Compositions and methods for treating cancer with Anti-CD22 immunotherapy

Abstract

To provide CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions.SOLUTION: Chimeric antigen receptors (CARs) containing CD22 antigen binding domains are provided. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the CARs are also provided. Methods of treating or preventing cancer in a subject, and methods of making CAR T cells are also provided.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 October 16, 2017, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy (created October 12, 2018) has the filename "Sequence Listing.txt" and is 234 kilobytes in size.

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

[0004] Field of the Disclosure 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 use thereof. [Background technology]

[0005] background Cancer is one of the most deadly threats to human health. With nearly 1.3 million new cases each year in the United States alone, it is the second leading cause of death after cardiovascular disease, accounting for one in four deaths. Most of these deaths are caused by solid tumors. While significant advances have been made in the medical treatment of some specific cancers, the five-year survival rate for all cancers combined has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrollably, making them extremely difficult to treat.

[0006] The current standard of care for B-lineage leukemias is thought to involve induction treatment with high-dose chemotherapy or radiation therapy followed by consolidation therapy, which may be characterized by stem cell transplantation and further chemotherapy if necessary (see cancer.gov on the World Wide Web). Because these treatments are toxic and carry the risk of complications such as relapse, secondary malignancies, or graft-versus-host disease (GVHD), better alternatives are being sought. 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 is lost in plasma cells (Nitschke L, 2009, Immunological Reviews, 230:128-143). CD22 contains an intracellular ITIM (immunoreceptor tyrosine-based inhibition motifs) domain, which acts to downregulate subsequent cell activation upon antigen binding by the B cell receptor. Antibody binding to CD22 results in phosphorylation (phosorylation). CD22 induces colocalization with SHP-1, an intracellular phosphatase that also acts to downregulate signaling via CD22 receptor activator (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, as CD22 is expressed in a tightly regulated manner on normal B cells but not on hematopoietic stem cells or mature plasma cells, making it a suitable target antigen for B-cell leukemias. CD22 expression on both adult and pediatric B-cell malignancies (pre-B-ALL) has led to the development of both antibody- and chimeric antigen receptor (CAR)-T cell-based therapies leveraging this target (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, 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 currently being evaluated in clinical trials as a single agent and in combination with chemotherapy (NCT01664910, funded by MD Anderson Cancer Center) (DiJoseph JF et al., 2004, Blood, 103:1807-1814). Outcomes as a single agent were favorable compared to those observed with standard therapy, although significant hepatotoxicity was observed (Kantarjian H et al., 2016, Inotuzumb ozogamicin versus standard therapy for acute lymphoblastic leukemia (ALL), New England Journal of Medicine, 375:740-753). Epratuzumab, a native CD22 therapeutic antibody, is also currently being tested in combination with chemotherapy (NCT01219816, funded by Nantes University Hospital). Epratuzumab is a chimeric protein constructed by grafting mouse CDRs onto a human antibody framework. Moxetumomab Although pasudotoxin (a type of antibody) is effective against certain types of leukemia, it has not undergone widespread clinical development due to issues with the immunogenicity of the bacterial toxin to which it is fused and its modest activity level, comparable to other agents (see NCT01829711, funded by MediImmune). Many of the CD22 binding sites used in CAR constructs to date utilize domains derived from the murine antibodies mentioned above, but do not effectively activate T cells targeting this CD22 domain (e.g., HA22 (an anti-CD22 binder) 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, Journal of Immunology 180:7028-7038). One anti-CD22 binder effective as an anti-CD22 CAR is This drug is currently undergoing clinical trials at the National Institutes of Health (NIH), 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 this application (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 was demonstrated to be effective as a CAR in research led by Dr. Rimas Orentas, another of the inventors of this 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 binding / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly active CD22-specific CAR. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is typically modeled after a single-chain fragment variable fragment (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs, such as receptor ligands (i.e., IL-13 was engineered to bind to the IL-13 receptor expressed in tumors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D), have also been engineered into CARs. Other cellular targets for expressing CARs, such as NK or gamma-delta T cells, are under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Substantial further effort remains to be expended in identifying the most active T cell populations to transduce with CAR vectors, in identifying optimal culture and expansion techniques, and in elucidating the molecular details of the CAR protein structure itself.

[0009] The binding motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed as a long, flexible linker. Structural motifs, such as those derived from the IgG constant domain, can be used to extend the ScFv binding domain far from the T cell membrane surface. This may be important for some tumor targets (e.g., disialoganglioside GD2; Orentas et al., this observation unpublished) whose binding domains are particularly close to the tumor cell surface membrane. All signaling motifs used in CARs to date include the CD3-zeta chain, as this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was used in third-generation CARs, which also contained the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the advent of new technologies, it is no longer necessary for the CAR itself to encode T cell activation by beads coupled to anti-CD3 and anti-CD28 antibodies, and the presence of the classical "signal 2" from CD28. Third-generation vectors using bead activation have been shown to be no superior to second-generation vectors in in vitro assays, and furthermore, in mouse models of leukemia. and did not offer a clear advantage over 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 sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which the CAR T cell population is cultured, such as the inclusion of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther. 2015;22(2):72-78).

[0010] Currently, the challenge to broadening and effectively applying CAR therapy to cancers relates to the lack of promising targets. While engineering binders that bind to cell surface antigens is now readily achievable, discovering cell surface antigens that are tumor-specific and spare normal tissue remains extremely challenging. Combining multiple CAR approaches can potentially confer stronger target cell specificity to CAR-expressing T cells. In one system, the CD3-zeta and CD28 signaling units are split into two separate CAR constructs expressed in the same cell. In another system, two CARs are expressed in the same T cell, but one CAR has lower affinity, requiring the other CAR to bind first to maximize its activity (Lanitis E et al. Cancer Immunol Res. 2013;1(1):43-53; Kloss CC et al. Nat Biotechnol. 2013;31(1):71-5). A second challenge in generating a single ScFv-based CAR as an immunotherapeutic agent is the heterogeneity of tumor cells. At least one group has developed a CAR-based treatment for glioblastoma, in which effector cell populations simultaneously target multiple antigens (HER2, IL-13Ra, and EphA2) in an attempt to avoid the proliferation of non-targeted populations (Hegde M et al. Mol Ther. 2013;21(11):2087-101).

[0011] T cell-based immunotherapy has become a new frontier in synthetic biology. Multiple promoters and gene products have been designed to target these highly potent cells to the tumor microenvironment, where they can circumvent negative regulatory signals and mediate effective tumor killing. Eliminating unwanted T cells through drug-induced dimerization of inducible caspase-9 constructs using dimerization-inducing chemicals such as AP1903 offers one way to pharmacologically trigger a powerful switch that can control T cell populations (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Furthermore, generating effector T cell populations that are resistant to the negative regulatory effects of transforming growth factor-β by expressing a decoy receptor demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, although CARs appear to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, the limited expansion of CAR+ T cells in vivo, their rapid loss after infusion, and poor clinical activity currently pose significant obstacles to the clinical application of this technology. This may be due in part to the murine origin of some of the CAR sequences used, and the limited clinical activity of the CARs disclosed herein. The invention presented here directly addresses this problem.

[0012] Thus, there is an urgent and long-felt need in the art to discover new compositions and methods for treating B-ALL, DLBCL, FL, and other CD22-expressing B-cell malignancies using approaches that can exhibit specific and highly potent anti-tumor effects without exhibiting the problems described above. Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention addresses the above-mentioned needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the invention disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with dysregulated expression of CD22, which contain a CD22 antigen-binding domain that has high surface expression on transduced T cells, a high degree of cytolysis of CD22-expressing cells, and in vivo proliferation and persistence of the transduced T cells. [Means for solving the problem]

[0014] overview Provided herein are novel anti-CD22 antibodies, or their antigen-binding domains, and chimeric antigen receptors (CARs) containing such CD22 antigen-binding domains, as well as host cells (e.g., T cells) that express the receptors and nucleic acid molecules encoding the receptors. The CARs exhibit high surface expression on transduced T cells, a high degree of cytolysis, and in vivo proliferation and persistence of the transduced T cells. Additionally, methods of using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject, are provided.

[0015] Thus, in one aspect, an isolated polynucleotide encoding a human anti-CD22 antibody or fragment thereof is provided, 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, an isolated polynucleotide encoding a fully human anti-CD22 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

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

[0018] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising, from N- to C-terminus, 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, at least one transmembrane domain, and at least one intracellular signaling domain.

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

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

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

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

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

[0024] In yet another embodiment, an isolated nucleic acid molecule is provided 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, wherein the CAR further encodes an extracellular antigen binding domain that targets an antigen including (but not limited to) CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[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, or an anti-EGFRvIII ScFv. The antigen-binding domain of the ScFv includes an anti-GD-2 ScFv, an anti-NY-ESO-1 TCR ScFv, an anti-MAGE A3 TCR ScFv, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0026] In one embodiment, 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, is 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 linked 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 the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination 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 costimulatory 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 costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0033] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided that further contains a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide 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 embodiment, provided herein is a CAR comprising, from N-terminus to 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 to the antigen, or at least one heavy-chain variable region of an antibody that binds to the antigen, or a combination thereof.

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

[0038] In some embodiments, a CAR is provided, wherein the CAR is selected from the group consisting of 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 The antigen-binding domain of the ScFv includes an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

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

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

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

[0043] In another embodiment, the nucleic acid sequence encoding the CAR comprises 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 comprising 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 comprises 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 comprises the amino acid sequence of SEQ ID NO: 24 (LTG 2247 LP-25P-CD8 TM-41BB-CD3 zeta It encodes a CAR containing the CAR amino acid sequence (Figure 2C).

[0045] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 33 (LTG 2248 LP-11S-CD8 TM-41BB-CD3 Zeta CAR nucleic acid In one embodiment, the nucleic acid sequence encodes a CAR comprising 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 comprises the amino acid sequence of SEQ ID NO: 34 (LTG 2204 LP-16P16-CD8 TM-41BB-CD3 zeta It encodes a CAR containing the 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 comprises the amino acid sequence of SEQ ID NO: 74 (LTG 2205 LP-16P20-CD8 TM-41BB-CD3 zeta It encodes a CAR containing the 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 comprises the amino acid sequence of SEQ ID NO: 114 (LTG 2209 LP-16P17-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2L)). The data encodes a CAR containing the 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 comprises the amino acid sequence of SEQ ID NO: 134 (LTG 2216 LP-16P1-CD8 TM-41BB-CD3 zeta It encodes a CAR containing the 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 comprises the amino acid sequence of SEQ ID NO: 154 (LTG 2218 LP-16P8-CD8 TM-41BB-CD3 zeta It encodes a CAR containing the 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 embodiment, a CAR disclosed herein is modified to express or contain a detectable marker for use in diagnosing, monitoring, and / or predicting treatment outcomes, such as progression-free survival, of cancer patients, or for monitoring the progress of such treatment.

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

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

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

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

[0065] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein 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 with cancer. Cancers include, inter alia, hematological cancers such as leukemia (e.g., CLL, ALL, AML, or CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma (NHL), or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

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

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

[0068] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of human T cells from a human with cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, ALL, minimal residual disease (MRD) in AML, adult B-cell malignancies including CLL, CML, NHL, pediatric B-cell malignancies (including B-lineage ALL), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0069] In another embodiment, a method of producing CAR-containing T cells (hereinafter "CAR-T cells") is provided, which method comprises transducing T cells with a vector or nucleic acid molecule encoding a CAR (as disclosed) that specifically binds to CD22, thereby producing CAR-T cells.

[0070] In yet another embodiment, a method for generating a population of RNA-engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed CAR into cells of a subject, thereby generating CAR cells.

[0071] In yet another aspect, a method for diagnosing a disease, disorder, or condition associated with expression of CD22 in a cell is provided, comprising the steps of: a) contacting the cell with a human anti-CD19 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from 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, wherein if CD19 is present, the disease, disorder, or condition associated with expression of CD22 is diagnosed.

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

[0073] In another embodiment, a method for diagnosing or prognosing or risk-judging a CD19-related disease in a mammal is provided, comprising detecting expression of CD22 in a sample from the mammal, the method comprising: a) contacting the sample with a 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 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, wherein if CD22 is present, diagnosing the mammal as having a CD22-related disease.

[0074] In another embodiment, a method of inhibiting CD22-dependent T cell inhibition is provided, comprising contacting a cell with a 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 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 a CD22-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0075] In another embodiment, a method is provided for blocking T cell inhibition mediated by CD22-expressing cells and altering the tumor microenvironment to inhibit tumor growth in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-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. In one embodiment, the cell is selected from the group consisting of a CD19-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0076] In another embodiment, a method of inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-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. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a CD22-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

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

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

[0079] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises 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 wherein the T cells are T cells of a subject with cancer.

[0080] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a 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 linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of the subject with cancer. In some embodiments of the methods described above, at least one transmembrane domain comprises a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0081] In yet another embodiment, a method is provided for generating a persistent population of genetically engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells genetically engineered to express a CAR, wherein the CAR is selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, and at least one CD22 antigen binding domain comprising the amino acid sequence of 122, 132, 142, 152, 162, or 172, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the persistent population of engineered T cells, or a population of progeny of the T cells, persists in humans 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 include 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 of the cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens described above can be treated or prevented or ameliorated using one or more of the CARs disclosed herein.

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

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

[0086] [Figure 1] Schematic diagram of the general domain structure of a CAR with the novel extracellular CD22 antigen-binding domain sequence. The CAR is composed of an extracellular CD22-binding ScFv domain, a CD8 spacer and transmembrane domain, an intracellular signaling CD137 costimulatory domain, and a CD3 zeta signaling domain. [Figure 2A] Figure 2A shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2202 16P CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 3) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 4). [Figure 2B] Figure 2B shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2246 24P CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 13) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 14). [Figure 2C]Figure 2C shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. The following lentiviral vector expresses a CAR containing the nucleotide sequence (SEQ ID NO: 23) and the encoded amino acid sequence (SEQ ID NO: 24): LTG 2247 25P CD22ScFv-CD8 TM-41BB-CD3 zeta. [Figure 2D] Figure 2D shows several CARs containing the novel extracellular CD22 antigen-binding domain sequence. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. The following figure shows the nucleic acid sequence (SEQ ID NO: 33) of LTG 2248 11s CD22ScFv-CD8 TM-41BB-CD3 zeta, and a lentiviral vector expressing a CAR containing the encoded amino acid sequence. [Figure 2E] Figure 2E shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2249 12s CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 43) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 44). [Figure 2F]Figure 2F shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2203 16P3 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 53) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 54) are shown. [Figure 2G] Figure 2G shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2204 16P16 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 63) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 64). [Figure 2H] Figure 2H shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2205 16P20 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 73) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 74). [Figure 2I]Figure 2I shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The general scheme of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2206 16P2 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 83) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 84). [Figure 2J] Figure 2J shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2207 16P6 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 93) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 94). [Figure 2K] Figure 2K shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2208 16P10 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 103) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 104) are shown. [Figure 2L]Figure 2L shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The general scheme of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2209 16P17 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 113) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 114). [Figure 2M] Figure 2M shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2210 16P20v2 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 123) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 124). [Figure 2N] Figure 2N shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2216 16P1 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 133) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 134). [Figure 2O]Figure 2O shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The general scheme of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2217 16P3v2 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 143) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 144). [Figure 2P] Figure 2P shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The general scheme of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2218 16P8 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 153) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 154) are shown. [Figure 2Q] Figure 2Q shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The general scheme of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2219 16P13 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 163) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 164). [Figure 2R]Figure 2R shows several CARs containing the sequence of the novel extracellular CD22 antigen-binding domain. The general scheme of the CAR includes, from N- to C-terminus, a signal peptide, an anti-CD22 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. LTG 2220 16P15 CD22ScFv-CD8 TM-41BB-CD3 zeta nucleic acid sequence (SEQ ID NO: 173) and a lentiviral vector expressing a CAR containing the encoded amino acid sequence (SEQ ID NO: 174). [Figure 3] Figure 1 shows anti-CD22 CAR T surface expression on primary human T cells. CAR T cells redirected to the CD22 tumor antigen using ScFv domains (as indicated in each column) were generated by lentiviral transduction of the CAR expression construct. CAR T detection was performed by flow cytometry. T cells were washed twice in cold PBS-EDTA buffer and stained with CD22-Fc peptide followed by anti-Fc-PE reagent. At least 20,000 cells were obtained and subjected to each analysis. Cells were gated based on forward and side scatter, singlet discrimination, and 7AAD negativity to allow analysis of only viable cells. Data were acquired using a MACSQuant 10 flow cytometer (Miltenyi Biotec). A vertical dotted line in the panel indicates the CAR-expressing population (to the right of this gate). The top panel shows untransduced cells (UTD) as a negative control, and the second row immediately below shows cells transduced with the m971 positive control. Subsequent rows show CAR expression for each vector construct, as specified on the left axis of the figure. Results represent T cell transduction in three donors. [Figure 4]Figure 1 shows the in vitro lysis of CD22-positive tumors mediated by anti-CD22 CAR T cells incorporating ScFv binders (16P, 16P1, 16P3v2, 16P8, 16P10, 16P13, 16P15, 16P17) versus utd = untransduced negative control and m971 = previously published anti-CD22 CAR positive control. CAR T cells expressing anti-CD22 constructs were incubated overnight with CD22-positive cell lines (Raji and Reh) or CD19-negative lines stably transduced with firefly luciferase (K562 and 293T) at effector-to-target ratios of 1.25, 2.5, 5, 10, 20, and 40 (x-axis). CAR T cytotoxic activity was then assessed by luciferase activity measurement as described in Materials and Methods. Each bar is the mean of three technical replicates, and error bars represent SD. At least three separate experiments are represented. [Figure 5] Figure 1 shows the production of three cytokines (interferon-gamma, TNF-alpha, and IL-2) by CD22-specific CAR T cells when cultured alone (medium gray, CAR only) or with a CD22-positive leukemia line (Raji, black bars; Reh, light gray) or a CD22-negative line (293T, light gray). The assay was performed overnight at an E:T ratio of 10:1, and then supernatants were analyzed for cytokine concentrations by ELISA. N=3 + SEM. Negative control was untransduced T cells (utd), and positive control was transduced m971 CD22 CAR-T cells. The LTG number of each LV used to transduce human T cells is specified on the x-axis. [Figure 6]Two-dimensional flow cytometry analysis of CAR expression on the surface of T cells transduced with LV to express (top to bottom, left to right): no CAR (UTD), or LTG2200, 2202, 2216, 2206, 2217, 2207, 2218, 2208, 2219, 2220, 2209, 2205, a control GFP-expressing vector, or control CAR-19 (LTG1538) (as indicated above each graph). The y-axis shows staining for CD4, and the x-axis shows CAR expression stained with the target antigen (CD22-Fc recombinant protein (R&D Biosystems) with a secondary anti-Fc PE antibody). [Figure 7A] Figure 7A shows cytolytic activity (CTL activity) as a percentage of lysis of target cell lines expressing luciferase, respectively: the CD22-positive cell lines Raji and Reh, and the CD22-non-expressing line K562. [Figure 7B] Figure 7B shows cytolytic activity (CTL activity) as a percentage of lysis of target cell lines expressing luciferase, respectively, for K562-CD19 and K562-CD22 cell lines specifically transfected to express the target antigen. Each LV-transduced T cell population, as specified on the x-axis, was tested at three effector-target ratios of 10:1, 5:1, and 2.5:1: utd (untransduced), GFP-LV, LTG1538 (anti-CD19), m971 (LTG2200, control anti-CD22), 16p (LTG2202), 16p1 (LTG2216), 16p2 (LTG2206), 16p3v2 (LTG2217), 16p6 (LTG2207), 16p8 (LTG2218), 16p10 (LTG2208), 16p13 (LTG2219), 16p15 (LTG2220), 16p17 (TG2209), and 16p20 (LTG2205). [Figure 8]Figure 1 shows the production of IFN-gamma (top), IL-2 (middle), and TNF-alpha (bottom graph) by anti-CD22 CAR T cells when incubated overnight with CD22-positive Raji and Reh leukemia cell lines (black or gray bars, respectively) or without target tumor cells (T cells alone) at an E:T ratio of 10:1, and then supernatant cytokine concentrations were analyzed by ELISA. A negative control group of CAR alone was used to assess spontaneous cytokine secretion by CAR T cells. At least three separate experiments are represented. CAR-T activity is illustrated for leukemia targets incubated with untransduced T cells (utd), GFP-LV (GFP), CD19-CAR (LTG1538), CD22 control CAR (LTG2220, m971), 16p (LTF2202), 16p1 (LTG2216), 16p2 (LTG2206), 16p3v2 (LTG2217), 16p6 (LTG2207), 16p8 (LTG2218), 16p10 (LTG2208), 16p13 (LTG2219), 16p15 (LTG2220), 16p17 (LTG2209), 16p20 (LTG2205), or no CAR T cells (tumor only), as specified on the x-axis. [Figure 9]This study demonstrates the ability of CD22-specific CAR T to control disease in an animal model. Immunocompromised mice (NSG) were intravenously injected with Raji leukemia cells stably expressing firefly luciferase on study day 0. Disease burden was measured along the x-axis after injection of the luciferase substrate luciferin and imaged for individual animals using an IVIS instrument, and is shown as the mean brightness for each group. On day 6, animals with comparable disease burden were assigned to the same groups (six mice per group) and injected with CAR T cells on day 7. Disease progression was tracked over time. Animals injected with Raji cells but not treated with T cells (TA, open circles) rapidly deteriorated and had to be sacrificed on day 21. Other groups received untransduced T cells (UTD, open squares), CAR-19 transduced T cells (1538 CAR 19, open triangles), control anti-CD22 CAR (2200 m971, -x-), new CAR LTG2209 (2209 16P17, open diamonds), and new CAR LTG2219 (2219 16P13, open inverted triangles). DETAILED DESCRIPTION OF THE INVENTION

[0087] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly contradicts otherwise. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" that does not exclude other elements. The word "and / or" means "and" or "or" and ... or." Furthermore, unless otherwise specified, it is understood that any and all base size or amino acid size and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, particularly preferred methods and materials are described below. In the event of a conflict, the present specification (including explanations of terms) will control. Additionally, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. In order to facilitate identification of the various embodiments, explanations of terms are provided below.

[0088] The term "about," when referring to measurable possible values ​​such as amounts and durations, is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for the practice of the disclosed methods.

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

[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 directly correlates with improving the functional activity of CAR-expressing T cells. As a result of one or more of these modifications, the CAR exhibits high levels of cytokine-induced cytolysis and cell surface expression in transduced T cells, and also leads to high levels of T cell proliferation and persistence of transduced CAR-expressing T cells in vivo.

[0091] The unique ability to combine functional moieties from different protein domains is an innovative feature of CARs. The choice of these protein domains, as well as the specific binding mode, are key design features. Individual design domains are essential components that can be used in any heterogeneous CAR platform to manipulate lymphocyte function. For example, the selection of extracellular binding domains can enable otherwise ineffective CARs.

[0092] The non-variable framework components of the immunoglobulin-derived protein sequence used to generate the extracellular antigen-binding domain of a CAR can be completely neutral, or they can be self-binding and drive T cells into a metabolically exhausted state, significantly reducing the efficacy of therapeutic T cells expressing the CAR. This phenomenon occurs independently of the antigen-binding function of the CAR domain. Furthermore, the selection of the intracellular signaling domain can also govern the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability to bind target antigens and transmit activation signals to T cells via the above-described extracellular and intracellular domains are important CAR design aspects, it has become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on CAR potency and therefore may play a crucial role in CAR function and clinical utility.

[0093] Surprisingly and unexpectedly, it was found that the use of a fully human antigen-binding domain in a CAR, rather than a mouse-derived antigen-binding fragment (which tends to induce an anti-mouse immune response and CAR T elimination in the host) (see: University of Pennsylvania-funded clinical trial using a mouse-derived SS1 ScFv sequence, NCT02159716), can determine the functional activity of CAR-expressing T cells.

[0094] The CARs disclosed herein are expressed at high levels in cells. Cells expressing these CARs have high in vivo proliferation rates, produce large amounts of cytokines, and exhibit high cytotoxicity against cells bearing the CD22 antigen to which the CAR binds. The use of a human extracellular CD22 antigen-binding domain results in the creation of CARs with improved in vivo function, while avoiding the induction of anti-CAR immunity and the extinction of the CAR T cell population in the host immune response. CARs expressing a fully human extracellular CD22 ScFv antigen-binding domain exhibit superior activity and / or properties, including: i) preventing the poor persistence and poor function of CAR T (as observed with murine-derived binding sequences); ii) lack of targeting of CARs to specific regions (i.e., intrapleural) for efficacy; and iii) the ability to design CAR T cells based on both high and low CD19 affinity binders. The latter property allows researchers to better control the efficacy versus toxicity and / or tissue specificity of CAR T products. This is because CD22 is more highly expressed in tumors than in normal tissues, so lower affinity binders may have higher specificity for tumors than normal tissues, thereby preventing on-target off tumor toxicity and bystander cell killing.

[0095] The CARs of the present invention are now described in detail, including a description of their extracellular CD22 antigen-binding domain, transmembrane domain, and intracellular domain, as well as further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, treatment methods, compositions, and kits using the disclosed CARs.

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

[0097] Chimeric antigen receptors (CARs) are artificially engineered hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity to selected targets in a manner that is not restricted by major histocompatibility complex (MHC) and to exploit the antigen-binding properties of monoclonal antibodies. Because of their ability to recognize antigens without MHC restriction, CAR-expressing T cells have the ability to recognize antigens independently of antigen processing, thereby circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs).

[0098] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to the portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to the intracellular domain of a costimulatory molecule. This refers to the portion of a CAR that contains a receptor, which is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to respond efficiently to antigens.

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

[0100] In one embodiment, CAR can be designed to target the tumor antigen of interest by designing a desired antigen binding domain that specifically binds to the antigen on tumor cells.Tumor antigens are proteins produced by tumor cells that elicit immune responses, particularly T cell-mediated immune responses.The choice of antigen binding domain can depend on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, 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 by way of example only. The list is not intended to be limiting, and other examples will be readily apparent to those skilled in the art.

[0101] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express multiple proteins that can serve as target antigens for immune attack. Such molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules include those belonging to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins represent truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, CD22, BCMA, ROR1, and CD37, are also potential target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, CD22, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but without much success.

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

[0103] The type of tumor antigen can be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells, but instead are expressed under conditions that do not induce immune tolerance to this antigen. In tumors, TAAs are also expressed on normal cells. Expression of this antigen in tumors can occur under conditions that allow the immune system to respond to this antigen. A TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is not yet mature and able to respond to antigens, or a TAA can be an antigen that is normally present at very 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 multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and the like; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, and 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 an antigen including, but not limited to, CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, and MAGE A3 TCR.

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

[0107] In one 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 thereto.

[0108] In one preferred embodiment, an 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 a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. The amino acid sequence is

[0109] In one 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 thereto.

[0110] In one 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 thereto.

[0111] In one 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42.

[0112] In one 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52.

[0113] In one 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 62.

[0114] In one preferred embodiment, an 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 thereto.

[0115] In one preferred embodiment, an 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 thereto.

[0116] In one 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 92.

[0117] In one 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 102.

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

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

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

[0121] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 ScFv15 antigen-binding domain has the nucleotide sequence of SEQ ID NO: 141, or an identical sequence thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD22 ScFv15 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 142.

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

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

[0124] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD22 ScFv18 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 171, or a sequence which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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 which is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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, identified as SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively) each individually contribute to creating the binding characteristics of the CD22-specific scFv1, collectively contribute to creating the light chain binding characteristics of the scFv (LCDR1, LCDR2, and LCDR3), collectively contribute to creating the heavy chain binding characteristics of the scFv (HCDR1, HCDR2, and HCDR3), and collectively, the six SEQ ID NOs contribute together to creating the binding characteristics of the 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, identified as SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively) and heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively) each individually contribute to generating the binding properties of the CD22-specific scFv2, collectively contribute to generating the light chain binding properties of the scFv (LCDR1 and LCDR2 and LCDR3), and collectively contribute to generating the heavy chain binding properties of the scFv (HCDR1, HCDR2, and LCDR3). R1 and HCDR2 and HCDR3), and the six SEQ ID NOs collectively contribute to creating the binding properties of the CD22-specific scFv2 by 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.

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

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

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

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

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

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

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

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

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

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

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

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

[0139] In one preferred embodiment, an isolated light chain complementarity determining region amino acid sequence (LCDR The heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150) and the heavy chain complementarity determining region amino acid sequences (HCDR1, HCDR2, HCDR3, identified as SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, respectively) each individually contribute to creating the binding properties of the CD22-specific scFv15, collectively contribute to creating the light chain binding properties of the scFv (LCDR1, LCDR2, and LCDR3), collectively contribute to creating the heavy chain binding properties of the scFv (HCDR1, HCDR2, and HCDR3), and collectively, the six SEQ ID NOs contribute together to creating the binding properties of the 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.

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

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

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

[0143] In various embodiments of the CD22-specific CAR disclosed herein, a general scheme is set forth in FIG. 1, which includes, from N-terminus to C-terminus, a signal or leader peptide, an anti-CD22 ScFv, an extracellular linker, a CD8 transmembrane segment, 4-1BB, Contains CD3 zeta, bold letters indicate cloning site of linking domain.

[0144] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4 [LTG 2202 LP-16P-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 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 with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2204 LP-16P16-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 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 Figure 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 with 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 with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2205 LP-16P20-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2H)].

[0160] In yet another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid 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 Figure 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 with 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 with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2206 LP-16P2-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 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 Figure 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 with 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 with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2207 LP-16P6-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 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 Figure 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 with 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 with 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 with 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 with 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 sequence column (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 with 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 with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2210 LP-16P20v2-CD8 TM-41BB-CD3zeta 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 CARs incorporating single-chain variable fragment (ScFv) sequences reactive to the CD22 antigen is shown in Example 2 below, and a summary is shown in Tables 2, 3, and Figure 6. The expression level of each ScFv-containing CAR was 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 fragments conjugated to PE, and detected by flow cytometry (see Figure 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 (indicated by the gated population) compared with the non-transduced T cell control (ungated cell population, UTD). Representative results from one donor are shown.

[0181] As shown in Example 2 and Figures 4, 7A, and 7B, lentiviral vectors (LVs) expressing the following CARs were generated and tested for anti-leukemia activity, demonstrating the high cytolytic activity of the CD22 CAR. All CARs used in the experiments contain the described 4-1BB / CD3 zeta chain signaling motif and specific anti-CD22 binding motif / domain. Leukemia target lines with various CD22 surface expression 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), scFv4(16P8), scFv5(16P10), scFv6(16P13), scFv7(16P15), and scFv8(16P17), respectively) were able to efficiently lyse the CD22-rich tumor lines Raji and Reh, but they had little or no specific lytic activity against K562 or 293T (Figures 4, 7A, and 7B). These results demonstrate the efficiency and specificity of the CAR constructs generated.

[0182] The cytokine secretion capacity of anti-CD22 CAR T cells was then assessed. Tumor cells were incubated overnight with CAR T cells or control T cells at an effector-target ratio of 10:1, and culture supernatants were analyzed by ELISA for IFN-gamma, TNF-alpha, and IFN-alpha. , 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), and scFv8 (16P17), respectively, produced high levels of IFN-gamma, whereas no cytokine induction was observed with the negative control (untransduced, utd). However, clear differences were observed in TNF-alpha and IL-2 production. Strikingly, the CD22 CAR LTG2202 produced significantly lower levels of TNF-alpha and IL-2 compared to the Reh tumor line, and each vector differed in its ability to produce IL-2 and TNF-alpha against the tumor line targets tested. These differences may result in different antitumor and toxicity profiles, which may be realized individually depending on the disease burden in specific disease settings. The positive control CAR, m971, was used to benchmark results and is currently in clinical trials and is safe for use in advanced disease settings.

[0183] While not intending to be limited to any particular mechanism of action, reasons for improved therapeutic function associated with exemplary CARs of the present invention may include, but are not limited to, for example, a) more efficient signaling due to improved lateral movement in the plasma membrane, b) better location in plasma membrane microdomains (such as lipid rafts) that allow for improved interaction with transmembrane signaling cascades associated with T cell activation, c) better location in the plasma membrane due to preferential movement away from reducing or down-regulating interactions, e.g., greater distance from or less interaction with phosphatases such as CD45, and d) better assembly into the T cell receptor signaling complex (i.e., immune synapse), or any combination thereof.

[0184] To this point, the present disclosure has been illustrated using the extracellular CD22 variable heavy chain alone and the ScFv antigen-binding domain as specific examples, however, 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 CD22 antigen-binding domains 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, if CD22 is the desired antigen to be targeted, an antibody to CD22 may be used as the antigen-binding domain to be incorporated into the CAR.

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

[0187] In one exemplary embodiment, the antigen binding domain portion of the CAR further comprises 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 embodiment of the present invention, CARs are provided that can bind to non-TSAs or non-TAAs, including, but not limited to, antigens derived from Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enteroviruses, human coxsackieviruses, rhinoviruses, and echoviruses), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., herpes simplex virus types 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes viruses), Poxviridae (e.g., smallpox virus, vaccinia virus, and poxviruses), or hepatitis C virus, or any combination thereof.

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

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

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

[0194] In some instances, the transmembrane domain can be selected or amino acid substituted to prevent the domain from binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interactions of the domain with other receptor complex components.

[0195] In one embodiment, the transmembrane domain in a CAR of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 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 that is 95-99% identical to the amino acid sequence of SEQ ID NO: 182 with at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions).

[0197] In some examples, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 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 is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

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

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

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

[0202] The spacer domain can be the entire or a portion of amino acids 137-206 (SEQ ID NO: 39) of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). A portion of the constant region of an antibody heavy or light chain can also be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0203] Furthermore, a signal peptide sequence may be attached to the N-terminus of the CAR. This signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above as intracellular domains have a signal peptide sequence, this signal peptide can be used as the signal peptide for the CAR. In one embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 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 an immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a protein portion that transmits an effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, it can be used in place of the entire chain, as long as the truncated portion is capable of transmitting the effector function signal. Thus, the meaning of the term "intracellular signaling domain" includes any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[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 cooperate to initiate signal transduction following binding of an antigen to the receptor, as well as any derivatives or variants of these sequences, and any synthetic sequence with the same functional capability.

[0206] It is known that signals emitted through the TCR alone are insufficient to fully activate T cells, and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct types of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (first cytoplasmic signaling sequence), and one that acts in an antigen-independent manner to provide a secondary or costimulatory signal (second cytoplasmic signaling sequence).

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

[0208] Examples of ITAMs containing a first cytoplasmic signaling sequence that are particularly useful in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP__932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP__004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP__000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP__000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP__000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP__000724.1), and CD5 (NCBI RefSeq: NP__000724.1). The amino acids 402 to 495 of CD79a (NCBI RefSeq:NP__055022.2), 707 to 847 of CD79a (NCBI RefSeq:NP__001762.2), 166 to 226 of CD79a (NCBI RefSeq:NP__001774.1), 182 to 229 of CD79b (NCBI RefSeq:NP__000617.1), and CD66d (NCBI RefSeq:NP__000617.1) were also included. RefSeq:NP_001806.2), as well as variants having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information in NCBI RefSeq ID or GenBank described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0209] In a preferred embodiment, the intracellular domain of the CAR may be designed to contain a CD3-zeta signaling domain by itself, or may be combined with any other desired cytoplasmic domain useful in the context of a CAR. For example, the intracellular domain of a CAR may contain a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. Specific examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP__001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP__000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP__055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP__006130.1), and CD137 (4-1BB, NCBI Peptides having the amino acid sequences of 214 to 255 of CD134 (NCBI RefSeq:NP__001552.2), 241 to 277 of CD134 (OX40, NCBI RefSeq:NP__003318.1), and 166 to 199 of ICOS (NCBI RefSeq:NP__036224.1), and These include, but are not limited to, variants that have the same function as the peptide. Thus, although the present disclosure has thus far been exemplified primarily using 4-1BB as a costimulatory signaling element, other costimulatory elements are within the scope of the present disclosure.

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

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

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

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

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

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

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

[0218] A functional variant may, for example, comprise at least one conservative amino acid substitution in the amino acid sequence of the parent CAR. Alternatively, or in addition, a functional variant may comprise at least one non-conservative amino acid substitution in the amino acid sequence of the parent CAR. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may improve the biological activity of the functional variant, such that the biological activity of the functional variant is superior to that of the parent CAR.

[0219] The amino acid substitutions in CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / charged polar amino acid (e.g., Asp or Glu) with another acidic / charged polar amino acid, the substitution of a non-polar side chain-containing amino acid (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another non-polar side chain-containing amino acid, the substitution of a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, the substitution of a polar side chain-containing uncharged amino acid (e.g., Asn, Gin, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, the substitution of a beta-branched side chain-containing amino acid (e.g., He, Thr, and Val) with another beta-branched side chain-containing amino acid, the substitution of 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] A CAR can consist essentially of one or more of the specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0221] CARs (including functional portions and functional variants) can be of any length, i.e., contain any number of amino acids, so long as the CAR (or functional portion or variant thereof) retains biological activity, such as, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal. For example, a CAR can be from about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more amino acids in length.

[0222] CARs (including functional portions and functional variants of the invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chloro ... Examples of amino acids include phenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0223] CARs (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized or conjugated.

[0224] CAR (including its functional part and functional variant) can be obtained by methods well known in the art. CAR can be produced by any suitable polypeptide or protein production method. Suitable methods for de novo synthesis of polypeptides and proteins are described in prior art documents such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional parts and functional variants thereof) may be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) may be commercially synthesized by companies. In this regard, 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 a CAR, an antibody, or an antigen-binding domain or portion thereof, that specifically binds to one or more of the antigens disclosed herein. As used herein, a "T cell expressing a CAR" or "CAR T cell" refers to a T cell that expresses a CAR and has antigen specificity, e.g., determined by the antibody-derived targeting domain of the CAR.

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

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

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

[0229] Each heavy and light chain contains 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., p. 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind to an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting only of heavy chains are functional and stable even in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH," or "VH," refer to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment, such as Fv, ScFv, dsFv, or Fab. References to "VL," or "VL," refer to the variable domain of an antibody light chain, including that of an Fv, ScFv, dsFv or Fab.

[0230] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity-determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Vol. 1, No. 1, pp. 111-114, 2002). (See, for example, the "International Framework for Antibody Development" (International Standards Board of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved among species. The framework regions of an antibody, i.e., the framework regions of the constituent light and heavy chains, together position and align the CDRs in three-dimensional space.

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

[0232] "Antigen-binding fragments" are portions of full-length antibodies, and various combinations of such portions, that retain the ability to specifically recognize their cognate antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments generated by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

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

[0234] In dsFvs, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the bond between the two chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but are connected to complementary domains on another chain using a linker that is too short to connect the two domains into a single chain, thereby forming two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

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

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

[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 a competition assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are well known, and exemplary competition assays are provided herein.

[0238] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Constant regions may be absent, but if present, may be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90% (e.g., about 95% or more) identical. Thus, all parts of the humanized antibody or antigen-binding fragment (possibly with the exception of the CDRs) are substantially identical to corresponding parts of natural human antibody sequences.

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

[0240] A "fully human antibody" or "human antibody" is an antibody that contains sequences from (or derived from) the human genome and no sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated by using antibody production techniques based on sequences derived from the human genome, for example, by phage display or the use of transgenic animals (see, e.g., Barbas et al., Phage Display). display:A Laboratory Manuel.1st edition New York:Cold Spring Harbor Laboratory Press,2004.Print.;Lonberg,Nat.Biotech.,23:1117-1 125, 2005; see 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, the binding sites may be identical to one another or may be different. For example, a native immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[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, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929, below).

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

[0244] C.conjugates CARs, CAR-expressing T cells, or monoclonal antibodies, or antigen-binding fragments thereof, specific for one or more of the antigens disclosed herein may be conjugated to agents such as effector molecules or detectable markers using any of a number of means well known to those skilled in the art. Both covalent and non-covalent means may be used. Conjugates include, but are not limited to, molecules in which an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein is covalently linked to an effector molecule or detectable marker. Those skilled in the art will be familiar with the use of conjugates that are specifically linked to chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C. 3 H, and 35 It will be understood that a variety of effector molecules and detectable markers can be used, including, but not limited to, radioactive agents such as S, as well as other labels, targeting moieties, and ligands.

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

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

[0247] In some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,28 Nos. 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated by reference in its entirety.

[0248] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolea). The linker can be, for example, a peptide linker cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. 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 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids in length. Proteases may include cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics). 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin-B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[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 be hydrolyzed in the lysosome. Linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, or ketals, etc.) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions (e.g., in blood), but are unstable below pH 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929)).

[0250] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[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 US Publication No. 2005 / 0238649, incorporated herein by reference in its entirety).

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

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

[0255] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to well-known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogs can be prepared synthetically according to well-known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, certain microorganisms were further discovered to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, and 4, Nos. 313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for their preparation, and therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and 6,441,163, and European Patent No. EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0256] Additional toxins can be used with CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Exemplary 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 are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of these toxins (see, e.g., U.S. Patent 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 lacks a mechanism for specific intracellular entry and therefore must bind to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein for efficient entry into cells.

[0258] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins has been mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0259] Ricin is found in Ricinus communis (castor bean) ) is a lectin RCA60 obtained from Ricinus communis. For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. There are two forms of Ricinus communis agglutinin (RCA), which have molecular weights of approximately 65 kD and 120 kD, respectively, and are therefore called RCA. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds lysine to cell surface galactose residues, facilitating transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

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

[0261] Abrin includes toxic lectins obtained from Abrus precatorius. The toxic components, abrins a, b, c, and d, have molecular weights of approximately 63-67 kD and consist of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis, while the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

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

[0263] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may also be conjugated with lanthanides (such as europium and dysprosium) and manganese. Antibodies or antigen-binding fragments may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a secondary antibody binding site, a metal binding domain, an epitope tag, etc.).

[0264] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may also be conjugated with radiolabeled amino acids. Radiolabels may be used for both diagnostic and therapeutic purposes. For example, radiolabels may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabels may be used in therapy as toxins to treat tumors in subjects, such as neuroblastoma. Examples of labels for polypeptides include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 These include, but are not limited to, radioisotopes such as I or radionucleotides.

[0265] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels may be detected using photographic film or a scintillation counter, fluorescent markers may be detected by detecting emitted light using a photodetector. Enzyme labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced 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

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

[0267] In some embodiments, the nucleotide sequence may be codon-modified.Without being bound by any theory, it is believed that the codon optimization of the nucleotide sequence can increase the translation efficiency of mRNA transcripts.The codon optimization of the nucleotide sequence may involve replacing natural codons with other codons that code for the same amino acid but can be translated by tRNAs that are more easily utilized in cells, thus increasing translation efficiency.The optimization of the nucleotide sequence may also reduce secondary mRNA structures that may interfere with translation, thus increasing translation efficiency.

[0268] In one embodiment of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the invention. , may comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0269] As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, may be synthetic or obtained from natural sources (e.g., by isolation and / or purification), may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages (such as phosphoramidate or phosphorothioate linkages instead of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides). In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be preferred for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0270] Recombinant nucleic acids may have sequences that do not occur in nature or sequences that are an artificial combination of two separate regions of a sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificially manipulating separate nucleic acid regions using genetic engineering techniques, such as those described in Sambrook et al., supra. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the duplex formed by hybridization.Examples of modified nucleotides that can be used in nucleic acid generation include 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 adenines, 7-methylguanine, 5-methylaminomethyl Examples of nucleic acids include, but are not limited to, uracil, 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, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention may be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

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

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

[0273] Nucleotide sequences that hybridize under stringent conditions may also hybridize under highly stringent conditions. "Highly stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount detectably greater than nonspecific hybridization. Highly stringent conditions include conditions under which polynucleotides with exactly complementary sequences or those with only a few scattered mismatches can be distinguished from random sequences that coincidentally contain a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more in length, and can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions include, for example, conditions under which a polynucleotide with an exactly complementary sequence or one with only a few scattered mismatches can be distinguished from a random sequence that coincidentally contains a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more in length, and can be easily distinguished by highly stringent hybridization. These conditions may include low salt and / or high temperature conditions, such as NaCl or its equivalent at a temperature of about 50-70°C. Such highly stringent conditions tolerate very little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0274] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical.

[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 purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and that is capable of causing a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with a host cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide in the host cell. Such vectors generally do not occur in nature.

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

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

[0279] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

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

[0281] In one embodiment, recombinant expression vectors may be prepared using standard recombinant DNA techniques, e.g., as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear constructs of expression vectors may be prepared to contain replication mechanisms that function in prokaryotic or eukaryotic host cells. Replication mechanisms may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, and bovine papilloma virus.

[0282] Recombinant expression vectors contain a variety of transcriptional and translational codons, including transcriptional and translational initiation and termination codons, that are appropriate for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, and take into account whether the vector is DNA or RNA based. The recombinant expression vector may contain restriction sites to facilitate cloning.

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

[0284] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding the CAR (including functional portions and functional variants thereof) or a nucleotide sequence complementary to or hybridizing to the CAR-encoding nucleotide sequence. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific, developmental-specific, etc.) is within the ordinary skill of those in the art. Similarly, the association of a nucleotide sequence with a promoter is also within the ordinary skill of those in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0285] Recombinant expression vectors may be designed for either transient expression, stable expression, or both, and may be engineered for constitutive or inducible expression.

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

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

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

[0289] In one embodiment, the CARs described herein can be used in suitable cells that are not T cells, such as those that have immune effector functions, such as NK cells and T-like cells developed from pluripotent stem cells.

[0290] One embodiment also provides a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population, comprising host cells comprising any of the described recombinant expression vectors, as well as at least one other cell type, e.g., host cells that do not comprise any of the recombinant expression vectors (e.g., T cells), or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogeneous population, comprising primarily (e.g., consisting essentially of) host cells comprising the recombinant expression vector. The population may also be a clonal cell population, in which all cells in the population are clones of a single host cell comprising the recombinant expression vector and therefore all cells in the population comprise this recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0291] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the host cells are more pure than they are in their natural environment in the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, or may be greater than about 60%, about 70%, or about 80%, or may be about 100%.

[0292] E. Treatment Method It is contemplated that the CARs disclosed herein may be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, The method includes administering the host cell, the population of cells, the antibody and / or antigen-binding portion thereof, and / or the pharmaceutical composition in an amount effective to treat or prevent cancer in a mammal.

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

[0294] For purposes of this method, in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells may be autologous to the mammal. As used herein, allogeneic refers to any material derived from an animal of the same species as the individual into which the material is introduced, but from a different individual. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci for those individuals are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically different to be able to interact antigenically. As used herein, "autologous" refers to any material derived from the same individual into whom the material will later be reintroduced.

[0295] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, Rodentia mammals, such as mice and hamsters, and Logomorpha mammals, such as rabbits. The mammal may be of the Order Carnivora, which includes Felidae (cats) and Canidae (dogs). The mammal may be of the Order Artiodactyla, which includes Bovinae (cattle) and Porcinae (pigs), or Persodactyla, which includes Equidae (horses). The mammal may be of the Order Primates, Ceboids, or Simoids (monkeys), or Apes (humans and apes). Preferably, the mammal is a human.

[0296] For the methods described above, the cancer may be ALL, AML, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic myeloid carcinoma (CML), colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma. The cancer may be any cancer, including any of the following: hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, NHL, B-chronic lymphocytic leukemia, hairy cell leukemia, Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0297] The terms "treatment" and "prevention," and derivatives thereof, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as potentially beneficial or therapeutic. In this regard, the method can provide any amount or level of cancer treatment or prevention in a mammal.

[0298] Furthermore, the treatment or prevention provided by the method may be effective in treating a variety of conditions, including the disease being treated or prevented. For purposes herein, "prevention" may include the treatment or prevention of one or more conditions or symptoms of a disease (e.g., cancer). For purposes herein, "prevention" may also 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, comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody, and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

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

[0301] For one embodiment of the method for detecting the presence of a proliferative disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a whole cell lysate fraction, e.g., a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction. When the sample contains whole cells, the cells can be any cells of the mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

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

[0303] In addition, detection of the complex may be performed by any of several methods known in the art. For example, the CAR disclosed herein, the polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or antibody described herein, or antigen-binding portion thereof, may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle), as disclosed above.

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

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

[0306] Any method of administration may be used for the disclosed therapeutic agents, including local and systemic administration, for example, topically, orally, intravascularly, such as intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intraspinal, intradermal, intrathecal, intramuscular, intravenous ...venous, Intrathecal and subcutaneous administration may be used. The specific mode of administration and dosing regimen may be selected by the attending clinician, taking into consideration the specifics of the case (e.g., the subject, the disease, any associated disease states, and whether the treatment is prophylactic). When more than one agent or composition is administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally, and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic and pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue area after tumor removal or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as an eye drop or intravitreously to the eye.

[0307] The disclosed therapeutic agents may be formulated in unit dosage forms suitable for administering precise dosage amounts one at a time. Additionally, the disclosed therapeutic agents may be administered in a single-dose or multiple-dose schedule. A multiple-dose schedule may involve an initial series of treatment in which more than one dose (e.g., 1-10 doses) may be administered separately, followed by subsequent doses at intervals, as needed, to maintain or enhance the effect of the composition. Treatment may involve administering the compound once daily or multiple times daily (multi-daily doses) for a period ranging from two to three days to several months or even years. Accordingly, the dosage regimen may be determined, at least in part, based on the specific requirements of the subject being treated and may be dependent on the judgment of the administering physician.

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

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

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

[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 crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenesis agent may be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. The methods and therapeutic dosages for such agents are well known to those skilled in the art and can be determined by skilled clinicians.

[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 folates (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 benzodiazepines (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), and vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antineoplastic antibiotics such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other agents such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are well known to those skilled in the art and can be determined by a skilled clinician.

[0313] Combination therapy can produce synergistic effects and can be proven to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the combined effect achieved when the same compounds are used separately. Synergistic effects can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit-dose preparation, (2) delivered alternately or in parallel as separate preparations, or (3) when some other regimen is used. In the case of alternate delivery, synergistic effects can occur when the compounds are administered or delivered sequentially, for example, by separate injections in separate syringes. Generally, in the case of alternate administration, an effective dosage of each active ingredient is administered sequentially, i.e., consecutively, while in combination therapy, effective dosages of two or more active ingredients are administered together.

[0314] In one embodiment, an antibody that specifically binds to one or more of the antigens disclosed herein Alternatively, an effective amount of an antigen-binding fragment or conjugate thereof is administered to a tumor-bearing subject after anti-cancer treatment. After a sufficient time has passed, the administered antibody, antigen-binding fragment, or conjugate forms an immune complex with the antigen expressed on the respective cancer cells, and the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control obtained before the treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control obtained before the treatment indicates that the treatment is effective.

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

[0316] The administration composition may comprise a solution of the CAR, or CAR-expressing T cells, conjugate, antibody, or antigen-binding fragment, 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 material. The composition may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and adjuvants, as needed to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the CAR, or CAR-expressing T cells, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected depending primarily on fluid volume, viscosity, and body weight, depending on the particular mode of administration selected and the requirements of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are well known or will be apparent to those skilled in the art.

[0317] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing compositions for administration will be well known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0318] The CAR, or T cells, antibodies, antigen-binding fragments, or conjugates expressing the CAR, may be provided in lyophilized form and administered by reconstitution with sterile water, although the CAR may also be administered in sterile solutions of known concentrations. The CAR, or CAR-expressing T cells, antibody, or antigen-binding fragment, or conjugate solution is then loaded into an infusion bag containing 0.9% sodium chloride (USP) and, in some cases, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art in administering antibody, antigen-binding fragment, and conjugate drugs; for example, antibody drugs have been on the U.S. market since the approval of Rituxan® in 1997. The CAR, or CAR-expressing T cells, antibody, or antigen-binding fragment, and conjugate thereof, may be administered by slow infusion rather than intravenous push or bolus. In one example, a higher loading dose is administered, followed by a maintenance dose at a lower rate. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or the corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) may be infused over approximately 90 minutes, and if this initial dose is well tolerated, then weekly maintenance doses of 2 mg / kg may be infused over 30 minutes for 4-8 weeks.

[0319] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particulate systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries are about 5 μm in diameter, only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, edited by A. Kydonieus, Marcel Dekker, Inc., New York, NY, pp. 315-339, (1992).

[0320] Polymers may be used for ion-controlled release of the CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugate compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are well known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous, mobile liquid at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the preparation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). In addition to these, numerous systems for controlled delivery of therapeutic proteins are known (U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 5,188,837). 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 embodiment, further provided is a kit that uses the CAR disclosed herein.For example, a kit for treating tumors in a subject or for generating CAR T cells that express one or more of the CARs disclosed herein.Such kits can typically include the antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cells that express CAR disclosed herein.More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cells that express CAR can be included in the kit.

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

[0323] The label or package insert may typically further include instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells, for example, in a method for treating or preventing tumors or in a method for generating CAR T cells. The package insert typically includes instructions customarily included in commercial packaging for therapeutic products, which include information about the indications, usage, dosage, administration, contraindications, and / or warnings associated with the use of the therapeutic product. The contents of the instructions may be written in electronic format (e.g., a floppy disk or compact disk) or in visual format (e.g., a video file). The kit may further include additional components to facilitate the specific use for which the kit is designed. Thus, for example, the kit may further include label detection means (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, or an appropriate secondary label such as a secondary antibody). The kit may further include buffers and other reagents routinely used in the practice of a particular method. Such kits and suitable contents are well known to those of skill in the art.

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

[0325] Isolation of CD22-specific antibodies from phage- and yeast-displayed fully human ScFv libraries material and method: a) Generation of human ScFv and CD22-specific antibodies A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library (approximately 10 unique specificities as diversity) constructed from peripheral blood B cells of 50 healthy donors was generated. 10 ) (ZYZhu and DSDimitrov, unpublished data) was used to select ScFv for recombinant human CD19 protein (Miltenyi Biotec, unpublished data). 12The amplified ScFv library was incubated with 5 μg, 3 μg, and 1 μg of coated CD22 in a volume of 5 × 100 μl (equally distributed among five wells of a 96-well plate) for 2 hours at room temperature during the first, second, and third rounds of biopanning. After each incubation, wells were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST) five times after the first round and ten times after subsequent rounds to remove nonspecifically bound phage. Bound phage were mixed with TG1 competent cells for 1 hour at 37°C, and the phage were amplified from the infected cells and used in the next round of biopanning. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells and individually inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density at 600 nm (OD600) of 0.5, helper phage M13K07 at a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) of kanamycin were added to the medium, and the plate was further incubated overnight at 30°C in a shaker at 250 rpm. The phage supernatant was mixed with 3% nonfat milk in PBS at a volume ratio of 4:1 and used in enzyme-linked immunosorbent assay (ELISA) to identify phage clones displaying ScFv or VH 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 five times with PBST (after overnight incubation at 4°C, blocking with 3% nonfat milk in PBS and washing three times with PBS containing 0.05% Tween 20). CD22-binding phage was detected using horseradish peroxidase-conjugated goat anti-M13 antibody.After incubation with the antibody, the wells were washed to remove nonspecifically 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 an A450 greater than 1.0 were selected for further characterization.

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

[0327] c) ELISA Binding Assay For ELISA analysis, 50 μl of recombinant human CD22 diluted at 2 μg / ml in PBS was coated onto a 96-well plate overnight at 4°C. Serial dilutions of purified ScFvs bearing His and Flag tags were added to the target protein-coated wells. After washing, a 1:3000 dilution of HRP-labeled anti-Flag antibody was added for 1 hour at room temperature. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated for 10 minutes at room temperature. The reaction was stopped by adding 1N H2SO4, and the OD at 450 nm was read to quantify the relative CD22-binding ability of the ScFvs.

[0328] d) Yeast display of scFv libraries The same ScFv starting material used for phage display was also incorporated into the yeast ScFv display system. To complement the phage-based scFv analysis, a yeast library expressing a human scFv library was also screened. Cell panning of CD22-transfected CHOK1 cells was performed to enrich for yeast expressing scFvs that bind to both recombinant CD22-Fc and CD19 expressed on the cell surface of CHOK1 cells. For the first round of panning on the cell surface, CHOK1-CD22 cells were seeded into 6-well plates and incubated in F12 Grown in K medium to 50% confluence, 5 × 10 yeast cells were then added. 7The cells were washed twice with PBSA buffer, resuspended in 3 mL of F12K medium, and then gently added dropwise to the 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 that had not bound to CHOK1-CD22. Then, 0.05% trypsin-EDTA (Gibco) was used to dissociate the CHOK1-CD22 cells from the bound yeast cells. The cell mixture containing both yeast and CHOK1 cells was then inoculated into 10 mL of SDCAA medium and expanded overnight at 30°C, followed by induction in SGCAA medium at 30°C for 16 hours. For the second round of cell panning, a similar protocol to that described above was performed, using more stringent washing conditions. This panning method yielded 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 a CAR T format revealed that only the 16P binders had specific tumor lytic activity in vitro, and this activity was lower than that of the CAR positive control. Furthermore, when 16P-based CAR T cells were tested in an in vivo xenograft model, their anti-tumor function was very weak (Example 2 below). Taken together, these results indicated that the biological characteristics of CARs generated from this binder set were still suboptimal, and therefore affinity maturation of the anti-CD22 ScFv binders was required.

[0329] To increase the affinity of 16P, a library of mutant scFvs displayed in yeast was generated using error-prone PCR to generate random point mutations in the scFv gene sequence. After electroporation, the resulting mutant library was then grown in SDCAA medium at 30°C overnight for 16 hours, and then switched to SGCAA medium for an additional 16 hours of growth at 30°C. The mutant library was then screened 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 to 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 selecting binders with the highest binding affinity and c-Myc expression level. This process was then repeated. After two additional rounds of this process, the average binding affinity of the mutant pool was higher than that of the starting construct in flow cytometry of yeast particles bearing fluorescently tagged antigen. Binding affinities were assessed by flow cytometry of the yeast pool with decreasing amounts of labeled CD22. This process resulted in an increase in EC50 (effective concentration at which 50% of labeled CD19 binds to yeast displaying ScFv) from 0.5 μg / ml for 16P to less than 0.01 μg / ml for the affinity-matured binders (16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P16, 16P17, 16P20, and 16P20v2).

[0330] result: Due to the inherent challenges of the CD22 structure, phage display candidates failed to produce fully functional CAR constructs with high biological activity and specificity. Therefore, ScFvs for binders with bioactivity and high specificity were generated by yeast display. Based on flow cytometry analysis of yeast-displayed ScFvs, 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 less than 0.01 μg / ml) 16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P17, 16P20, and 16P20v2, respectively. The generation of CARs expressing the 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 the anti-CD22 fully human binding sequence. Homo sapiens CD22 (SIGLEC-2, Leu14) is a well-studied cell surface glycoprotein expressed in B-cell leukemias and lymphomas. At least two anti-CD22 antibody drugs (inotuzumab ozogamicin) or immunotoxin conjugates (moxetumomab pasudotox) have been investigated in clinical trials (NCT02981628, NCT00659425). This approach has met with some success and is currently being investigated, for example, in combination with other chemotherapeutic agents (Muller F, Stookey S, Cunningham T, Pastan I, 2017, Paclitaxel synergizes with exposure time adjusted CD22-targeted immunotoxins against B-cell malignancies, Oncotarget 8:30644-30655). However, given the current advances in T cell-based therapies using CD19 CARs, cell-based immunotherapy appears to be the best approach for targeting CD22-expressing malignancies. Therapies featuring m971-based anti-CD22 CARs are currently in clinical trials at the National Cancer Institute (NCT02315612, PI: Terry Fry, MD), but results have not yet been published. The CAR constructs presented here represent an innovative and novel approach to generating and implementing novel CD22 binding sites derived from human sequences, potentially resulting in significantly different activity profiles in vivo, given the range of cytotoxicity and cytokine production capabilities of individual constructs.

[0332] The novel anti-CD22 CAR-T constructs described herein have high levels of cell surface expression on primary human T cells, specificity against CD22-positive tumor cells, and potent cytotoxic and cytokine function. As in Example 1, phage display Using CD22-binding sequences derived from the ScFv candidates initially identified by Ray, CD22 CARs were designed and cloned under the control of the EF1a promoter into lentiviral expression vectors containing selected structural and signaling domains for characterization and tested in vitro for transduction efficiency, killing function, and cytokine production in both model cell lines and primary human T cells. Table 1 provides an overview of the nomenclature used. CAR constructs LTG1538, anti-CD19 CAR is used as a positive control and comparison. m971 CAR LTG2200 is used as an anti-CD22 CAR positive control.

[0333] [Table 1]

[0334] material and method: (a) Cell line The Burkitt lymphoma cell line Raji and the chronic myeloid leukemia line K562 were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). The 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 luciferase-expressing cell lines were purchased from Fisher Scientific, Grand Island, NY. Luciferase-positive clones 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 donor's 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) according to the manufacturer's protocol.

[0335] (b) Construction of chimeric antigen receptor (CAR) expression vectors The sequence of the CAR antigen-binding domain ScFv was derived from the human anti-CD22 ScFv or heavy chain variable fragment. The CAR T construct was generated by combining an in-frame binder sequence with the CD8a binding and transmembrane domain (aa 123-191, reference sequence number NP_001759.3), followed by 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). The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology). HEK293T cells were transiently transfected to obtain supernatants containing lentiviral vectors (LVs), which were then pelleted by centrifugation of the LV-containing supernatants and stored at -80°C.

[0336] (c) Purification and transduction of primary T cells CD4 + and CD8 + Human primary T cells from healthy volunteers were purified from whole blood or buffy coats using immunomagnetic bead selection of cells (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's protocol. T cells were grown at densities of 0.3–2 × 10 in TexMACS medium supplemented with 200 IU / ml IL-2. 6 Cultured at 100 cells / ml and CD3 / CD28 MACS® GMP T cells were activated using T cell TransAct reagent (Miltenyi Biotec) and transduced overnight with a lentiviral vector encoding a CAR construct on day 2 in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), with the medium changed on day 3. Cultures were propagated in TexMACS medium supplemented with 200 IU / ml IL-2 and then 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). Target-only wells (maximum CPS) and target-only wells with 1% Tween-20 (minimum CPS) were used to determine the assay range. The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants were removed from co-cultures at an E:T ratio of 10:1 and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0338] (e) Flow cytometry analysis For cell staining, 500,000 CAR T-transduced cells were harvested from culture and washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with CD22-Fc peptide followed by anti-Fc-PE conjugate (Jackson ImmunoResearch, West Grove, PA). Anti-CD4 antibody conjugated with VioBlue fluorophore (Miltenyi Biotec) was used where indicated, according to the manufacturer's protocol. Untransduced cells served as a negative control. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice, resuspended in 200 μl of staining buffer, and then quantitatively analyzed by flow cytometry. 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). Five hundred thousand mouse-adapted Raji-luc cells were cultured in NSG (NOD.Cg-Prkdc) mice. scid Il2rg tm1Wjl Raji-luc was injected into the tail vein of 1000 (SzJ) mice. Six days after Raji-luc injection, tumor engraftment was measured by intraperitoneal injection of 150 mg / kg luciferin and 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 for each mouse was calculated as mean brightness (photons / second / cm). 2The tumor growth and eradication kinetics were expressed as mean tumor volume / steradian. On day 7, CAR T cells were administered to mice by tail vein injection. Imaging was performed at the indicated days after CAR T injection to establish the kinetics of tumor growth and eradication by CAR T cells.

[0340] result: To evaluate novel anti-CD22 fully human ScFv binding sequences, we designed Set 1 CAR constructs incorporating constructs 2246-2249 (ScFv sequences derived from the phage display library, Table 1; ScFv1(16P), ScFv2(24P), ScFv3(25P), ScFv4(11S), ScFv5(12S), and CAR construct 2202 (m971 positive control) as tumor antigen-binding domains). Each CAR was designed with a tumor-targeting domain followed by a linker and transmembrane domain from the human CD8 protein, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain (Table 2 below).

[0341] [Table 2]

[0342] T cells transduced with anti-CD22 chimeric antigen receptors exhibit 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. Primary human T cells derived from healthy donors were transduced with the lentiviral vector encoding the CAR. Untransduced cells (referred to as UTD or mock) from the same donor or GFP-transduced cells from the same donor were used as negative controls. Data represent results from at least three assays from different donors.

[0344] [Table 3]

[0345] As described in Materials and Methods, T cells were activated in the presence of IL-2 on day 0 using TransAct T cell reagent (Miltenyi Biotec, Inc.). On days 8–10 of culture, anti-CD22 CAR expression on the surface of transduced T cells was detected by staining with protein L conjugated to biotin followed by streptavidin-PE reagent. Alternatively, CAR T cells were stained with CD22-Fc peptide (R&D Systems, Inc.) followed by anti-Fc-PE antibody, and data were acquired by flow cytometry (Figure 3). All CAR constructs, except for CAR2247, which consists of a lambda light chain (and therefore does not react with protein L), showed surface CAR expression of greater than 50–70% as detected by protein L staining. In contrast, the anti-CD22 ScFv antigen-binding site When stained with CD22-Fc peptide that specifically binds to CAR16P, only CAR16P demonstrated CAR expression (up to 30%) (CAR24P not shown). These data indicate that although the majority of CAR constructs were expressed on the T cell surface, only the CAR16P construct exhibited an ScFv configuration that maintained CD22 protein binding.

[0346] b) Cytolytic and cytokine assays of anti-CD22 CAR To demonstrate the cytolytic function of the generated CAR T cells, a luciferase-based killing assay was performed by combining CAR-T with CD22-positive Raji-luc cells, CD22-positive Reh-luc cells, and CD22-negative K562-luc cells at E:T ratios of 20:1, 10:1, 5:1, or 2.5:1 in an overnight cell killing assay, as described in Materials and Methods (Figure 4). The anti-CD19 CAR construct 1538 inhibited Raji and Reh (CD19 +) but reacts with K562 (CD19 - ) line, which has previously been shown to be insensitive to CD22 and was used as a positive control. Only CAR2202 (binder 16P) demonstrated dose-dependent, CD22-specific tumor killing; CARs 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 Materials and Methods. Tumors were implanted via the tail vein on day 0, staged on day 6, and mice were intravenously injected with 4 x 10 CAR T cells on day 7. 6 Treatment groups included 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 study day 18, whereas the test construct, CAR 2202(16P), only moderately slowed tumor progression, and its effect was indistinguishable from the negative control treatment (UTD) after study day 32. Thus, the antitumor activity of the ScFv binder 2202(16P) in vitro and in vivo was weak, necessitating the creation of additional CAR constructs incorporating improved ScFv binder sequences.

[0348] Set 2 CAR constructs (LTG numbers 2203-2220) (Table 1, Set 2, below) were constructed incorporating ScFv binder sequences to improve affinity for CD22, as described in Materials and Methods. The derivation of affinity-improved ScFv binders is described in Example 1. LVs encoding Set 2 CAR constructs under the control of the human EF1a promoter were generated and tested for expression and function in vitro as described above. Briefly, T cells were activated on day 0 of culture using TransAct T cell reagent (active association of CD3 and CD28 antigens, Miltenyi Biotec, Inc.) in the presence of IL-2, as described in Materials and Methods. On days 8-10 of culture, CAR T cells were harvested and CAR surface expression was assessed by flow cytometry. CTL activity was assessed by co-incubation assay, and secretion of inflammatory cytokines was assessed by ELISA. A comparative summary of the functional results 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] Testing of Set 2 CAR constructs (LTG numbers 2203-2220) This was performed in at least three separate experiments by transducing LV-encoded CAR sequences into cells from independent donors. Transduction of CAR T constructs from Set 2 into donor cells typically resulted in CAR expression ranging from 20% to 80%, as detected by CD22-Fc staining, consisting mostly of CD4+ T cells (Figure 6, selected constructs).

[0350] The CTL activity of CARs was determined by co-incubating CAR T cells with luciferase-expressing tumor cells at E:T ratios ranging from 10:1 to 2.5:1 overnight (Figure 7). Residual luciferase activity 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 exhibited robust killing activity in the CD22+ Raji lymphoma and Reh leukemia lines, whereas the negative control constructs GFP and UTD did not produce lysis (Figure 7A). The exception was construct 2202 (16P), which killed the tumor lines to a relatively moderate extent. The CAR19-targeting control 1538 and the CD22-targeting control 2200 (m971) killed Raji and Reh tumors, as expected. The K562 lines (CD22-K562 and CD19-K562 lines) exhibited background killing activity for the positive control constructs 2202 and 1538, resulting in 40% and 60% tumor cell lysis, respectively, at an E:T ratio of 10:1. This activity is likely due to the indirect action of inflammatory cytokines secreted by CAR T cells or the activity of contaminating NK / NKT. The killing activity of the test CAR22 constructs against K56 was comparable to or slightly higher than that of the control 2200 (m971). To further demonstrate the specificity of the novel CAR22 constructs, we used K562 cells engineered to stably express CD22 or CD19 (Figure 7B).

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

[0352] In contrast, in the K562-CD22 line, the CD22 CAR control CAR22 2200 (m971) and most of the novel CAR22 constructs exhibited 80% specific CTL activity, whereas the nonspecific killing activity of the CAR19 control 1538 was low at 20%. Thus, despite the sensitivity of the K562 line to the CAR constructs, all novel CD22 CARs tested exhibited specific lytic activity against CD22-expressing targets.

[0353] We then measured the concentrations of the proinflammatory cytokines IFN-gamma, TNF-alpha, and IL-2 secreted by CAR T cells transduced with the CAR22 constructs when challenged with the CD22-positive cell lines Raji and REH (Figure 8). A CAR T cell-alone control was included with each construct to examine basal levels of cytokine production. + T cells exposed to Raji cells strongly induced levels of TNF-alpha, IFN-gamma, and IL-2, whereas for most CAR22 constructs, Reh tumors only affected the induction of IFNg and TNFa, but not IL-2. For a subset of constructs, T cell-alone controls also showed cytokine induction, raising the possibility that the constructs are activated in the absence of specific ligand. Thus, CAR design and binder selection are not trivial, as they must be active in soluble IgG or ScFv formats and expressible on the T cell surface in CAR T formats. However, some binders applicable to this study have low killing or cytokine production efficiency when incubated with CD22-positive tumors. For example, in the case of LTG2217 (16P3v2 binder) and LTG2220 (16P15), these constructs exhibited autoactivation of T cells in the absence of tumor targets, resulting in sustained production of IL-2 and TNF-alpha. This may have adverse effects on clinical use, and therefore disqualify LTG2217 and LTG2220 as therapeutic candidates.

[0354] To avoid non-specific activation of CAR22 cells, Applicants identified constructs that secrete no or minimal 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 an NSG Raji xenograft tumor model. Constructs 2200 (m971) and 1538 (FMC63) were used as positive controls, and tumor alone (TA) and untransduced T cells (UTD) were used as negative controls. Experimental procedures were performed as detailed in Materials and Methods. Mice were implanted with Raji-luciferase-expressing tumor cells on day 0, followed by CAR T treatment on day 7. Weekly bioluminescence measurements were performed beginning on study day 6 to monitor tumor progression and CAR T activity (Figure 9). Compared to the negative control groups TA and UTD, which progressed unabated after day 6, CAR test constructs 2209 and 2209 inhibited tumor progression, reducing tumor bioluminescence to baseline levels at the start of treatment (day 6) by day 21. The antitumor activity of test constructs 2209 and 2209 was equal to or greater than the positive control CD22 CAR construct 2200(m971).

[0356] In summary, novel affinity-improved fully human anti-CD22 CAR constructs LTG Nos. 2203-2220 (Table 2 below) derived from a yeast screening library were shown to be highly functional. In particular, CAR constructs 2209, 2219, and 2218 exhibited activity profiles superior to or distinct from the positive control LTG2220 (m971), and are therefore expected to have high therapeutic activity.

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

[0358] The foregoing description of several specific embodiments provides sufficient information to enable others, by application of their current knowledge, to easily modify or adapt the specific embodiments for various uses without departing from the general concept, and such adaptations and modifications should therefore be understood to be within the meaning and range of equivalents of the disclosed embodiments. It is understood that the words or terminology used herein are for purposes of description and not of limitation. In the drawings and description, exemplary embodiments are disclosed. Although specific terminology may be employed, these are used generically and for descriptive purposes only, unless otherwise indicated, and not for purposes of limitation, and are not intended to limit the scope of the claims. Moreover, those of ordinary skill in the art will understand that certain steps of the methods disclosed herein may be performed in an alternate order or steps may be combined. Accordingly, it is not intended that the claims appended hereto be limited to the specific embodiments disclosed herein. Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the inventive embodiments described herein. Such equivalents are encompassed by the following claims.

[0359] Sequences according to the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strand is included when referring to the displayed strand. In the accompanying sequence listing: SEQ ID NO: 1 is the nucleic acid sequence of CD22-specific binder (scFv1) 16P: CAAGTACAACTCCAGCAAAGCGGGCCTGGTCTGGTGAAGCCGTCACAGACGCTTTCACTTACGTGTGCGATCTCCGGTGACTCCGTGAGTTCTAATAGCGCGGCTTGGAACTGGATTAGGCAGTCTCCATCCCGAGGATTGGAATGGCTCGGCAGGACTTATTATAGAAGTAAGTGGTACAAC GATTATGCAGTCTCTGTGAAATCTCGCATCACCATTAACCCAGACACGTCTAAGAATCAGTTCAGTCTTCAACTCAACTCTGTAACCCCCGAAGATACAGCGGTCTACTACTGTGCTCAGGAGGTGCAACCCCACGATGCTTTTGATATCTGGGGCCAGGGTACCATGGTTACGGTGTCTTCT GGGGGAGGGGGGTCCGGTGGGGGAGGATCAGGGGGTGGGGGCAGCGACATACAAATGACGCAATCCCCGTCTTCTGTTTCTGCGTCTGTCGGAGATAAAGTAACAATAACCTGTCGAGCGTCACAGGACGTTAGTGGCTGGCTTGCGTGGTATCAGCAAAAACCGGGGCTCGCCCCGCAATTG CTTATATTTGGAGCGAGTACTCTTCAGGGCGAGGTACCTAGCAGATTTTCTGGGTCCGGCTCAGGTACGGACTTCACCCTGACCATATCTAGCTTGCAGCCTGAAGATTTCGCCACCTACTATTGTCAACAGGCGAAGAACTTTCCATATACGTTCGGGCAGGGTACGAAATTGGAGATAAAA SEQ ID NO: 2 is the amino acid sequence of CD22-specific binder (scFv1) 16P: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPHDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKR SEQ ID NO: 3 is the nucleic acid sequence of CD22 CAR LTG2202 (LP-scFv1-CD8TM-41BB-CD3zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTACAACTCCAGCAAAGCGGGCCTGGTCTGGTGAAGCCGTCACAGACGCTTTCACTTACGTGTGCGATCTCCGGTGACTCCGTGAGTTCTAATAGCGCGGCTTGGAACTGGATTAGGCAGTCTCCATCCCGAGGATTGGAATGGCTCGGCAGGACTTATTATAGAAGTAA SEQ ID NO: 4 is the amino acid sequence of CD22 CAR LTG2202 (LP-scFv1-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVQPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 5 is the amino acid sequence of scFv1(16P)LCDR1: QDVSGW SEQ ID NO: 6 is the amino acid sequence of scFv1(16P)LCDR2: GAS SEQ ID NO: 7 is the amino acid sequence of scFv1(16P)LCDR3: QQAKNFPYT SEQ ID NO: 8 is the amino acid sequence of scFv1(16P)HCDR1: GDSVSSNSAA SEQ ID NO: 9 is the amino acid sequence of scFv1(16P)HCDR2: TYYRSKWYN SEQ ID NO: 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: CAAGTACAGCTGCAACAATCTGGCCCTGGGCTTGTGAAACCTCTCAGACTTTGTCCTTGACGTGCGCGATAAGTGGCGATTCAGTTAGTTCTAACAGCGCCGCTTGGAACTGGATTAGACAGAGCCCCAGTCGGGGACTCGAATGGCTTGGCCGGACTTATTATCGCAGTAAATGGTATAAT GATTATGCTGTGAGTGTGAAAAGTAGGATCACAATCAACCCCGATACGAGCAAGAATCAATTCTCATTGCAACTGAACAGCGTCACTCCCGAGGATACAGCTGTATATTATTGTGCAAGAGAAGGTGGGTGGTATGGCGATGGATGTATGGGGGAAAGGAACTACGGTAACTGTGTCCAGT GGCGGAGGCGGTTCAGGTGGTGGAGGCTCTGGAGGAGGAGGGTCCGAAATCGTGCTTACCCAGTCTCCGGCTACTCTGAGCGTTAGTCCGGGTGAAAGGGCCTCACTCTCTTGTCGAGCTTCACAGTCAGTCTCTTCCTACTTGGCTTGGTATCAGCAGAAGCCAGGTCAGGGCCCCGCTTG CTCATTTACGACGCAAGCACACGAGCGACAGGCATTCCAGACAGATTTTCTGGGTTCTGGTTCTGGCACGGACTTTACTCTTACTATAAACTCACTTGAGGCAGAGGATGCTGCGACTTACTATTGTCACCAATCAAGCTCTCTGCCTTACACCTTTGGGCAAGGCACCAAACTCGAAATCAAG SEQ ID NO: 12 is the amino acid sequence of CD22-specific binder (scFv2) 24P: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGGWYGEMDVWGKGTTVTVSS GGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERASLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLPYTFGQGTKLEIKR SEQ ID NO: 13 is the nucleic acid sequence of CD22 CAR LTG2246 (LP-scFv2-CD8TM-41BB-CD3zeta): ATGCTGCTGTTGGTGACATCACTTCTGCTCTGTGAACTCCCCCATCCAGCCTTTCTGCTTATACCGCAAGTACAGCTGCAACAATCTGGCCCTGGGCTTGTGAAACCTCTCAGACTTTGTCCTTGACGTGCGCGATAAGTGGCGATTCAGTTAGTTCTAACAGCGCCGCTTGGAACTGGATTAGACAGAGCCCCAGTCGGGGACTCGAATGGCTTGGC CGGACTTATTATCGCAGTAAATGGTATAATGATTATGCTGTGAGTGTGAAAAGTAGGATCACAATCAACCCCGATACGAGCAAGAATCAATTCTCATTGCAACTGAACAGCGTCACTCCCGAGGATACAGCTGTATATTATTGTGCAAGAGAAGGTGGGTGGTATGGCGAGATGGATGTATGGGGGAAAGGAACTACGGTAACTGTGTCCAGTGGCGGAG SEQ ID NO: 14 is the amino acid sequence of CD22 CAR LTG2246 (LP-scFv2-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARE GGWYGEMDVWGKGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERASLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGTDFTLTINSLEAEDAATYYCH QSSSLPYTFGQGTKLEIKVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 15 is the amino acid sequence of scFv2(24P)LCDR1: QSVSSY SEQ ID NO: 16 is the amino acid sequence of scFv2(24P)LCDR2: DAS SEQ ID NO: 17 is the amino acid sequence of scFv2(24P)LCDR3: HQSSSLPYT SEQ ID NO: 18 is the amino acid sequence of scFv2(24P)HCDR1: GDSVSSNSAA SEQ ID NO: 19 is the amino acid sequence of scFv2(24P)HCDR2: TYYRSKWYN SEQ ID NO: 20 is the amino acid sequence of scFv2(24P)HCDR3: AREGGWYGEMDV SEQ ID NO: 21 is the nucleic acid sequence of CD22-specific binder (scFv3) 25P: CAAGTACAGCTCCAACAGAGTGGACCTGGTCTCGTTAAGCCGTCCCAAACACTGTCTTTGACGTGCGCTATTAGTGGCGACAGCGTATCATCCAATTCTGCTGCTTGGAACTGGATTAGACAGTCACCGTCCAGAGGCTTGGAATGGCTGGGCAGGACGTACTACCGCTCAAAATGGTATAACGAT TACGCGGTTAGTGTCAAATCCAGGATTACCATTAACCCTGACACAAGTAAGAATCAGTTTTCTCTTCAGCTGAATTCCTGACTCCTGAGGATACGGCCGTTTACTACTGTGCCCGAGAACACCAGAATGAGGCGGCTTTTGATATTTGGGGGCAAGGAACAATGGTCACAGTTAGCAGTGGGGGG GGTGGCTCCGGGGGAGGTGGTTCCGGCGGCGGTGGTTCTCAATCCGTCCTGACACAACCTCCCTCAGCGAGCGGGACTCCCGGTCAAAGGGTGACCATCTCTTGTTCTGGGGGAGGTAGTAACATCGGGACAAATACTGCGTCCTGGTATCAGCAACTCCCTGGGACCGCTCCCAAGTTGTTGATA TATCGCAATACGCAACGACCTAGTGGATACCTGATAGATTCAGCGGAAGCAAAAGTGGTACGAGTGCGTCTTTGGCAATATCTGGCCTCCAGTCCGAGGACGAAGCGGATTACTATTGTGCGGCCTGGGATGACTCACTGAATGGTTATGTGTTCGGTGCAGGTACTCAACTCACCGTACTTGGT SEQ ID NO: 22 is the amino acid sequence of CD22-specific binder (scFv3) 25P: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSLTPEDTAVYYCAREHQNEAAFDIWGQGTMVTVSSGG GGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGGGSNIGTNTASWYQQLPGTAPKLLIYRNTQRPSGIPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGAGTQLTVLG SEQ ID NO: 23 is the nucleic acid sequence of CD22 CAR LTG2247 (LP-scFv3-CD8TM-41BB-CD3zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTACAGCTCCAACAGAGTGGACCTGGTCTCGTTAAGCCGTCCCAAACACTGTCTTTGACGTGCGCTATTAGTGGCGACAG CGTATCATCCAATTCTGCTGCTTGGAACTGGATTAGACAGTCACCGTCCAGAGGCTTGGAATGGCTGGGCAGGACGTACTACCGCTCAAAATGGTATAACGATTACGCGGTTAGTGTCAAATCCAGGATTACCATTAACCCTGACACAAG TAAGAATCAGTTTTCTCTTCAGCTGAATTCCTGACTCCTGAGGATACGGCCGTTTACTACTGTGCCCGAGAACACCAGAATGAGGCGGCTTTTGATATTTGGGGGCAAGGAACAATGGTCACAGTTAGCAGTGGGGGGGGTGGCTCCGG GGGAGGTGGTTCCGGCGGCGGTGGTTCTCAATCCGTCCTGACACAACCTCCCTCAGCGAGCGGGACTCCCGGTCAAAGGGTGACCATCTCTTGTTCTGGGGGAGGTAGTAACATCGGGACAAATACTGCGTCCTGGTATCAGCAACTCCC TGGGACCGCTCCCAAGTTGTTGATATATCGCAATACGCAACGACCTAGTGGGATACCTGATAGATTCAGCGGAAGCAAAAGTGGTACGAGTGCGTCTTTGGCAATATCTGGCCTCCAGTCCGAGGACGAAGCGGATTACTATTGTGCGGCCTGGGATGACTCACTGAATGGTTATGTGTTCGGTGCAGGTACTCAACTCACCGTACTTGGTGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 24 is the amino acid sequence of CD22 CAR LTG2247 (LP-scFv3-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSLTPEDTAVYYCARE HQNEAAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSASGTPGQRVTISCSGGGSNIGTNTASWYQQLPGTAPKLLIYRNTQRPSGIPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGYVFGAGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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 CD22-specific binder (scFv4) 11s: CAAGTCCAGTTGCAACAGTCCGGGCCAGGTCTGGTTAAGCCATCCCAAACTCTGAGTTTGACGTGCGCTATTAGCGGAGATTCCGTGTCCAGCAATTCTGCAACCTGGAATTGGATCCGGCAGAGTCCGAGTGGCGGTTTGGAATGGCTCGGACGCACTTACTACAGGAGCAAATGGTACGATGATTATGCTGTTTCTGTGCGCTCTCGAATCACCATGAATCCTGATACTTCTAAGAACCAATTTTCTTTGCAGTTGAACTCCGTCACGCCTGAAGATACTGCGGTCTACTATTGCGCACGCGAAGGCGTAGCCGGCGATTTTGATTACTGGGGGCAAGGAACATTGGTCACGGTCTCCTCTGGTGGAGGAGGATCAGGAGGCGGGGGTTCAGGTGGAGGTGGGAGCGATATTCAACTTACGCAGTCTCCGAGCAGTCTTTCTGCTTCCGTGGGAGACCGAGTGACGATTACTTGTAGGGCATCTCAGTCAATAAGTTCCTATCTTAACTGGTATCAGCAGAAGCCTGGAAAGGCTCCAAAACTTCTTATTTATGCCGCATCCTCATTGCAATCCGGCGTGCCTTCCCGATTTTCCGGATCTGGCTCAGGCACTGACTTTACCTTGACTATTAGTTCCCTTCAACCAGAAGATTTTGCTACCTATTACTGCCAACAATCATACAGTACCCCATATACATTCGGCCAAGGCACGAAATTGGAGATTAAA SEQ ID NO: 32 is the amino acid sequence of CD22-specific binder (scFv4)11s: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSGGLEWLGRTYYRSKWYDDYAVSVRSRITMNPDTSKNQFSLQLNSVTPEDTAVYYCAREGVAGDFDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIKR SEQ ID NO: 33 is the nucleic acid sequence of CD22 CAR LTG2248 (LP-scFv4-CD8TM-41BB-CD3zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTCCAGTTGCAACAGTCCGGGCCAGGTCTGGTTAAGCCATCCCAAACTCTGAGTTTGACGTGCGCTATTAGCGGAGATTCCGTGTCCAGCAATTCTGCAACCTGGAATTGGATCCGGCAGAGTCCGAGTGGCGGTTTGGAATGGCTCGGACGCACTTACTACAGGAGCAAATGGTACGATGATTATGCTGTTTCTGTGCGCTCTCGAATCACCATGAATCCTGATACTTCTAAGAACCAATTTTCTTTGCAGTTGAACTCCGTCACGCCTGAAGATACTGCGGTCTACTATTGCGCACGCGAAGGCGTAGCCGGCGATTTTGATTACTGGGGGCAAGGAACATTGGTCACGGTCTCCTCTGGTGGAGGAGGATCAGGAGGCGGGGGTTCAGGTGGAGGTGGGAGCGATATTCAACTTACGCAGTCTCCGAGCAGTCTTTCTGCTTCCGTGGGAGACCGAGTGACGATTACTTGTAGGGCATCTCAGTCAATAAGTTCCTATCTTAACTGGTATCAGCAGAAGCCTGGAAAGGCTCCAAAACTTCTTATTTATGCCGCATCCTCATTGCAATCCGGCGTGCCTTCCCGATTTTCCGGATCTGGCTCAGGCACTGACTTTACCTTGACTATTAGTTCCCTTCAACCAGAAGATTTTGCTACCTATTACTGCCAACAATCATACAGTACCCCA TATACATTCGGCCAAGGCACGAAATTGGAGATTAAAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 34 is the amino acid sequence of CD22 CAR LTG2248 (LP-scFv4-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSGGLEWLGRTYYRSKWYDDYAVSVRSRITMNPDTSKNQFSLQLNSVTPEDTAVYYCA REGVAGDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQSYSTPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREGDDALDIWGQGTMVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISHFLNWYQQKPGTAPKLLITTASGLGSGVPSRFSGSGSGTDFTLTINSLQPEDLATYYCQQSYTTPLTFGGGTKLEIKR SEQ ID NO: 43 is the nucleic acid sequence of CD22 CAR LTG2249 (LP-scFv5-CD8TM-41BB-CD3zeta): ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACAAGTTCAGTTGCAGCAGAGTGGCCCTGGGCTTGTTAAACCATCACAGACGCTCTCACTGACCTGTGCCATCTCTGGAGACAGTGTAAGTTCTAACTCAGCCGCGTGGAATTGGATTAGACAATCACCAAGCCGGGGACTTGAATGGCTTGGTCGGACGTACTATAGATCTAAGTGGTATAATGACTACGCAGTGTCAGTGAAATCACGGATAACCATAAACCCTGACACCAGCAAAAACCAATTTTCTCTTCAGCTTAATTCCGTCACGCCAGAAGATACGGCCGTTTACTACTGTGCGAGGGAAGGTGATGACGCATTGGACATCTGGGGTCAGGGGACCATGGTGACTGTCTCTTCCGGCGGGGGGGGTAGTGGAGGGGGTGGCTCAGGTGGTGGCGGGTCAGATATACAAATGACACAGAGCCCTAGTAGTCTGAGTGCTTCAGTGGGCGACCGCGTAACTATAACCTGTAGAGCATCCCAAAGCATTTCCCACTTCCTTAATTGGTACCAGCAGAAGCCGGGCACAGCGCCCAAACTCCTGATCACCACTGCGAGCGGACTTGGTTCAGGTGTTCCTAGCCGGTTTAGTGGGTCAGGTAGCGGTACAGATTTCACTCTCACGATAAACTCCCTTCAGCCTGAGGACCTGGCGACATATTACTGTCAACAATCCTATACCACCCCACTGACATTCGGAGGGGGCACAAAACTGGAGATCAAAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGC CGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 44 is the amino acid sequence of CD22 CAR LTG2249 (LP-scFv5-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCA REGDDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISHFLNWYQQKPGTAPKLLITTASGLGSGVPSRFSGSGSGTDFTLTINSLQPEDLATYYC QQSYTTPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 45 is the amino acid sequence of scFv5(12s)LCDR1: QSISHF SEQ ID NO: 46 is the amino acid sequence of scFv5(12s)LCDR2: TAS SEQ ID NO: 47 is the amino acid sequence of scFv5(12s)LCDR3: QQSYTTPLT SEQ ID NO: 48 is the amino acid sequence of scFv5(12s)HCDR1: GDSVSSNSAA SEQ ID NO: 49 is the amino acid sequence of scFv5(12s)HCDR2: TYYRSKWYN SEQ ID NO: 50 is the amino acid sequence of scFv5(12s)HCDR3: AREGDDALDI SEQ ID NO: 51 is the nucleic acid sequence of the CD22-specific binder (scFv6) 16P3: CAGATACAGTTGCAGCAGTCAGGTCCAGGACTAGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTACAACCTGATGATGCTTTAGATATCTGGGGCCAAGGGACAATGGTCACCGT CTCTTCAGGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTCTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAAAATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 52 is the amino acid sequence of CD22-specific binder (scFv6) 16P3: QIQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDALDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIK SEQ ID NO: 53 is the nucleic acid sequence of CD22 CAR LTG2203 (LP-scFv6-CD8TM-41BB-CD3zeta): CCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTAC AACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 54 is the amino acid sequence of CD22 CAR LTG2203 (LP-scFv6-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQIQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVQPDDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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 the CD22-specific binder (scFv7) 16P16: CAAGTACAGTTGCAGCAGTCAGGACCTGGCCTTGTGAAACCATCCCAAACTCTCAGCCTCACGTGTGCTATTTCTGGTGACTCAGTAAGTAGCAATAGCGCTGCTTGGAACTGGATCAGACAATCTCCC TCCAGGGGTCTCGAATGGCTGGGGCGAACCTATTACCGATCTAAATGGTATAACGATTATGCAGTATCCGTGAAATCCAGGATTACAATCAACCCAGATACGTTCAAGAATCAATTCTCTCTTCAGCTCA ACTCCGTAACTCCAGAGGACACTGCGGTATATTATTGCGCCCAAGAAGTCGAGCCACACGATGCCCTCGATATCTGGGGTCAAGGTACCATGGTTACAGTTAGTAGTGGGGGTGGGGGAAGCGGGGGCGG TGGGTCCGGTGGCGGGGGTTCAGACATCAAGATGACCCAATCCCCAAGCTCTGTTTCAGCATCCGTGGGCGATAAGGTAACCATTACATGCAGAGCGAGTCAGGACGTTTCAGGGTGGCTGGCTTGGTAC CAGCAAAACCGGGACTCGCACCGCAGCTGTTGATTTTCGGCGCCAGTAGCTTCAGGGCGAAGTACCGTCCAGGTTCAGTGGGTCAGGTTCTGGCACCGATTTTACGCTCACGATATCCAGTCTCCAACCGGAGGATTTTGCTACTTATTACTGCCAGCAGGCTAAGTATTTTCCATACACATTTGGCCAGGGGACAAAGTTGGAGATCAAA SEQ ID NO: 62 is the amino acid sequence of CD22-specific binder (scFv7) 16P16: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVYYYCAQEVEPHDALDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 63 is the nucleic acid sequence of CD22 CAR LTG2204 (LP-scFv7-CD8TM-41BB-CD3zeta): CGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 64 is the amino acid sequence of CD22 CAR LTG2204 (LP-scFv7-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVYYCAQ EVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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 SEQ ID NO: 68 is the amino acid sequence of scFv7(16P16)HCDR1: GDSVSSNSAA SEQ ID NO: 69 is the amino acid sequence of scFv7(16P16) HCDR2: TYYRSKWYN SEQ ID NO: 70 is the amino acid sequence of scFv7(16P16) HCDR3: AQEVEPHDALDI SEQ ID NO: 71 is the nucleic acid sequence of CD22-specific binder (scFv8) 16P20: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGT CCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCATGATGCTCTTGATATCTGGGGC CAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGCGGAGGCGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACGGCAGTCTCCATCATCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTT AGCCTGGTATCAACAGAAACCAGGGCTAGCCCCTCAGCTCCTGATCTTTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAAC AGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 72 is the amino acid sequence of CD22-specific binder (scFv8) 16P20: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 73 is the nucleic acid sequence of CD22 CAR LTG2205 (LP-scFv8-CD8TM-41BB-CD3zeta): SEQ ID NO: 74 is the amino acid sequence of CD22 CAR LTG2205 (LP-scFv8-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 75 is the amino acid sequence of scFv8(16P20)LCDR1: QDVSGW SEQ ID NO: 76 is the amino acid sequence of scFv8(16P20)LCDR2: GAS SEQ ID NO: 77 is the amino acid sequence of scFv8(16P20)LCDR3: QQAKYFPYT SEQ ID NO: 78 is the amino acid sequence of scFv8(16P20)HCDR1: GDSVSSNSAA SEQ ID NO: 79 is the amino acid sequence of scFv8(16P20) HCDR2: TYYRSKWYN SEQ ID NO: 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: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAT GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATTCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCCATGATGCTCTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA GGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCAAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTC CTGATCTTTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 82 is the amino acid sequence of CD22-specific binder (scFv9) 16P2: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWI RQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVYYCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIKM TQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 83 is the nucleic acid sequence of CD22 CAR LTG2206 (LP-scFv9-CD8TM-41BB-CD3zeta): SEQ ID NO: 84 is the amino acid sequence of CD22 CAR LTG2206 (LP-scFv9-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTFKNQFSLQLNSVTPEDTAVY YCAQEVEPHDALDIWGQGTMVTVSSGGGGSGGGGSGGGSDIKMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 85 is the amino acid sequence of scFv9(16P2)LCDR1: QDVSGW SEQ ID NO: 86 is the amino acid sequence of scFv9(16P2)LCDR2: GAS SEQ ID NO: 87 is the amino acid sequence of scFv9(16P2)LCDR3: QQAKYFPYT SEQ ID NO: 88 is the amino acid sequence of scFv9(16P2)HCDR1: GDSVSSNSAA SEQ ID NO: 89 is the amino acid sequence of scFv9(16P2) HCDR2: TYYRSKWYN SEQ ID NO: 90 is the amino acid sequence of scFv9(16P2) HCDR3: AQEVEPHDALDI SEQ ID NO: 91 is the nucleic acid sequence of CD22-specific binder (scFv10) 16P6: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAT GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGATACGGCTGTGTATTACTGTGCCCAAGAGGTACAACCTGATGATGCTTTTGATATCTGGGGCCAAGGGACAATGATCACCGTCTCTTCA GGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTC CTGATCTCTGGTGCATCCACTTTGCAAGGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAAAATTTCCCTTACACTTTTGGTCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 92 is the amino acid sequence of CD22-specific binder (scFv10) 16P6: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYC AQEVQPDDAFDIWGQGTMITVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIK SEQ ID NO: 93 is the nucleic acid sequence of CD22 CAR LTG2207 (LP-scFv10-CD8TM-41BB-CD3zeta): SEQ ID NO: 94 is the amino acid sequence of CD22 CAR LTG2207 (LP-scFv10-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDAFDIWGQGTM ITVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 95 is the amino acid sequence of scFv10(16P6)LCDR1: QDVSGW SEQ ID NO: 96 is the amino acid sequence of scFv10(16P6)LCDR2: GAS SEQ ID NO: 97 is the amino acid sequence of scFv10(16P6)LCDR3: QQAKNFPYT SEQ ID NO: 98 is the amino acid sequence of scFv10(16P6)HCDR1: GDSVSSNSAA SEQ ID NO: 99 is the amino acid sequence of scFv10(16P6) HCDR2: TYYRSKWYN SEQ ID NO: 100 is the amino acid sequence of scFv10(16P6) HCDR3: AQEVQPDDAFDI SEQ ID NO: 101 is the nucleic acid sequence of CD22-specific binder (scFv11) 16P10: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAT GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCAGGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA GGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTC CTGATCTTTGGTGCATCCACTCTGCAAGGTGAAGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCCGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 102 is the amino acid sequence of CD22-specific binder (scFv11) 16P10: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGPGTKLEIK SEQ ID NO: 103 is the nucleic acid sequence of CD22 CAR LTG2208 (LP-scFv11-CD8TM-41BB-CD3zeta): SEQ ID NO: 104 is the amino acid sequence of CD22 CAR LTG2208 (LP-scFv11-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLN SVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGPGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 105 is the amino acid sequence of scFv11(16P10)LCDR1: QDVSGW SEQ ID NO: 106 is the amino acid sequence of scFv11(16P10)LCDR2: GAS SEQ ID NO: 107 is the amino acid sequence of scFv11(16P10)LCDR3: QQAKYFPYT SEQ ID NO: 108 is the amino acid sequence of scFv11(16P10)HCDR1: GDSVSSNSAA SEQ ID NO: 109 is the amino acid sequence of scFv11 (16P10) HCDR2: TYYRSKWYN SEQ ID NO: 110 is the amino acid sequence of scFv11 (16P10) HCDR3: AQEVEPQDAFDI SEQ ID NO: 111 is the nucleic acid sequence of CD22-specific binder (scFv12) 16P17: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCACTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAT GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA GGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTATGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTC CTGATCTCTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 112 is the amino acid sequence of CD22-specific binder (scFv12) 16P17: QVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 113 is the nucleic acid sequence of CD22 CAR LTG2209 (LP-scFv12-CD8TM-41BB-CD3zeta): ATGCTTCTTTTGGTGACTTCCCTTTTGCTGTGCGAGTTGCCACACCCCGCCTTCCTGCTTATTCCCCAGGTACAGCTTCAACAGAGTGGGCCGGGACTGGTGAAACACTCCCAAACACTTTCTCTGACGTGCGCTATATCAGGTGACTCTGTTTCATCT SEQ ID NO: 114 is the amino acid sequence of CD22 CAR LTG2209 (LP-scFv12-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 115 is the amino acid sequence of scFv12(16P17)LCDR1: QDVSGW SEQ ID NO: 116 is the amino acid sequence of scFv12(16P17)LCDR2: GAS SEQ ID NO: 117 is the amino acid sequence of scFv12(16P17)LCDR3: QQAKYFPYT SEQ ID NO: 118 is the amino acid sequence of scFv12(16P17)HCDR1: GDSVSSNSAA SEQ ID NO: 119 is the amino acid sequence of scFv12(16P17) HCDR2: TYYRSKWYN SEQ ID NO: 120 is the amino acid sequence of scFv12(16P17) HCDR3: AQEVEPHDAFDI SEQ ID NO: 121 is the nucleic acid sequence of CD22-specific binder (scFv13) 16P20v2: CAAGTACAACTTCAACAGTCTGGGCCTGGGCTTGTAAAACCTAGCCAAACTCTGTCCCTCACGTGCGCGATTTCAGGGACAGTGTAAGTTCCAACTCAGCCGCATGGAACTGGATCAGGCAGTCACCTTCAAGGGGGCTCGAATGGCTTGGCCGAACGTACTACAGGAGTAAGTGGTACAAC GATTATGCAGTGTCTGTGAAATCACGGATTACTATCAATCCCGACACGTCCAAGAGAACCAGTTCTCTCTGCAACTCAACTCAGTGACACCAGAGGATACGGCCGTTTACTATTGTGCACAGGAAGTGCAACCTGATGATGCCTTTGACATTTGGGGTCAGGGCACGATGGTTACGGTAAGCTCT GGGGGAGGCGGCAGTGGAGGGGGAGGTAGTGGGGGAGGGGGATCTGATATACAGATGACACAAAGCCCGTCATCCGTCAGTGCTTCAGTTGGTGATAAAGTAACCATTACGTGCCGCGCTTCCCAAGACGTTAGCGGATGGTTGGCTTGGTATCAACAAAAACCGGGGTTGGCTCCGCAACTC CTCATATCCGGTGCGAGTACGCTCCAAGGCGAAGTCCCTAGCAGATTTTCCGGGAGCGGTTCCGGTACAGATTTCACGTTGACCATTAGCTCTCTCCAGCCCGAAGATTTTGCAACCTACTATTGCCAACAGGCCAAAAATTTTCCATATACATTTGGTCAAGGCACTAAGCTCGAAATCAAA SEQ ID NO: 122 is the amino acid sequence of CD22-specific binder (scFv13) 16P20v2: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPDDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKNFPYTFGQGTKLEIK SEQ ID NO: 123 is the nucleic acid sequence of CD22 CAR LTG2210 (LP-scFv13-CD8TM-41BB-CD3zeta): ATGCTTCTTTTGGTGACTTCCCTTTTGCTGTGCGAGTTGCCACACCCCGCCTTCCTGCTTATTCCCCAAGTACAACTTCAACAGTCTGGGCCTGGGCTTGTAAAACCTAGCCAAACTCTGTCCCTCACGTGCGCGATTTCAGGGGACAGTGTAAGTTCC AACTCAGCCGCATGGAACTGGATCAGGCAGTCACCTTCAAGGGGGCTCGAATGGCTTGGCCGAACGTACTACAGGAGTAAGTGGTACAACGATTATGCAGTGTCTGTGAAATCACGGATTACTATCAATCCCGACACGTCCAAGAACCAGTTCTCTCTGC SEQ ID NO: 124 is the amino acid sequence of CD22 CAR LTG2210 (LP-scFv13-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVQPDDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKNFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 125 is the amino acid sequence of scFv13(16P20v2)LCDR1: QDVSGW SEQ ID NO: 126 is the amino acid sequence of scFv13(16P20v2)LCDR2: GAS SEQ ID NO: 127 is the amino acid sequence of scFv13(16P20v2)LCDR3: QQAKNFPYT SEQ ID NO: 128 is the amino acid sequence of scFv13(16P20v2)HCDR1: GDSVSSNSAA SEQ ID NO: 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: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGACATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAT GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGATAGAACCTCATGATGCTTTTGATATCTGGGACCAAGGGACAATGGTCACCGTCTCTTCA GGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGTCATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTC CTGATCTCTGGTGCATCCTCTTTGCAAGGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 132 is the amino acid sequence of CD22-specific binder (scFv14) 16P1: QVQLQQSGPGLVKPSQTLSLTCDISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEIEPHDAFDIWDQGTMVTVSS GGGGSGGGGSGGGGSVIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 133 is the nucleic acid sequence of CD22 CAR LTG2216 (LP-scFv14-CD8TM-41BB-CD3zeta): ATGTTGCTGCTCGTGACCTCGCTCCTTCTGTGCGAGCTGCCCCATCCGGCTTTTCTGCTCATCCCTCAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCCGTCCCAGACTCTGAGCCTGACTTGCGATATTAGCGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAA SEQ ID NO: 134 is the amino acid sequence of CD22 CAR LTG2216 (LP-scFv14-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCDISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EIEPHDAFDIWDQGTMVTVSSGGGGSGGGGSGGGGSVIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 135 is the amino acid sequence of scFv14(16P1)LCDR1: QDVSGW SEQ ID NO: 136 is the amino acid sequence of scFv14(16P1)LCDR2: GAS SEQ ID NO: 137 is the amino acid sequence of scFv14(16P1)LCDR3: QQAKYFPYT SEQ ID NO: 138 is the amino acid sequence of scFv14(16P1)HCDR1: GDSVSSNSAA SEQ ID NO: 139 is the amino acid sequence of scFv14(16P1)HCDR2: TYYRSKWYN SEQ ID NO: 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: CAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCACTCCCAGACTCTGAGCCTGGCCTGCGCGATTACGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTATAAC GACTACGCCGTGTCCGTGAAGTCCCGGATCACCATTAACCCCGACACCTCGAAGAACCAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATCCGCGGTGTACTACTGCGCACAAGAAGTGCAGCCGCAGGACGCCCTGGACATTTGGGGGCAGGGAACGATGGTCACAGTGTCGTCC GGTGGAGGAGGTTCCGGAGGCGGTGGATCTGGAGGCGGAGGTTCGGATATCCAGATGACCCAGAGCCCCTCCTTCGTGTCCGCATCCGTGGGCGATAAGGTCATTATTATTACCTGTAGAGCGTCCCAGGACGTGTCCGGATGGCTGGCCTGGTACCAGCAGAAGCCAGGCTTGGCTCCTCAACTG CTGATCTCCGGCGCCAGCACTCTTCAGGGGGAAGTGCCATCACGCTTCTCCGGATCCGGTTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCTGAGGACTTCGCCACTTACTACTGCCAACAGGCCAAGTACTTCCCCTATACCTTCGGACAAGGCACTAAGCTGGAAATCAAG SEQ ID NO: 142 is the amino acid sequence of CD22-specific binder (scFv15) 16P3v2: QVQLQQSGPGLVKHSQTLSLACAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVQPQDALDIWGQGTMVTVSS GGGGSGGGGSGGGGSDIQMTQSPSFVSASVGDKVIITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK SEQ ID NO: 143 is the nucleic acid sequence of CD22 CAR LTG2217 (LP-scFv15-CD8TM-41BB-CD3zeta): ATGTTGCTGCTCGTGACCTCGCTCCTTCTGTGCGAGCTGCCCCATCCGGCTTTTCTGCTCATCCCTCAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCACTCCCAGACTCTGAGCCTGGCCTGCGCGATTACGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAA GGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTATAACGACTACGCCGTGTCCGTGAAGTCCCGGATCACCATTAACCCCGACACCTCGAAGAACCAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATACCGCGGTGTACTACTGCGCACAAGAAGTGCAGCCGCAGGACGCCCTGGACATT SEQ ID NO: 144 is the amino acid sequence of CD22 CAR LTG2217 (LP-scFv15-CD8TM-41BB-CD3zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKHSQTLSLACAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVQPQDALDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSFVSASVGDKVIITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 145 is the amino acid sequence of scFv15(16P3v2)LCDR1: QDVSGW SEQ ID NO: 146 is the amino acid sequence of scFv15 (16P3v2) LCDR2: GAS SEQ ID NO: 147 is the amino acid sequence of scFv15 (16P3v2) LCDR3: QQAKYFPYT SEQ ID NO: 148 is the amino acid sequence of scFv15 (16P3v2) HCDR1: GDSVSSNSAA SEQ ID NO: 149 is the amino acid sequence of scFv15 (16P3v2) HCDR2: TYYRSKWYN SEQ ID NO: 150 is the amino acid sequence of scFv15 (16P3v2) HCDR3: AQEVQPQDALDI SEQ ID NO: 151 is the nucleic acid sequence of CD22-specific binder (scFv16) 16P8: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATACT GATTATGCAGTATCTGTGAAAAATCGAATAACCATCAACCCAGACACATCCAAGAATCAGTTCTCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCCCAAGAGGTAGAACCTCAGGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA GGAGGTGGCGGGTCTGGCGGTGGAGGTAGCGGTGGTGGCGGATCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGTCGGGCGAGTCAGGATGTTAGCGGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGCTAGCCCCTCAGCTC CTGATCTTTGGTGCATCCACTTTGCAAGGTGAAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGCTAAATATTTCCCTTACACTTTTGGCCGGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 152 is the amino acid sequence of CD22-specific binder (scFv16) 16P8: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYTDYAVSVKNRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGRGTKLEIK Sequence number 153 is the nucleic acid sequence of CD22 CAR LTG2218 (LP-scFv16-CD8TM-41BB-CD3 zeta): ATGTTGCTGCTCGTGACCTCGCTCCTTCTGTGCGAGCTGCCCCATCCGGCTTTTCTGCTCATCCCTCAAGTGCAGCTGCAGCAGTCCGGTCCTGGACTGGTCAAGCCGTCCCAGACTCTGAGCCTGACTTGCGCAATTAGCGGGGACTCAGTCTCGTCCAATTCGGCGGCCTGGAACTGGATCCGGCAGTCACCATCAAGGGGCCTGGAATGGCTCGGGCGCACTTACTACCGGTCCAAATGGTATACCGACTACGCCGTGTCCGTGAAGAATCGGATCACCATTAACCCCGACACCTCGAAGAACCAGTTCTCACTCCAACTGAACAGCGTGACCCCCGAGGATACCGCGGTGTACTACTGCGCACAAGAAGTGGAACCGCAGGACGCCTTCGACATTTGGGGACAGGGAACGATGGTCACAGTGTCGTCCGGTGGAGGAGGTTCCGGAGGCGGTGGATCTGGAGGCGGAGGTTCGGATATCCAGATGACCCAGAGCCCCTCCTCGGTGTCCGCATCCGTGGGCGATAAGGTCACCATTACCTGTAGAGCGTCCCAGG ACGTGTCCGGATGGCTGGCCTGGTACCAGCAGAAGCCAGGCTTGGCTCCTCAACTGCTGATCTTCGGCGCCAGCACTCTTCAGGGGGAAGTGCCATCACGCTTCTCCGGATCCGGTTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCTGAGGACTTCGCCACTTACTACTGCCAACAGGCCAAGTACTTCCCCTATACCTTCGGAAGAGGCACTAAGCTGGAAATCAAGGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 154 is the amino acid sequence of CD22 CAR LTG2218 (LP-scFv16-CD8TM-41BB-CD3 zeta): MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYTDYAVSVKNRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPQDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLIFGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGRGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 155 is the amino acid sequence of scFv16(16P8)LCDR1: QDVSGW SEQ ID NO: 156 is the amino acid sequence of scFv16(16P8)LCDR2: GAS SEQ ID NO: 157 is the amino acid sequence of scFv16(16P8)LCDR3: QQAKYFPYT SEQ ID NO: 158 is the amino acid sequence of scFv16 (16P8) HCDR1: GDSVSSNSAA SEQ ID NO: 159 is the amino acid sequence of scFv16 (16P8) HCDR2: TYYRSKWYT SEQ ID NO: 160 is the amino acid sequence of scFv16 (16P8) HCDR3: AQEVEPQDAFDI SEQ ID NO: 161 is the nucleic acid sequence of CD22-specific binder (scFv17) 16P13: CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCAGGGAACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGAC...

Claims

1. 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 encoded by a nucleotide sequence comprising SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, or 171, at least one transmembrane domain, and at least one intracellular signaling domain.

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

4. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one 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. 5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to the transmembrane domain.

6. The isolated nucleic acid molecule of claim 1 , wherein the encoded extracellular CD22 antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

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

191.

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

9. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular CD22 antigen-binding domain comprises a nucleotide sequence comprising SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, or 171, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

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

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

12. The encoded at least one costimulatory domain may be OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS ( 12. The isolated nucleic acid molecule of claim 11, comprising a functional signaling domain of DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

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

14. The CAR of claim 13, comprising 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 transmembrane domain, and at least one intracellular signaling domain.

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

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

18. 18. The CAR of claim 17, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 182, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

182.

19. The CAR of claim 14, wherein 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, and the at least one intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

20. The CAR of claim 19, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to the transmembrane domain.

21. The CAR of claim 14, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

22. The CAR of claim 21, wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

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

24. 24. The vector of claim 23, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

25. 24. The vector of claim 23, further comprising a promoter.

26. 26. The vector of claim 25, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

27. A cell comprising the vector of claim 23.

28. 28. The cell of claim 27, wherein the cell is a T cell.

29. 29. The cell of claim 28, wherein the T cell is a CD8+ T cell.

30. 28. The cell of claim 27, wherein the cell is a human cell.

31. A method for producing cells, comprising the step of transducing a T cell with the vector of claim 23.

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

33. 30. A method of providing anti-tumor immunity to a mammal, comprising administering to said mammal an effective amount of the cells of claim 27.

34. A method for treating or preventing cancer in a mammal, comprising a step of administering to the mammal the CAR of claim 13 in an amount effective for treating or preventing cancer in the mammal.

35. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, the 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 from a human with cancer.

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

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

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

39. 39. The pharmaceutical composition of claim 38, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML).

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

41. 38. The pharmaceutical composition of claim 37, wherein the hematological cancer is multiple myeloma.

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

43. 1. A method for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, the T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, including 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 or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of the subject with cancer.

44. 1. A method of treating cancer in a subject in need thereof, the method comprising: administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, the 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 or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cells are T cells of the subject with cancer.

45. 45. The method of claim 43 or 44, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

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

47. 47. The process for producing a chimeric antigen receptor (CAR)-expressing cell of claim 46, wherein the cell is a T cell or a cell population containing a T cell.

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