may be any molecule used to connect the antibody or antigen binding fragment to an effector molecule or a detectable marker. The linker is capable of forming a covalent bond with both the antibody or antigen binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched 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 connected to the constituent amino acids through its side groups (e.g., through a disulfide bond with cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0218] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill and include those listed in the following: 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, 4,879,278; 4,816,444; and 4,486,414, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0219] In some embodiments, the joint is cleavable under intracellular conditions, so that the cutting of the joint in the intracellular environment releases the effector molecule or detectable marker from the antibody or antigen binding fragment. In other embodiments, the joint is non-cleavable and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the joint can be cut by a cutting agent present in the intracellular environment (for example, in a lysosome or endosome or caveolea). The joint can be, for example, a peptide joint cut by an intracellular peptidase or protease (including but not limited to a lysosome or endosomal protease). In some embodiments, the peptide joint is at least 2 amino acids long or at least 3 amino acids long. However, the joint can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, 1 to 15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives so that active drugs are released within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptide linker (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker) that can be cleaved by the sulfhydryl-dependent protease cathepsin-B can be used. Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker that can be cleaved by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0220] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Generally speaking, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661.). Such linkers are relatively stable under neutral pH conditions (e.g., those in blood), but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0221] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogeled., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Pat. No. 4,880,935).

[0222] In other specific embodiments, 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(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0223] In other embodiments, the linker is non-cleavable and the effector molecule or detectable marker is released by antibody degradation. (See U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety).

[0224] 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), 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 in the conjugate sample is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period of time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours), and then quantifying the amount of free effector molecules or detectable markers present in the plasma. A variety of exemplary linkers that can be used for conjugates are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0225] In some embodiments, a conjugate of a CAR, a T cell expressing a CAR, an antibody, or an antigen binding portion thereof, and one or more small molecule toxins is provided, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecene, and CC1065, as well as derivatives of these toxins having toxin activity.

[0226] Maytansine compounds suitable for use as maytansine alkaloid toxin parts are well known in the art, and can be separated from natural sources according to known methods, can be produced using genetic engineering techniques (see Yu et al (2002) PNAS 99: 7968-7973), or can be maytansinol and maytansinol analogs prepared according to known methods. Maytansinol alkaloids are mitotic inhibitors that play a role by inhibiting tubulin polymerization. Maytansine was first separated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). Subsequently, it was found that some microorganisms also produced maytansinol alkaloids, such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 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 incorporated herein by reference. Conjugates comprising maytansinoids, methods for their preparation and their therapeutic uses are disclosed, for example, in US Pat. Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 Bl, the disclosures of which are expressly incorporated herein by reference.

[0227] Other toxins can be used with CAR, T cells expressing CAR, antibodies or their antigen binding portions.Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, nuclear toxins (ribotoxin), ribonucleases, saporin and calicheamicin, and botulinum toxin A to F. These toxins are well known in the art and many can be easily obtained from commercial sources (such as Sigma Chemical Company, St.Louis, MO).Contemplated toxins also include variants of toxins (see, for example, referring to U.S. Patent Nos. 5,079,163 and 4,689,401).

[0228] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosome complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, the toxin does not have a mechanism for specific entry into cells, and therefore needs to be conjugated to an antibody or antigen-binding fragment that recognizes a cell surface protein that is internalized in order to be efficiently taken up by cells.

[0229] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Generally, diphtheria toxins used for immunotoxins are mutated to reduce or eliminate nonspecific toxicity. A mutant called CRM107 with full enzyme activity but significantly reduced nonspecific toxicity has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.

[0230] Ricin is a lectin RCA60 from Ricinus communis (castor bean). For some examples of ricin, see U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, which are denoted RCA based on their molecular weights of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to galactose residues on the cell surface and promotes 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).

[0231] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary nuclear toxins (e.g., α-sarcin and restrictocin) are discussed, 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 double-strand breaks in DNA, leading to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0232] Abrin includes toxic lectins from Abrus precatorius. The toxic components abrin a, b, c and d have molecular weights of about 63 kD to 67 kD and consist of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem. 52: 1095, 1988; and Olsnes, Methods Enzymol. 50: 330-335, 1978).

[0233] CARs, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof that are specific for one or more antigens disclosed herein can also be conjugated to a detectable marker; for example, a detectable marker that can be detected by: ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, detectable markers that can be used include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), are also used. CAR, T cells expressing CAR, antibodies or their antigen-binding portions may also be conjugated with enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When CAR, T cells expressing CAR, antibodies or their antigen-binding portions are conjugated with detectable enzymes, they can be detected by adding additional reagents used by the enzyme to produce a distinguishable reaction product. For example, when there is a reagent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. CAR, T cells expressing CAR, antibodies or their antigen-binding portions may also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself may be conjugated with an enzyme or a fluorescent marker.

[0234] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof can be conjugated with paramagnetic agents (e.g., gadolinium). Paramagnetic agents (e.g., superparamagnetic iron oxide) are also used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope (e.g., a leucine zipper pair sequence, a binding site of a second antibody, a metal binding domain, an epitope tag) recognized by a second reporter.

[0235] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof may also be conjugated to radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect one or more antigens and antigen-expressing cells disclosed herein by x-rays, emission spectra or other diagnostic techniques. In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, for example, for treating neuroblastoma. Some examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I.

[0236] Methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using film or scintillation counters, and fluorescent markers can be detected using a photodetector to detect the emitted radiation. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme acting on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0237] D. Nucleotides, expression, vectors and host cells

[0238] One embodiment of the present invention also provides a nucleic acid comprising a nucleotide sequence encoding any CAR, antibody, or antigen binding portion thereof described herein (including functional portions and functional variants thereof). The nucleic acid of the present invention may include a nucleotide sequence encoding any leader sequence, antigen binding domain, transmembrane domain, and / or intracellular T cell signaling domain described herein.

[0239] In some embodiments, the nucleotide sequence can be codon-modified. Without being bound by a particular theory, it is believed that codon optimization of nucleotide sequences improves the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences may involve replacing natural codons with another codon, which encodes the same amino acid but can be translated by tRNA that is more easily obtained in the cell, thereby improving translation efficiency. Optimization of nucleotide sequences can also reduce secondary mRNA structures that may interfere with translation, thereby improving translation efficiency.

[0240] In one embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding the antigen binding domains of the CAR of the present invention. In another embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding any CAR described herein (including its functional parts and functional variants).

[0241] As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a DNA or RNA polymer that may be single-stranded or double-stranded, synthetic, or obtained from a natural source (e.g., isolated and / or purified), that may contain natural, non-natural, or altered nucleotides, and that may contain natural, non-natural, or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, replacing the phosphodiester present between the nucleotides of an unmodified oligonucleotide. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, in some cases for nucleic acids, it may be appropriate to contain one or more insertions, deletions, inversions, and / or substitutions.

[0242] Recombinant nucleic acid can be a nucleic acid having a non-natural sequence or having a sequence prepared by artificially combining two otherwise separated sequence fragments. This artificial combination is usually achieved by chemical synthesis or more commonly by artificially operating separated nucleic acid fragments, such as by genetic engineering techniques, such as those described in Sambrook et al (supra). Nucleic acid can be constructed based on chemical synthesis and / or enzymatic ligation using operations known in the art. See, for example, Sambrook et al (supra) and Ausubel et al. (supra). For example, nucleic acid can be chemically synthesized using naturally occurring nucleotides or multiple modified nucleotides (such as thiophosphate derivatives and acridine substituted nucleotides) designed to improve the biological stability of the molecule or improve the physical stability of the doublet formed after hybridization. Some examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0243] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR or a functional part or functional variant thereof. Alternatively, the nucleotide sequence may comprise a degenerate nucleotide sequence of any sequence or a combination of degenerate sequences.

[0244] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0245] Nucleotide sequences hybridized under stringent conditions can hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes with the target sequence (nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that can distinguish polynucleotides with precise complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that occasionally have some small regions (e.g., 3 to 10 bases) that match the nucleotide sequence. Compared with full-length complements with 14 to 17 bases or more bases, such small complementary regions are more easily melted, and high stringency hybridization makes it easily distinguishable. Relatively high stringency conditions may include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalents at a temperature of about 50°C to 70°C. Such high stringency conditions allow for few mismatches between nucleotide sequences and template strands or target strands (if any), and are particularly suitable for detecting the expression of any CAR of the present invention. It is generally appreciated that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0246] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

[0247] In one embodiment, nucleic acid can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any nucleic acid. For purposes herein, the term "recombinant expression vector" means the following genetically modified oligonucleotide or polynucleotide construct, which allows host cells to express mRNA, protein, polypeptide or peptide when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and the vector is contacted with a cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector is not naturally occurring as a whole.

[0248] However, portions of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-natural internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0249] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. Vectors can be selected from pUC series (Fermentas Life Sciences, GlenBurnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).

[0250] Phage vectors may also be used, e.g. 1, λZapII (Stratagene), EMBL4 and λNMI149. Some examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121 and pBIN19 (Clontech). Some examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector or a lentiviral vector. A lentiviral vector is a vector derived from at least a portion of a lentiviral genome, particularly including a self-inactivating lentiviral vector, such as provided in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but not limited to, LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica plc, LENTIMAX.TM. vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0251] A variety of transfection techniques are 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)).

[0252] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high-speed microprojectiles (see, e.g., Klein et al, Nature, 327:70-73 (1987)).

[0253] In one embodiment, the recombinant expression vector can be prepared using standard recombinant DNA techniques such as described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0254] Recombinant expression vectors may contain regulatory sequences, such as transcriptional and translational start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0255] The recombinant expression vector may comprise one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, such as antibiotics, heavy metal resistance, etc., provide prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0256] The recombinant expression vector may include a natural or non-natural promoter operably connected to a nucleotide sequence encoding CAR (including its functional part and functional variant) or a nucleotide sequence complementary to or hybridized with the nucleotide sequence encoding CAR. The selection of promoters (e.g., strong, weak, inducible, tissue-specific and development-specific) is within the ordinary skills of technicians. Similarly, the combination of nucleotide sequence and promoter is also within the skills of technicians. The promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, an RSV promoter, or a promoter present in the long terminal repeat of a mouse stem cell virus.

[0257] The recombinant expression vector can be designed for transient expression, for stable expression or for both. In addition, the recombinant expression vector can be made for constitutive expression or for inducible expression.

[0258] In addition, the recombinant expression vector can be made to contain suicide genes. The term "suicide gene" used herein refers to a gene that causes cell death expressing the suicide gene. The suicide gene can be a gene that gives cells expressing the gene sensitivity to reagents (e.g., drugs), and causes cell death when cells are contacted with or exposed to the reagent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0259] One embodiment also provides a host cell comprising any recombinant expression vector described herein. The term "host cell" used herein refers to any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, an animal, a fungus or an algae, or may be a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, that is, directly separated from an organism (e.g., a human). The host cell may be an adherent cell or a suspended cell (i.e., a cell grown in suspension). Suitable host cells are known in the art, and include, for example, DH5a Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. When the host cell can be any cell type, can be derived from any type of tissue, and can be at any developmental stage, the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell can be a T cell.

[0260] For purposes herein, T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells separated from people. T cells can be any type of T cells and can be in any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+T cells (for example, cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), initial T cells, etc. T cells can be CD8+T cells or CD4+T cells.

[0261] In one embodiment, the CAR described herein can be used for suitable non-T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells produced by pluripotent stem cells.

[0262] One embodiment also provides a cell group comprising at least one host cell described herein.Cell group can be a heterogeneous colony, which includes a host cell containing any recombinant expression vector and at least one other cell, such as a host cell (e.g., T cell) or a cell different from a T cell that does not include any recombinant expression vector, such as a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a myocyte, a brain cell, etc. Alternatively, a cell group can be a substantially homogeneous colony, wherein the colony mainly includes a host cell (e.g., substantially consisting of) containing a recombinant expression vector. The colony can also be a clonal cell group, wherein all cells in the colony are clones of a single host cell comprising a recombinant expression vector, so that all cells in the colony include a recombinant expression vector. In one embodiment of the invention, a cell group is a clonal colony comprising a host cell containing a recombinant expression vector as described herein.

[0263] CAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including its population) and antibodies (including its antigen binding portion) can be separated and / or purified. For example, a purified (or separated) host cell preparation is a host cell preparation in which the host cell has a higher purity than the cell in its natural environment in vivo. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the preparation of the host cell is purified so that the host cell represents at least about 50% of the total cell content of the preparation, for example, at least about 70%. For example, the purity may be at least about 50%, may be greater than about 60%, about 70% or about 80%, or may be about 100%.

[0264] E. Treatment methods

[0265] It is contemplated that the CAR disclosed herein can be used in a method for treating or preventing a disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof, and / or a pharmaceutical composition to the mammal in an amount effective to treat or prevent cancer in the mammal.

[0266] One embodiment also includes lymphodepleting the mammal prior to administering the CAR disclosed herein. Some examples of lymphodepleting include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, whole body irradiation, etc.

[0267] For the purpose of the method for administering a host cell or cell group, the cell can be a mammalian allogeneic or autologous cell. Preferably, the cell is a mammalian autologous cell. Allogeneic as used herein means any of the following materials: it and the individual into which the material is introduced are derived from different animals of the same species. When the genes of one or more loci are not the same, two or more individuals are considered to be allogeneic to each other. In some aspects, the allogeneic material from the individual of the same species can be genetically sufficiently dissimilar to interact antigenically. "Autologous" as used herein means any of the following materials: it is derived from the same individual into which the material is reintroduced later.

[0268] The mammals mentioned herein can be any mammal. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. The mammal can be from the order Carnivora, including Felidae (cats) and Canidae (dogs). The mammal can be from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs) or the order Persistodactyla, including Equine (horses). The mammal can be of the order Primates, Ceboids or Simoids (monkeys), or the order Anthropoids (humans and apes). Preferably, the mammal is a human.

[0269] With respect to the methods, the cancer can be any cancer, including any of the following: acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, Cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, chronic B lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0270] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that are considered by one of ordinary skill in the art to have a potential benefit or therapeutic effect. In this aspect, the method can provide any amount or level of cancer treatment or prevention in a mammal.

[0271] In addition, the treatment or prevention provided by the method may include treating or preventing one or more disorders or symptoms of the disease (e.g., cancer) being treated or prevented. In addition, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or disorders.

[0272] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof, or a pharmaceutical composition, thereby forming a complex, (b) and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0273] The sample can be obtained by any suitable method (e.g., biopsy or autopsy). A biopsy is the removal of tissue and / or cells from an individual. Such removal can be for the purpose of collecting tissue and / or cells from an individual to perform experiments on the removed tissue and / or cells. The experiment may include an experiment to determine whether the individual has and / or suffers from a particular condition or disease state. The condition or disease may be, for example, cancer.

[0274] For one embodiment of a method for detecting the presence of a proliferative disorder (e.g., cancer) in a mammal, the sample comprising cells of the mammal can be a sample comprising whole cells, whole cell lysates, or fractions of whole cell lysates (e.g., nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions). If the sample comprises whole cells, the cells can be any cells of the mammal, such as cells of any organ or tissue, including blood cells or endothelial cells.

[0275] For mammals, contacting can occur in vitro or in vivo. Preferably, contacting is in vitro.

[0276] In addition, the detection of the complex can be carried out by any number of methods known in the art. For example, CAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell group, or antibody or its antigen binding portion described herein can be labeled with a detectable marker, such as a radioisotope, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles) as disclosed above.

[0277] The method for testing the ability of CAR to recognize target cells and antigen specificity is known in the art. For example, Clay et al., J.Immunol, 163: 507-513 (1999) teaches the method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2). In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, such as Zhao et al, J.Immunol, 174: 4415-4423 (2005).

[0278] Another embodiment provides the CAR of the present invention, nucleic acid, recombinant expression vector, host cell, cell group, antibody or its antigen binding portion thereof, and / or pharmaceutical composition for treating or preventing proliferative disorders (eg, cancer) in mammals. Cancer can be any cancer described herein.

[0279] Any method of administration can be used for the disclosed therapeutic agents, including local and systemic administration. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific mode of administration and dosage regimen will be selected by the attending clinician considering the specific circumstances of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). In the case where more than one agent or composition is administered, one or more routes of administration can be used; for example, a chemotherapeutic agent can be administered orally and an antibody or antigen binding fragment or conjugate or composition can be administered intravenously. The method of administration includes injection, in which case CAR, CAR T cells, conjugates, antibodies, antigen binding fragments, or compositions are provided in, for example, the following non-toxic pharmaceutically acceptable carriers: water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compounds may be used, for example, by applying the antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed, or to an area suspected of being prone to tumorigenesis. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical formulation comprising a therapeutically effective amount of the antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops, or intravitreally to the eye.

[0280] The disclosed therapeutic agents can be formulated into a unit dosage form suitable for single administration of precise dosage. In addition, the disclosed therapeutic agents can be administered in a single dose or in a multiple dose regimen. A multiple dose regimen is a regimen in which the primary course of treatment may have more than one single dose, e.g., 1 to 10 doses, followed by other doses given as needed at subsequent time intervals to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound over a period of days to months or even years. Therefore, the dosage regimen will also be determined at least in part based on the specific needs of the subject to be treated and will depend on the judgment of the administering physician.

[0281] A typical dosage of the antibody or conjugate may be from about 0.01 mg / kg to about 30 mg / kg, for example from about 0.1 mg / kg to about 10 mg / kg.

[0282] In some specific examples, a therapeutic composition comprising one or more conjugates, antibodies, compositions, CARs, CAR T cells, or other agents is administered to a subject for a period of, for example, weeks, months, or years, based on a multiple daily dosing regimen (e.g., at least 2 consecutive days, 10 consecutive days, etc.). In one example, a conjugate, antibody, composition, or other agent is administered to a subject for at least 30 days, for example, 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.

[0283] In some embodiments, the disclosed method includes providing surgery, radiotherapy and / or chemotherapy to the subject in combination with a disclosed antibody, antigen binding fragment, conjugate, CAR or T cell expressing CAR (e.g., sequentially, substantially simultaneously or simultaneously). Such medicaments and treatment methods and therapeutic doses are known to those skilled in the art and can be determined by skilled clinicians. The preparation and dosing regimen for other medicaments can be used according to the manufacturer's instructions or determined empirically by skilled technicians. Preparations and dosing regimens for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., MCPerry, Williams & Wilkins, Baltimore, Md.

[0284] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Non-limiting examples of additional therapeutic agents that can be used for combination therapy include microtubule binders, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered in a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anticancer agent or anti-angiogenic agent can be administered in combination with CAR, CAR-T cells, antibodies, antigen binding fragments, or conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and can be determined by skilled clinicians.

[0285] Additional chemotherapeutic agents include, but are not limited to, 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 uracil mustard; antimetabolites, such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), 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, alkali, vindesine, and vinorelbine); cytotoxic / antitumor 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 other agents such as alitretinoin, altretinoin, 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 and therapeutic dosage of such agents are known to those skilled in the art and can be determined by the skilled clinician.

[0286] Combination therapy may provide a synergistic effect and demonstrate synergy, i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. Synergy is achieved when the active ingredients are: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered in separate formulations in alternation or in parallel; or (3) by some other regimen. When delivered in alternation, synergy is achieved when the compounds are administered or delivered sequentially, such as by different injections in separate syringes. Generally, during alternation, an effective dose of each active ingredient is administered sequentially, i.e., one after the other, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0287] In one embodiment, an effective amount of an antibody or antigen binding fragment or its conjugate that specifically binds to one or more antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed to allow the administered antibody or antigen binding fragment or conjugate to form an immune complex with the antigen expressed on the corresponding cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, compared to the control obtained before the treatment, the immune complex increases and indicates that the treatment is ineffective, whereas compared to the control obtained before the treatment, the immune complex decreases and indicates that the treatment is effective.

[0288] F. Biopharmaceutical Compositions

[0289] Provided herein are biopharmaceutical compositions or biopharmaceutical compositions (hereinafter "compositions") for gene therapy, immunotherapy and / or cell therapy, including one or more 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 in a carrier (e.g., a pharmaceutically acceptable carrier). The composition can be prepared in a unit dose form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired result. The composition can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing CARs, antibodies, antigen binding fragments, and conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising CARs disclosed herein, or T cells expressing CARs, conjugates, antibodies, or antigen binding fragments are used for, for example, the treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some examples, the composition can be used for the treatment or detection of cancer. Compositions comprising a CAR, or a T cell expressing a CAR, a conjugate, an antibody or an antigen-binding fragment as disclosed herein are also used, for example, in the detection of pathological angiogenesis.

[0290] The composition for administration may include a solution of CAR, or T cells expressing CAR, conjugates, antibodies or antigen binding fragments dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional known sterilization techniques. These compositions may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR, or T cells expressing CAR, antibodies or antigen binding fragments or conjugates in these preparations may vary widely, and will be selected mainly based on fluid volume, viscosity, body weight, etc. according to the selected specific mode of administration and the needs of the subject. The actual method of preparing such a dosage form for gene therapy, immunotherapy and / or cell therapy is known or will be obvious to those skilled in the art.

[0291] Typical compositions for intravenous administration include about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate / subject / day (or a corresponding dose of CAR or T cells expressing CAR, a conjugate comprising an antibody or antigen-binding fragment). Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art, and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0292] CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates can be provided in lyophilized form and rehydrated with sterile water before administration, but they are also provided as sterile solutions of known concentrations. The CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugate solutions are then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 to 15 mg / kg body weight. There is considerable experience in the art in the administration of antibodies or antigen binding fragments and conjugate drugs; for example, since 1997, Since its approval, antibody drugs have been sold in the United States. CAR, or T cells expressing CAR, antibodies, antigen binding fragments and conjugates thereof can be administered by slow infusion rather than intravenous push or rapid injection (intravenous push or bolus). In one example, a higher loading dose is administered, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg antibody or antigen binding fragment (or a corresponding dose of a conjugate comprising an antibody or antigen binding fragment) can be infused within about 90 minutes, followed by a weekly maintenance dose of 2 mg / kg infused within 30 minutes if the previous dose is well tolerated for 4 to 8 weeks.

[0293] Controlled release parenteral formulations can be prepared as implants, oily injections, or as particle systems. For an overview of protein delivery systems, see Banga, AJ, 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 (e.g., cytotoxins or drugs) as a central core. In microspheres, therapeutic agents are dispersed throughout the particles. Particles, microspheres, and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. The diameter of the capillary is about 5 μm so that only nanoparticles are administered intravenously. The diameter of the microparticles is generally about 100 μm and is administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0294] Polymers can be used for CAR disclosed herein or T cells expressing CAR, antibodies or antigen binding fragments or conjugate compositions for ion controlled release. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer poloxamer 407 exists as a viscous but movable liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown that it is an effective carrier for the formulation 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 controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). On the other hand, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). A number of additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S. Pat. No. 5,534,496).

[0295] G.Medicine box

[0296] In one aspect, a kit for using the CAR disclosed herein is also provided. For example, a kit for treating a tumor in a subject, or for preparing a CAR T cell expressing one or more CARs disclosed herein. As disclosed herein, the kit will generally contain the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. More than one disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing CAR may be included in the kit.

[0297] The medicine box may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed of a variety of materials (e.g., glass or plastic). The container typically contains a composition comprising one or more disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. In some embodiments, the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0298] The label or package insert will generally also include instructions for using the disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or T cell expressing CAR in a method for treating or preventing tumors or preparing CAR T cells. The package insert is generally included in the instructions generally included in the commercial packaging of the therapeutic product, which contains information about the indications, usage, dosage, administration, contraindications and / or warnings related to the use of such therapeutic products. The illustrative material can be written in electronic form (such as a computer disk or a compact disc) or can be visual (such as a video file). The kit may also include additional components that are conducive to the specific application for which the kit is designed. Therefore, for example, the kit may additionally include tools for detecting the label (such as enzyme substrates for enzyme labeling, filter devices for detecting fluorescent labels, suitable second labels (such as second antibodies), etc.). The kit may additionally include buffers and other reagents commonly used in the practice of a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0299] Example

[0300] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting the scope of the present invention. On the contrary, it should be clearly understood that a number of other embodiments, modifications thereof and equivalents may be sought, which may be apparent 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.

[0301] Example 1

[0302] Isolation of CD19-specific antibodies from fully human phage and yeast displayed ScFv libraries

[0303] Materials and methods:

[0304] a) Generation of human ScFv and CD19-specific antibodies

[0305] A naive human ScFv (recombinant single-chain fragment variant of immunoglobulin) phage display library (approximate diversity, 10 10 ScFv (Miltenyi Biotec, unpublished) was used to select for recombinant human CD19 protein. 12The amplified library of ScFv displayed by phage was incubated with 5 μg, 3 μg and 1 μg of coated CD19 in a 5×100-μl volume and evenly distributed in 5 wells of a 96-well plate for 2 hours at room temperature during the first, second and third rounds of bio-panning, respectively. After each round of incubation, the wells were washed 5 times in the first round with phosphate-buffered saline (PBST) containing 0.05% Tween 20 and 10 times in subsequent rounds to remove non-specifically bound phages, the bound phages were mixed with TG1 competent cells at 37°C for 1 hour, and the phages were amplified from the infected cells and used for the next round of bio-panning. After the third round of bio-panning, 380 clones were randomly picked from infected TG1 cells, and each clone was inoculated into 150 μl 2YT culture medium containing 100 μg / ml carbenicillin and 0.2% glucose in 96-well plates by using automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density of 0.5 at 600nm (OD600), the kanamycin of helper phage M13K07 and 50 μg / ml (final concentration) with a multiplicity of infection (multiplicity of infection, MOI) of 10 was added to the culture medium, and the plate was further incubated overnight at 30 ° C in a shaking table under 250 rpm. The phage supernatant was mixed with 3% skim milk in PBS at a volume ratio of 4: 1, and used for enzyme-linked immunosorbent assay (ELISA) to identify the phage clones showing ScFv or VH with high CD19 binding affinity. The supernatant was incubated at room temperature for 2 hours, recombinant human CD19 was coated at 50 ng / well in a 96-well plate and washed five times with PBST, (after incubation overnight at 4°C, blocked with 3% skim milk in PBS and washed three times with PBS containing 0.05% Tween 20), and CD19-bound phage was detected using horseradish peroxidase-conjugated goat anti-M13 antibody. After incubation with the antibody, non-specifically bound antibodies were removed by washing the wells, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and the absorbance of the solution at 450 nm (A450) was measured. Clones that bound to CD19 with A450>1.0 were selected for further characterization.

[0306] b) Expression and purification of selected soluble ScFvs.

[0307] DNA sequencing was performed on the VH and VL of the selected clones, and the ScFv encoded by the clones with unique sequences was expressed and purified as described below. Plasmids extracted from these clones were used to transform HB2151 cells. Single colonies were picked from the plates containing freshly transformed cells, inoculated into 200 ml 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture reached 0.90 at OD at 600 nm, 0.5 mM final concentration of isopropyl-β-d-thiogalactopyranoside was added, and the culture was further incubated overnight at 30°C. The bacterial pellet was collected after 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 xg for 25 minutes at 4°C and the supernatant was used for ScFv purification using Ni-NTA resin according to the supplier's protocol (Qiagen).

[0308] c) ELISA binding assay: 50 μl of recombinant human CD19 diluted at 2 μg / ml in PBS was coated in a 96-well plate overnight at 4°C. Purified ScFv with His and Flag tags were serially diluted and added to the target protein coated wells. After washing, HRP-conjugated anti-Flag antibody was added at a dilution of 1:3000 for 1 hour at room temperature. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes before adding 1N H 2 SO 4 The reaction was stopped and the OD was read at 450 nm to quantify the relative ability of the ScFv to bind CD19.

[0309] d) Display of yeast scFv libraries.

[0310] The same ScFv starting material as used for phage display was also incorporated into the yeast ScFv display system. To complement the phage-based scFv analysis, a yeast library expressing a human scFv library was also screened. To enrich for yeast expressing scFvs that bind to both recombinant CD19-Fc and CD19 expressed on the cell surface of CHOK1 cells, cell panning was performed on CHOK1 transfected with CD19 cells. For the first round of panning on the cell surface, CHOK1-CD19 cells were seeded into 6-well plates two days prior to panning and grown to 50% confluence in F12 K medium. Then 5×10 7The yeast cells were washed twice with PBSA buffer and resuspended in 3mL F12K culture medium, and then gently added dropwise to CHOK1-CD19 cells. After being gently shaken on ice for 2 hours, CHOK1-CD19 cells were then washed 3 times with ice-cold PBSA to remove yeast cells that were not bound to CHOK1-CD19, and then .05% trypsin-EDTA (Gibco) was used to separate CHOK1-CD19 cells and bound yeast cells from the plate. The cell mixture containing both yeast and CHOK1 cells was then inoculated into 10mL SDCAA culture medium and amplified overnight at 30°C, and then induced in SGCAA culture medium at 30°C for 16 hours. For the second round of cell panning, a similar scheme as described above was performed except that more stringent washing conditions were used. This panning method produced m19217 conjugates. Further characterization of the conjugate and other conjugates from phage display showed that affinity maturation was needed because the biological properties of the CAR created from this success were still not optimal.

[0311] In order to improve the affinity of m19217, yeast display m19217 mutant scFv library was created by creating random point mutations in the scFv gene sequence using error-prone PCR. After electroporation, the resulting mutant library was then grown overnight in SDCAA culture medium at 30°C for 16 hours, and then transferred to SGCAA culture medium and grown again at 30°C for 16 hours. The mutant library was then classified by MACS (immunomagnetic column, Miltenyi Biotec) as the capture antigen using CD19-Fc to reduce the library size and increase the population of mutants that can be combined with CD19-Fc. The strongest binder was then selected by double staining the library (pool) with anti-c-Myc-Alexa 488 and CD19-Fc / anti-Hu-Fc and selecting the binder with the highest binding affinity and c-Myc expression level. Then repeat this process more than twice, until the flow cytometry of yeast particles with fluorescent tag antigen produces the average binding affinity of the mutant library improved than the starting construct. Using the CD19 of the reduced amount of labeling, the binding affinity is evaluated by the flow cytometry of the yeast library. This process causes the EC50 of M19217 (effective concentration for CD19 50% binding marked on the yeast displaying ScFv) to increase by 0.5 μg / ml so that the affinity of the affinity matured conjugate (M19217-1, 19217-2, M19217-7, M19217-23, M19217-29, M19217-38, M19217-40) is <0.01 μg / ml.

[0312] result:

[0313] Due to the unique challenges of the CD19 structure, phage display candidates did not yield biologically functional CAR constructs and therefore ScFv identification that produced biologically active binders was generated via yeast display. Based on flow cytometric analysis of yeast-displayed ScFvs, eight ScFv clones specific for recombinant human CD19 were identified and labeled as the human anti-CD19 ScFv binder M19217 (LTG2050, founder clone, EC50 of 0.5 μg / ml) and the following affinity matured binders (EC50 < 0.01 μg / ml): M19217-1 (LTG2065), M19217-2 (LTG2066), M19217-7 (LTG2067), M19217-23 (LTG2068), M19217-29 (LTG2069), M19217-38 (LTG2070) and M19217-40 (LTG2071). The generation of chimeric antigen receptors expressing the LTG2050, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070 and LTG2071 human anti-CD19 binders is outlined in Example 2 below.

[0314] Example 2

[0315] CAR expressing anti-CD19 fully human binding sequence.

[0316] Homo sapiens CD19 (B4, CVID3, Leu-12) is a well-studied cell surface glycoprotein expressed on B-cell leukemias and lymphomas. At least two antibody drug conjugates are being evaluated in Phase II clinical trials, (SGN-CD19A, Denintuzumab Mafodotin (Seattle Genetics) and SAR3419, Coltuximab Ravtansine. A Phase I study of SGN-19A showed a CRc of 35% in the best treatment group (Fathi AT, Borate U, DeAngelo DJ, O'Brien MM, Trippett T, Shah BD, Hale GA, Foran JM, Silverman LB, Tibes R, Cramer S, Pauly M, Kim S, Kostic A, Huang X, Pan Y, Chen R, 2015, Blood 126:1328). However, development was suspended due to disappointing Phase II results of SAR3419 (Coiffer B, Thieblemont C, de Guibert S, Dupuis J, Ribrag V, Bouabdallah R, Morschhauser F, Navarro R, Le Gouill S, HaiounC, Houot R, Cassasnovas O, Holte H, Lamy T, Broussais F, Payrard S, Hatteville L, Tilly H, 2016, Bt J Haematolo 173: 722-30). The use of bispecific anti-CD3 / anti-CD19 antibodies (Blinotumomab) is discussed above. In view of the current progress of T cell-based therapy using CD19 CAR, the best method is of course cell-based immunotherapy and the CAR construct proposed here is a new method for creating and implementing a new CD19 binding part derived from a human sequence.

[0317] The new anti-CD19 CAR-T construct described herein has high cell surface expression levels and specificity for CD19-positive tumor cells and effective cytotoxicity and cytokine function in primary human T cells.CD19 CAR is designed using a CD19 binding sequence derived from a ScFv candidate identified by phage display as in Example 1, and for characterization, it is cloned into a lentiviral expression vector and tested in vitro for transduction efficiency, killing function and cytokine production in both model cell lines and primary human T cells, and the lentiviral expression vector contains a selected structure and signaling domain under the control of the EF1a promoter.Table 1 summarizes the terms used.CAR construct LTG#1538 is a relevant comparator because the mouse-derived sequence is a conjugate currently used in commercial development (see KTE-C19, Kite Pharma and CTL019, Novartis).

[0318] Table 1 - Construct LTG numbers and corresponding ScFv binder names for fully human CD19 CAR design

[0319] CAR construct LTG# huCAR19 ScFv conjugate UTD Untransduced T cell control 1538 FMC63 mouse CAR19 control 2050 M19217 2065 M19217-1 2066 M19217-2 2067 M19217-7 2068 M19217-23 2069 M19217-29 2070 M19217-38 2071 M19217-40

[0320] Materials and methods:

[0321] (a) Cell lines

[0322] The Burkitt lymphoma cell line Raji and the chronic myeloid leukemia line K562 were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and propagated in CD FortiCho medium (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single cell clones expressing luciferase cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD) and then cloning and selecting luciferase-positive clones.

[0323] (b) Creation of chimeric antigen receptor (CAR)-expression vector

[0324] The CAR antigen binding domain and ScFv sequence are derived from human anti-CD19 ScFv fragments. The CAR T construct is generated by connecting the in-frame binder sequence to the CD8a linker and transmembrane domain (UniProt sequence ID P01732, amino acids 138 to 206), and then to the 4-1BB (CD137, amino acids 214 to 255, UniProt sequence ID Q07011) signaling domain and CD3ζ signaling domain (CD247, amino acids 52 to 163, reference sequence ID: NP_000725.1). The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK 293T cells, and the vectors were precipitated by centrifugation of the supernatant containing the lentiviral vectors and stored at -80°C.

[0325] (c) Primary T cell purification and transduction

[0326] CD4 + and CD8 + Human primary T cells from healthy volunteers were purified from whole blood or buffy coat (purchased from commercial providers with written consent from donors) by immunomagnetic selection of cells. 0.3 × 10 6 Up to 2×10 6 T cells were cultured at a density of cells / ml and CD3 / GMP T cells were activated with TransAct reagent (Miltenyi Biotec) and transduced with lentiviral vector encoding CAR constructs overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 2, and the medium was changed on day 3. Cultures were propagated in TexMACS medium supplemented with 200 IU / ml IL-2 until harvest on days 8 to 12.

[0327] (d) Immune effector assays (CTL and cytokines)

[0328] To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells in a variety of effector / target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence was quantified as counts per second (sample CPS). Only the target wells (maximum CPS) and only the target wells plus 1% Tween 20 (minimum CPS) were used to determine the range of the assay. The percentage of lysis was calculated as (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)). The supernatant was removed from a co-culture with an E:T ratio of 10:1 and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0329] (e) Flow cytometry analysis

[0330] For cell staining, 500,000 CAR T-transduced cells were harvested from culture, washed twice in cold AutoMACS buffer supplemented with 0.5% fetal bovine serum albumin (Miltenyi Biotec), and CAR surface expression was detected by staining with CD 19-Fc peptide (R&D, Minneapolis, MN) followed by anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). Non-transduced cells were used as negative controls. Dead cells were excluded from all studies by 7AAD staining (BD Biosciences, San Jose, CA). The cells were washed twice and resuspended in 200 μl of staining buffer and subsequently quantified by flow cytometry. Flow cytometric analysis was performed on a 10 analyzer (Miltenyi Biotec), and data graphs were generated using FloWJo software (Ashland, OR).

[0331] result:

[0332] In order to evaluate the new anti-CD19 fully human ScFv binding sequence, a CAR construct incorporating each of the ScFv sequences in Table 1 below was designed: ScFv1 (M19217), ScFv2 (M19217-1), ScFv3 (M19217-2), ScFv4 (M19217-7), ScFv5 (M19217-23), ScFv6 (M19217-29), ScFv7 (M19217-38), ScFv8 (M19217-40) as tumor antigen binding domains. In each CAR design, the tumor targeting domain is followed by a linker and transmembrane domain derived from human CD8 protein, a 4-1BB costimulatory domain, and a CD3ζ signaling domain (Table 2 below). A CAR construct encoding a murine immunoglobulin-derived FMC63 conjugate sequence was used as a positive control.

[0333] Table 2: List of CD19-targeting CAR constructs

[0334] LTG1538: FMC63-CD8 TM-41BB-CD3ζ, control LTG2050: ScFv1-CD8 TM-41BB-CD3ζ LTG2065: ScFv2-CD8TM-4-1BB-CD3ζ LTG2066: ScFv3-CD8TM-4-1BB-CD3ζ LTG2067: ScFv4-CD8TM-4-1BB-CD3ζ LTG2068: ScFv5-CD8TM-4-1BB-CD3ζ LTG2069: ScFv6-CD8TM-4-1BB-CD3ζ LTG2070: ScFv7-CD8TM-4-1BB-CD3ζ LTG2071: ScFv8-CD8TM-4-1BB-CD3ζ

[0335] T cells transduced with anti-cD19 chimeric antigen receptors exhibit surface expression and cytolytic activity. a) Surface expression of anti-CD19 CAR

[0336] In order to evaluate the new anti-CD19 CAR, as described in Materials and Methods, a lentiviral vector (LV) encoding a CAR construct under the control of the human EF1a promoter was generated. Human primary T cells derived from healthy donors were then transduced with a lentiviral vector encoding CAR. Non-transduced cells from the same donor (NT) or GFP-transduced cells from the same donor were used as negative controls. The data represent the results of at least 3 assays from different donors.

[0337] As described in the materials and methods, T cells were activated with TransAct T cell reagent (active engagement of CD3 and CD28 antigens, Miltenyi Biotec, Inc.) on day 0 of culture in the presence of IL-2. On days 8 to 10 of culture, the expression of anti-CD19 CAR on the surface of T cells was detected by CD19-Fc peptide followed by anti-Fc-AF647 and analyzed by flow cytometry. Each anti-CD19 CAR construct selected showed surface CAR expression. Using untransduced T cells as negative controls (0%), the following CAR expression levels were observed: LTG1538, 60%; LTG2050, 46%; LTG2065, 64%; LTG2066, 21%; LTG2067, 81%; LTG2068, 54%; LTG2069, 68%; LTG2070, 47%; LTG2071, 21%.

[0338] b) Cell lysis assay and cytokine assay of anti-CD19 CAR

[0339] To demonstrate the cytolytic function of the generated CAR T cells, luciferase-based killing assays were performed as described in Materials and Methods by combining CAR-T with CD19-positive Raji-luc cells, CD19-positive Reh-luc cells, CD19-negative K562-luc or CD19-negative 293T-luc cells at an E:T ratio of 40:1, 20:1 or 10:1 in an overnight cell killing assay ( Figure 4 ). The selected constructs (LTG2050, LTG2065-2071) showed dose-dependent CD19-specific tumor killing. We then measured the concentrations of inflammatory cytokines IFN-γ, TNF-α, and IL-2 secreted by CART cells transduced with CAR19 constructs when attacked by CD19-positive tumor cells ( Figure 5 To test basal levels of cytokine production, each construct contained a single CAR T cell control. + Raji cell T cells strongly induced levels of TNF-α, IFN-γ, and IL-2. In addition, in the absence of tumor cell targets, none of the constructs showed cytokine production above baseline. Therefore, the CAR T constructs LTG2050, LTG2065-2071 had a significant effect on CD19 + Tumor lines have specificity. Unexpectedly, construct LTG2050, although effectively killing CD19 positive cell lines in vitro, shows low IL-2 levels, as detected by ELISA. Therefore, CAR design and binder selection are not insignificant, because some binders are active in the form of soluble IgG or ScFv and tend to be expressed on the surface of T cells in the form of CAR T, although when incubated with CD19 positive tumors, it is inefficient in killing or producing cytokines. Although many binders were found during the screening, the following binders are not included in this article: those that are not expressed on the surface of T cells in our CAR expression vector, or those that have no strong lytic activity against CD19 positive cell lines.

[0340] In summary, the high functionality of the new fully human anti-CD19 CAR constructs LTG2050, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070 and LTG2071 was shown (Table 1 below). Except for LTG2050, all CAR constructs are superior to the positive control mouse FMC63 (LTG1538), and are therefore expected to have stronger therapeutic activity.

[0341] Each application and patent cited herein, and each document or reference cited in each of said applications and patents (including during the pendency of each issued patent; "application cited documents"), and each PCT and foreign application or patent corresponding to and / or claiming priority from any of these applications and patents, and each document cited or referenced in each application cited document are hereby expressly incorporated herein by reference and may be used in the practice of the present invention. More generally, several documents or references are cited herein, whether in a reference list preceding the claims or in the text itself; and each of these documents or references ("herein cited references") and each document or reference cited in each herein cited reference (including any manufacturer's instructions, guides, etc.) are hereby expressly incorporated herein by reference.

[0342] The foregoing description of some specific embodiments provides sufficient information so that other personnel can easily modify or adjust such specific embodiments for different applications by applying current knowledge without departing from the general concept, and therefore such adjustments and modifications should and are intended to be included in the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for description rather than limitation purposes. Exemplary embodiments have been disclosed in the drawings and the specification, and although specific terms may have been used, unless otherwise stated, they are only used in a general descriptive sense, rather than for limitation purposes, and the scope of the claims is therefore not limited thereto. In addition, those skilled in the art will understand that some steps of the methods discussed herein can be sorted in an alternative order or the steps can be combined. Therefore, this means that the attached claims are not limited to the specific embodiments disclosed herein. Those skilled in the art will recognize or be able to determine many equivalents of the embodiments of the present invention described herein using only routine experiments. Such equivalents are covered in the following claims.

[0343] Sequence of the present disclosure

[0344] The nucleic acid sequences and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.FR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. In the attached sequence listing:

[0345] SEQ ID NO: 1 Nucleotide sequence of CD19-reactive ScFv1 binding domain (LTG2050)

[0346]

[0347] SEQ ID NO:2 Amino acid sequence of CD19-reactive ScFv1 binding domain (LTG2050)

[0348]

[0349] SEQ ID NO:3 Nucleotide sequence of CD19-reactive ScFv2 binding domain (LTG2065)

[0350]

[0351]

[0352] SEQ ID NO:4 Amino acid sequence of CD19-reactive ScFv2 binding domain (LTG2065)

[0353]

[0354] SEQ ID NO:5 Nucleotide sequence of CD19-reactive ScFv3 binding domain (LTG2066)

[0355]

[0356] SEQ ID NO:6 Amino acid sequence of CD19 reactive ScFv3 binding domain (LTG2066)

[0357]

[0358] SEQ ID NO:7 Nucleotide sequence of CD19-reactive ScFv4 binding domain (LTG2067)

[0359]

[0360]

[0361] SEQ ID NO:8 Amino acid sequence of CD19 reactive ScFv4 binding domain (LTG2067)

[0362]

[0363] SEQ ID NO: 9 Nucleotide sequence of CD19-reactive ScFv5 binding domain (LTG2068)

[0364]

[0365] SEQ ID NO: 10 Amino acid sequence of CD19 reactive ScFv5 binding domain (LTG2068)

[0366]

[0367] SEQ ID NO: 11 Nucleotide sequence of CD19-reactive ScFv6 binding domain (LTG2069)

[0368]

[0369] SEQ ID NO: 12 Amino acid sequence of CD19-reactive ScFv6 binding domain (LTG2069)

[0370]

[0371] SEQ ID NO: 13 Nucleotide sequence of CD19-reactive ScFv7 binding domain (LTG2070)

[0372]

[0373] SEQ ID NO: 14 Amino acid sequence of CD19-reactive ScFv7 binding domain (LTG2070)

[0374]

[0375] SEQ ID NO: 15 Nucleotide sequence of CD19-reactive ScFv8 binding domain (LTG2071)

[0376]

[0377] SEQ ID NO: 16 Amino acid sequence of CD19-reactive ScFv8 binding domain (LTG2071)

[0378]

[0379] SEQ ID NO: 17 Nucleotide sequence of leader / signal peptide sequence (LP)

[0380]

[0381] SEQ ID NO:18 Amino acid sequence of leader / signal peptide sequence (LP)

[0382]

[0383] SEQ ID NO: 19 Nucleotide sequence of LTG2050_(LP-M19217-CD8 TM-41BB-CD3ζ)

[0384]

[0385] SEQ ID NO:20 Amino acid sequence of LTG2050_(LP-M19217-CD8 TM-41BB-CD3ζ)

[0386]

[0387] SEQ ID NO:21 Nucleotide sequence of LTG2065 (LP-M19217-1-CD8 TM-41BB-CD3ζ)

[0388]

[0389] SEQ ID NO:22 Amino acid sequence of LTG2065_(LP-M19217-1-CD8 TM-41BB-CD3ζ)

[0390]

[0391] SEQ ID NO:23 Nucleotide sequence of LTG2066_(LP-M19217-2-CD8 TM-41BB-CD3ζ)

[0392]

[0393] SEQ ID NO:24 Amino acid sequence of LTG2066_(LP-M19217-2-CD8 TM-41BB-CD3ζ)

[0394]

[0395]

[0396] SEQ ID NO:25 Nucleotide sequence of LTG2067_(LP-M19217-7-CD8 TM-41BB-CD3ζ)

[0397]

[0398] SEQ ID NO:26 Amino acid sequence of LTG2067_(LP-M19217-7-CD8 TM-41BB-CD3ζ)

[0399]

[0400] SEQ ID NO:27 Nucleotide sequence of LTG2068_(LP-M19217-23-CD8 TM-41BB-CD3ζ)

[0401]

[0402] SEQ ID NO:28 Amino acid sequence of LTG2068_(LP-M19217-23-CD8 TM-41BB-CD3ζ)

[0403]

[0404]

[0405] SEQ ID NO:29 Nucleotide sequence of LTG2069_(LP-M19217-29-CD8 TM-41BB-CD3ζ)

[0406]

[0407] SEQ ID NO:30 Amino acid sequence of LTG2069_(LP-M19217-29-CD8 TM-41BB-CD3ζ)

[0408]

[0409]

[0410] SEQ ID NO:31 Nucleotide sequence of LTG2070_(LP-M19217-38-CD8 TM-41BB-CD3ζ)

[0411]

[0412] SEQ ID NO:32 Amino acid sequence of LTG2070_(LP-M19217-38-CD8 TM-41BB-CD3ζ)

[0413]

[0414]

[0415] SEQ ID NO:33 Nucleotide sequence of LTG2071_(LP-M19217-40-CD8 TM-41BB-CD3ζ)

[0416]

[0417] SEQ ID NO:34 Amino acid sequence of LTG2071_(LP-M19217-40-CD8 TM-41BB-CD3ζ)

[0418]

[0419]

[0420] SEQ ID NO:35 Nucleotide sequence of DNACD8 transmembrane domain

[0421]

[0422] SEQ ID NO:36 Amino acid sequence of CD8 transmembrane domain

[0423]

[0424] SEQ ID NO:37 Nucleotide sequence of DNACD8 hinge domain

[0425]

[0426] SEQ ID NO:38 Amino acid sequence of CD8 hinge domain

[0427]

[0428] SEQ ID NO:39 Amino acid sequence of hinge and transmembrane region of CD8.α (NCBI RefSeq: NP.sub.--001759.3) from amino acid 137 to 206

[0429]

[0430] SEQ ID NO:40 Nucleotide sequence of the DNA signaling domain of 4-1BB

[0431]

[0432] SEQ ID NO:41 Amino acid sequence of the signaling domain of 4-1BB

[0433]

[0434] SEQ ID NO:42 Nucleotide sequence of the intracellular signaling domain of CD3-ζ

[0435]

[0436]

[0437] SEQ ID NO:43 Amino acid sequence of CD3-ζ

[0438]

[0439] SEQ ID NO:44 Nucleotide sequence of the intracellular signaling domain of CD3-ζ variant

[0440]

[0441] SEQ ID NO:45 Amino acid sequence of CD3-zeta signaling domain variant

[0442]

[0443] SEQ ID NO:46 Nucleotide sequence of ScFv CD19 (FMC63)

[0444]

[0445] SEQ ID NO:47 Amino acid sequence of ScFv CD19 (FMC63)

[0446]

[0447] SEQ ID NO:48 Nucleotide sequence of anti-CD33 ScFv (LTG1936)

[0448]

[0449] SEQ ID NO:49 Amino acid sequence of anti-CD33 ScFv (LTG1936)

[0450]

[0451] SEQ ID NO: 50 Nucleotide sequence of anti-mesothelin ScFv (LTG1904)

[0452]

[0453]

[0454] SEQ ID NO:51 Amino acid sequence of anti-mesothelin ScFv (LTG1904)

[0455]

[0456] The original claims of the parent case of this application are incorporated herein as part of this specification:

[0457] 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises at least one extracellular antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the extracellular antigen binding domain comprises a CD19 antigen binding domain encoded by a nucleotide sequence comprising SEQ ID NO. 1, 3, 5, 7, 9, 11, 13 or 15.

[0458] 2. The isolated nucleic acid molecule of claim 1 , wherein the encoded at least one CD19 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD19.

[0459] 3. The isolated nucleic acid molecule of claim 1 , wherein the encoded at least one CD19 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19.

[0460] 4. The isolated nucleic acid molecule of claim 1 , wherein the encoded at least one CD19 antigen binding domain, the at least one intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0461] 5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is connected to the transmembrane domain.

[0462] 6. The isolated nucleic acid molecule of claim 1 , wherein the encoded extracellular CD19 antigen binding domain is preceded by a leading nucleotide sequence encoding a leading peptide.

[0463] 7. The isolated nucleic acid molecule of item 6, wherein the leading nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 17, and the nucleotide sequence encodes the leading amino acid sequence of SEQ ID NO: 18.

[0464] 8. The isolated nucleic acid molecule of claim 1 , wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of a T cell receptor; CD8; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD83; CD86; CD134; CD137; CD154; and TNFRSF19; or any combination thereof.

[0465] 9. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular CD19 antigen binding domain comprises a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0466] 10. The isolated nucleic acid molecule of claim 1 , wherein the encoded at least one intracellular signaling domain further comprises a CD3ζ intracellular domain.

[0467] 11. The isolated nucleic acid molecule of claim 10, wherein the encoded at least one intracellular signaling domain is arranged at the N-terminal side relative to the CD3ζ intracellular domain.

[0468] 12. The isolated nucleic acid molecule of claim 1 , wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

[0469] 13. The isolated nucleic acid molecule of claim 12, 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 any combination thereof.

[0470] 14. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of item 1.

[0471] 15. The CAR of claim 14, comprising at least one extracellular antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the extracellular antigen binding domain comprises a CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16.

[0472] 16. The CAR of claim 15, wherein the CD19 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD19.

[0473] 17. The CAR of claim 15, wherein the CD19 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19.

[0474] 18. The CAR of claim 15, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: α, β or ζ chain of a T cell receptor; CD8; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; and TNFRSF19; or any combination thereof.

[0475] 19. The CAR of claim 18, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO:35, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:28.

[0476] 20. The CAR of claim 15, wherein the at least one extracellular antigen binding domain and the at least one intracellular signaling domain or both are connected to the transmembrane domain via a linker or a spacer domain, and the extracellular antigen binding domain comprises a CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16.

[0477] 21. The CAR of claim 20, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is connected to the transmembrane domain.

[0478] 22. The CAR of claim 17, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0479] 23. The CAR of claim 22, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, wherein the co-stimulatory domain comprises 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.

[0480] 24. A vector comprising the nucleic acid molecule described in item 1.

[0481] 25. The vector of item 24, wherein the vector is selected from: a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

[0482] 26. The vector described in claim 24 further comprises a promoter.

[0483] 27. The vector of claim 26, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

[0484] 28. A cell comprising the vector of item 24.

[0485] 29. The cell of claim 28, wherein the cell is a T cell.

[0486] 30. The cell of claim 28, wherein the T cell is a CD8+ T cell.

[0487] 31. The cell of claim 28, wherein the cell is a human cell.

[0488] 32. A method for preparing cells, comprising transducing T cells with the vector described in item 24.

[0489] 33. A method for producing an RNA-modified cell population, comprising introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule described in item 1.

[0490] 34. A method for providing anti-tumor immunity in a mammal, comprising administering an effective amount of the cell of item 28 to the mammal.

[0491] 35. A method for treating or preventing cancer in a mammal, comprising administering the CAR of item 15 to the mammal in an amount effective to treat or prevent cancer in the mammal.

[0492] 36. A pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16, and wherein the T cells are T cells of a person suffering from cancer.

[0493] 37. A pharmaceutical composition as described in claim 36, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of the T cell receptor; CD8; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; and CD154; or any combination thereof.

[0494] 38. The pharmaceutical composition of claim 36, wherein the T cells are T cells of a human suffering from a hematological cancer.

[0495] 39. The pharmaceutical composition of claim 38, wherein the hematological cancer is leukemia or lymphoma.

[0496] 40. The pharmaceutical composition of claim 39, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myeloid leukemia (CML).

[0497] 41. The pharmaceutical composition of claim 39, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma.

[0498] 42. The pharmaceutical composition of claim 38, wherein the hematological cancer is multiple myeloma.

[0499] 43. The pharmaceutical composition of claim 36, wherein the human cancer comprises adult cancers comprising oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinomas), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), as well as breast cancer, reproductive system cancers (cervix, uterine body, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system cancers (bladder, kidney and renal pelvis, ureter), eye cancer and orbital cancer, endocrine system cancers (thyroid), as well as brain cancer and other nervous system cancers, or any combination thereof.

[0500] 44. A method for treating a mammal suffering from a disease, disorder or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one comprising an extracellular antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the extracellular antigen binding domain comprising a CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, wherein the T cells are T cells of a subject suffering from cancer.

[0501] 45. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO.: 2, 4, 6, 8, 10, 12, 14 or 16, wherein the T cells are T cells of a subject suffering from cancer.

[0502] 46. ​​The method of claim 44 or 45, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of the T cell receptor; CD8; CD28; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; and CD154; or any combination thereof.

[0503] 47. A method for producing a cell expressing a chimeric antigen receptor, the method comprising introducing into the cell the isolated nucleic acid of item 1.

[0504] 48. A method for producing a cell expressing a chimeric antigen receptor according to item 47, wherein the cell is a T cell or a cell population comprising a T cell.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32 or 34.

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

3. A pharmaceutical composition comprising a population of isolated human T cells, wherein the isolated human T cells comprise a nucleic acid sequence encoding the chimeric antigen receptor (CAR) of claim 2, and wherein the isolated human T cells are isolated from a human suffering from cancer.

4. The pharmaceutical composition of claim 3, wherein the cancer is a hematological cancer.

5. The pharmaceutical composition of claim 4, wherein the hematological cancer is leukemia or lymphoma.

6. The pharmaceutical composition of claim 5, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myeloid leukemia (CML).

7. The pharmaceutical composition of claim 5, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma.

8. The pharmaceutical composition of claim 4, wherein the hematological cancer is multiple myeloma.

9. The pharmaceutical composition of claim 3, wherein the cancer is selected from the group consisting of oral and pharyngeal cancer, digestive system cancer, respiratory system cancer, bone and joint cancer, soft tissue cancer, skin cancer, central nervous system cancer, breast cancer, reproductive system cancer, urinary system cancer, eye cancer and orbital cancer, endocrine system cancer, and brain cancer.

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

11. The vector of claim 10, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector, a retroviral vector, and a combination thereof.

12. The vector of claim 10, further comprising a promoter.

13. The vector of claim 12, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

14. An isolated cell comprising the vector of claim 10.

15. The isolated cell of claim 14, wherein the isolated cell is a T cell.

16. The isolated cell of claim 14, wherein the isolated T cell is a CD8+ T cell.

17. The isolated cell of claim 14, wherein the isolated cell is a human cell.

Images