Compositions and methods for treating cancer by anti-CD22 immunotherapy

Abstract

To provide chimeric antigen receptors (CARs) containing CD22 antigen binding domains, and nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the CARs.SOLUTION: The present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen binding domain comprising a CD22 antigen binding domain comprising an amino acid sequence having a specific sequence, at least one transmembrane domain, and at least one intracellular signaling domain.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 572,926, filed on October 16, 2017. The entire disclosure of the U.S. Provisional Patent Application is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format, and the entire sequence listing is incorporated herein by reference. The file name of this ASCII copy (created on October 12, 2018) is "Sequence Listing.txt", and the size is 234 kilobytes.

[0003] Description of Research or Development Sponsored by the Federal Government This invention was made by the realization of a Cooperative Research and Development Agreement between the National Institutes of Health and an agency of the U.S. Department of Health and Human Services. The U.S. government has certain rights in this invention.

[0004] Field of the Present Disclosure This application relates to the field of cancer, in particular, to a CD22 antigen - binding domain, a chimeric antigen receptor (CAR) containing this CD22 antigen - binding domain, and methods of using the same.

Background Art

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

[0006] Current standard treatment for B-cell leukemia is thought to involve consolidation therapy following remission induction with high-dose chemotherapy or radiation therapy, and may feature stem cell transplantation and additional chemotherapy as needed (see cancer.gov on the World Wide Web). Such treatments are highly toxic and carry a risk of complications such as relapse, secondary malignancies, or graft-versus-host disease (GVHD), so better alternative treatments are being explored. CD22, also known as SIGLEC-2 (sialic acid-binding immunoglobulin-like lectin-2), is a 95 kDa transmembrane surface glycoprotein containing six Ig-like C2-type domains and one Ig-like V-type domain (uniprot.org / uniprot / P20273#structure, accessed July 12, 2017). During B-cell ontogeny, CD22 is expressed on the surface of B cells at the pre-B cell stage, persists until mature B cells, and disappears in plasma cells (Nitschke L, 2009, Immunological Reviews, 230:128-143). CD22 contains an intracellular ITIM (immunoreceptor tyrosine-based inhibition motifs) domain, which functions to downregulate subsequent cell activation when the B-cell receptor binds an antigen. When an antibody binds to CD22, phosphorylation (phosoryla Co-localization with SHP-1, an intracellular phosphatase that also acts to downregulate signaling based on (Lumb S, Fleishcer SJ, Wiedemann A, Daridon C, Maloney A, Shock A, Dorner T, 2016, Journal of Cell Communication and Signaling, 10:143-151). This is important in the context of treating B-cell malignancies. This is because CD22 is expressed on normal B cells in a tightly regulated manner but not on hematopoietic stem cells or mature plasma cells, and thus is a suitable target antigen for B-cell leukemia. Since CD22 is expressed in both adult B-cell malignancies and pediatric B-cell malignancies (pre-B-ALL), therapies based on both antibodies and chimeric antigen receptor (CAR)-T cells have been found by exploiting this target (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerlad DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ, 2013, Blood, 121:1165-1174)(Wayne AS, Kreitman RJ, Findley HW, Lew G, Delbrook C, Steinberg SM, Stetler-Stevenson M, FitzGerald DJ, Pastan I, 2010, Clinical Cancer Research, 16:1894-1903).

[0007] Many new approaches have been developed to treat B-cell leukemia and lymphoma, including anti-CD22 antibodies conjugated to bacterial toxins or chemotherapeutic agents (Wayne AS, FitzGerald DJ, Kreitman RJ, Pastan I, 2014, Immunotoxins for leukemia, Blood, 123:2470-2477). Inotuzumab ozogamicin (CMC-544, humanized murine monoclonal antibody G5 / 44) is an antibody-drug conjugate and is currently being evaluated in clinical trials as a single agent or in combination with chemotherapy (NCT01664910, funding: MD Anderson Cancer Center) (DiJoseph JF et al., 2004, Blood, 103:1807-1814). The outcomes as a single agent were favorable compared to those observed with standard therapies, but significant hepatotoxicity was noted (Kantarjian H et al., 2016, Inotuzumb ozogamicin versus standard therapy for acute lymphoblastic leukemia (ALL), New England Journal of Medicine, 375:740-753). For epratuzumab, an unmodified CD22-targeting antibody, trials in combination with chemotherapy are also ongoing (NCT01219816, funding: Nantes University Hospital). Epratuzumab is a chimeric protein composed of murine CDRs grafted onto the framework of a human antibody. Moxetumomab pasudotox (Moxetumomab Pasudotox) is effective against certain types of leukemia, but widespread clinical development has not been carried out due to the problem of the immunogenicity of the bacterial toxin to which the antibody is fused and the problem that the activity level is moderate or equivalent to other agents (see NCT01829711, funding: MedImmune). Many of the CD22 binding sites used in CAR constructs to date utilize domains derived from the mouse antibodies described above and are not effective in activating T cells targeting this CD22 domain (such as HA22 (anti-CD22 binder), which is used as the backbone of moxetumomab pasudotox. See James SE, Greenberg PD, Jensen MC, Lin Y, Wang J, Till BG, Raubitschek AA, Forman SJ, Press OW, 2008, Jounral of Immunology 180:7028-7038). One anti-CD22 binder effective as an anti-CD22 CAR is , and is currently in clinical trials at the National Institutes of Health (NIH) in the United States, but the results have not yet been published (ClinicalTrials.gov Identifier: NCT02315612, Anti-CD22 Chimeric Receptor T Cells in Pediatric and Young Adults with Recurrent or Refractory CD22-expressing B Cell Malignancies, sponsor: NCI). This binder is based on the m971 fully human antibody developed in the laboratory of Dr. Dimiter Dimitrov, one of the inventors of the present invention (Xiao X, Ho M, Zhu Z, Pastan I, Dimitrov D, 2009, Identification and characterization of fully human anti-CD22 monoclonal antibodies, MABS, 1:297-303). This m971 domain has been demonstrated to be effective as a CAR in studies conducted under the guidance of Dr. Rimas Orentas, another one of the inventors of the present application (Haso W et al., 2013, Anti-CD22-CARs targeting B-cell precursor ALL, Blood, 121:1165-1174).

[0008] Chimeric antigen receptors (CARs) are hybrid molecules consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a hinge / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific CAR. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of a CAR is generally engineered to mimic the single chain Fragment variable (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs, such as receptor ligands (i.e., IL-13 engineered to bind to the IL-13 receptor expressed in tumors), full-length immunoreceptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D) have also been engineered into CARs by design. Other cell targets for CAR expression (such as NK or gamma-delta T cells) are also under development (Brown CE et al Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al PLoS One. 2012;7(2):e31210). Considerable additional effort is required to identify the most highly active T cell populations to transduce with CAR vectors, find optimal culture and expansion techniques, and elucidate in detail the structure of the CAR protein itself at the molecular level.

[0009] The CAR binding motif may be a relatively stable structural domain such as the constant domain of IgG, or can be designed as a long flexible linker. Structural motifs such as those derived from the IgG constant domain can be used to extend the ScFv binding domain to a position far from the T cell membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the surface membrane of tumor cells (such as disialoganglioside GD2; Orentas et al., this observation is unpublished). All of the signaling motifs used in CARs to date include the CD3-zeta chain, because this core motif is an important signal for T cell activation. The first reported second-generation CARs were characterized by the CD28 signaling domain and the CD28 transmembrane sequence. This motif was used in third-generation CARs that further contained the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). The emergence of new technologies has eliminated the need for the CAR itself to encode T cell activation by beads conjugated to anti-CD3 and anti-CD28 antibodies, as well as the presence of the classical "signal 2" from CD28. Third-generation vectors using bead activation were found not to outperform second-generation vectors in in vitro assays, and furthermore, in a leukemia mouse model It did not offer benefits clearly exceeding those of the second-generation vectors (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-CARs targeting B cell precursor ALL, Blood. 2013;121(7):1165-74; Kochenderfer JN et al. Blood. 2012;119(12):2709-20). In addition to CD137, other members of the tumor necrosis factor receptor superfamily, such as OX40, can also provide important persistence signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions for culturing the CAR T cell population, such as including the cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther. 2015;22(2):72-78).

[0010] At present, the challenges in more widely and effectively applying CAR therapy to cancer are related to the lack of promising targets. The production of binders that bind to cell surface antigens can now be easily achieved, but the discovery of cell surface antigens that are tumor-specific and do not affect normal tissues remains very difficult. As a method that can endow CAR-expressing T cells with stronger target cell specificity than before, there is a method of using a combination of multiple CAR approaches. In one system, the CD3-zeta signal unit and the CD28 signal unit are divided into each of two different CAR constructs expressed in the same cell. In another system, two CARs are expressed in the same T cell, but since one of them has a lower affinity, it is necessary to first bind the other CAR to maximize the activity of the latter CAR (Lanitis E et al. Cancer 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 the heterogeneity of tumor cells. At least one group has developed a treatment method for glioblastoma using CAR, and this method targets multiple antigens (HER2, IL-13Ra, EphA2) simultaneously in the effector cell population and attempts to avoid the growth of populations without target antigens (Hegde M et al. Mol Ther. 2013;21(11):2087-101).

[0011] T cell-based immunotherapy has become a new cutting-edge area in synthetic biology. Multiple promoters and gene products have been conceived for the purpose of inducing these highly potent cells into the tumor microenvironment, and T cells can mediate effective tumor killing by avoiding negative control signals in the tumor microenvironment. The method of removing unwanted T cells by dimerization of an inducible caspase 9 construct (drug-induced dimerization) using a dimerization-inducing chemical such as AP1903 presents one strategy that can pharmacologically initiate a powerful switch for controlling the T cell population (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Furthermore, the method of creating an effector T cell population that does not respond to the negative regulatory action of transforming growth factor β by expressing a decoy receptor indicates the extent to which effector T cells can be manipulated to obtain optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Therefore, although CAR seems to be able to trigger T cell activation in a manner similar to the endogenous T cell receptor, at present, the limited proliferation of CAR+ T cells in vivo, the disappearance of these cells soon after infusion, and the poor clinical activity are major obstacles, and the clinical application of this technology has not advanced. It may be partly due to the fact that some of the CAR sequences used are of mouse origin, and the invention disclosed herein directly addresses this problem.

[0012] Therefore, by using a method that can exhibit specific and highly potent antitumor effects without presenting the problems described above, it is urgently and long-term needed in the art to discover new compositions and methods for treating B-ALL, DLBCL, FL, and other CD22-expressing B cell malignancies. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0013] The present invention addresses the above-described need by providing CAR compositions and methods of treatment that can be used for the treatment of cancer as well as other diseases and / or conditions. In particular, the invention disclosed and described herein provides a CAR that can be used for the treatment of diseases, disorders, or conditions associated with dysregulated expression of CD22, the CAR containing a CD22 antigen-binding domain with high surface expression on transduced T cells, having a high degree of cell lysis of CD22-expressing cells, and the transduced T cells proliferating and persisting in vivo.

Means for Solving the Problems

[0014] Overview A novel anti-CD22 antibody, or an antigen-binding domain thereof, a chimeric antigen receptor (CAR) containing such a CD22 antigen-binding domain, a host cell (e.g., a T cell) expressing the receptor, and a nucleic acid molecule encoding the receptor are provided herein. The CAR has high surface expression on transduced T cells, a high degree of cell lysis, and the transduced T cells proliferate and persist in vivo. Further provided are methods of using the disclosed CAR, host cells, and nucleic acid molecules, for example, for treating cancer in a subject.

[0015] Thus, in one aspect, there is provided an isolated polynucleotide encoding a human anti-CD22 antibody or a fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, and 171.

[0016] In one embodiment, there is provided an isolated polynucleotide encoding a fully human anti-CD22 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, an F(ab’)2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

[0017] In one embodiment, there is provided an isolated polynucleotide encoding a fully human anti-CD22 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, and 172.

[0018] In one aspect, there is provided an isolated nucleic acid molecule encoding a CAR comprising at least one CD22 antigen-binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, and 171 from the N-terminus towards the C-terminus, at least one transmembrane domain, and at least one intracellular signaling domain.

[0019] In one embodiment, there is provided an isolated nucleic acid molecule encoding a CAR, wherein the extracellular CD22 antigen-binding domain encoded comprises at least one single-chain variable fragment of an antibody that binds CD22.

[0020] In another embodiment, there is provided an isolated nucleic acid molecule encoding a CAR, wherein the extracellular CD22 antigen-binding domain encoded comprises at least one heavy chain variable region of an antibody that binds CD22.

[0021] In yet another embodiment, there is provided an isolated nucleic acid molecule encoding a CAR, wherein the extracellular CD22 antigen-binding domain of the encoded CAR further comprises at least one lipocalin-based antigen-binding antigen (antikarin) that binds CD22.

[0022] In one embodiment, there is provided an isolated nucleic acid molecule, wherein the extracellular CD22 antigen-binding domain encoded is linked to the transmembrane domain by a linker domain.

[0023] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular antigen-binding domain of the encoded CD22 is located behind the sequence encoding a leader or signal peptide.

[0024] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR comprising at least one CD22 antigen-binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, and 171 is provided, wherein the CAR further encodes an extracellular antigen-binding domain targeting an antigen comprising (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.

[0025] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the further encoded extracellular antigen-binding domain is an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-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-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0026] In one aspect, the CAR provided herein further comprises a linker or spacer domain.

[0027] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular CD22 antigen-binding domain, the intracellular signaling domain, or both are linked to the transmembrane domain by a linker or spacer domain.

[0028] 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 bound to the transmembrane domain.

[0029] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or combinations thereof.

[0030] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0031] 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.

[0032] In a further embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or combinations thereof.

[0033] In one embodiment, an isolated nucleic acid molecule encoding a CAR further containing a leader sequence or signal peptide is provided, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 190.

[0034] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 191.

[0035] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from the N-terminus to the C-terminus, at least one CD22 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0036] In one embodiment, a CAR is provided, wherein the extracellular CD22 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds the antigen, or at least one heavy-chain variable region of an antibody that binds the antigen, or a combination thereof.

[0037] In another embodiment, a CAR is provided, wherein the at least one transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0038] In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen-binding domain comprising CD19, CD20, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD1 38, 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.

[0039] In one embodiment, a CAR is provided, wherein the extracellular antigen-binding domain is an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-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-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0040] In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0041] In yet another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a / CD18), ICOS(CD278), DAP10, DAP12, and 4-1BB(CD137), or a combination thereof.

[0042] In one embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 3 (LTG It includes the 2202 LP-16P-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2A). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 4 (LTG 2202 LP-16P-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2A)).

[0043] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 13 (LTG 2246 LP-24P-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2B)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 14 (LTG 2246 LP-24P-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2B)).

[0044] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 23 (LTG 2247 LP-25P-CD8 TM-41BB-CD3 zeta CAR nucleotide sequence (Figure 2C)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 24 (LTG 2247 LP-25P-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2C)).

[0045] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 33 (LTG 2248 LP-11S-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2D)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 34 (LTG 2248 LP-11S-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2D)).

[0046] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 43 (LTG 2249 LP-12S-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2E)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 28 (LTG 2208 LP-12S-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2E)).

[0047] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 53 (LTG 2203 LP-16P3-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2F)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 54 (LTG 2203 LP-16P3-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2F)).

[0048] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 63 (LTG 2204 LP-16P16-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2G)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 34 (LTG 2204 LP-16P16-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2G)).

[0049] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73 (LTG 2205 LP-16P20-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2H)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 (LTG 2205 LP-16P20-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2H)).

[0050] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 83 (LTG 2206 LP-16P2-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2I)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 84 (LTG 2206 LP-16P2-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2I)).

[0051] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93 (LTG 2207 LP-16P6-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2J)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94 (LTG 2205 LP-16P20-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2J)).

[0052] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 103 (LTG 2208 LP-16P10-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2K)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 104 (LTG 2208 LP-16P10-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2K)).

[0053] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113 (LTG 2209 LP-16P17-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2L)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114 (LTG 2209 LP-16P17-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2L)). zeta CAR amino acid sequence (Figure 2L)).

[0054] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 123 (LTG 2210 LP-16P20v2-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2M)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 124 (LTG 2210 LP-16P20v2-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2M)).

[0055] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 133 (LTG 2216 LP-16P1-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2N)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 134 (LTG 2216 LP-16P1-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2H)).

[0056] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 143 (LTG 2217 LP-16P3v2-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2O)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 144 (LTG 2217 LP-16P3v2-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2O)).

[0057] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 153 (LTG 2218 LP-16P8-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2P)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 154 (LTG 2218 LP-16P8-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2P)).

[0058] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 163 (LTG 2219 LP-16P13-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2Q)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 164 (LTG 2219 LP-16P13-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2Q)).

[0059] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 173 (LTG 2220 LP-16P15-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2R)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 174 (LTG 2220 LP-16P15-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2R)).

[0060] In one aspect, the CARs disclosed herein are modified to express or contain a detectable marker for use in the diagnosis, monitoring, and / or prediction of treatment outcomes such as progression-free survival of cancer patients, or for monitoring the progress of such treatments.

[0061] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector such as a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0062] In certain embodiments, the vector further comprises a promoter, which is , an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0063] In yet another embodiment, the vector expressing the CAR may be further modified to include one or more operable elements for controlling the expression of CAR T cells or for removing CAR-T cells by a suicide switch. This suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0064] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is further provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.

[0065] In yet another aspect, a pharmaceutical composition comprising a population of anti-tumor effective amount of human T cells is provided, wherein the T cells comprise a nucleic acid sequence encoding a CAR, and the CAR comprises at least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a human having cancer. The cancer includes, inter alia, blood cancers such as leukemia (e.g., CLL, ALL, AML, or CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma), or multiple myeloma, or combinations thereof.

[0066] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR contains the transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0067] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer includes adult cancers such as oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung, and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, genital system (cervix, corpus uteri, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous system cancers, or any combination thereof.

[0068] In yet another embodiment, a pharmaceutical composition is provided that contains a population of human T cells from a human having cancer in an anti-tumor effective amount, wherein the cancer is a refractory cancer that does not respond to one or more chemotherapeutic agents. The cancer includes hematopoietic cancers, myelodysplastic syndromes, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in ALL, AML, adult B cell malignancies including CLL, CML, NHL, pediatric B cell malignancies (including B-line ALL), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other blood cancers and solid tumors, or any combination thereof.

[0069] In another aspect, a method for producing CAR-containing T cells (hereinafter referred to as "CAR-T cells") is provided. This method includes the step of transducing a T cell with a vector or nucleic acid molecule encoding a CAR (disclosed) that specifically binds to CD22, thereby producing CAR-T cells.

[0070] In yet another aspect, a method for generating a population of RNA-engineered cells is provided, which includes the step of introducing an in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding the disclosed CAR into a cell of interest, thereby generating CAR cells.

[0071] In yet another aspect, a method for diagnosing a disease, disorder, or condition associated with the expression of CD22 in a cell is provided, which includes: a) contacting the cell with a human anti-CD19 antibody or a fragment thereof, wherein the antibody or the fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172; and b) detecting the presence of CD22, and diagnosing that it is a disease, disorder, or condition associated with the expression of CD22 when CD19 is present.

[0072] In one embodiment, the disease, disorder, or condition associated with the expression of CD22 is a cancer including hematopoietic malignancies, myelodysplastic syndromes, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in ALL, AML, adult B-cell malignancies including CLL, CML, NHL, pediatric B-cell malignancies (including B-line ALL), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other blood cancers and solid tumors, or any combination thereof.

[0073] In another embodiment, a method for diagnosing, prognosticating, or determining the risk of a CD19-related disease in a mammal is provided, which includes the step of detecting the expression of CD22 in a sample derived from the mammal, and this step includes: a) contacting the sample with a human anti-CD22 antibody or a fragment thereof, wherein the antibody or the fragment thereof includes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172; and b) detecting the presence of CD22, and diagnosing that the mammal has a CD22-related disease when CD22 is present.

[0074] In another embodiment, a method for inhibiting CD22-dependent T cell inhibition is provided, which includes the step of contacting a cell with a human anti-CD22 antibody or a fragment thereof, and the antibody or the fragment thereof includes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172. In one embodiment, the cell is selected from the group consisting of CD22-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0075] In another embodiment, a method for blocking T cell inhibition mediated by CD22-expressing cells and changing the tumor microenvironment to inhibit tumor growth in a mammal is provided, which includes the step of administering a composition containing an isolated anti-CD22 antibody or a fragment thereof to the mammal in an effective amount, and the antibody or the fragment thereof includes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, and 172. In one embodiment, the cell is selected from the group consisting of CD19-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0076] In another embodiment, a method is provided for inhibiting, suppressing, or preventing immunosuppression of an antitumor or anticancer immune response in a mammal, which comprises administering to the mammal, in an effective amount, a composition comprising an isolated anti-CD22 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, and 172. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, and the first cell is selected from the group consisting of CD22-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0077] In another aspect, a method is provided for inducing antitumor immunity in a mammal, which comprises administering to the mammal, in a therapeutically effective amount, T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0078] In another embodiment, a method for treating or preventing cancer in a mammal is provided, which comprises administering to the mammal one or more of the disclosed CARs in an amount effective for treating or preventing cancer in the mammal. The method comprises administering to the subject, in a therapeutically effective amount, host cells expressing a CAR (disclosed) that specifically binds to CD22 and / or one or more of the antigens described above, under conditions sufficient to form an immune complex consisting of the antigen-binding domain of the CAR, the extracellular domain of CD22, and / or one or more of the antigens described above in the subject.

[0079] In yet another embodiment, a method for treating a mammal having a disease, disorder, or condition associated with increased expression of a tumor antigen is provided, the method comprising administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, the T cells comprising a nucleic acid sequence encoding a CAR, the CAR comprising at least one extracellular CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172 or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer.

[0080] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, which comprises administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, the T cells comprising a nucleic acid sequence encoding a CAR, the CAR comprising at least one CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172 or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer. In some embodiments of the methods described above, the at least one transmembrane domain comprises the transmembrane of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or combinations thereof.

[0081] In yet another embodiment, a method for generating a population of genetically engineered T cells that are persistent in a human diagnosed with cancer is provided. In one embodiment, the method includes administering to the human T cells that have been genetically engineered to express a CAR, wherein the CAR comprises at least one CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and the population of genetically engineered T cells that are persistent, or the population of progeny T cells, persist in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0082] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0083] In all aspects and embodiments of the methods described herein, any cancer, disease, disorder, or condition associated with increased expression of a tumor antigen, as described above, can be treated or prevented or remitted using one or more of the CARs disclosed herein.

[0084] In yet another aspect, a kit for generating the CAR T cells described above, or for preventing, treating, or remitting any of the cancers, diseases, disorders, or conditions associated with increased expression of a tumor antigen in a subject, as described above, is provided, which comprises any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, or a container containing instructions for use of the kit.

[0085] It is understood that the CARs, host cells, nucleic acids, and methods described above are useful beyond the scope of the specific embodiments and implementations described in detail herein. The features and advantages of the present disclosure described above will become even more apparent from the following detailed description, which is described with reference to the accompanying drawings.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0087] Detailed Description Definitions As used herein, the singular forms "a", "an", and "the" refer to both the singular and plural forms unless there is an obvious contradiction in context. For example, the term "an antigen" can include one or more antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". Thus, "comprising an antigen" means "including an antigen" without excluding other elements. The phrase "and / or" means "and" or "or". "Or" means. Further, unless otherwise specified, any and all base sizes or amino acid sizes and all molecular weight or molecular mass values given to a nucleic acid or polypeptide are approximate values and are understood to be provided for illustrative purposes. Many methods and materials similar or equivalent to those described herein can be used, but particularly preferred methods and materials are described below. In case of any conflict, this specification (including explanations of terms) shall govern. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. To facilitate the identification of various embodiments, explanations of terms are provided below.

[0088] The term "about", when referring to measurable possible values such as amounts and durations, means including a variation 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 value explicitly stated. This is because such variations are appropriate for the practice of the disclosed methods.

[0089] Unless otherwise specified, scientific terms in this specification are used in their conventional sense. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science, 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, 1995; and other similar references.

[0090] The present disclosure provides a CD22 antibody or fragment thereof, and a CAR having such a CD22 antigen-binding domain. Improving the functional activity of the CAR is directly related to improving the functional activity of CAR-expressing T cells. As a result of making one or more of such modifications, the CAR exhibits both cytokine-induced cytolysis and cell surface expression in transduced T cells at high levels, and high levels of T cell proliferation and persistence of transduced CAR-expressing T cells in vivo.

[0091] The unique ability to combine functional sites derived from different protein domains is an innovative feature of the CAR. Which of these protein domains to select is an important design feature, just as the manner in which they specifically bind. The individual design domains are essential components that can be used in any heterologous CAR platform for the purpose of manipulating lymphocyte function. For example, the selection of the extracellular binding domain can render a CAR that is otherwise ineffective effective.

[0092] The non-variable framework components of the sequence of the immunoglobulin-derived protein used to make the extracellular antigen-binding domain of the CAR may be completely neutral or may be such that they self-associate and significantly reduce the effectiveness of the therapeutic T cells expressing this CAR by causing the T cells to enter a metabolically depleted state. This phenomenon occurs independently of the antigen-binding function of this CAR domain. Furthermore, the selection of the intracellular signaling domain can also govern the activity and durability of the therapeutic lymphocyte population used in immunotherapy. Here, the ability to bind the target antigen and to transmit an activation signal to the T cell via each of the extracellular and intracellular domains described above are important CAR design aspects, but it has also become clear that the choice of the source of the extracellular antigen-binding fragment can have a significant effect on the efficacy of the CAR and thus potentially play a decisive role in the function and clinical utility of the CAR.

[0093] Surprisingly and unexpectedly, instead of using a mouse-derived antigen-binding fragment (which tends to induce an anti-mouse immune response and CAR T elimination in the host; see: Clinical trial using mouse-derived SS1 ScFv sequence with funding from the University of Pennsylvania, NCT02159716), it has been found that using a fully human antigen-binding domain in the CAR can determine the functional activity of CAR-expressing T cells.

[0094] The CARs disclosed herein are expressed at high levels in cells. The cells expressing this CAR have a high proliferation rate in vivo, produce a large amount of cytokines, and have high cytotoxic activity against cells having the CD22 antigen to which the CAR binds on their surface. As a result of using a human extracellular CD22 antigen-binding domain, CARs with improved in vivo function were produced, and at the same time, induction of anti-CAR immunity in the host immune response and death of the CAR T cell population were avoided. CARs expressing a fully human extracellular CD22 ScFv antigen-binding domain exhibit excellent activity and / or properties, which include: i) prevention of poor persistence and poor function of CAR T (seen with mouse-derived binding sequences); ii) lack of delivery of the CAR to a specific region (i.e., intrapleural) for efficacy; and iii) the ability to enable the design of CAR T cells based on both high-affinity and low-affinity CD19 binders. Due to the latter property, researchers can better regulate the efficacy-to-toxicity and / or tissue specificity of CAR T products. This is because CD22 is highly expressed in tumors compared to normal tissues, so the lower-affinity binder may have higher specificity for tumors than for normal tissues, thereby preventing on-target off-tumor toxicity and bystander cell killing.

[0095] Next, the CAR of the present invention will be described in detail. This description includes an explanation of its extracellular CD22 antigen-binding domain, transmembrane domain, and intracellular domain, as well as further explanations regarding CARs, antibodies, and their antigen-binding fragments, conjugates, nucleotides, expression, vectors, and host cells, treatment methods using the disclosed CARs, compositions, and kits.

[0096] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein include at least one CD22 antigen-binding domain capable of binding to CD22, at least one transmembrane domain, and at least one intracellular domain.

[0097] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. A characteristic of CARs is the ability to redirect the specificity and reactivity of T cells to a selected target in a manner not restricted by the Major Histocompatibility Complex (MHC), and to utilize the antigen-binding properties of monoclonal antibodies. Since CAR-expressing T cells can recognize antigens independently of antigen processing, they have the ability to avoid a major mechanism of tumor escape. Also, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0098] 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) the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain is the intracellular domain of a co-stimulatory molecule Refers to a part of the CAR that includes a non-antigen receptor or its ligand cell surface molecule other than those necessary for lymphocytes to efficiently respond to antigens.

[0099] 1. Extracellular domain In one embodiment, the CAR comprises a target-specific binding element, also referred to as an antigen-binding domain or site. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in a CAR include those associated with viral infections, bacterial infections, and parasitic infections, autoimmune diseases, and cancer cells.

[0100] In one embodiment, the CAR can be designed to target a desired tumor antigen by designing a desired antigen-binding domain that specifically binds to an antigen on the tumor cell. A tumor antigen is a protein produced by a tumor cell that elicits an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain can depend on the specific type of cancer being treated. Tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, Ephrin B2, CD19, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD22. The tumor antigens disclosed herein are included merely by way of example. The listing is not intended to be limiting, and other examples will be readily recognized by those skilled in the art.

[0101] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express multiple proteins that can serve as target antigens for immune attack. Such molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules include those belonging to the group of transformation-related molecules such as the cancer gene HER-2 / Neu / ErbB-2. Still another group of target antigens includes cancer fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin corresponds to a truly tumor-specific immunoglobulin antigen that is unique to each tumor. B-cell differentiation antigens such as CD19, CD20, CD22, BCMA, ROR1, and CD37 are also candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) are used as targets for passive immunotherapy using monoclonal antibodies, but have not achieved sufficient success.

[0102] In a preferred embodiment, the tumor antigen is CD22, and tumors associated with the expression of CD22 include mesothelioma of the lung, ovarian and pancreatic cancers that express the extracellular protein CD22 at high levels, or any combination thereof.

[0103] The type of tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur on other cells of the body. TAA is not unique to tumor cells, but rather, under conditions where immune tolerance to this antigen is not induced Moreover, it is also expressed on normal cells. The expression of this antigen in tumors can occur under conditions that enable the immune system's response to this antigen. A TAA can be an antigen that is expressed on normal cells during fetal development when the immune system is immature and unable to respond to the antigen, or a TAA can be an antigen that is normally present at extremely low levels on normal cells but is expressed at significantly higher levels on tumor cells.

[0104] Examples of TSAs or TAAs include, but are not limited to, differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed cancer genes and mutant tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, etc.; as well as 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0105] In one embodiment, the antigen-binding domain portion of the CAR targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, and MAGE A3 TCR.

[0106] In a preferred embodiment, the antigen-binding domain portion of the CAR targets the extracellular CD22 antigen.

[0107] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 scFv1 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 CD22 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 to the amino acid sequence of SEQ ID NO: 2.

[0108] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 scFv2 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 CD22 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 to the amino acid sequence of SEQ ID NO: 12.

[0109] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv3 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 21, 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 CD22 ScFv3 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 22.

[0110] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv4 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 31, 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 CD22 ScFv4 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 32.

[0111] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv5 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 41, 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 CD22 ScFv5 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 42.

[0112] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv6 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 51, 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 CD22 ScFv6 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 52.

[0113] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv7 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 61, 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 CD22 ScFv7 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 62.

[0114] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv8 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 71, 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 CD22 ScFv8 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 72.

[0115] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv9 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 81, 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 CD22 ScFv9 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 82.

[0116] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv10 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 91, 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 CD22 ScFv10 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 92.

[0117] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv11 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 101, 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 CD22 ScFv102 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 102.

[0118] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv12 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 111, 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 CD22 ScFv112 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 112.

[0119] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv13 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 121, 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 CD22 ScFv13 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 122.

[0120] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv14 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 131, 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 CD22 ScFv14 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 132.

[0121] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv15 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 141, or the same It includes sequences with an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In one embodiment, an isolated nucleic acid molecule is provided, wherein the extracellular CD22 ScFv15 antigen-binding domain encoded thereby includes the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence having an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO: 142.

[0122] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv16 antigen-binding domain includes the nucleotide sequence of SEQ ID NO: 151, or a sequence having an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% therewith. In one embodiment, an isolated nucleic acid molecule is provided, wherein the extracellular CD22 ScFv16 antigen-binding domain encoded thereby includes the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence having an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO: 152.

[0123] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv17 antigen-binding domain includes the nucleotide sequence of SEQ ID NO: 161, or a sequence having an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% therewith. In one embodiment, an isolated nucleic acid molecule is provided, wherein the extracellular CD22 ScFv17 antigen-binding domain encoded thereby includes the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO: 162.

[0124] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv18 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 171, 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 CD22 ScFv18 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 172, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 172.

[0125] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv1, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding characteristics of CD22-specific scFv1 by co-expression of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 in one ScFv amino acid sequence.

[0126] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv2, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCD R1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv2 by the co-expression of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 in one amino acid sequence, together as a group.

[0127] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv3, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv3 by the co-expression of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 in one amino acid sequence, together as a group.

[0128] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv4, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv4 by the co-expression of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40 in one amino acid sequence, all together.

[0129] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv5, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv5 by the co-expression of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50 in one amino acid sequence, all together.

[0130] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv6, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv6 by co-expression of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60 in one amino acid sequence, together as a group.

[0131] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv7, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv7 by co-expression of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70 in one amino acid sequence, together as a group.

[0132] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv8, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv8 by the co-expression of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80 in one amino acid sequence, all together.

[0133] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv9, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv9 by the co-expression of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90 in one amino acid sequence, all together.

[0134] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv10, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv10 by co-expression of SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100 in one amino acid sequence, all together.

[0135] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv11, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv11 by co-expression of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110 in one amino acid sequence, all together.

[0136] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv12, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv12 by co-expression of SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120 in one amino acid sequence, all together.

[0137] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv13, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv13 by co-expression of SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130 in one amino acid sequence, all together.

[0138] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv14, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv14 by co-expression of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140 in one amino acid sequence, all together.

[0139] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequence (LCDR 1, LCDR2, LCDR2, specified as SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv15, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv15 by co-expression of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150 in one amino acid sequence, all together.

[0140] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 155, SEQ ID NO: 156, and SEQ ID NO: 157, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv16, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv16 by co-expression of SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160 in one amino acid sequence, all together.

[0141] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 165, SEQ ID NO: 166, and SEQ ID NO: 167, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv17, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv17 by co-expression of SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170 in one amino acid sequence, all together.

[0142] In a preferred embodiment, the isolated light chain complementarity determining region amino acid sequences (LCDR1, LCDR2, LCDR2, specified as SEQ ID NO: 175, SEQ ID NO: 176, and SEQ ID NO: 177, respectively) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, specified as SEQ ID NO: 178, SEQ ID NO: 179, and SEQ ID NO: 180, respectively) each individually contribute to the creation of the binding properties of CD22-specific scFv18, and together as a group contribute to the creation of the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and together as a group contribute to the creation of the heavy chain binding properties of the scFv (HCDR1 and HCDR2 and HCDR3), and the six SEQ ID NOs together as a group contribute to the creation of the binding properties of CD22-specific scFv18 by the co-expression of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, and SEQ ID NO: 180 in one amino acid sequence, all together.

[0143] In various embodiments of the CD22-specific CARs disclosed herein, a schematic scheme is described in FIG. 1, which, from the N-terminus to the C-terminus, includes a signal or leader peptide, an anti-CD22 ScFv, an extracellular linker, a CD8 transmembrane portion, 4-1BB, CD3 zeta, where the bolded letters represent the cloning sites of the linking domain.

[0144] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 3 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4 [LTG 2202 LP-16P-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2A)].

[0145] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG 2202 LP-16P-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2A)].

[0146] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 13 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 14 [LTG 2246 LP-24P-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2B)].

[0147] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 13 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 14 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG 2246 LP-24P-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2B)].

[0148] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 24 [LTG 2247 LP-25P-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (shown in Figure 2C)].

[0149] In another embodiment, the nucleic acid sequence encoding the 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 a CAR comprising the amino acid sequence of SEQ ID NO: 24 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG 2247 LP-25P-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (shown in Figure 2C).

[0150] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 33 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 34 [LTG2248 LP-11S-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2D)].

[0151] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 33 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 34 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2248 LP-11S-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2D)].

[0152] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 43 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 44 [LTG2249 LP-12S-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2E)].

[0153] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 43 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 44 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2249 LP-12S-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2E)].

[0154] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 53 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 54 [LTG2203 LP-16P3-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2F)].

[0155] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 53 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 54 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2203 LP-16P3-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2F)].

[0156] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 63 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 64 [LTG2204 LP-16P16-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2G)].

[0157] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 63 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 64 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2204 LP-16P16-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2G)].

[0158] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 [LTG2205 LP-16P20-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2H)].

[0159] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2205 LP-16P20-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2H)].

[0160] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 83 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 84 [LTG2206 LP-16P2-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2I)].

[0161] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 83 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 84 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2206 LP-16P2-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2I)].

[0162] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94 [LTG2207 LP-16P6-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2J)].

[0163] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2207 LP-16P6-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2J)].

[0164] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 103 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 104 [LTG2208 LP-16P10-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2K)].

[0165] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 103 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 104 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2208 LP-16P10-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2K)].

[0166] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114 [LTG2209 LP-16P17-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2L)].

[0167] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2209 LP-16P17-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2L)].

[0168] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 123 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 124 [LTG2210 LP-16P20v2-CD8 TM-41BB-CD3 zeta amino acid se quence (shown in Figure 2M)].

[0169] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 123 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 124 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2210 LP-16P20v2-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2M)].

[0170] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 133 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 134 [LTG2216 LP-16P1-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2N)].

[0171] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 133 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 134 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2216 LP-16P1-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2N)].

[0172] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 143 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 144 [LTG2217 LP-16P3v2-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2O)].

[0173] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 143 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 144 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2217 LP-16P17-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2O)].

[0174] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 153 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 154 [LTG2218 LP-16P8-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2P)].

[0175] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 153 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 154 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2218 LP-16P8-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2P)].

[0176] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 163 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 164 [LTG2219 LP-16P13-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2Q)].

[0177] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 163 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 164 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2219 LP-16P13-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2Q)].

[0178] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 173 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 174 [LTG2220 LP-16P15-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2R)].

[0179] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 173 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 174 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2220 LP-16P15-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2R)].

[0180] The surface expression of anti-CD22 CAR incorporating a single-chain variable fragment (ScFv) sequence reactive with CD22 antigen is shown in Example 2 below, and its overview is shown in Tables 2, 3, and FIG. 6. The expression levels of each ScFv-containing CAR were determined by flow cytometry analysis of LV-transduced T cells from healthy donors using recombinant CD22-Fc peptide followed by anti-human Fc F(ab’)2 fragment conjugated to PE and detected by flow cytometry (see FIG. 6). The ScFv-based anti-CD22 CAR constructs LTG2202, LTG2216, LTG2217, LTG2218, LTG2208, LTG2219, LTG2220, and LTG2209 were highly expressed in human primary T cells (shown by the gated population) compared to non-transduced T cell controls (ungated cell population, UTD). Representative results from one donor are shown.

[0181] As shown in Example 2 and FIGS. 4, 7A, and 7B, a lentiviral vector (LV) expressing the following CARs was prepared and tested for anti-leukemia activity, and high cytolytic activity of the CD22 CAR was shown. All of the CARs used in the experiment contained the described 4-1BB / CD3 zeta chain signaling motif and specific anti-CD22 binding motif / domain. Leukemia target strains with various CD22 surface expressions were used: Raji and Reh; and CD19-negative K562 and 293T. The ScFv-based anti-CD22 CAR constructs LTG2202, LTG2216, LTG2217, LTG2218, LTG2208, LTG2219, LTG220, and LTG2209 (expressing scFv1(16P), ScFv2(16P1), scFv3(16P3v2), scFv3(16P3v2), scFv4(16P8), scFv5(16P10), scFv6(16P13), scFv7(16P15), scFv8(16P17), respectively) could efficiently lyse the high CD22 tumor strains Raji and Reh, but they had little or no specific lytic activity against K562 or 293T (see FIGS. 4, 7A, 7B). These results demonstrate the efficiency and specificity of the prepared CAR constructs.

[0182] Next, the cytokine secretion ability of anti-CD22 CAR T cells was evaluated. Tumor cells were incubated overnight at an effector-target ratio of 10:1 with CAR T cells or control T cells, and the culture supernatant was analyzed for IFN gamma, TNF alpha by ELISA. and IL-2 were analyzed (see Figure 8). Notably, CAR T cells transduced with LTG2202, LTG2216, LTG2217, LTG2218, LTG2208, LTG2219, LTG2220, and LTG2209 and expressing scFv1(16P), ScFv2(16P1), scFv3(16P3v2), scFv3(16P3v2), scFv4(16P8), scFv5(16P10), scFv6(16P13), scFv7(16P15), scFv8(16P17) produced high levels of IFN gamma, but cytokine induction by the negative control (non-transduced, utd) was not observed. However, distinct differences were seen in TNF-alpha production and IL-2 production. Surprisingly, CD22 CAR LTG2202 had significantly lower output levels of TNF-alpha and IL-2 compared to the Reh tumor line, and each vector had a different ability to produce IL-2 and TNF-alpha against the tumor line targets tested. These differences are thought to result in differences in anti-tumor and toxicity profiles and are thought to be realized individually depending on the disease burden in a specific disease situation. m971, a CAR used as a positive control, was used to evaluate the results based on certain criteria and is currently in clinical trials and can be safely used at present in an advanced disease situation.

[0183] Although not intended to be limited to any particular mechanism of action, reasons for the improved therapeutic function associated with the exemplary CARs according to the present invention include, for example, a) signal transduction became more efficient because the lateral movement in the cell membrane was improved, b) the ability to interact with transmembrane signal transduction cascades related to T cell activation was improved because of the excellent position in the cell membrane microdomain (such as lipid rafts), c) the position in the cell membrane was excellent because there was a preferential movement away from inhibitory or downregulatory interactions, for example, the distance from phosphatases such as CD45 was relatively far or the interaction with the phosphatase was relatively small, and d) the assembly into the T cell receptor signal transduction complex (i.e., immune synapse) was excellent, or any combination of these is considered, but not limited thereto.

[0184] So far, the present disclosure has been illustrated using the extracellular CD22 variable heavy chain alone and the ScFv antigen-binding domain as specific examples, but other nucleotide and / or amino acid variants in the CD22 variable heavy chain alone and the ScFv antigen-binding domain can also be used to derive a CD22 antigen-binding domain for use in the CARs described herein.

[0185] Depending on the desired antigen to be targeted, the CAR may be further engineered to include an appropriate antigen-binding domain specific for the desired antigen target. For example, when CD22 is the desired antigen to be targeted, an antibody against CD22 may be used as the antigen-binding domain incorporated into the CAR.

[0186] In an exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD33. Preferably, the antigen-binding domain in the CAR is an anti-CD33 heavy chain-only binder VH-4, wherein the nucleic acid sequence of the anti-CD33 heavy chain-only binder comprises the sequence of SEQ ID NO: 202. In one embodiment, the anti-CD33 heavy chain-only binder comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 202. In another embodiment, the anti-CD33 heavy chain-only portion of the CAR comprises the amino acid sequence of SEQ ID NO: 203. In another exemplary embodiment, the nucleic acid sequence of CAR LTG1906 expressing the anti-CD33 heavy chain-only binder consists of SEQ ID NO: 204. In another embodiment, the amino acid sequence of CAR LTG1906 expressing the anti-CD33 heavy chain-only binder consists of SEQ ID NO: 205.

[0187] In an exemplary embodiment, the antigen-binding domain portion of the CAR further targets mesothelin Preferably, the antigen-binding domain in the CAR is an anti-mesothelin ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the sequence of SEQ ID NO: 198. In one embodiment, the anti-mesothelin ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 199. In another embodiment, the anti-mesothelin ScFv portion of the CAR comprises the amino acid sequence of SEQ ID NO: 199. In another exemplary embodiment, the nucleic acid sequence of the CAR expressing the anti-mesothelin scFv consists of SEQ ID NO: 200. In another embodiment, the amino acid sequence of the anti-mesothelin CAR LTG1904 is SEQ ID NO: 201.

[0188] In one aspect of the present invention, for example, antigens derived from the Retroviridae family (such as human immunodeficiency viruses like HIV-1 and HIV-LP), Picornaviridae family (such as poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae family, Herpesviridae family (such as herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus), Poxviridae family (such as smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof (not limited thereto)) are provided with CARs that can bind other than TSA or other than TAA.

[0189] In another aspect of the present invention, CARs are provided that can bind to antigens derived from bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, for example, Helicobacter pylori, Legionella pneumophilia, Mycobacterium species (such as Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria, Streptococcus pyogenes, group A Streptococcus, group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or antigens derived from infectious bacteria such as combinations thereof are provided with CARs that can bind. pneumophilia), Mycobacterium species (such as Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides meningitides), Listeria, Streptococcus pyogenes, group A Streptococcus, group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or antigens derived from infectious bacteria such as combinations thereof are provided with CARs that can bind.

[0190] 2. Transmembrane domain With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD22 antigen-binding domain of the CAR.

[0191] The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, this domain may be derived from any membrane-bound or transmembrane protein.

[0192] Transmembrane regions particularly useful in the CARs described herein may be derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19 (i.e., comprising at least their transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it may predominantly contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2 to 10 amino acids in length, may form a bond between the transmembrane domain of the CAR and the cytoplasmic signaling domain. A doublet of glycine and serine provides a particularly suitable linker.

[0193] In one embodiment, a transmembrane domain originally associated with one of the domains in the CAR is used in addition to the transmembrane domains described above.

[0194] In some examples, the transmembrane domain can be selected or amino acid-substituted so that the domain avoids binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize the interaction of the domain with other receptor complex components.

[0195] In one embodiment, the transmembrane domain in the CAR according to the present invention is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 181. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 182. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 182.

[0196] In one embodiment, the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 182, or a sequence having at least 1, 2, or 3 modifications (such as substitutions) added to the amino acid sequence of SEQ ID NO: 182 with 95-99% identity thereto, provided that the modifications (such as substitutions) are 20, 10, or 5 or less.

[0197] In some examples, the transmembrane domain of the CAR comprises the CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 183. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 184. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 184 or a sequence having 95-99% identity thereto.

[0198] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and binds to the transmembrane CD8 domain, the transmembrane CD28 domain, or a combination thereof.

[0199] 3. Spacer domain In a CAR, a spacer domain may be disposed between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0200] In some embodiments, the linker may include a spacer element. When the spacer element is present, the linker is enlarged by the spacer element, increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art and include those listed in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as U.S. Patent Publication Nos. 20110212088 and and 20110070248 (the entireties of which are incorporated herein by reference).

[0201] The spacer domain preferably has a sequence that promotes the binding of the CAR to the antigen and increases signal transduction into the cell. Examples of amino acids expected to promote binding include cysteine, charged amino acids, and serine and threonine at sites where glycosylation is possible, and these amino acids can be used as the amino acids constituting the spacer domain.

[0202] As this spacer domain, the whole or a part of amino acid numbers 137 to 206 (SEQ ID NO: 39) of the hinge region of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acid numbers 135 to 195 of CD8 beta (GenBank: AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: NP__000607.1), or amino acid numbers 137 to 152 of CD28 (NCBI RefSeq: NP__006130.1) can be used. Also, as this spacer domain, a part of the constant region of the H chain or L chain of an antibody can be used. Furthermore, this spacer domain may be an artificially synthesized sequence.

[0203] Furthermore, in the CAR, a signal peptide sequence may be bound to the N-terminus. This signal peptide sequence is present at the N-terminus of many secreted proteins and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above as the intracellular domain have a signal peptide sequence, this signal peptide can be used as the signal peptide for the CAR. In one embodiment, the signal peptide contains the amino acid sequence of SEQ ID NO: 191.

[0204] 4. Intracellular domain The cytoplasmic domain or intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to the specialized functions of a cell. For example, the effector functions of T cells can be cytolytic activity or helper activity including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the protein portion that transmits effector function signals and directs the cell to perform specialized functions. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, this truncated portion may be used in place of the full chain as long as it can transmit effector function signals. Thus, the meaning of the term "intracellular signaling domain" includes any truncated portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[0205] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling after antigen-receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional ability.

[0206] It is known that signals emanating only through the TCR are insufficient to fully activate T cells and that a second or co-stimulatory signal is further required. Thus, it can be said that two distinct types of cytoplasmic signaling sequences are involved in T cell activation: those that initiate antigen-dependent first activation via the TCR (first cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a second or co-stimulatory signal (second cytoplasmic signaling sequences).

[0207] The first cytoplasmic signaling sequence regulates the first activation of the TCR complex in either a stimulatory or inhibitory manner. The first cytoplasmic signaling sequence that functions in a stimulatory manner may contain a signaling motif known as an immunoreceptor activation tyrosine motif or ITAM.

[0208] Examples of ITAMs containing a first cytoplasmic signaling sequence particularly useful in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP__932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP__004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP__000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP__000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP__000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP__000724.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP__055022.2), 0022 (NCBI RefSeq: NP__001762.2) amino acids 707-847, amino acids 166-226 of CD79a (NCBI RefSeq: NP__001774.1), amino acids 182-229 of CD79b (NCBI RefSeq: NP__000617.1), and CD66d (NCBI Peptides having the sequence of amino acid numbers 177 to 252 of RefSeq:NP__001806.2), and variants having the same function as these peptides, including but not limited to these. The amino acid numbers based on the NCBI RefSeq ID or amino acid sequence information of GenBank described in this specification are numbered based on the full length of the precursor (including signal peptide sequence, etc.) of each protein. In one embodiment, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling sequence derived from CD3 zeta.

[0209] In a preferred embodiment, the intracellular domain of the CAR may be designed to itself include a CD3-zeta signaling domain or may be combined with any other desirable cytoplasmic domain useful in the context of the CAR. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for lymphocytes to efficiently respond to an antigen. 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. Specific examples of such co-stimulatory molecules include peptides having the sequences of amino acids 236 to 351 of CD2 (NCBI RefSeq:NP__001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq:NP__000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq:NP__055022.2), amino acids 207 to 235 of CD8 alpha (NCBI RefSeq:NP__001759.3), amino acids 196 to 210 of CD83 (GenBank:AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq:NP__006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBI RefSeq:NP__001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq:NP__003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq:NP__036224.1), as well as variants having the same function as these peptides, but not limited thereto. Thus, although the present disclosure has thus far been exemplified mainly using 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present disclosure. Including, but not limited to, variants having the same function as these peptides. Thus, although the present disclosure has thus far been exemplified mainly using 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present disclosure.

[0210] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR may be bound to each other in a random or specific order. Optionally, a short oligo or polypeptide linker, preferably 2 to 10 amino acids in length, may form this bond. A doublet of glycine and serine provides a particularly suitable linker.

[0211] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta, as well as the signaling domains of CD28 and 4-1BB.

[0212] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the nucleic acid sequence of SEQ ID NO: 186, and the signaling domain of CD3-zeta includes the nucleic acid sequence of SEQ ID NO: 188.

[0213] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 187, and the signaling domain of CD3-zeta includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 189.

[0214] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the amino acid sequence of SEQ ID NO: 187, and the signaling domain of CD3-zeta includes the amino acid sequence of SEQ ID NO: 189.

[0215] 5. Further Explanation of CAR The functional portions of the CARs disclosed herein are also clearly included within the scope of the present invention. When the term "functional portion" is used in reference to a CAR, it refers to any one or more portions or fragments of the CARs disclosed herein, and such portions or fragments retain the biological activity of the CAR (parent CAR). Functional portions include, for example, CAR portions that retain the ability to recognize target cells, or detect, treat, or prevent disease, to a similar extent, to the same extent, or to a higher extent than the parent CAR. With respect to a parent CAR, a functional portion may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0216] A functional portion may include additional amino acids not found in the amino acid sequence of the parent CAR at the amino terminus or carboxy terminus or both termini of the portion. Desirably, these additional amino acids do not interfere with the biological function of the functional portion, such as, for example, recognition of target cells, detection of cancer, treatment or prevention of cancer. More desirably, these additional amino acids improve such biological activity over the biological activity of the parent CAR.

[0217] Functional variants of the CARs disclosed herein are included within the scope of the present disclosure. The term "functional variant" as used herein refers to sequence identity with the parent CAR Refers to a CAR, polypeptide, or protein having a substantial or significant degree of identity or similarity, and this functional variant retains the biological activity of the CAR from which the variant is derived. Functional variants include, for example, variants of a CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar extent, to the same extent, or to a higher extent than the parent CAR. With respect to the parent CAR, the functional variant may have, for example, an amino acid sequence identity with the parent CAR of at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0218] Functional variants may include, for example, those in which at least one conservative amino acid substitution is added to the amino acid sequence of the parent CAR. Alternatively or additionally, functional variants may include those in which at least one non-conservative amino acid substitution is added to the amino acid sequence of the parent CAR. In this case, it is preferred that this non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. This non-conservative amino acid substitution may improve the biological activity of the functional variant such that the biological activity of the functional variant is superior to that of the parent CAR.

[0219] Amino acid substitutions of the CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include substituting an acidic / negatively charged polar amino acid (e.g., Asp or Glu) with another acidic / negatively charged polar amino acid, substituting a nonpolar side chain-containing amino acid (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another nonpolar side chain-containing amino acid, substituting a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, substituting a polar side chain-containing uncharged amino acid (e.g., Asn, Gln, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, substituting a beta-branched side chain-containing amino acid (e.g., Ile, Thr, and Val) with another beta-branched side chain-containing amino acid, substituting an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid, and the like.

[0220] The CAR may essentially consist of one or more of the specified amino acid sequences described herein, such that the biological activity of the functional variant is not substantially changed by other components (e.g., other amino acids).

[0221] A CAR (including functional parts and functional variants) may be of any length, i.e., may contain any number of amino acids, as long as the CAR (or its functional part or functional variant) retains a biological activity such as, for example, the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent a disease in a mammal. For example, the CAR may be of a length of about 50 to about 5000 amino acids, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids in length.

[0222] A CAR (including the functional parts and functional variants according to the present invention) may contain synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chloro phenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, Ν’,Ν’-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentane carboxylic 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.

[0223] A CAR (including functional parts and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridging), or converted to an acid addition salt, and / or optionally dimerized or polymerized or conjugated.

[0224] CAR (including its functional parts and functional variants) can be obtained by methods well known in the art. CAR may be made by any suitable polypeptide or protein production method. Suitable methods for newly synthesizing polypeptides and proteins are described in prior art documents such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Also, polypeptides and proteins may be recombinantly produced using the nucleic acids described herein and 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. Further, some of the CARs (including their functional parts and functional variants) may be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Isolation and purification methods are well known in the art. Alternatively, the CARs (including their functional parts and functional variants) described herein may be commercially synthesized by a company. In this regard, the CARs may be synthetic, recombinant, isolated, and / or purified.

[0225] B. Antibodies and antigen-binding fragments One embodiment further provides a CAR, a T cell expressing the CAR, an antibody, or an antigen-binding domain or portion thereof that specifically binds to one or more of the antigens disclosed herein. As used herein, "T cell expressing a CAR" or "CAR T cell" means a T cell that expresses a CAR and has antigen specificity, for example, as determined by the antibody-derived targeting domain of the CAR.

[0226] As used herein, "antigen-binding domain" may include an antibody and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and includes diverse antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, provided that they exhibit the desired antigen-binding activity. Examples of antibodies include, but are not limited to, intact immunoglobulins well known in the art that retain binding affinity for an antigen, as well as variants and fragments thereof. As used herein, "antigen-binding domain" may include an antibody and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and includes diverse antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, provided that they exhibit the desired antigen-binding activity. Examples of antibodies include, but are not limited to, intact immunoglobulins well known in the art that retain binding affinity for an antigen, as well as variants and fragments thereof.

[0227] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up this population are identical except for natural mutations that may be present in very small amounts. Monoclonal antibodies are highly specific and are directed against a single antigen epitope. The modifier "monoclonal" indicates the property that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted to mean that the antibody must be produced by any particular method. In some examples, monoclonal antibodies are antibodies produced by a single clone of B lymphocytes or by cells or their progeny transfected with nucleic acids encoding the light and heavy chain variable regions of the antibody for a single antibody (or an antigen-binding fragment thereof). In some examples, monoclonal antibodies are isolated from a subject. Monoclonal antibodies may have conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are well known, for example, see Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition See Cold Spring Harbor Publications, New York (2013).

[0228] Typically, an immunoglobulin has heavy (H) and light (L) chains that are linked to each other by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five main classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule (IgM, IgD, IgG, IgA, and IgE).

[0229] The heavy and light chains each contain a constant region (or constant domain) and a variable region (or variable domain) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind an antigen. In additional embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable without a light chain (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH" or "VH" refer to the variable region of an antibody heavy chain that includes the variable region of an antigen-binding fragment, such as Fv, ScFv, dsFv, or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain that includes those of Fv, ScFv, dsFv, or Fab.

[0230] The variable regions of the light and heavy chains contain "framework" regions interrupted by three hypervariable regions (also called "complementary determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved among species. The framework regions of an antibody, i.e., those of the constituent light and heavy chains together, position and align the CDRs in three-dimensional space.

[0231] CDRs are primarily responsible for binding to antigen epitopes. The boundaries of the amino acid sequences 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,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are further typically identified by the chain on which the CDR is located. Thus, VH CDR3 is the CDR3 from the variable domain of the heavy chain of an antibody that contains it, and VL CDR1 is the CDR1 from the variable domain of the light chain of an antibody that contains it. Light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3.

[0232] An "antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize cognate antigens, as well as various combinations of such portions. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments made by modification of whole antibodies or newly synthesized using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0233] A single-chain antibody (ScFv) is a genetically engineered molecule containing a VH domain and a VL domain of one or more antibodies linked by a suitable polypeptide linker to form a gene-fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains within an ScFv typically does not determine the ScFv. Thus, ScFvs having both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0234] In dsFv, the variable chains of the heavy and light chains have had disulfide bonds introduced by mutation to stabilize the binding of the two chains. Also included is the diabody, which is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to join the two domains into a single chain is used, so the two domains are joined to the complementary domains of another chain, forming two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0235] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized mouse antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0236] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, or can be produced recombinantly, or can be obtained, for example, by screening a combinatorial library consisting of variable heavy and variable light chains as described by Huse et al., Science 246:1275-1281 (1989) (incorporated herein by reference). These methods, as well as other methods for making, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies, are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0237] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in an antagonistic assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in an antagonistic assay. Antibody antagonistic assays are well known, and exemplary antagonistic assays are provided herein.

[0238] A "humanized" antibody or antigen-binding fragment thereof comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment that provides the CDR is called the "donor," and the human antibody or antigen-binding fragment that provides the framework is called the "acceptor." In one embodiment, all of the CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region may be absent, but if present, may be substantially identical to a human immunoglobulin constant region, e.g., at least about 85-90% (such as about 95% or more) identical. Thus, all parts of the humanized antibody or antigen-binding fragment (possibly excluding the CDRs) are substantially identical to the corresponding parts of a native human antibody sequence.

[0239] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies (typically of different species). In some examples, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0240] A "fully human antibody" or "human antibody" is an antibody that contains sequences from the human genome (or is derived therefrom) and does not contain sequences from another species. In some embodiments, a human antibody comprises CDRs, framework regions, and (if present) an Fc region from the human genome (or is derived therefrom). Human antibodies can be identified and isolated, for example, by using antibody production techniques based on human genome-derived sequences, such as by phage display or the use of recombinant animals (e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1 (see 125,2005; Lonenberg, Curr. Opin. Immunol., 20:450 - 459, 2008).

[0241] An antibody may have one or more binding sites. If there is more than one binding site, these binding sites may be the same as each other or different. For example, a natural immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0242] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are well known in the art and include any antibody - antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, for example, Janeway et al. below, US Patent Application Publication No. 2002 / 0197266 A1, and US Patent No. 7,338,929).

[0243] Also, a CAR, a T cell expressing the CAR, an antibody, or an antigen - binding portion thereof may be modified to include a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0244] C. Conjugates A CAR, a T cell expressing the CAR, a monoclonal antibody, or an antigen-binding fragment thereof, specific for one or more of the antigens disclosed herein, may be conjugated to an agent such as an effector molecule or a detectable marker using any of a number of means well known to those of skill in the art. Either covalent or non-covalent means may be used. The conjugate includes, but is not limited to, a molecule in which an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein is covalently bound to an effector molecule or a detectable marker. It is understood by those of skill in the art that a variety of effector molecules and detectable markers can be used, including, but not limited to, chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C, 3 H, and 35 radioactive agents such as S, as well as other labels, target sites, and ligands, etc.

[0245] The choice of a specific effector molecule or detectable marker depends on the specific target molecule or cell and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin used to cause the death of a particular target cell (such as a tumor cell).

[0246] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which can be utilized in reactions with suitable functional groups on the antibody, resulting in the attachment of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may be associated with the attachment of any of a plurality of well-known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). A linker may be any molecule used to connect an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker can form covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker may be attached to the constituent amino acids via their side chains (e.g., to cysteine via a disulfide bond) or to the amino and carboxy groups of the alpha carbon of the terminal amino acids.

[0247] In some embodiments, the linker may include a spacer element, and when the spacer element is present, the spacer element increases the size of the linker and increases the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art and include those listed in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (each of which is hereby incorporated by reference in its entirety).

[0248] In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet another embodiment, the linker is not cleavable and the effector molecule or detectable marker is released, for example, by degradation of the antibody. In some embodiments, the linker is cleavable by a cleaving agent present within the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker may be, for example, a peptide linker cleaved by an intracellular peptidase, or a protease enzyme including but not limited to lysosomal or endosomal proteases. In some embodiments, the peptide linker is at least two amino acids in length, or at least three amino acids in length. However, the linker may be four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acids in length, for example, one to two, one to three, two to five, three to ten, three to fifteen, one to five, one to ten, or one to fifteen amino acids in length. The proteases may include cathepsin B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by a thiol-dependent protease such as cathepsin-B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is hereby incorporated by reference herein. In one specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0249] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, such pH-sensitive linkers hydrolyze under acidic conditions. For example, acid-labile linkers that can hydrolyze within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amide, orthoesters, acetals, or ketals, etc.) can be used. (See, e.g., U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions (such as in blood), but are unstable at a pH of about 5.5 or less than 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bonded to the therapeutic agent via an acylhydrazone linkage (see, e.g., U.S. Patent No. 5,622,929, etc.)).

[0250] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are well known in the art and include, for example, those formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008). See also U.S. Patent No. 4,880,935.

[0251] In yet another specific embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0252] In yet another embodiment, the linker is not cleavable and the effector molecule or detectable marker is released by degradation of the antibody (see U.S. Publication No. 2005 / 0238649, which is hereby incorporated by reference in its entirety).

[0253] In some embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), in a sample of the conjugate, no more than about 20%, about 15%, about 10%, about 5%, about 3%, or about 1% of the linker is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating a conjugate containing the linker of interest with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of effector molecule or detectable marker that has been released into the plasma. A variety of exemplary linkers that can be used in conjugates are described in WO2004-010957, US Publication No. 2006 / 0074008, US Publication No. 20050238649, and US Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0254] In some embodiments, provided are conjugates of a CAR, a T cell expressing the CAR, an antibody, or an antigen-binding portion thereof, and one or more small molecule toxins such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene, and CC1065, and derivatives of these toxins that have toxin activity.

[0255] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art, can be isolated from natural sources according to well-known methods, can be produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968 - 7973), or maytansinol and maytansinol analogs can be prepared synthetically according to well-known methods. Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from Maytenus serrata, a shrub native to East Africa (U.S. Patent No. 3,896,111). Subsequently, it was further discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is hereby incorporated by reference herein. Conjugates containing maytansinoids, methods for their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163, and European Patent No. EP0425235 B1, the disclosures of which are hereby expressly incorporated by reference herein.

[0256] Additional toxins can be used in combination with a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof. Examples of toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A-F. Such toxins are well known in the art and many of them are readily available from commercial suppliers (e.g., Sigma Chemical Company, St. Louis, MO). The intended toxins also include variants of such toxins (see, e.g., U.S. Pat. Nos. 5,079,163 and 4,689,401).

[0257] Saporin is a toxin derived from Saponaria officinalis that inhibits protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin does not have a mechanism for specifically entering cells and thus needs to be conjugated to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein in order to efficiently enter cells.

[0258] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins has reduced or eliminated non-specific toxicity by mutation. A mutant known as CRM107 has sufficient enzymatic activity but significantly reduced non-specific toxicity and has been well known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Pat. No. 5,792,458 and U.S. Pat. No. 5,208,021.

[0259] Ricin is derived from Ricinus communis (castor bean) It is lectin RCA60 obtained from ). For examples of ricin, see U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) has two forms, and since their molecular weights are approximately 65 kD and approximately 120 kD respectively, RCA 60 and RCA 120 are so-called (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for the inactivation of protein synthesis and cell death. The B chain binds ricin to cell surface galactose residues and 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).

[0260] Ribonucleases are also used as immunotoxins by binding to target molecules (see Suzuki et al., Nat. Biotech. 17:265 - 70, 1999). Exemplary ribotoxins such as α - sarcin and restrictocin are described, for example, in Rathore et al., Gene 190:31 - 5, 1997, and Goyal and Batra, Biochem. 345 Pt 2:247 - 54, 2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes DNA double - strand breaks and induces apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070 - 87, 1989). This drug is, in clinical trials, the toxic site of immunotoxins (see, for example, Gillespie et al., Ann. Oncol. 11:735 - 41, 2000).

[0261] Abrin contains a toxic lectin obtained from Abrus precatorius. Its toxic components, abrin a, b, c, and d, have a molecular weight of approximately 63-67 kD and are composed of two polypeptide chains A and B linked by a disulfide bond. Chain A inhibits protein synthesis, and chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0262] CARs, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof that are specific for one or more of the antigens disclosed herein may also be conjugated to a detectable marker, such as ELISA, spectrophotometry, flow cytometry, microscopy, or imaging diagnostic techniques (computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound diagnosis, fiber optic examination, and laparoscopy, etc.) that can be detected by a detectable marker. Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. CARs, T cells expressing CARs, antibodies, or antigen-binding portions thereof may also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof is conjugated to a detectable enzyme, a reaction distinguishable when used for the enzyme It can be detected by adding additional reagents that produce a response product. For example, in the presence of horseradish peroxidase, which is an agent, a coloring reaction product can be obtained by adding hydrogen peroxide and diaminobenzidine, and this can be detected visually. A CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof may also be bound to biotin and may be detected by indirectly measuring the binding of avidin or streptavidin. Notably, avidin itself may be bound to an enzyme or a fluorescent label.

[0263] A CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof may be bound to a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxides are also useful as labels. Antibodies may also be bound to lanthanides (such as europium and dysprosium) and manganese. An antibody or antigen-binding fragment may also be labeled with a predefined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a binding site for a secondary antibody, a metal-binding domain, an epitope tag, etc.).

[0264] A CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof may also be bound to a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For example, the radiolabel may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, emission spectrum, or other diagnostic techniques. Further, the radiolabel may be used as a toxin in the treatment of treating tumors in a subject, for example, for treating neuroblastoma. Examples of labels for polypeptides are 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 radioisotopes or radiolabeled nucleotides such as I, but are not limited thereto.

[0265] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, a radioactive label may be detected using a photographic film or a scintillation counter, and a fluorescent marker may be detected by detecting the emitted light using a photodetector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0266] D. Nucleotides, Expression, Vectors, and Host Cells According to one embodiment of the present invention, there is further provided a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants) described herein. The nucleic acids according to the present invention may include a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0267] In some embodiments, the nucleotide sequence may have codons modified. Without being bound by any theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing natural codons with other codons that encode the same amino acid but are more readily translated by tRNAs that are more readily available within the cell, and thus the translation efficiency may be increased. Optimization of the nucleotide sequence may also be one that reduces secondary mRNA structures that may interfere with translation, and thus the translation efficiency may be increased.

[0268] In one embodiment of the present invention, the nucleic acid may include a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the present invention. In another embodiment of the present invention, the nucleic acid is It may include a codon-optimized nucleotide sequence encoding any of the CARs (including its functional parts and functional variants) described herein.

[0269] As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally may be single-stranded or double-stranded, may be obtained from synthetic or natural sources (e.g., by isolation and / or purification), may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages (such as phosphoramidate linkages or phosphorothioate linkages instead of the phosphodiester found between nucleotides of unmodified oligonucleotides), meaning a polymer of DNA or RNA. In some embodiments, the nucleic acid contains no insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0270] A recombinant nucleic acid may have a sequence that does not exist naturally or may have a sequence in which two regions that are distant in the sequence are artificially combined. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificially manipulating distant nucleic acid regions using genetic engineering techniques such as those described in the references of Sambrook et al. mentioned above. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, the references of Sambrook et al. and Ausubel et al. mentioned above. For example, nucleic acids may be chemically synthesized using natural nucleotides or nucleotides modified in various ways (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the double-strands formed by hybridization.Examples of modified nucleotides that can be used for nucleic acid generation 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, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids according to the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0271] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any of the CARs described above or a functional portion or functional variant thereof. Alternatively, the nucleotide sequence may be a nucleotide sequence degenerate to any of the sequences described above or a combination of degenerate sequences and may comprise.

[0272] One embodiment further provides an isolated or purified nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein, or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0273] The nucleotide sequence that hybridizes under stringent conditions may hybridize under highly stringent conditions. "Highly stringent conditions" means that the nucleotide sequence specifically hybridizes to the target sequence (the nucleotide sequence of any of the nucleic acids described herein), and the amount thereof is detectably more than non-specific hybridization. Highly stringent conditions include conditions that can distinguish a polynucleotide having a strictly complementary sequence or having only 2 to 3 scattered mismatches from a random sequence having 2 to 3 small regions (for example, 3 to 10 bases) that happen to match the nucleotide sequence by chance. Such small complementary regions are more easily melted than the full-length complementary regions of 14 to 17 bases or longer in length, and these can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions can include, for example, low salt and / or high temperature conditions such as about 0.02 to 0.1 M NaCl or equivalent and a temperature of about 50 to 70 °C. Thus, highly stringent conditions allow very little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any of the CARs of the present invention. It is generally understood that the conditions can be made more stringent by increasing the amount of formamide added.

[0274] Also provided are nucleic acids comprising nucleotide sequences that have at least about 70% or more identity with any of the nucleic acids described herein, for example, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0275] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids described above. For the purposes described herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector and a host cell are contacted under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the host cell, the host cell is capable of expressing the mRNA, protein, polypeptide, or peptide. Such vectors are generally not naturally occurring.

[0276] However, some of such vectors may occur naturally. The recombinant expression vector may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, may be synthetic or obtained in part from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may comprise natural or non-natural nucleotide linkages, or both types of linkages. Preferably, the non-natural or modified nucleotides or nucleotide linkages do not interfere with the transcription or replication of the vector.

[0277] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell It can be used. Suitable vectors include those designed for the purpose of propagation and growth, or for expression, or for both purposes (such as plasmids and viruses). The vector may be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA).

[0278] Bacteriophage vectors such as λΤΙΟ, λΤΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, for example, a retroviral vector or a lentiviral vector. The lentiviral vector is a vector derived from at least a part of the lentiviral genome, including, in particular, a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used in clinics include, for example, but not limited to, the LENTIVECTOR® gene transfer technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc. Lentiviral vectors that are not of clinical type are also available and may be well known to those skilled in the art.

[0279] Multiple transfection techniques are generally well-known in the art (see, for example, 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)).

[0280] Transfection methods include calcium phosphate co-precipitation (see, for example, Graham et al. supra), direct microinjection into cultured cells (see, for example, Capecchi, Cell, 22:479-488 (1980)), electroporation (see, for example, Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, for example, Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transfection (see, for example, Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid introduction using a high-velocity microparticle gun (see, for example, Klein et al., Nature, 327:70-73 (1987)).

[0281] In one embodiment, the recombinant expression vector may be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., supra, and Ausubel et al., supra. The circular or linear construct of the expression vector may be prepared to contain a replication mechanism that functions in a prokaryotic or eukaryotic host cell. The replication mechanism may be derived from, for example, ColE1, 2μ plasmid, λ, SV40, and bovine papillomavirus, etc.

[0282] The recombinant expression vector is appropriately specific for the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), and transcription codons, translation initiation codons, and stop codons, etc., are considered in view of whether the vector is based on DNA or RNA. It may contain a regulatory sequence. The recombinant expression vector may contain restriction sites for facilitating cloning.

[0283] The recombinant expression vector may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include, for example, biocide resistance such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0284] The recombinant expression vector may contain a natural or non-natural promoter operably linked to a nucleotide sequence encoding a CAR (including its functional parts and functional variants), or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a CAR. The selection of the promoter (e.g., strong, weak, inducible, tissue-specific, and developmental-specific, etc.) is within the scope of the ordinary knowledge of those skilled in the art. Similarly, the binding of the nucleotide sequence and the promoter is also within the scope of the ordinary knowledge of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, for example, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or the promoter found in the terminal repeat sequence of the murine stem cell virus.

[0285] The recombinant expression vector may be designed for either transient expression, or stable expression, or both. Also, the recombinant expression vector may be prepared for constitutive expression or inducible expression.

[0286] Furthermore, the recombinant expression vector may be constructed to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, on the cells expressing the gene, causing the death of the cells when the cells are contacted or exposed to the agent. Suicide genes are well known in the art (see, e.g., Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0287] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector according to the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or alga, or a prokaryotic cell, such as a bacterium or protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism, such as a human). The host cell may be an adherent cell or a suspension cell (i.e., a cell that grows in suspension). Suitable host cells are well known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is the amplification or replication of the recombinant expression vector For this purpose, the host cell may be a prokaryotic cell, such as a DH5a cell. When the production of a recombinant CAR is the goal, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be any cell type, may be derived from any type of tissue, and may be at any stage of development, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0288] For the purposes described herein, the T cell may be any T cell, a cultured T cell (such as a primary T cell, etc.), or a T cell from a cultured T cell line (such as Jurkat, SupTl, etc.), or a T cell obtained from a mammal. When obtained from a mammal, the T cell may be obtained from a wide variety of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell may be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be any type of T cell, may be at any stage of development, and includes, but is not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD8+ T cells (such as cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm, and naive-type T cells. The T cell may be a CD8+ T cell or a CD4+ T cell.

[0289] In one embodiment, the CAR described herein can be used in suitable cells that are not T cells. Such cells are those having immune effector functions, such as, for example, NK cells and T-like cells generated from pluripotent stem cells.

[0290] In addition, one embodiment also provides a population of cells comprising at least one host cell described herein. This population of cells, in addition to host cells containing any of the described recombinant expression vectors, may be a heterogeneous population comprising at least one other type of cell, for example, a host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, such as a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells may be a substantially homogeneous population that mainly comprises (e.g., consists essentially of) host cells containing the recombinant expression vector. Also, the population may be a clonal cell population in which all cells of the population are clones of a single host cell containing the recombinant expression vector, and thus all cells of the population contain this recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells containing the recombinant expression vector described herein.

[0291] CARs (including their functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including their populations), and antibodies (including their antigen-binding parts) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the purity of the host cells is higher than that in their natural environment in the body. Such host cells may be prepared, for example, by standard purification techniques. In some embodiments, the preparation of host cells is purified such that the host cells occupy at least about 50%, for example, at least about 70% of the total cell content of this preparation. For example, this purity may be at least about 50%, or may exceed about 60%, about 70%, or about 80%, or may be about 100%.

[0292] E. Method of treatment It is intended that the CARs disclosed herein may be used in methods for treating or preventing diseases in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, which comprises administering to the mammal a CAR, nucleic acid, recombinant expression vecto - Administering a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition, in an amount effective for treating or preventing cancer in a mammal.

[0293] One embodiment further includes the step of lymphodepleting the mammal prior to administering the CAR disclosed herein. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0294] For the purposes of the method in which the host cell or population of cells is administered, the cell may be a xenogeneic-derived cell of the mammal or an autologous-derived cell thereof. Preferably, the cell may be an autologous-derived cell of the mammal. As used herein, "xenogeneic-derived" means any material that is derived from an animal that is of the same species as, but a different individual from, the individual into which the material is introduced. Two or more individuals are said to be xenogeneic-derived from each other if the genes at one or more loci are not identical. In some embodiments, xenogeneic-derived materials from individuals of the same species may be genetically different enough to interact antigenically with each other. As used herein, "autologous-derived" means any material that is derived from the same individual as the individual into which the material will later be re-introduced.

[0295] As used herein, the mammal may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals such as mice and hamsters and lagomorph mammals such as rabbits. The mammal may be of the order Carnivora, including the families Felidae (cats) and Canidae (dogs). The mammal may be of the order Artiodactyla, including the subfamily Bovinae (cows) and swine (pigs), or of the order Perissodactyla, including the family Equidae (horses). The mammal may be of the order Primates, Ceboids, or Simoids (monkeys), or of the order Anthropoidea (humans and apes). Preferably, the mammal is a human.

[0296] With respect to the methods described above, the cancer may be any cancer, including but not limited to ALL, AML, alveolar rhabdomyosarcoma, bladder cancer (e.g., urothelial carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia (CLL), chronic myeloid cancer (CML), colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, NHL, B-chronic lymphocytic leukemia, hairy cell leukemia, Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omentum, and mesentery cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0297] As used herein, the terms "treatment" and "prevention" and their derivatives do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that would be recognized by one of ordinary skill in the art as having the potential for a benefit or therapeutic effect. In this regard, the present method can provide any amount or any level of cancer treatment or prevention in a mammal.

[0298] Furthermore, the treatment or prevention provided by the present method may include the treatment or prevention of one or more conditions or symptoms of the disease (e.g., cancer). Also, for the purposes described herein, "prevention" may include delaying the onset of a disease or its symptoms or conditions.

[0299] Another embodiment provides a method for detecting the presence of cancer in a mammal, which comprises: (a) forming a complex by contacting a sample containing one or more cells from the mammal with a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody, and / or antigen-binding portion thereof, or a pharmaceutical composition; and (b) detecting the complex, wherein the detection of the complex suggests the presence of cancer in the mammal.

[0300] The sample may be obtained by any suitable method, such as a biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may be for the purpose of subjecting the removed tissue and / or cells to laboratory methods, which may include experiments to determine whether the individual has or is suffering from a particular condition or disease state. The condition or disease may be, for example, cancer.

[0301] Regarding one embodiment of a method for detecting the presence of a proliferative disorder, such as cancer, in a mammal, a sample containing mammalian cells may be a sample containing whole cells, their lysates, or whole cell lysate fractions, such as nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions. When the sample contains whole cells, these cells may be any cells of a mammal, such as cells of any organ or tissue (including blood cells or endothelial cells).

[0302] The contact described above may occur in vitro or in vivo for a mammal. Preferably, the contact occurs in vitro.

[0303] Also, the detection of the complex may be performed by any of a plurality of methods well known in the art. For example, the CARs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies, or antigen-binding portions thereof, disclosed herein may be labeled with detectable labels such as, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles) as disclosed above.

[0304] Methods for testing the target cell recognition ability and antigen specificity of CARs are well known in the art. For example, Clay et al., J. Immunol, 163:507-513 (1999) teach a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a), or interleukin 2 (IL-2)). In addition, the function of CARs may be evaluated by measuring the cytotoxicity of cells as described in Zhao et al., J. Immunol, 174:4415-4423 (2005).

[0305] Another embodiment provides for the use of a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody, or antigen-binding portion thereof, and / or pharmaceutical composition according to the invention for treating or preventing a proliferative disorder, such as cancer, in a mammal. The cancer can be any of the cancers described herein.

[0306] Any method of administration, including local and systemic administration, may be used with the disclosed therapeutic agents. For example, intravascular (such as topical, oral, intravenous), intramuscular, intraperitoneal, intranasal, intradermal, subarachnoid, and subcutaneous administrations may be used. Specific modes of administration and dosing regimens may be selected by the attending clinician taking into account the details of the case (such as the subject, disease, associated disease state, and whether the treatment is prophylactic). When more than one agent or composition is administered, more than one route of administration may be used. For example, a chemotherapeutic agent may be administered orally, and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. The method of administration includes injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic and pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, non-volatile oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue area after tumor removal or an area suspected of having a tendency to develop tumors. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea or intravitreally as an eye drop.

[0307] ​The disclosed therapeutic agent may be formulated in unit dosage forms suitable for administering the exact dosage each time. In addition, the disclosed therapeutic agent may be administered according to a single-dose or multiple-dose schedule. The multiple-dose schedule may be such that more than one administration (e.g., 1 to 10 administrations) is performed separately each time in the first series of treatments, and subsequently, the remaining administrations may be performed at time intervals as necessary to maintain or increase the effect of the composition. The treatment may involve administering the compound once or multiple times a day (multi-daily doses) over a period of 2 to 3 days to several months, or even several years. Thus, the dosing regimen may be determined based at least in part on the specific requirements of the subject to be treated and may depend on the judgment of the administering physician.

[0308] Typical dosages of the antibody or conjugate may range from about 0.01 to about 30 mg / kg, such as from about 0.1 to about 10 mg / kg.

[0309] In a specific example, the subject is administered a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or an additional agent according to a multiple daily dosing schedule, such as for at least 2 consecutive days and up to 10 consecutive days, over a period of, for example, several weeks, several months, or several years. In one example, the subject is administered a conjugate, an antibody, a composition, or an additional agent over a period of at least 30 days, such as over a period of at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0310] In some embodiments, the disclosed methods include providing to a subject surgery, radiation therapy, and / or chemotherapy, in combination with (e.g., sequentially, substantially simultaneously, or simultaneously) the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR. Methods and therapeutic dosages for such agents and treatments are well known to those of skill in the art and may be determined by a skilled clinician. Preparations and dosing schedules for additional agents may be used according to the manufacturer's instructions or based on the judgment of a skilled physician's experience. Also, preparations and dosing schedules for such chemotherapy are described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

[0311] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Examples of additional therapeutic agents that can be used in combination therapy include, but are not limited to, microtubule-binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered at therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent may be administered in combination with a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages for such agents are well known to those of skill in the art and may be determined by a skilled clinician.

[0312] Additional chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antineoplastic antibiotics such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other agents including, but not limited to, alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib, and tretinoin. The selection of such agents and the therapeutic dosage are well known to those of ordinary skill in the art and may be determined by a skilled clinician.;

[0313] Combination therapies can be synergistic and can be shown to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the sum of the effects obtained when the same compounds are used separately. Synergistic effects can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit dosage preparation, (2) delivered separately, alternately or in parallel as separate preparations, or (3) when some other regimen is used. When delivered alternately, for example, by separate injection with separate syringes, a synergistic effect can occur when the compounds are administered or delivered sequentially. Generally, in the case of alternation, the effective dosage of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the effective dosages of two or more active ingredients are administered together.

[0314] In one embodiment, an antibody that specifically binds to one or more of the antigens disclosed herein An effective amount of an antibody or antigen-binding fragment thereof, or a conjugate thereof, is administered to a subject having a tumor after anti-cancer treatment. After sufficient time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on the respective cancer cells, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, if the immune complex has increased compared to a control obtained prior to the treatment, it is shown that the treatment is ineffective, and if the immune complex has decreased compared to a control obtained prior to the treatment, it is shown that the treatment is effective.

[0315] F. Biopharmaceutical Compositions A biopharmaceutical composition or biological composition (hereinafter, "composition") containing one or more of the disclosed CARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR that specifically bind to one or more antigens disclosed herein in a carrier (such as a pharmaceutically acceptable carrier) is provided herein for use in gene therapy, immunotherapy, and / or cell therapy. The composition may be prepared in unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome. The composition may be formulated for systemic (such as intravenous) or local (such as intratumoral) administration. In one example, the disclosed CAR, or T cell expressing a CAR, antibody, antigen-binding fragment, conjugate is formulated for parenteral administration such as intravenous administration. Compositions containing the CARs, or T cells expressing a CAR, conjugates, antibodies, or antigen-binding fragments disclosed herein are useful, for example, in the treatment and detection of tumors (such as, but not limited to, neuroblastoma). In some examples, the composition is useful in the treatment or detection of cancer. Compositions containing the CARs, or T cells expressing a CAR, conjugates, antibodies, or antigen-binding fragments disclosed herein are also useful, for example, in the detection of pathological angiogenesis.

[0316] This composition for administration may comprise a solution in which a CAR, or a T cell expressing a CAR, conjugate, antibody, or antigen-binding fragment is dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. Such solutions are sterile and generally free of undesirable substances. This composition may be sterilized by conventional well-known sterilization techniques. This composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as needed to approximate physiological conditions. The concentration of the CAR, or T cell expressing a CAR, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected according to the particular mode of administration and the requirements of the subject selected, mainly based on factors such as the volume of the fluid, viscosity, and body weight. The actual method of preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy is well-known or will be apparent to those skilled in the art.

[0317] A typical composition for intravenous administration comprises from about 0.01 to about 30 mg / kg per day per subject of an antibody or antigen-binding fragment or conjugate (or the corresponding dosage of a CAR, or a T cell expressing a CAR, an antibody or antigen-binding fragment-containing conjugate). The actual method of preparing the composition for administration may be well-known or apparent to those skilled in the art and is described in more detail in publications such as Remington’s Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0318] The CAR, or T cell expressing a CAR, antibody, antigen-binding fragment, or conjugate may be provided in lyophilized form and reconstituted with sterile water for administration, but in a sterile solution of a well-known concentration It is also provided in a dissolved form. The solution of the CAR, or T cells expressing the CAR, antibody, or antigen-binding fragment, or conjugate is then filled into an infusion bag containing 0.9% sodium chloride (USP) and is optionally administered at a dosage of 0.5 to 15 mg / kg body weight. In the administration of the antibody or antigen-binding fragment and conjugate drugs, a significant amount of experience can be found in the art. For example, antibody drugs have been on the US market since the approval of Rituxan® in 1997. The CAR, or T cells expressing the CAR, antibody, its antigen-binding fragment, and conjugate may be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher loading dose is administered, followed by a maintenance dose administered at a lower level. For example, an antibody or antigen-binding fragment has an initial loading dose of 4 mg / kg (or the corresponding dose of the conjugate containing the antibody or antigen-binding fragment) infused over about 90 minutes, and if this initial dose is well tolerated, subsequently, a maintenance dose of 2 mg / kg once a week may be infused for 30 minutes each time over 4 to 8 weeks.

[0319] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particulate systems. For an extensive overview of protein delivery systems, see Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain therapeutic proteins, such as cytotoxins or drugs, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since capillaries are about 5 μm in diameter, only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, 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).

[0320] The polymer may be used for ion-controlled release of the CAR, or T cells expressing the CAR, antibody, or antigen-binding fragment, or conjugate compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are well known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a mobile liquid with viscosity at low temperatures but forms a semi-solid gel at body temperature. This has been shown to be an effective vehicle for the preparation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and, Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for the controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). In addition to these, a very large number of systems for the controlled delivery of therapeutic proteins are well known (U.S. Patent No. 5,055,303, U.S. Patent No. 5,188,837, U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028, U.S. Patent No. 4,957,735, U.S. Patent No. 5,019,369, U.S. Patent No. 5,055,303, U.S. Patent No. 5,514,670, U.S. Patent No. 5,413,797, U.S. Patent No. 5,268,164, U.S. Patent No. 5,004,697, U.S. Patent No. 4,902,505, U.S. Patent No. 5,506,206, U.S. Patent No. 5,271,961, U.S. Patent No. 5,254,342, and U.S. Patent No. 5,534,496).

[0321] G. Kit In one aspect, a kit using the CAR disclosed herein is further provided. For example, a kit for treating a tumor in a subject or for generating CAR T cells that express one or more of the CARs disclosed herein. Such kits may typically include an antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR disclosed herein. More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR may be included in the kit.

[0322] The kit may include a container and a label or package insert attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials such as glass or plastic. The container typically contains a composition comprising one or more of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is to be used for the treatment of a particular condition.

[0323] The label or accompanying document may typically further include, for example, a description of the use of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR in a method for treating or preventing a tumor or a method for producing CAR T cells. The accompanying document typically includes the instructions customarily included in the commercial package of the therapeutic product, which includes information on indications, usage, dosage, administration, contraindications, and / or warnings related to the use of the therapeutic product. The content of the instructions may be described in electronic form (such as a floppy disk or a compact disk) or visual form (such as a video file). The kit may further include additional components to facilitate a particular use for which the kit is designed. Thus, for example, the kit may further include means for detecting a label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, or a suitable secondary label such as a secondary antibody). The kit may further include buffers and other reagents customarily used in the implementation of a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0324] Examples The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the present invention. On the contrary, reliance must be placed on various other embodiments, modifications, and equivalents, and it is clearly understood that such other embodiments, modifications, and equivalents may occur to those skilled in the art after reading the description herein without departing from the spirit and / or scope of the appended claims.

Examples

[0325] Isolation of CD22-Specific Antibodies from Phage Display and Yeast Displayed Full-Human ScFv Libraries Materials and Methods: a) Preparation of Human ScFv and CD22-Specific Antibodies A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library (with an approximate diversity of 10 unique specificities of 10 10 )(Z.Y. Zhu and D.S. Dimitrov, data not published) constructed from peripheral blood B cells of 50 healthy donors was used to select ScFvs for recombinant human CD19 protein (Miltenyi Biotec, not published). 10 phage-displayed 12The amplification library of individual ScFvs was incubated with CD22 coated at 5 μg, 3 μg, and 1 μg, in a volume of 5 × 100 μl (equally distributed among five wells of a 96-well plate), for 2 hours each time, at room temperature, during the first, second, and third rounds of biopanning. After each incubation, the wells were washed 5 times after the first round and 10 times after subsequent rounds with phosphate-buffered saline containing 0.05% Tween 20 (PBST) to remove non-specifically bound phages. The bound phages were mixed with TG1 competent cells at 37°C for 1 hour to amplify the phages from the infected cells for use in the next round of biopanning. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells, and each one was inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the optical density (OD600) at 600 nm of this bacterial culture reached 0.5, helper phage M13K07 with a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) of kanamycin were added to the medium, and the plates were incubated at 30°C overnight in a shaker at 250 rpm. The phage supernatant was mixed with 3% skim milk (in PBS) at a volume ratio of 4:1 and used in an enzyme-linked immunosorbent assay (ELISA) to identify clones of phages presenting ScFvs or VHs with high CD22 binding affinity. The supernatant was incubated with recombinant human CD22 coated at 50 ng per well of a 96-well plate for 2 hours at room temperature and washed 5 times with PBST (blocked with 3% skim milk (in PBS) after incubation overnight at 4°C and washed 3 times with PBS containing 0.05% Tween 20). CD22-binding phages were detected using horseradish peroxidase-labeled goat anti-M13 antibody.After incubation with this antibody, the wells were washed to remove non-specifically bound antibody, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added, and the absorbance of the solution was measured at 450 nm (A450). CD22-binding clones with A450 exceeding 1.0 were selected for further characterization.

[0326] b) Expression and purification of the selected soluble ScFv The DNA sequences of VH and VL of the selected clones were determined, and ScFv with unique sequences encoded by the clones was expressed and purified as described below. HB2151 cells were transformed using the plasmid extracted from the clone. A single colony was selected from the plate containing the newly transformed cells and inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37 °C with shaking at 250 rpm. When the OD at 600 nm of this culture reached 0.90, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was incubated at 30 °C overnight. The bacterial pellet was collected by centrifugation at 8,000×g for 20 minutes and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation at room temperature for 30 minutes with rotation at 50 rpm, the resuspended pellet was centrifuged at 25,000×g at 4 °C for 25 minutes, and the supernatant was used to purify ScFv using Ni-NTA resin according to the vendor's protocol (Qiagen).

[0327] c) ELISA binding assay For ELISA analysis, 50 μl of recombinant human CD22 diluted to 2 μg / ml in PBS was used to coat a 96-well plate overnight at 4°C. Purified ScFv with His-tag and Flag-tag was serially diluted and added into the wells coated with the target protein. After washing, HRP-labeled anti-Flag antibody diluted at 1:3000 was added at room temperature for 1 hour. After washing, 3,3,5,5’-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes, then 1N H2SO4 was added to stop the reaction, and the O.D. at 450 nm was read to quantify the relative CD22-binding ability of ScFv.

[0328] d) Yeast display of scFv library The same ScFv starting material used for phage display was also incorporated into the yeast ScFv display system. To complement phage-based scFv analysis, screening was also performed on a yeast library expressing a human scFv library. To enrich yeast expressing scFv that binds to both recombinant CD22-Fc and CD19 expressed on the cell surface of CHO-K1 cells, cell panning of CHO-K1 cells transfected with CD22 was performed. For the first panning against the cell surface, 2 days before panning, CHO-K1-CD22 cells were seeded in a 6-well plate and grown in F12 K medium to 50% confluence. Then, 5×10 yeast cells 7The cells were washed twice with PBSA buffer, resuspended in 3 mL of F12 K medium, and then gently dropped into CHOK1-CD22 cells. After gently rocking on ice for 2 hours, the CHOK1-CD22 cells were washed three times with ice-cold PBSA to remove yeast cells not bound to CHOK1-CD22, and then 0.05% trypsin-EDTA (Gibco) was used to dissociate the CHOK1-CD22 cells and the bound yeast cells from the plate. Subsequently, the cell mixture containing both yeast and CHOK1 cells was inoculated into 10 mL of SDCAA medium and amplified overnight at 30 °C, followed by induction in SGCAA medium at 30 °C for 16 hours. For the second cell panning, a protocol similar to that described above was performed using more stringent washing conditions. This panning method generated 16P, 24P, 25P, 11S, and 12S binders. The binder sequences were incorporated into the CAR T constructs described in Example 2 below in a series of in vitro CAR T functional assays. Characterization of such binders from phage display in the CAR T format revealed that only the 16P binder had specific tumor lysis activity in vitro, and this activity was lower than that of the CAR positive control. Furthermore, when CAR T cells based on 16P were tested in an in vivo xenograft model, their anti-tumor function was very weak (Example 2 below). Collectively, these results indicated that the biological characteristics of CARs made from such a binder set were not yet optimal, demonstrating the need for affinity maturation of the anti-CD22 ScFv binder.

[0329] To increase the affinity for 16P, a library of mutant scFvs displayed on yeast was generated using error-prone PCR to introduce random point mutations in the scFv gene sequence. After electroporation, the resulting mutant library was then grown overnight at 30 °C for 16 h in SDCAA medium and then switched to SGCAA medium and grown for an additional 16 h at 30 °C. The mutant library was then sorted by MACS (immunomagentic column, Miltenyi Biotec) using CD22-Fc as the capture antigen to reduce the library size and enrich the population of mutants that could bind CD22-Fc. The pool was then double stained with anti-c-Myc-Alexa 488 and CD19-Fc / anti-Hu-Fc, and the strongest binders were selected by choosing the binders with the highest binding affinity and c-Myc expression levels. This process was repeated two more times, and the average binding affinity of the mutant pool became higher than that of the starting construct in flow cytometry of yeast particles with fluorescently tagged antigen. Flow cytometry of the yeast pool was performed while reducing the amount of labeled CD22 to evaluate the binding affinities (Binidng affinities). As a result of this process, the EC50 (effective concentration at which 50% of the labeled CD19 binds to yeast presenting the ScFv) for 16P increased from 0.5 μg / ml to less than 0.01 μg / ml affinity for the affinity matured binders (16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P16, 16P17, 16P20, 16P20v2).

[0330] Results: Due to the inherent challenges of the CD22 structure, no fully functional CAR constructs with high biological activity and specificity could be obtained from phage display candidates. Therefore, ScFvs for binders with bioactivity and high specificity were generated by yeast display. Based on flow cytometry analysis of ScFvs presented on yeast, 13 ScFv clones specific for recombinant human CD22 were identified and classified as human anti-CD22 ScFv binder 16P (LTG2202, founder clone, EC50 0.5 μg / ml), and affinity matured binders (EC50 < 0.01 μg / ml) 16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P17, 16P20, 16P20v2, respectively. The generation of CARs expressing LTG2203, LTG2205, LTG2206, LTG2207, LTG2208, LTG2209, LTG2210, LTG2216, LTG2217, LTG2218, LTG2219, and LTG2220 human anti-CD22 binders is outlined in Example 2 below.

Example

[0331] CAR expressing an anti-CD22 fully human binding sequence. Homo sapiens CD22 (SIGLEC-2, Leu14) is a cell surface glycoprotein expressed in B cell leukemia and lymphoma and has been well studied. Clinical trials have been conducted for at least two anti-CD22 antibody drugs (inotuzumab ozogamicin) or immunotoxin conjugates (moxetumomab pasudotox) (NCT02981628, NCT00659425). This approach has been somewhat successful and research continues, for example, as a combination with other chemotherapeutic agents (Muller F, Stookey S, Cunningham T, Pastan I, 2017, Paclitaxel synergizes with exposure tume adjusted CD22-targeted immunotoxins against B-cell malignancies, Oncotarget 8:30644-30655). However, considering the current progress of T cell-based therapies using CD19 CAR, the best approach for targeting CD22-expressing malignancies is thought to be cell-based immunotherapy. A therapy featuring an anti-CD22 CAR based on m971 is currently in clinical trials at the National Cancer Institute in the United States (NCT02315612, P.I.: Terry Fry, M.D.), but the results have not yet been published. The CAR constructs presented here are an innovative and novel approach for creating and implementing a new CD22 binding site derived from human sequences, and considering the range of cytotoxicity and cytokine production capabilities of the individual constructs, there may be significantly different activity profiles in vivo.

[0332] The novel anti-CD22 CAR-T constructs described herein have high levels of cell surface expression in primary human T cells, specificity for CD22-positive tumor cells, and potential cytotoxicity and cytokine function. As in Example 1, a CD22 CAR was designed using a CD22 binding sequence derived from a ScFv candidate first identified by phage display, and for characterization, it was cloned under the control of the EF1a promoter into a lentiviral expression vector containing the selected structural and signaling domains and tested in vitro for transduction efficiency, killing function, and cytokine production in both model cell lines and primary human T cells. Table 1 shows an overview of the academic terms used. The CAR construct LTG1538, anti-CD19 CAR is used as a positive control and for comparison. The m971 CAR LTG2200 is used as an anti-CD22 CAR positive control.

[0333]

Table 1

[0334] material and method: (a) Cell line The Burkitt's lymphoma cell line Raji and chronic myeloid leukemia line K562 were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). The REH and NALM-6 leukemia lines were purchased from DSMZ (Leibniz Institute DSMZ, Braunschwieg, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC (Gibco / Thermo Single cell clones of the luciferase-expressing cell lines were purchased from Sigma-Aldrich (Fishery Scientific, Grand Island, NY). Luciferase-positive T cells were generated by stable transduction of wild-type tumor lines with lentiviral vectors (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones. Whole blood or buffy coats were collected from healthy volunteers at the Oklahoma Blood Institute (OBI, Oklahoma City, OK) with the donors' written consent. CD4+ and CD8+ human T cells were purified from the buffy coats by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as per the manufacturer's protocol.

[0335] (b) Construction of chimeric antigen receptor (CAR) expression vector The sequence of the CAR antigen-binding domain ScFv was derived from a human anti-CD22 ScFv or heavy chain variable fragment. The binder sequence in the framework was ligated to the CD8a binding and transmembrane domains (aa 123 - 191, reference sequence number NP_001759.3), and then ligated to the 4-1BB (CD137, aa 214 - 255, UniProt sequence number Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa 52 - 163, reference sequence number: NP_000725.1) to generate a CAR T construct. The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). By transient transfection of HEK293T cells, a supernatant containing the lentiviral vector (LV) was obtained. The LV was pelleted by centrifuging the LV-containing supernatant and stored at -80°C.

[0336] (c) Purification and transduction of primary T cells CD4 + and CD8 + cells were purified from whole blood or buffy coat of healthy volunteers using immunomagnetic bead selection (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's protocol. T cells were cultured in TexMACS medium supplemented with 200 IU / ml of IL-2 at a density of 0.3 - 2×10 6 cells / ml, activated using CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), and on day 2, transduced overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) using the lentiviral vector encoding the CAR construct. The medium was changed on day 3. The cultures were expanded in TexMACS medium supplemented with 200 IU / ml of IL-2 and harvested on days 8 - 13.

[0337] (d) Immune effector assays (CTL and cytokines) To examine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various effector: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). The assay range was determined using target-only wells (maximum CPS) and target-only wells with 1% Tween-20 added (minimum CPS). The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants were removed from co-cultures at an E:T ratio of 10:1 and the concentrations of IFNγ, TNFα, and IL-2 were analyzed by ELISA (eBioscience, San Diego, CA).

[0338] (e) Flow cytometry analysis For cell staining, 500,000 cells transduced with CAR T were recovered from the culture, washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and stained with CD22-Fc peptide followed by anti-Fc-PE conjugate to detect CAR surface expression (Jackson ImmunoResearch, West Grove, PA). An anti-CD4 antibody labeled with VioBlue fluorophore (Miltenyi Biotec) was used at the designated location according to the manufacturer's protocol. Un-transduced cells were used as negative controls. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). The cells were washed twice and resuspended in 200 μl of staining buffer, and then quantitatively analyzed by flow cytometry. Flow cytometry analysis was performed using a MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR).

[0339] (f) In vivo analysis of CAR function All animal experiments were approved by the MI Bioresearch Animal Care and Use Committee (Ann Arbor, MI). 500,000 mouse-adapted Raji-luc cells were injected into the tail vein of NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) mice. On day 6 after Raji-luc injection, 150 mg / kg of luciferin was injected intraperitoneally, and tumor engraftment was measured by performing imaging using a Xenogen IVIS-200 instrument (Caliper Biosciences, now Perkin Elmer, Shelton, Connecticut). Images were analyzed using Living Image (version 4.1) software (Perkin Elmer), and the bioluminescence signal flux of each mouse was measured as the average luminance (photons / sec / cm 2 / Stelladian) and expressed. On the 7th day, CAR T cells were administered to mice by tail vein injection. Image diagnosis was performed on the designated days after CAR T injection to establish the reaction rate of tumor growth and eradication by CAR T cells.

[0340] Results: To evaluate the novel anti-CD22 fully human ScFv binding sequences, CAR constructs of Set 1 were designed by incorporating constructs 2246 - 2249 (ScFv sequences from phage display libraries, Table 1, ScFv1(16P), ScFv2(24P), ScFv3(25P), ScFv4(11S), ScFv5(12S), and CAR construct 2202 (m971 positive control) as the tumor antigen binding domains). For the design of each CAR, a linker and transmembrane domain derived from human CD8 protein, 4-1BB co-stimulatory domain, and CD3 zeta signaling domain were placed behind the tumor target domain (Table 2 below).

[0341]

Table 2

[0342] T cells transduced with anti-CD22 chimeric antigen receptors show surface expression and cytolytic activity.

[0343] a) Surface expression of anti-CD22 CAR To evaluate the novel anti-CD22 CAR, a lentiviral vector (LV) encoding the CAR construct was generated under the control of the human EF1a promoter as described in Materials and Methods. Human primary T cells derived from healthy donors were transduced with the lentiviral vector encoding the CAR. Non-transduced cells (referred to as UTD or mock) from the same donor or GFP-transduced cells from the same donor were used as negative controls. The data represent results obtained from at least 3 assays from different donors.

[0344]

Table 3

[0345] As described in the Materials and Methods, on day 0 of culture, T cells were activated using the TransAct T cell reagent (Miltenyi Biotec, Inc.) in the presence of IL-2. On days 8 - 10 of culture, the expression of anti-CD22 CAR on the surface of transduced T cells was detected by using protein L conjugated to biotin and subsequently staining with streptavidin-PE reagent. Alternatively, CAR T was stained by using CD22-Fc peptide (R&D Systems, Inc.) and subsequently staining with anti-Fc-PE antibody, and data were obtained by flow cytometry (Figure 3). All CAR constructs except CAR2247, which consists of lambda light chain (and thus does not react with protein L), showed surface CAR expression exceeding 50 - 70% by detection using protein L staining. In contrast, when stained with CD22-Fc peptide that specifically binds to the anti-CD22 ScFv antigen-binding site only CAR16P showed CAR expression (up to 30%) (CAR 24P is not shown). Such data indicate that most of the CAR constructs were expressed on the T cell surface, but only the CAR16P construct presented an ScFv configuration that maintained CD22 protein binding.

[0346] b) Cytotoxicity assay and cytokine assay of anti-CD22 CAR To demonstrate the cytotoxic function of the generated CAR T cells, a luciferase-based killing assay was performed, and in the overnight cell killing assay, CAR-T was combined with CD22-positive Raji-luc cells, CD22-positive Reh-luc cells, CD22-negative K562-luc cells at an E:T ratio of 20:1, 10:1, 5:1, or 2.5:1 as described in the Materials and Methods (Figure 4). The anti-CD19 CAR construct 1538 was used for Raji and Reh (CD19 +) reacts with but not with K562 (CD19 - ) strain has been previously shown not to react, but this was used as a positive control. Only CAR2202 (Binder 16P) showed dose-dependent and CD22-specific tumor killing, while CAR 2247 (25P), 2248 (11S), and 2249 (12S) had no tumor-specific activity.

[0347] After determining that the novel human construct CAR2202 (16P) was functional in vitro, its antitumor activity was tested in vivo in an established NSG mouse xenograft model of Raji Burkitt lymphoma as described in the Materials and Methods. Tumors were transplanted via the tail vein on day 0, progression was diagnosed on day 6, and on day 7, the mice were treated by intravenous injection with 4×10 6 CAR T cells. The treatment groups were CAR16P (2202), CAR19 (1538) positive control, CAR22 (2200 m971) positive control, and UTD (untransduced T cells) negative control. As shown in Figure 5, the positive control CAR T preparations CAR22 (2200 m971) and CAR19 (1538) effectively inhibited tumor growth after day 18 of the study, but the test construct CAR 2202 (16P) only mildly decelerated tumor progression and, after day 32 of the study, its effect became indistinguishable from that of the negative control treatment (UTD). Therefore, the in vitro and in vivo antitumor activities of the ScFv binder 2202 (16P) were weak, and it was necessary to generate additional CAR constructs incorporating improved ScFv binder sequences.

[0348] As described in Materials and Methods, a set 2 of CAR constructs (LTG numbers 2203 - 2220) with improved affinity for CD22 by incorporating ScFv binder sequences (Table 1 below, set 2) was constructed. Induction of ScFv binders with improved affinity is described in Example 1. LV encoding the set 2 of CAR constructs was generated under the control of the human EF1a promoter and tested for expression and function in vitro as described above. Briefly, as described in Materials and Methods, on day 0 of culture, T cells were activated using TransAct T cell reagent (active engagement of CD3 and CD28 antigens, Miltenyi Biotec, Inc.) in the presence of IL-2. On days 8 - 10 of culture, CAR T cells were harvested and CAR surface expression was evaluated by flow cytometry. CTL activity was evaluated by co-incubation assay and secretion of inflammatory cytokines was evaluated by ELISA. An overview of the results comparing the functions of all CAR22 constructs is shown in Table 3. The positive control CAR construct and the novel CAR22 candidate with the most favorable functional profile are shown in bold.

[0349] Tests on the set 2 of CAR constructs (LTG numbers 2203 - 2220) were performed in at least three separate experiments by transducing the CAR sequences encoded by the LV into cells from independent donors. Transduction of CAR T constructs from set 2 into donor cells typically resulted in CAR expression in the range of 20% - 80%, which was detected by CD22-Fc staining and consisted mostly of CD4+ T cells (Figure 6, selected constructs).

[0350] The CTL activity of the CAR was determined by co-incubating CAR T cells with luciferase-expressing tumor cells at an E:T ratio ranging from 10:1 to 2.5:1 and assaying overnight (Figure 7). Residual luciferase activity derived from the surviving portion of the tumor cell population was determined at the end of the culture period, and % lysis was calculated as described in Materials and Methods. The novel CD22-targeting CAR cells showed strong killing activity in the CD22+ Raji lymphoma and Reh leukemia cell lines, while the negative control groups GFP and UTD did not cause lysis (Figure 7A). An exception was construct 2202(16P), which killed the tumor lines to a relatively moderate extent. Control 1538 targeting CAR19 and control 2200(m971) targeting CD22 killed Raji and Reh tumors as expected. The K562 cell lines (CD22-K562 and CD19-K562) showed background killing activity for the positive control constructs 2202 and 1538, which was 40% and 60% tumor cell lysis, respectively, at an E:T ratio of 10:1. This activity is thought to be due to the indirect action of inflammatory cytokines secreted by the CAR T cells or the activity of contaminating NK / NKT. The killing activity of the test CAR22 construct against K56 was equivalent to or slightly higher than that of control 2200(m971). To show the specificity of the novel CAR22 construct in more detail, the applicants used K562 cells engineered to stably express CD22 or CD19 (Figure 7B).

[0351] In the K562-CD19 cell line, the positive control CAR19 (1538) showed up to 70% lysis at an E:T of 10:1, while the background killing activity of control CAR22 2200(m971) was low at up to 20% at an E:T ratio of 10:1. In contrast, the % lysis resulting from most of the test CAR22 constructs at the same E:T ratio was between 20% and 60%.

[0352] As a comparison, in the K562-CD22 strain, the specific CTL activity between the CD22 CAR control CAR22 2200 (m971) and most of the novel CAR22 construct was 80%, and the non-specific killing activity of the CAR19 control 1538 was as low as 20%. Therefore, despite the susceptibility of the K562 strain to the CAR construct, all the novel CD22 CARs tested showed lysis activity specific to the CD22-expressing target.

[0353] Next, when induced by the CD22-positive cell lines Raji and REH (Figure 8), the concentrations of the inflammatory cytokines IFN-gamma, TNF-alpha, and IL-2 secreted by CAR T cells transduced with the CAR22 construct were measured. To examine the basal levels of cytokine production, controls of CAR T cells alone were included for each construct. CD22 + T cells exposed to Raji cells strongly induced the levels of TNF-alpha, IFN-gamma, and IL-2. However, for most of the CAR22 constructs, the Reh tumor only affected the induction of IFNg and TNFa, but not the induction of IL-2. For a subset of constructs, the T cell-alone control group also showed cytokine induction, suggesting that the constructs were likely to be activated in the absence of specific ligands. From this, the design of the CAR and the selection of the binder are not trivial, as they are active in the soluble IgG or ScFv format and expressed on the T cell surface in the CAR T format Some binders applicable to are, nevertheless, of low killing efficiency or cytokine production efficiency when incubated with CD22-positive tumors. For example, in the case of LTG2217 (16P3v2 binder) and LTG2220 (16P15), IL-2 and TNF-alpha were continuously produced by this construct showing self-activation of T cells in the absence of tumor targets, which may have an adverse effect on clinical use. Therefore, LTG2217 and LTG2220 are ineligible as therapeutic candidates.

[0354] To avoid non-specific activation of CAR22 cells, the present applicants identified constructs that do not secrete or secrete minimally inflammatory cytokines in the absence of specific tumors (LTG2209 (16P17), LTG2218 (16P8), and LTG2219 (16P13)).

[0355] Next, the novel CAR22 constructs 2219 and 2209 were tested in the NSG Raji xenograft tumor model. Constructs 2200 (m971) and 1538 (FMC63) were used as positive controls, and tumor alone (TA) and non-transduced T cells (UTD) were used as negative controls. The experimental procedure was carried out as detailed in the Materials and Methods. On day 0, Raji-luciferase-expressing tumor cells were transplanted into mice, followed by CAR T treatment on day 7. Bioluminescence measurements were started on day 6 of the study once a week to examine tumor progression and CAR T activity (Figure 9). Compared with the negative control groups TA and UTD in which tumor growth progressed without decline after day 6, the CAR test constructs 2209 and 2209 inhibited tumor progression and reduced tumor bioluminescence to the baseline at the start of treatment (day 6) by day 21. The antitumor effects of the test constructs 2209 and 2209 were equal to or greater than those of the positive control CD22 CAR construct 2200 (m971).

[0356] In summary, it was shown that the novel fully human anti-CD22 CAR constructs LTG numbers 2203 to 2220 (Table 2 below) with improved affinity derived from a yeast screening library have high functionality. In particular, CAR constructs 2209, 2219, and 2218 are expected to have high therapeutic activity because they showed an excellent or different activity profile from the positive control LTG2220 (m971).

[0357] Each application and patent cited in this text, as well as each document or prior art document cited in each application and patent (including each issued patent "application cited document" in litigation), and each PCT application or patent and foreign application or patent corresponding to and / or claiming priority from any of the said applications and patents, and each document cited or referenced in each application cited document are hereby expressly incorporated by reference into this specification, and these may be used in the practice of the present invention. More generally, a document or prior art document is cited in any of the text, the list of prior art documents before the claims, or the text itself, and each of the said document or prior art document (each "prior art document cited in this specification"), and each document or prior art document cited in each prior art document cited in this specification (including manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference into this specification.

[0358] The above-described description of some specific embodiments provides sufficient information for others to easily modify or adapt the specific embodiments for various uses without departing from the general concept by applying the latest knowledge. Therefore, such adaptations and modifications should be understood to be within the meaning and scope of the equivalents of the disclosed embodiments. It is understood that the terms or technical terms used in this specification are for illustrative purposes and not for limiting purposes. In the drawings and the description, exemplary embodiments are disclosed Although certain terms may be used, unless otherwise specified, these are used only in a general and illustrative sense and not for purposes of limitation, and thus do not limit the scope of the claims. Also, those skilled in the art will understand that the specific steps of the methods disclosed herein may be performed in a different order or combined. Accordingly, it is intended that the claims appended to the specific embodiments disclosed herein not be limited. Those skilled in the art may recognize many equivalents to the embodiments of the invention described herein or may be able to grasp them using only routine experimental methods. Such equivalents are encompassed by the following claims.

[0359] The sequences according to the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and the three-letter codes for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strand is included when referring to the strand shown. In the accompanying Sequence Listing: SEQ ID NO: 1 is the nucleic acid sequence of CD22-specific binder (scFv1) 16P: CAAGTACAACTCCAGCAAAGCGGGCCTGGTCTGGTGAAGCCGTCACAGACGCTTTCACTTACGTGTGCGATCTCCGGTGACTCCGTGAGTTCTAATAGCGCGGCTTGGAACTGGATTAGGCAGTCTCCATCCCGAGGATTGGAATGGCTCGGCAGGACTTATTATAGAAGTAAGTGGTACAACGATTATGCAGTCTCTGTGAAATCTCGCATCACCATTAACCCAGACACGTCTAAGAATCAGTTCAGTCTTCAACTCAACTCTGTAACCCCCGAAGATACAGCGGTCTACTACTGTGCTCAGGAGGTGCAACCCCACGATGCTTTTGATATCTGGGGCCAGGGTACCATGGTTACGGTGTCTTCTGGGGGAGGGGGGTCCGGTGGGGGAGGATCAGGGGGTGGGGGCAGCGACATACAAATGACGCAATCCCCGTCTTCTGTTTCTGCGTCTGTCGGAGATAAAGTAACAATAACCTGTCGAGCGTCACAGGACGTTAGTGGCTGGCTTGCGTGGTATCAGCAAAAACCGGGGCTCGCCCCGCAATTGCTTATATTTGGAGCGAGTACTCTTCAGGGCGAGGTACCTAGCAGATTTTCTGGGTCCGGCTCAGGTACGGACTTCACCCTGACCATATCTAGCTTGCAGCCTGAAGATTTCGCCACCTACTATTGTCAACAGGCGAAGAACTTTCCATATACGTTCGGGCAGGGTACGAAATTGGAGATAAAA SEQ ID NO: 2 is the amino acid sequence of CD22-specific binder (scFv1) 16P: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKR Sequence number 3 is the nucleic acid sequence of CD22 CAR LTG2202 (LP-scFv1-CD8TM-41BB-CD3 zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTACAACTCCAGCAAAGCGGGCCTGGTCTGGTGAAGCCGTCACAGACGCTTTCACTTACGTGTGCGATCTCCGGTGACTCCGTGAGTTCTAATAGCGCGGCTTGGAACTGGATTAGGCAGTCTCCATCCCGAGGATTGGAATGGCTCGGCAGGACTTATTATAGAAGTAA Accession number 4 is the amino acid sequence of CD22 CAR LTG2202 (LP-scFv1-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 5 is the amino acid sequence of scFv1(16P)LCDR1: QDVSGW Accession number 6 is the amino acid sequence of scFv1(16P)LCDR2: GAS Accession number 7 is the amino acid sequence of scFv1(16P)LCDR3: QQAKNFPYT Accession number 8 is the amino acid sequence of scFv1(16P)HCDR1: GDSVSSNSAA Accession number 9 is the amino acid sequence of scFv1(16P)HCDR2: TYYRSKWYN Accession number 10 is the amino acid sequence of scFv1(16P)HCDR3: AQEVQPHDAFDI SEQ ID NO: 11 is the nucleic acid sequence of CD22-specific binder (scFv2)24P: CAAGTACAGCTGCAACAATCTGGCCCTGGGCTTGTGAAACCCTCTCAGACTTTGTCCTTGACGTGCGCGATAAGTGGCGATTCAGTTAGTTCTAACAGCGCCGCTTGGAACTGGATTAGACAGAGCCCCAGTCGGGGACTCGAATGGCTTGGCCGGACTTATTATCGCAGTAAATGGTATAATGATTATGCTGTGAGTGTGAAAAGTAGGATCACAATCAACCCCGATACGAGCAAGAATCAATTCTCATTGCAACTGAACAGCGTCACTCCCGAGGATACAGCTGTATATTATTGTGCAAGAGAAGGTGGGTGGTATGGCGAGATGGATGTATGGGGGAAAGGAACTACGGTAACTGTGTCCAGTGGCGGAGGCGGTTCAGGTGGTGGAGGCTCTGGAGGAGGAGGGTCCGAAATCGTGCTTACCCAGTCTCCGGCTACTCTGAGCGTTAGTCCGGGTGAAAGGGCCTCACTCTCTTGTCGAGCTTCACAGTCAGTCTCTTCCTACTTGGCTTGGTATCAGCAGAAGCCAGGTCAGGCGCCCCGCTTGCTCATTTACGACGCAAGCACACGAGCGACAGGCATTCCAGACAGATTTTCTGGTTCTGGTTCTGGCACGGACTTTACTCTTACTATAAACTCACTTGAGGCAGAGGATGCTGCGACTTACTATTGTCACCAATCAAGCTCTCTGCCTTACACCTTTGGGCAAGGCACCAAACTCGAAATCAAG SEQ ID NO: 12 is the amino acid sequence of CD22-specific binder (scFv2)24P: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGGWYGEMDVWGKGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERASLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLPYTFGQGTKLEIKR Sequence number 13 is the nucleic acid sequence of CD22 CAR LTG2246 (LP-scFv2-CD8TM-41BB-CD3 zeta): ATGCTGCTGTTGGTGACATCACTTCTGCTCTGTGAACTCCCCCATCCAGCCTTTCTGCTTATACCGCAAGTACAGCTGCAACAATCTGGCCCTGGGCTTGTGAAACCCTCTCAGACTTTGTCCTTGACGTGCGCGATAAGTGGCGATTCAGTTAGTTCTAACAGCGCCGCTTGGAACTGGATTAGACAGAGCCCCAGTCGGGGACTCGAATGGCTTGGCCGGACTTATTATCGCAGTAAATGGTATAATGATTATGCTGTGAGTGTGAAAAGTAGGATCACAATCAACCCCGATACGAGCAAGAATCAATTCTCATTGCAACTGAACAGCGTCACTCCCGAGGATACAGCTGTATATTATTGTGCAAGAGAAGGTGGGTGGTATGGCGAGATGGATGTATGGGGGAAAGGAACTACGGTAACTGTGTCCAGTGGCGGAG Accession number 14 is the amino acid sequence of CD22 CAR LTG2246 (LP-scFv2-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGGWYGEMDVWGKGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERASLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLPYTFGQGTKLEIKVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 15 is the amino acid sequence of scFv2(24P)LCDR1: QSVSSY Accession number 16 is the amino acid sequence of scFv2(24P)LCDR2: DAS Accession number 17 is the amino acid sequence of scFv2(24P)LCDR3: HQSSSLPYT Accession number 18 is the amino acid sequence of scFv2(24P)HCDR1: GDSVSSNSAA Accession number 19 is the amino acid sequence of scFv2(24P)HCDR2: TYYRSKWYN Accession number 20 is the amino acid sequence of scFv2(24P)HCDR3: AREGGWYGEMDV SEQ ID NO: 21 is the nucleic acid sequence of the CD22-specific binder (scFv3)25P: CAAGTACAGCTCCAACAGAGTGGACCTGGTCTCGTTAAGCCGTCCCAAACACTGTCTTTGACGTGCGCTATTAGTGGCGACAGCGTATCATCCAATTCTGCTGCTTGGAACTGGATTAGACAGTCACCGTCCAGAGGCTTGGAATGGCTGGGCAGGACGTACTACCGCTCAAAATGGTATAACGATTACGCGGTTAGTGTCAAATCCAGGATTACCATTAACCCTGACACAAGTAAGAATCAGTTTTCTCTTCAGCTGAATTCCCTGACTCCTGAGGATACGGCCGTTTACTACTGTGCCCGAGAACACCAGAATGAGGCGGCTTTTGATATTTGGGGGCAAGGAACAATGGTCACAGTTAGCAGTGGGGGGGGTGGCTCCGGGGGAGGTGGTTCCGGCGGCGGTGGTTCTCAATCCGTCCTGACACAACCTCCCTCAGCGAGCGGGACTCCCGGTCAAAGGGTGACCATCTCTTGTTCTGGGGGAGGTAGTAACATCGGGACAAATACTGCGTCCTGGTATCAGCAACTCCCTGGGACCGCTCCCAAGTTGTTGATATATCGCAATACGCAACGACCTAGTGGGATACCTGATAGATTCAGCGGAAGCAAAAGTGGTACGAGTGCGTCTTTGGCAATATCTGGCCTCCAGTCCGAGGACGAAGCGGATTACTATTGTGCGGCCTGGGATGACTCACTGAATGGTTATGTGTTCGGTGCAGGTACTCAACTCACCGTACTTGGT SEQ ID NO: 22 is the amino acid sequence of the CD22-specific binder (scFv3)25P: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSLTPEDTAVYYCAREHQNEAAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGGGSNIGTNTASWYQQLPGTAPKLLIYRNTQRPSGIPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGAGTQLTVLG Sequence number 23 is the nucleic acid sequence of CD22 CAR LTG2247 (LP-scFv3-CD8TM-41BB-CD3 zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTACAGCTCCAACAGAGTGGACCTGGTCTCGTTAAGCCGTCCCAAACACTGTCTTTGACGTGCGCTATTAGTGGCGACAGCGTATCATCCAATTCTGCTGCTTGGAACTGGATTAGACAGTCACCGTCCAGAGGCTTGGAATGGCTGGGCAGGACGTACTACCGCTCAAAATGGTATAACGATTACGCGGTTAGTGTCAAATCCAGGATTACCATTAACCCTGACACAAGTAAGAATCAGTTTTCTCTTCAGCTGAATTCCCTGACTCCTGAGGATACGGCCGTTTACTACTGTGCCCGAGAACACCAGAATGAGGCGGCTTTTGATATTTGGGGGCAAGGAACAATGGTCACAGTTAGCAGTGGGGGGGGTGGCTCCGGGGGAGGTGGTTCCGGCGGCGGTGGTTCTCAATCCGTCCTGACACAACCTCCCTCAGCGAGCGGGACTCCCGGTCAAAGGGTGACCATCTCTTGTTCTGGGGGAGGTAGTAACATCGGGACAAATACTGCGTCCTGGTATCAGCAACTCCC TGGGACCGCTCCCAAGTTGTTGATATATCGCAATACGCAACGACCTAGTGGGATACCTGATAGATTCAGCGGAAGCAAAAGTGGTACGAGTGCGTCTTTGGCAATATCTGGCCTCCAGTCCGAGGACGAAGCGGATTACTATTGTGCGGCCTGGGATGACTCACTGAATGGTTATGTGTTCGGTGCAGGTACTCAACTCACCGTACTTGGTGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 24 is the amino acid sequence of CD22 CAR LTG2247 (LP-scFv3-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSLTPEDTAVYYCAREHQNEAAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGGGSNIGTNTASWYQQLPGTAPKLLIYRNTQRPSGIPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGAGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25 is the amino acid sequence of scFv3(25P) LCDR1: GSNIGTNT SEQ ID NO: 26 is the amino acid sequence of scFv3(25P) LCDR2: RNT SEQ ID NO: 27 is the amino acid sequence of scFv3(25P) LCDR3: AAWDDSLNGYV SEQ ID NO: 28 is the amino acid sequence of scFv3(25P) HCDR1: GDSVSSNSAA SEQ ID NO: 29 is the amino acid sequence of scFv3(25P) HCDR2: TYYRSKWYN SEQ ID NO: 30 is the amino acid sequence of scFv3(25P) HCDR3: AREHQNEAAFDI SEQ ID NO: 31 is the nucleic acid sequence of the CD22-specific binder (scFv4) 11s: CAAGTCCAGTTGCAACAGTCCGGGCCAGGTCTGGTTAAGCCATCCCAAACTCTGAGTTTGACGTGCGCTATTAGCGGAGATTCCGTGTCCAGCAATTCTGCAACCTGGAATTGGATCCGGCAGAGTCCGAGTGGCGGTTTGGAATGGCTCGGACGCACTTACTACAGGAGCAAATGGTACGATGATTATGCTGTTTCTGTGCGCTCTCGAATCACCATGAATCCTGATACTTCTAAGAACCAATTTTCTTTGCAGTTGAACTCCGTCACGCCTGAAGATACTGCGGTCTACTATTGCGCACGCGAAGGCGTAGCCGGCGATTTTGATTACTGGGGGCAAGGAACATTGGTCACGGTCTCCTCTGGTGGAGGAGGATCAGGAGGCGGGGGTTCAGGTGGAGGTGGGAGCGATATTCAACTTACGCAGTCTCCGAGCAGTCTTTCTGCTTCCGTGGGAGACCGAGTGACGATTACTTGTAGGGCATCTCAGTCAATAAGTTCCTATCTTAACTGGTATCAGCAGAAGCCTGGAAAGGCTCCAAAACTTCTTATTTATGCCGCATCCTCATTGCAATCCGGCGTGCCTTCCCGATTTTCCGGATCTGGCTCAGGCACTGACTTTACCTTGACTATTAGTTCCCTTCAACCAGAAGATTTTGCTACCTATTACTGCCAACAATCATACAGTACCCCATATACATTCGGCCAAGGCACGAAATTGGAGATTAAA SEQ ID NO: 32 is the amino acid sequence of CD22-specific binder (scFv4)11s: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSGGLEWLGRTYYRSKWYDDYAVSVRSRITMNPDTSKNQFSLQLNSVTPEDTAVYYCAREGVAGDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIKR SEQ ID NO: 33 is the nucleic acid sequence of CD22 CAR LTG2248 (LP-scFv4-CD8TM-41BB-CD3 zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTCCAGTTGCAACAGTCCGGGCCAGGTCTGGTTAAGCCATCCCAAACTCTGAGTTTGACGTGCGCTATTAGCGGAGATTCCGTGTCCAGCAATTCTGCAACCTGGAATTGGATCCGGCAGAGTCCGAGTGGCGGTTTGGAATGGCTCGGACGCACTTACTACAGGAGCAAATGGTACGATGATTATGCTGTTTCTGTGCGCTCTCGAATCACCATGAATCCTGATACTTCTAAGAACCAATTTTCTTTGCAGTTGAACTCCGTCACGCCTGAAGATACTGCGGTCTACTATTGCGCACGCGAAGGCGTAGCCGGCGATTTTGATTACTGGGGGCAAGGAACATTGGTCACGGTCTCCTCTGGTGGAGGAGGATCAGGAGGCGGGGGTTCAGGTGGAGGTGGGAGCGATATTCAACTTACGCAGTCTCCGAGCAGTCTTTCTGCTTCCGTGGGAGACCGAGTGACGATTACTTGTAGGGCATCTCAGTCAATAAGTTCCTATCTTAACTGGTATCAGCAGAAGCCTGGAAAGGCTCCAAAACTTCTTATTTATGCCGCATCCTCATTGCAATCCGGCGTGCCTTCCCGATTTTCCGGATCTGGCTCAGGCACTGACTTTACCTTGACTATTAGTTCCCTTCAACCAGAAGATTTTGCTACCTATTACTGCCAACAATCATACAGTACCCCA TATACATTCGGCCAAGGCACGAAATTGGAGATTAAAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 34 is the amino acid sequence of CD22 CAR LTG2248 (LP-scFv4-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSGGLEWLGRTYYRSKWYDDYAVSVRSRITMNPDTSKNQFSLQLNSVTPEDTAVYYCAREGVAGDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 35 is the amino acid sequence of scFv4(11s) LCDR1: QSISSY SEQ ID NO: 36 is the amino acid sequence of scFv4(11s) LCDR2: AAS SEQ ID NO: 37 is the amino acid sequence of scFv4(11s) LCDR3: QQSYSTPYT SEQ ID NO: 38 is the amino acid sequence of scFv4(11s) HCDR1: GDSVSSNSAT SEQ ID NO: 39 is the amino acid sequence of scFv4(11s) HCDR2: TYYRSKWYD SEQ ID NO: 40 is the amino acid sequence of scFv4(11s) HCDR3: AREGVAGDFDY SEQ ID NO: 41 is the nucleic acid sequence of CD22-specific binder (scFv5) 12s: CAAGTTCAGTTGCAGCAGAGTGGCCCTGGGCTTGTTAAACCATCACAGACGCTCTCACTGACCTGTGCCATCTCTGGAGACAGTGTAAGTTCTAACTCAGCCGCGTGGAATTGGATTAGACAATCACCAAGCCGGGGACTTGAATGGCTTGGTCGGACG TACTATAGATCTAAGTGGTATAATGACTACGCAGTGTCAGTGAAATCACGGATAACCATAAACCCTGACACCAGCAAAAACCAATTTTCTCTTCAGCTTAATTCCGTCACGCCAGAAGATACGGCCGTTTACTACTGTGCGAGGGAAGGTGATGACGCATTGGACATCTGGGGTCAGGGGACCATGGTGACTGTCTCTTCCGGCGGGGGGGGTAGTGGAGGGGGTGGCTCAGGTGGTGGCGGGTCAGATATACAAATGACACAGAGCCCTAGTAGTCTGAGTGCTTCAGTGGGCGACCGCGTAACTATAACCTGTAGAGCATCCCAAAGCATTTCCCACTTCCTTAATTGGTACCAGCAGAAGCCGGGCACAGCGCCCAAACTCCTGATCACCACTGCGAGCGGACTTGGTTCAGGTGTTCCTAGCCGGTTTAGTGGGTCAGGTAGCGGTACAGATTTCACTCTCACGATAAACTCCCTTCAGCCTGAGGACCTGGCGACATATTACTGTCAACAATCCTATACCACCCCACTGACATTCGGAGGGGGCACAAAACTGGAGATCAAA SEQ ID NO: 42 is the amino acid sequence of CD22-specific binder (scFv5) 12s: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGDDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISHFLNWYQQKPGTAPKLLITTASGLGSGVPSRFSGSGSGTDFTLTINSLQPEDLATYYCQQSYTTPLTFGGGTKLEIKR SEQ ID NO: 43 is the nucleic acid sequence of CD22 CAR LTG2249 (LP-scFv5-CD8TM-41BB-CD3 zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTTCAGTTGCAGCAGAGTGGCCCTGGGCTTGTTAAACCATCACAGACGCTCTCACTGACCTGTGCCATCTCTGGAGACAGTGTAAGTTCTAACTCAGCCGCGTGGAATTGGATTAGACAATCACCAAGCCGGGGACTTGAATGGCTTGGTCGGACGTACTATAGATCTAAGTGGTATAATGACTACGCAGTGTCAGTGAAATCACGGATAACCATAAACCCTGACACCAGCAAAAACCAATTTTCTCTTCAGCTTAATTCCGTCACGCCAGAAGATACGGCCGTTTACTACTGTGCGAGGGAAGGTGATGACGCATTGGACATCTGGGGTCAGGGGACCATGGTGACTGTCTCTTCCGGCGGGGGGGGTAGTGGAGGGGGTGGCTCAGGTGGTGGCGGGTCAGATATACAAATGACACAGAGCCCTAGTAGTCTGAGTGCTTCAGTGGGCGACCGCGTAACTATAACCTGTAGAGCATCCCAAAGCATTTCCCACTTCCTTAATTGGTACCAGCAGAAGCCGGGCACAGCGCCCAAACTCCTGATCACCACTGCGAGCGGACTTGGTTCAGGTGTTCCTAGCCGGTTTAGTGGGTCAGGTAGCGGTACAGATTTCACTCTCACGATAAACTCCCTTCAGCCTGAGGACCTGGCGACATATTACTGTCAACAATCCTATACCACCCCACTGACATTCGGAGGGGGCACAAAACTGGAGATCAAAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGC CGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Accession number 44 is the amino acid sequence of CD22 CAR LTG2249 (LP-scFv5-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGDDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISHFLNWYQQKPGTAPKLLITTASGLGSGVPSRFSGSGSGTDFTLTINSLQPEDLATYYCQQSYTTPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence number 45 is the amino acid sequence of scFv5(12s)LCDR1: QSISHF Sequence number 46 is the amino acid sequence of scFv5(12s)LCDR2: TAS Sequence number 47 is the amino acid sequence of scFv5(12s)LCDR3: QQSYTTPLT Sequence number 48 is the amino acid sequence of scFv5(12s)HCDR1: GDSVSSNSAA Sequence number 49 is the amino acid sequence of scFv5(12s)HCDR2: TYYRSKWYN Sequence number 50 is the amino acid sequence of scFv5(12s)HCDR3: AREGDDALDI Sequence number 51 is the nucleic acid sequence of CD22-specific binder (scFv6) 16P3: CAGATACAGTTGCAGCAGTCAGGTCCAGGACTAGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTACAACCTGATGATGCTTTAGATATCTGGGGCCAAGGGACAATGGTCACCGT CTCTTCAGGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTCTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAAAATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 52 is the amino acid sequence of CD22-specific binder (scFv6) 16P3: QIQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIK Sequence number 53 is the nucleic acid sequence of CD22 CAR LTG2203 (LP-scFv6-CD8TM-41BB-CD3 zeta): CCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 54 is the amino acid sequence of CD22 CAR LTG2203 (LP-scFv6-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQIQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 55 is the amino acid sequence of scFv6 (16P3) LCDR1: QDVSGW SEQ ID NO:56 is the amino acid sequence of scFv6(16P3) LCDR2: GAS SEQ ID NO:57 is the amino acid sequence of scFv6(16P3) LCDR3: QQAKNFPYT SEQ ID NO:58 is the amino acid sequence of scFv6(16P3) HCDR1: GDSVSSNSAA SEQ ID NO:59 is the amino acid sequence of scFv6(16P3) HCDR2: TYYRSKWYN SEQ ID NO:60 is the amino acid sequence of scFv6(16P3) HCDR3: AQEVQPDDALDI SEQ ID NO:61 is the nucleic acid sequence of CD22-specific binder (scFv7) 16P16: CAAGTACAGTTGCAGCAGTCAGGACCTGGCCTTGTGAAACCATCCCAAACTCTCAGCCTCACGTGTGCTATTTCTGGTGACTCAGTAAGTAGCAATAGCGCTGCTTGGAACTGGATCAGACAATCTCCCTCCAGGGGTCTCGAATGGCTGGGGCGAACCTATTACCGATCTAAATGGTATAACGATTATGCAGTATCCGTGAAATCCAGGATTACAATCAACCCAGATACGTTCAAGAATCAATTCTCTCTTCAGCTCAACTCCGTAACTCCAGAGGACACTGCGGTATATTATTGCGCCCAAGAAGTCGAGCCACACGATGCCCTCGATATCTGGGGTCAAGGTACCATGGTTACAGTTAGTAGTGGGGGTGGGGGAAGCGGGGGCGGTGGGTCCGGTGGCGGGGGTTCAGACATCAAGATGACCCAATCCCCAAGCTCTGTTTCAGCATCCGTGGGCGATAAGGTAACCATTACATGCAGAGCGAGTCAGGACGTTTCAGGGTGGCTGGCTTGGTAC CAGCAAAAACCGGGACTCGCACCGCAGCTGTTGATTTTCGGCGCCAGTACGCTTCAGGGCGAAGTACCGTCCAGGTTCAGTGGGTCAGGTTCTGGCACCGATTTTACGCTCACGATATCCAGTCTCCAACCGGAGGATTTTGCTACTTATTACTGCCAGCAGGCTAAGTATTTTCCATACACATTTGGCCAGGGGACAAAGTTGGAGATCAAA SEQ ID NO: 62 is the amino acid sequence of CD22-specific binder (scFv7) 16P16: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 63 is the nucleic acid sequence of CD22 CAR LTG2204 (LP-scFv7-CD8TM-41BB-CD3 zeta): CGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 64 is the amino acid sequence of CD22 CAR LTG2204 (LP-scFv7-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 65 is the amino acid sequence of scFv7 (16P16) LCDR1: QDVSGW SEQ ID NO: 66 is the amino acid sequence of scFv7 (16P16) LCDR2: GAS SEQ ID NO: 67 is the amino acid sequence of scFv7 (16P16) LCDR3: QQAKYFPYT Sequence number 68 is the amino acid sequence of scFv7(16P16) HCDR1: GDSVSSNSAA Sequence number 69 is the amino acid sequence of scFv7(16P16) HCDR2: TYYRSKWYN Sequence number 70 is the amino acid sequence of scFv7(16P16) HCDR3: AQEVEPHDALDI Sequence number 71 is the nucleic acid sequence of CD22-specific binder (scFv8) 16P20: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCATGATGCTCTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGCGGAGGCGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACGCAGTCTCCATCATCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAACAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTTTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAAC AGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA Sequence number 72 is the amino acid sequence of CD22-specific binder (scFv8) 16P20: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK Sequence number 73 is the nucleic acid sequence of CD22 CAR LTG2205 (LP-scFv8-CD8TM-41BB-CD3 zeta): Accession number 74 is the amino acid sequence of CD22 CAR LTG2205 (LP-scFv8-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 75 is the amino acid sequence of scFv8 (16P20) LCDR1: QDVSGW Accession number 76 is the amino acid sequence of scFv8 (16P20) LCDR2: GAS Accession number 77 is the amino acid sequence of scFv8 (16P20) LCDR3: QQAKYFPYT Accession number 78 is the amino acid sequence of scFv8 (16P20) HCDR1: GDSVSSNSAA Accession number 79 is the amino acid sequence of scFv8 (16P20) HCDR2: TYYRSKWYN Accession number 80 is the amino acid sequence of scFv8 (16P20) HCDR3: AQEVEPHDALDI SEQ ID NO: 81 is the nucleic acid sequence of CD22-specific binder (scFv9) 16P2: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATTCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCATGATGCTCTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCAAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTTTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 82 is the amino acid sequence of CD22-specific binder (scFv9) 16P2: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWI RQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK Sequence number 83 is the nucleic acid sequence of CD22 CAR LTG2206 (LP-scFv9-CD8TM-41BB-CD3 zeta): Accession number 84 is the amino acid sequence of CD22 CAR LTG2206 (LP-scFv9-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVY YCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 85 is the amino acid sequence of scFv9 (16P2) LCDR1: QDVSGW Accession number 86 is the amino acid sequence of scFv9 (16P2) LCDR2: GAS Accession number 87 is the amino acid sequence of scFv9 (16P2) LCDR3: QQAKYFPYT Accession number 88 is the amino acid sequence of scFv9 (16P2) HCDR1: GDSVSSNSAA Accession number 89 is the amino acid sequence of scFv9 (16P2) HCDR2: TYYRSKWYN Accession number 90 is the amino acid sequence of scFv9 (16P2) HCDR3: AQEVEPHDALDI SEQ ID NO: 91 is the nucleic acid sequence of the CD22-specific binder (scFv10) 16P6: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGATACGGCTGTGTATTACTGTGCCCAAGAGGTACAACCTGATGATGCTTTTGATATCTGGGGCCAAGGGACAATGATCACCGTCTCTTCAGGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTCTGGTGCATCCACTTTGCAAGGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAAAATTTCCCTTACACTTTTGGTCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 92 is the amino acid sequence of the CD22-specific binder (scFv10) 16P6: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDAFDIWGQGTMITVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIK Sequence number 93 is the nucleic acid sequence of CD22 CAR LTG2207 (LP-scFv10-CD8TM-41BB-CD3 zeta): Accession number 94 is the amino acid sequence of CD22 CAR LTG2207 (LP-scFv10-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDAFDIWGQGTMITVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 95 is the amino acid sequence of scFv10(16P6) LCDR1: QDVSGW Accession number 96 is the amino acid sequence of scFv10(16P6) LCDR2: GAS Accession number 97 is the amino acid sequence of scFv10(16P6) LCDR3: QQAKNFPYT Accession number 98 is the amino acid sequence of scFv10(16P6) HCDR1: GDSVSSNSAA Accession number 99 is the amino acid sequence of scFv10(16P6) HCDR2: TYYRSKWYN Accession number 100 is the amino acid sequence of scFv10(16P6) HCDR3: AQEVQPDDAFDI SEQ ID NO: 101 is the nucleic acid sequence of the CD22-specific binder (scFv11) 16P10: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCAGGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTTTGGTGCATCCACTCTGCAAGGTGAAGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCCGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 102 is the amino acid sequence of the CD22-specific binder (scFv11) 16P10: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGPGTKLEIK Sequence number 103 is the nucleic acid sequence of CD22 CAR LTG2208 (LP-scFv11-CD8TM-41BB-CD3 zeta): Accession number 104 is the amino acid sequence of CD22 CAR LTG2208 (LP-scFv11-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGPGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 105 is the amino acid sequence of scFv11 (16P10) LCDR1: QDVSGW Accession number 106 is the amino acid sequence of scFv11 (16P10) LCDR2: GAS Accession number 107 is the amino acid sequence of scFv11 (16P10) LCDR3: QQAKYFPYT Accession number 108 is the amino acid sequence of scFv11 (16P10) HCDR1: GDSVSSNSAA Accession number 109 is the amino acid sequence of scFv11 (16P10) HCDR2: TYYRSKWYN Sequence number 110 is the amino acid sequence of scFv11(16P10) HCDR3: AQEVEPQDAFDI Sequence number 111 is the nucleic acid sequence of CD22-specific binder (scFv12) 16P17: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCACTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTATGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTCTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA Sequence number 112 is the amino acid sequence of CD22-specific binder (scFv12) 16P17: QVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK Sequence number 113 is the nucleic acid sequence of CD22 CAR LTG2209 (LP-scFv12-CD8TM-41BB-CD3 zeta): ATGCTTCTTTTGGTGACTTCCCTTTTGCTGTGCGAGTTGCCACACCCCGCCTTCCTGCTTATTCCCCAGGTACAGCTTCAACAGAGTGGGCCGGGACTGGTGAAACACTCCCAAACACTTTCTCTGACGTGCGCTATATCAGGTGACTCTGTTTCATCT Accession number 114 is the amino acid sequence of CD22 CAR LTG2209 (LP-scFv12-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 115 is the amino acid sequence of scFv12(16P17) LCDR1: QDVSGW Accession number 116 is the amino acid sequence of scFv12(16P17) LCDR2: GAS Accession number 117 is the amino acid sequence of scFv12(16P17) LCDR3: QQAKYFPYT Accession number 118 is the amino acid sequence of scFv12(16P17) HCDR1: GDSVSSNSAA Accession number 119 is the amino acid sequence of scFv12(16P17) HCDR2: TYYRSKWYN Accession number 120 is the amino acid sequence of scFv12(16P17) HCDR3: AQEVEPHDAFDI SEQ ID NO: 121 is the nucleic acid sequence of the CD22-specific binder (scFv13) 16P20v2: CAAGTACAACTTCAACAGTCTGGGCCTGGGCTTGTAAAACCTAGCCAAACTCTGTCCCTCACGTGCGCGATTTCAGGGGACAGTGTAAGTTCCAACTCAGCCGCATGGAACTGGATCAGGCAGTCACCTTCAAGGGGGCTCGAATGGCTTGGCCGAACGTACTACAGGAGTAAGTGGTACAACGATTATGCAGTGTCTGTGAAATCACGGATTACTATCAATCCCGACACGTCCAAGAACCAGTTCTCTCTGCAACTCAACTCAGTGACACCAGAGGATACGGCCGTTTACTATTGTGCACAGGAAGTGCAACCTGATGATGCCTTTGACATTTGGGGTCAGGGCACGATGGTTACGGTAAGCTCTGGGGGAGGCGGCAGTGGAGGGGGAGGTAGTGGGGGAGGGGGATCTGATATACAGATGACACAAAGCCCGTCATCCGTCAGTGCTTCAGTTGGTGATAAAGTAACCATTACGTGCCGCGCTTCCCAAGACGTTAGCGGATGGTTGGCTTGGTATCAACAAAAACCGGGGTTGGCTCCGCAACTCCTCATATCCGGTGCGAGTACGCTCCAAGGCGAAGTCCCTAGCAGATTTTCCGGGAGCGGTTCCGGTACAGATTTCACGTTGACCATTAGCTCTCTCCAGCCCGAAGATTTTGCAACCTACTATTGCCAACAGGCCAAAAATTTTCCATATACATTTGGTCAAGGCACTAAGCTCGAAATCAAA SEQ ID NO: 122 is the amino acid sequence of the CD22-specific binder (scFv13) 16P20v2: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIK Sequence number 123 is the nucleic acid sequence of CD22 CAR LTG2210 (LP-scFv13-CD8TM-41BB-CD3 zeta): ATGCTTCTTTTGGTGACTTCCCTTTTGCTGTGCGAGTTGCCACACCCCGCCTTCCTGCTTATTCCCCAAGTACAACTTCAACAGTCTGGGCCTGGGCTTGTAAAACCTAGCCAAACTCTGTCCCTCACGTGCGCGATTTCAGGGGACAGTGTAAGTTCCAACTCAGCCGCATGGAACTGGATCAGGCAGTCACCTTCAAGGGGGCTCGAATGGCTTGGCCGAACGTACTACAGGAGTAAGTGGTACAACGATTATGCAGTGTCTGTGAAATCACGGATTACTATCAATCCCGACACGTCCAAGAACCAGTTCTCTCTGC Accession number 124 is the amino acid sequence of CD22 CAR LTG2210 (LP-scFv13-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 125 is the amino acid sequence of scFv13 (16P20v2) LCDR1: QDVSGW Accession number 126 is the amino acid sequence of scFv13 (16P20v2) LCDR2: GAS Accession number 127 is the amino acid sequence of scFv13 (16P20v2) LCDR3: QQAKNFPYT Accession number 128 is the amino acid sequence of scFv13 (16P20v2) HCDR1: GDSVSSNSAA Accession number 129 is the amino acid sequence of scFv13 (16P20v2) HCDR2: TYYRSKWYN SEQ ID NO: 130 is the amino acid sequence of scFv13(16P20v2) HCDR3: AQEVQPDDAFDI SEQ ID NO: 131 is the nucleic acid sequence of CD22-specific binder (scFv14) 16P1: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGACATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGATAGAACCTCATGATGCTTTTGATATCTGGGACCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGTCATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTCTGGTGCATCCTCTTTGCAAGGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 132 is the amino acid sequence of CD22-specific binder (scFv14) 16P1: QVQLQQSGPGLVKPSQTLSLTCDISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEIEPHDAFDIWDQGTMVTVSSGGGGSGGGGSGGGGSVIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK Sequence number 133 is the nucleic acid sequence of CD22 CAR LTG2216 (LP-scFv14-CD8TM-41BB-CD3 zeta): ATGTTGCTGCTCGTGACCTCGCTCCTTCTGTGCGAGCTGCCCCATCCGGCTTTTCTGCTCATCCCTCAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCCGTCCCAGACTCTGAGCCTGACTTGCGATATTAGCGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAA Accession number 134 is the amino acid sequence of CD22 CAR LTG2216 (LP-scFv14-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCDISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEIEPHDAFDIWDQGTMVTVSSGGGGSGGGGSGGGGSVIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Accession number 135 is the amino acid sequence of scFv14(16P1) LCDR1: QDVSGW Accession number 136 is the amino acid sequence of scFv14(16P1) LCDR2: GAS Accession number 137 is the amino acid sequence of scFv14(16P1) LCDR3: QQAKYFPYT Accession number 138 is the amino acid sequence of scFv14(16P1) HCDR1: GDSVSSNSAA Accession number 139 is the amino acid sequence of scFv14(16P1) HCDR2: TYYRSKWYN Accession number 140 is the amino acid sequence of scFv14(16P1) HCDR3: AQEIEPHDAFDI SEQ ID NO: 141 is the nucleic acid sequence of CD22-specific binder (scFv15) 16P3v3: CAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCACTCCCAGACTCTGAGCCTGGCCTGCGCGATTAGCGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTATAACGACTACGCCGTGTCCGTGAAGTCCCGGATCACCATTAACCCCGACACCTCGAAGAACCAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATACCGCGGTGTACTACTGCGCACAAGAAGTGCAGCCGCAGGACGCCCTGGACATTTGGGGGCAGGGAACGATGGTCACAGTGTCGTCCGGTGGAGGAGGTTCCGGAGGCGGTGGATCTGGAGGCGGAGGTTCGGATATCCAGATGACCCAGAGCCCCTCCTTCGTGTCCGCATCCGTGGGCGATAAGGTCATTATTACCTGTAGAGCGTCCCAGGACGTGTCCGGATGGCTGGCCTGGTACCAGCAGAAGCCAGGCTTGGCTCCTCAACTGCTGATCTCCGGCGCCAGCACTCTTCAGGGGGAAGTGCCATCACGCTTCTCCGGATCCG...

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain having the amino acid sequence of SEQ ID NO: 2, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one transmembrane domain, and at least one intracellular signaling domain.

2. The isolated nucleic acid molecule according to claim 1, wherein the CD22 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD22.

3. The isolated nucleic acid molecule according to claim 1, wherein the CD22 antigen-binding domain comprises at least one heavy-chain variable region of an antibody that binds to CD22.

4. The isolated nucleic acid molecule according to claim 1, wherein the CD22 antigen-binding domain, the at least one intracellular signaling domain, or both are linked to the transmembrane domain by a linker or spacer domain.

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

6. The isolated nucleic acid molecule according to claim 1, wherein the CD22 antigen-binding domain is located downstream of a leader nucleotide sequence encoding a leader peptide.

7. The isolated nucleic acid molecule according to claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 190 encoding the leader amino acid sequence of SEQ ID NO:

191.

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

9. The nucleic acid sequence encoding the CD22 antigen-binding domain comprises a nucleotide sequence comprising SEQ ID NO: 1, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, or 171, the isolated nucleic acid molecule according to claim 1.

10. The at least one intracellular signaling domain encoded by the isolated nucleic acid molecule of claim 1 further comprises a CD3 zeta intracellular domain.

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

12. The 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, the isolated nucleic acid molecule according to claim 11.

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

14. At least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, at least one transmembrane domain, and at least one intracellular signaling domain, the CAR according to claim 13.

15. The CAR according to claim 14, wherein the CD22 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD22.

16. The CAR according to claim 14, wherein the CD22 antigen-binding domain comprises at least one heavy-chain variable region of an antibody that binds to CD22.

17. The transmembrane domain of the CAR according to claim 14 comprises the transmembrane domain of a protein comprising alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

18. The transmembrane domain of a protein comprising CD8 comprises the amino acid sequence of SEQ ID NO: 182, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 182, the CAR according to claim 17.

19. At least one extracellular antigen-binding domain comprising a CD22 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, or 172, and at least one intracellular signaling domain, or both are bound to the transmembrane domain by a linker or spacer domain, the CAR according to claim 14.

20. The CAR according to claim 19, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is bound to the transmembrane domain. **Claim 21** The CAR according to claim 14, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain. **Claim 22** The CAR according to claim 21, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

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