cells

Abstract

The present invention provides a cell that co-expresses a first chimeric antigen receptor (CAR) and a second CAR on the cell surface, each CAR comprising an antigen binding domain, wherein the antigen binding domain of the first CAR binds CD19 and the antigen binding domain of the second CAR binds CD22.

Description

Field of the Invention

[0001] The present invention relates to cells comprising more than one chimeric antigen receptor (CAR). Background of the Invention

[0003] Several immunotherapeutic agents have been described for cancer treatment, including therapeutic monoclonal antibodies (mAbs), immunoconjugated mAbs, radioconjugated mAbs, and bispecific T cell engagers.

[0004] Typically, these immunotherapeutic agents target a single antigen: for example, Rituximab targets CD20; Myelotarg targets CD33; and Alemtuzumab targets CD52.

[0005] The human CD19 antigen is a 95kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is expressed very early in B cell differentiation and is lost only when the terminal B cells differentiate into plasma cells. Therefore, CD19 is expressed on all B cell malignancies except multiple myeloma. Since the loss of the normal B cell compartment is an acceptable toxicity, CD19 is an attractive CAR target, and clinical studies using CAR targeting CD19 have seen promising results.

[0006] A particular problem in the field of oncology is posed by the Goldie-Coldman hypothesis: it describes that targeting only a single antigen can lead to tumor escape through modulation of said antigen due to the inherently high mutation rate in most cancers. Modulation of the expression of this antigen can reduce the efficacy of known immunotherapies, including those targeting CD19.

[0007] Therefore, a problem with immunotherapies targeting CD19 is that B-cell malignancies can mutate and become CD19 negative. This can lead to relapses of CD19-negative cancers that do not respond to CD19-targeted therapies. For example, in a pediatric study, Grupp et al. reported that half of all relapses following CD19-targeted chimeric antigen receptor therapy for B-type acute lymphoblastic leukemia were due to CD19-negative disease (56 th American Society of Hematology Annual Meeting and Exposition).

[0008] Therefore, there is a need for immunotherapeutic agents that can target more than one cell surface structure to reflect the complex patterns of marker expression associated with various cancers, including CD19-positive cancers.

[0009] Chimeric Antigen Receptor (CAR)

[0010] Chimeric antigen receptors are proteins that graft the specificity of, for example, a monoclonal antibody (mAb) onto the effector function of a T cell. Their general form is a type I transmembrane domain protein with an antigen recognition amino terminus, a spacer region, a transmembrane domain, all connected to a complex intracellular domain that transmits T cell survival and activation signals (see Figure 1 A).

[0011] The most common form of these molecules is a single-chain variable fragment (scFv) derived from a monoclonal antibody that recognizes a target antigen, which is fused to the fusion of a signal transduction intracellular domain via a spacer and a transmembrane domain. In response to the recognition of its target by scFv, such molecules lead to the activation of T cells. When T cells express such CARs, they identify and kill target cells expressing the target antigen. Several CARs for tumor-associated antigens have been developed, and the adoptive transfer method of T cells using such expression CARs has been used in clinical studies for the treatment of various cancers.

[0012] It has been observed that using CAR methods for cancer treatment, tumor heterogeneity and immune editing can lead to escape from CAR treatment. For example, in the study described by Grupp et al. (2013; New Eng. J. Med 368: 1509-1518, paper No 380, ASH 2014), CAR-modified T cell methods are used for the treatment of acute B lymphoblastic leukemia. In this clinical trial, it was found that 10 patients with complete regression did relapse after one month, and 5 of them relapsed CD19 negative diseases.

[0013] Therefore, there is a need for alternative CAR therapeutic approaches that address the issues of cancer escape and tumor heterogeneity.

[0014] Expression of two CAR binding specificities

[0015] Bispecific CARs, called tandem CARs or TanCARs, have been developed to attempt to simultaneously target multiple cancer-specific markers. In TanCARs, the extracellular domain contains two antigen-binding specificities in series, connected by a linker. Thus, both binding specificities (scFvs) are attached to a single transmembrane portion; one scFv is juxtaposed to the membrane, while the other is located at a distal position.

[0016] Grada et al. (2013, Mol Ther Nucleic Acids 2:e105) described a TanCAR that includes a CD19-specific scFv followed by a Gly-Ser linker and then a HER2-specific scFv. The HER2-scFv is located near the membrane, while the CD19-scFv is located distally. This Tan CAR was shown to induce different T cell activity against each of the two tumor-restricted antigens. This arrangement was chosen because HER2 (632 ) and CD19 (280aa, ) makes it suitable for specific spatial arrangements. It is also known that HER2 scFv binds to the four most distal loops of HER2.

[0017] The problem with this approach is that due to the presence of distal scFv, especially its binding to antigen, the membrane-proximal scFv may not be accessible. Given the need to consider the spatial arrangement of antigens on target cells to select the relative positions of the two scFvs, it may not be possible to use this method for all scFv binding pairs. In addition, the TanCar approach is unlikely to be used for more than two scFvs. TanCARs with three or more scFvs will be very large molecules, and scFVs may fold back on each other, covering up the antigen binding site. It is also questionable whether antigen binding by the most distal scFv (which is separated from the transmembrane domain by two or more other scFvs) will be able to trigger T cell activation.

[0018] Therefore, there is a need for alternative methods to express two CAR binding specificities on the surface of cells, such as T cells.

[0019] Summary of the Invention

[0020] The present inventors have developed CAR T cells that express two CARs on the cell surface, one specific for CD19 and one specific for CD22.

[0021] Thus, a first aspect of the invention provides a cell that co-expresses a first chimeric antigen receptor (CAR) and a second CAR on the cell surface, each CAR comprising an antigen binding domain, wherein the antigen binding domain of the first CAR binds CD19 and the antigen binding domain of the second CAR binds CD22.

[0022] The fact that one CAR binds CD19 and the other binds CD22 is advantageous because some lymphomas and leukemias become CD19 negative after CD19 targeting (or possibly CD22 negative after CD22 targeting), so when this happens it provides a "backup" antigen.

[0023] The cell may be an immune effector cell, such as a T cell or a natural killer (NK) cell. The features mentioned herein regarding T cells are equally applicable to other immune effector cells, such as NK cells.

[0024] Each CAR can contain:

[0025] (i) an antigen-binding domain;

[0026] (ii) a spacer; and

[0027] (iii) transmembrane domain.

[0028] Each CAR can contain:

[0029] (i) antigen binding domain;

[0030] (ii) a spacer region;

[0031] (iii) a transmembrane domain; and

[0032] (iv) Intracellular domain.

[0033] The spacer of the first CAR may be different from the spacer of the second CAR such that the first and second CARs do not form heterodimers.

[0034] The spacer of the first CAR can have a different length and / or configuration than the spacer of the second CAR such that each CAR is tailored for recognition of its respective target antigen.

[0035] The antigen binding domain of the second CAR can bind to a membrane distal antigen on CD22. The antigen binding domain of the second CAR can bind to an epitope on Ig domain 1, 2, 3 or 4 of CD22, such as an epitope on Ig domain 3 of CD22.

[0036] The antigen binding domain of the first CAR can bind to an epitope on CD19 encoded by exon 1, 3, or 4.

[0037] The intracellular domain of one CAR may comprise a costimulatory domain and an ITAM-containing domain; and the intracellular domain of another CAR may comprise a TNF receptor family domain and an ITAM-containing domain.

[0038] For example, a CAR (which can be specific for CD19 or CD22) can have the following structure:

[0039] AgB1-spacer1-TM1-costim-ITAM

[0040] in:

[0041] AgB1 is the antigen-binding domain;

[0042] Spacer 1 is the spacer;

[0043] TM1 is the transmembrane domain;

[0044] Costim is the costimulatory domain; and

[0045] ITAM is an intracellular domain containing ITAM;

[0046] And another CAR (which can be CD22 or CD19 specific) can have the following structure:

[0047] AgB2-Spacer2-TM2-TNF-ITAM

[0048] in:

[0049] AgB2 is the antigen-binding domain;

[0050] Spacer 2 is a spacer;

[0051] TM2 is the transmembrane domain;

[0052] TNF is the intracellular domain of the TNF receptor; and

[0053] ITAM is an intracellular domain containing ITAM.

[0054] In a second aspect, the present invention provides nucleic acid sequences encoding both the first and second chimeric antigen receptors (CARs) as defined in the first aspect of the invention.

[0055] The nucleic acid sequence may have the following structure:

[0056] AgB1-Spacer1-TM1-coexpr-AbB2-Spacer2-TM2

[0057] in

[0058] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0059] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0060] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0061] coexpr is a nucleic acid sequence that enables co-expression of two CARs;

[0062] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0063] Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

[0064] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0065] The nucleic acid sequence, when expressed in a T cell, encodes a polypeptide that is cleaved at the cleavage site, such that the first and second CARs are co-expressed on the surface of the T cell.

[0066] The nucleic acid sequence may have the following structure:

[0067] AgB1-Spacer1-TM1-endo1-coexpr-AbB2-Spacer2-TM2-endo2

[0068] in

[0069] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0070] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0071] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0072] endo1 is a nucleic acid sequence encoding the intracellular domain of the first CAR;

[0073] coexpr is a nucleic acid sequence that enables co-expression of two CARs;

[0074] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0075] Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

[0076] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0077] endo2 is a nucleic acid sequence encoding the intracellular domain of the second CAR;

[0078] The nucleic acid sequence, when expressed in a T cell, encodes a polypeptide that is cleaved at the cleavage site, such that the first and second CARs are co-expressed on the surface of the T cell.

[0079] The nucleic acid sequence allowing co-expression of two CARs may encode a self-cleaving peptide or sequence that allows alternative ways of co-expressing two CARs, such as an internal ribosome entry sequence or a 2 nd Promoter or other means, so that those skilled in the art can express two proteins from the same vector.

[0080] To avoid homologous recombination, alternative codons can be used in sequence regions encoding the same or similar amino acid sequences, such as transmembrane and / or intracellular T cell signaling domains (intracellular domains). For example, alternative codons can be used in sequence portions encoding spacers, transmembrane domains, and / or all or part of the intracellular domain, such that the two CARs have the same or similar amino acid sequences for this or these portions, but are encoded by different nucleic acid sequences.

[0081] In a third aspect, the present invention provides a kit comprising

[0082] (i) a first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), the nucleic acid sequence having the following structure:

[0083] AgB1-spacer 1-TM1

[0084] in

[0085] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0086] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0087] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0088] and

[0089] (ii) a second nucleic acid sequence encoding a second chimeric antigen receptor, the nucleic acid sequence having the following structure:

[0090] AgB2-Spacer2-TM2

[0091] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0092] Spacer 2 is a nucleic acid sequence encoding a spacer for a second CAR; and

[0093] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR.

[0094] The kit may contain

[0095] (i) a first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), the nucleic acid sequence having the following structure:

[0096] AgB1-spacer1-TM1-endo1

[0097] in

[0098] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0099] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0100] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0101] endo1 is a nucleic acid sequence encoding the intracellular domain of the first CAR; and

[0102] and

[0103] (ii) a second nucleic acid sequence encoding a second chimeric antigen receptor (CAR), the nucleic acid sequence having the following structure:

[0104] AgB2-spacer2-TM2-endo2

[0105] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0106] Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

[0107] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0108] endo2 is a nucleic acid sequence encoding the intracellular domain of the second CAR.

[0109] In a fourth aspect, the present invention provides a kit comprising: a first vector comprising a first nucleic acid sequence; and a second vector comprising a second nucleic acid sequence.

[0110] The vector may be a plasmid vector, a retroviral vector or a transposon vector. The vector may be a lentiviral vector.

[0111] In a fifth aspect, the present invention provides a vector comprising the nucleic acid sequence according to the second aspect of the present invention. The vector may be a lentiviral vector.

[0112] The vector may be a plasmid vector, a retroviral vector or a transposon vector.

[0113] In a sixth aspect of the invention, a method for preparing a cell according to the first aspect of the invention is provided, which comprises introducing into a T cell one or more nucleic acid sequences encoding a first and a second CAR; or one or more vectors, as defined above.

[0114] The cells can be from a sample isolated from the patient, a related or unrelated hematopoietic transplant donor, a completely unrelated donor, from umbilical cord blood, differentiated from an embryonic cell line, differentiated from an inducible progenitor cell line, or derived from a transformed cell line.

[0115] In a seventh aspect, the pharmaceutical composition of the present invention comprises a plurality of cells according to the first aspect of the present invention.

[0116] In an eighth aspect, the present invention provides a method for treating and / or preventing a disease, comprising the step of administering to a subject the pharmaceutical composition according to the seventh aspect of the present invention.

[0117] The method may include the following steps:

[0118] (i) isolating a cell-containing sample from a subject;

[0119] (ii) transducing or transfecting the cell with one or more nucleic acid sequences encoding the first and second CARs, or one or more vectors comprising such nucleic acid sequences; and

[0120] (iii) administering the cells from (ii) to a subject.

[0121] The disease can be cancer. The cancer can be a B-cell malignancy.

[0122] In a ninth aspect, the present invention provides a pharmaceutical composition according to the seventh aspect of the present invention for use in treating and / or preventing a disease.

[0123] In a tenth aspect, the present invention provides use of the cell according to the first aspect of the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0124] The present invention also provides a nucleic acid sequence comprising:

[0125] a) a first nucleotide sequence encoding a first chimeric antigen receptor (CAR);

[0126] b) a second nucleotide sequence encoding a second CAR;

[0127] One of the CARs binds CD19 and the other binds CD22; and

[0128] c) a sequence encoding a self-cleaving peptide, which is positioned between the first and second nucleotide sequences such that the two CARs are expressed as separate entities.

[0129] In one or more of the first and second nucleotides, alternative codons may be used in the region encoding the same or similar amino acid sequence.

[0130] The invention also provides vectors and cells comprising such nucleic acids.

[0131] The present invention also develops new anti-CD19 and anti-CD22 CARs with improved properties.

[0132] Thus, in an eleventh aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a CD19 binding domain, comprising

[0133] a) a heavy chain variable region (VH) having a complementarity determining region (CDR) having the following sequence:

[0134] CDR1–SYWMN (SEQ ID No. 15);

[0135] CDR2–QIWPGDGDTNYNGKFK (SEQ ID No. 16);

[0136] CDR3–RETTTVGRYYYAMDY (SEQ ID No. 17); and

[0137] b) a light chain variable region (VL) having CDRs having the following sequences:

[0138] CDR1–KASQSVDYDGDSYLN(SEQ ID No.18);

[0139] CDR2–DASNLVS(SEQ ID No.19);

[0140] CDR3-QQSTEDPWT (SEQ ID No. 20).

[0141] The CD19 binding domain may comprise a VH domain having a sequence as shown in SEQ ID No. 23 or SEQ ID NO 24; or a VL domain having a sequence as shown in SEQ ID No. 25, SEQ ID No. 26 or SEQ ID No. 40, or a variant thereof having at least 90% sequence identity that retains the ability to bind to CD19.

[0142] The CD19 binding domain may comprise a sequence as shown in SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 39, or a variant thereof having at least 90% sequence identity that retains the ability to bind to CD19.

[0143] In a twelfth aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a CD22 binding domain, comprising

[0144] a) a heavy chain variable region (VH) having a complementarity determining region (CDR) having the following sequence:

[0145] CDR1–NYWIN (SEQ ID No. 27);

[0146] CDR2–NIYPSDSFTNYNQKFKD(SEQ ID No.28)

[0147] CDR3–DTQERSWYFDV (SEQ ID No. 29); and

[0148] b) a light chain variable region (VL) having CDRs having the following sequences:

[0149] CDR1–RSSQSLVHSNGNTYLH(SEQ ID No.30);

[0150] CDR2–KVSNRFS (SEQ ID No. 31)

[0151] CDR3-SQSTHVPWT (SEQ ID No. 32).

[0152] The CD22 binding domain may comprise a VH domain having a sequence as shown in SEQ ID No. 35 or SEQ ID NO 36; or a VL domain having a sequence as shown in SEQ ID No. 37 or SEQ ID No. 38, or a variant thereof having at least 90% sequence identity that retains the ability to bind to CD22.

[0153] The CD22 binding domain may comprise the sequence shown as SEQ ID No. 33 or SEQ ID No. 34, or a variant thereof having at least 90% sequence identity, said variant retaining the ability to bind CD22.

[0154] In a thirteenth aspect, a cell is provided which expresses the chimeric antigen receptor according to the eleventh aspect of the present invention and the chimeric antigen receptor according to the twelfth aspect of the present invention on the cell surface.

[0155] In a fourteenth aspect, nucleic acid sequences encoding the chimeric antigen receptor according to the eleventh aspect of the present invention and the chimeric antigen receptor according to the twelfth aspect of the present invention are provided.

[0156] In a fifteenth aspect, the present invention provides a vector comprising the nucleic acid sequence according to the fourteenth aspect of the present invention. The vector may be a lentiviral vector.

[0157] The vector may be a plasmid vector, a retroviral vector or a transposon vector.

[0158] In a sixteenth aspect, the present invention provides a method for preparing a cell according to the thirteenth aspect of the present invention, comprising the step of introducing one or more nucleic acid sequences; or one or more vectors as defined above into the cell.

[0159] The cell can be a T cell or a natural killer (NK) cell. The cell can be derived from a sample isolated from a patient, a related or unrelated hematopoietic transplant donor, a completely unrelated donor, from umbilical cord blood, differentiated from an embryonic cell line, differentiated from an inducible progenitor cell line, or derived from a transformed cell line.

[0160] In a seventeenth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells according to the thirteenth aspect of the present invention.

[0161] In an eighteenth aspect, the present invention provides a method for treating and / or preventing a disease, comprising the step of administering to a subject the pharmaceutical composition according to the seventeenth aspect of the present invention.

[0162] The method may include the following steps:

[0163] (i) isolating a cell-containing sample from a subject;

[0164] (ii) transducing or transfecting the cell with one or more nucleic acid sequences encoding the first and second CARs, or one or more vectors comprising such nucleic acid sequences; and

[0165] (iii) administering the cells from (ii) to a subject.

[0166] The disease can be cancer. The cancer can be a B-cell malignancy.

[0167] In a nineteenth aspect, the present invention provides a pharmaceutical composition according to the seventeenth aspect of the present invention, for use in treating and / or preventing a disease.

[0168] In a twentieth aspect, the present invention provides use of the cell according to the thirteenth aspect of the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0169] Also provided is a cell according to the first aspect of the invention, comprising a first CAR as defined in the eleventh aspect of the invention and a second CAR as defined in the twelfth aspect of the invention.

[0170] Also provided is a nucleic acid sequence according to the second aspect of the invention, which encodes a first CAR as defined in the eleventh aspect of the invention and a second CAR as defined in the twelfth aspect of the invention.

[0171] Also provided is a kit according to the third aspect of the invention, wherein the first nucleic acid sequence encodes a first CAR as defined in the eleventh aspect of the invention and the second nucleic acid sequence encodes a second CAR as defined in the twelfth aspect of the invention.

[0172] Also provided is a vector according to the fifth aspect of the present invention, comprising a nucleic acid sequence encoding a first CAR as defined in the eleventh aspect of the present invention and a second CAR as defined in the twelfth aspect of the present invention.

[0173] The present inventors have also discovered that in an OR gate system, performance is improved if the co-stimulatory domain and the domain generating the survival signal are "split" between two (or more) CARs.

[0174] Thus, in a twenty-first aspect, a cell is provided that co-expresses a first chimeric antigen receptor (CAR) and a second CAR on the cell surface, each CAR comprising an intracellular signaling domain, wherein the intracellular signaling domain of the first CAR comprises a costimulatory domain; and the intracellular signaling domain of the second CAR comprises a TNF receptor family intracellular domain.

[0175] The costimulatory domain may be a CD28 costimulatory domain.

[0176] The TNF receptor family intracellular domain can be, for example, an OX-40 or 4-1BB intracellular domain.

[0177] The intracellular signaling domains of the first and second CARs can also comprise an ITAM-containing domain, such as a CD3 zeta intracellular domain.

[0178] The first CAR may have the following structure

[0179] AgB1-spacer1-TM1-costim-ITAM

[0180] in:

[0181] AgB1 is the antigen-binding domain of the first CAR;

[0182] Spacer 1 is the spacer of the first CAR;

[0183] TM1 is the transmembrane domain of the first CAR;

[0184] Costim is the costimulatory domain; and

[0185] ITAM is an intracellular domain containing ITAM.

[0186] The second CAR may have the following structure:

[0187] AgB2-Spacer2-TM2-TNF-ITAM

[0188] in:

[0189] AgB2 is the antigen-binding domain of the second CAR;

[0190] Spacer 2 is the spacer for the second CAR;

[0191] TM2 is the transmembrane domain of the second CAR;

[0192] TNF is the intracellular domain of the TNF receptor; and

[0193] ITAM is an intracellular domain containing ITAM.

[0194] One of the first and second CARs may target CD19 and the other CAR may target CD22.

[0195] In a twenty-second aspect, there is provided a nucleic acid sequence encoding both the first and second chimeric antigen receptor (CAR) as defined in the twenty-first aspect of the invention.

[0196] The nucleic acid sequence may have the following structure:

[0197] AgB1-Spacer 1-TM1-costim-ITAM1-coexpr-AbB2-Spacer 2-TM2-TNF-ITAM2

[0198] in

[0199] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0200] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0201] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0202] costim is a nucleic acid sequence encoding a costimulatory domain;

[0203] ITAM1 is a nucleic acid sequence encoding the ITAM-containing intracellular domain of the first CAR;

[0204] coexpr is a nucleic acid sequence that enables co-expression of two CARs;

[0205] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0206] Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

[0207] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0208] TNF is a nucleic acid sequence encoding the intracellular domain of the TNF receptor;

[0209] ITAM2 is a nucleic acid sequence encoding the ITAM-containing intracellular domain of the second CAR;

[0210] When the nucleic acid sequence is expressed in a cell, it encodes a polypeptide that is cleaved at the cleavage site, such that the first and second CARs are co-expressed on the cell surface.

[0211] In a twenty-third aspect, the present invention provides a kit comprising

[0212] (i) a first nucleic acid sequence encoding a first chimeric antigen receptor (CAR) as defined in the twenty-first aspect of the present invention, wherein the nucleic acid sequence has the following structure:

[0213] AgB1-spacer1-TM1-costim-ITAM1

[0214] in

[0215] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0216] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0217] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0218] costim is a nucleic acid sequence encoding a costimulatory domain;

[0219] ITAM1 is a nucleic acid sequence encoding the ITAM-containing intracellular domain of the first CAR;

[0220] and

[0221] (ii) a second nucleic acid sequence encoding the second chimeric antigen receptor (CAR) as defined in the twenty-first aspect of the present invention, wherein the nucleic acid sequence has the following structure:

[0222] AbB2-Spacer2-TM2-TNF-ITAM2

[0223] in

[0224] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0225] Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

[0226] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0227] TNF is a nucleic acid sequence encoding the intracellular domain of the TNF receptor;

[0228] ITAM2 is a nucleic acid sequence encoding the ITAM-containing intracellular domain of the second CAR.

[0229] In a twenty-fourth aspect, there is provided a vector comprising a nucleic acid sequence according to the twenty-second aspect of the invention or as defined in the twenty-third aspect of the invention.

[0230] In the twenty-fifth aspect, a method for preparing a cell according to the twenty-first aspect of the present invention is provided, which comprises the steps of introducing into the cell: a nucleic acid sequence according to the twenty-second aspect of the present invention; a first nucleic acid sequence and a second nucleic acid sequence as defined in the twenty-third aspect of the present invention; or a vector according to the twenty-fourth aspect of the present invention.

[0231] In a twenty-sixth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells according to the twenty-first aspect of the present invention.

[0232] Also provided is a method for treating and / or preventing a disease, comprising the step of administering the pharmaceutical composition according to the twenty-sixth aspect of the present invention to a subject.

[0233] Also provided is a pharmaceutical composition according to the twenty-sixth aspect of the present invention for use in treating and / or preventing a disease.

[0234] Also provided is use of the cell according to the twenty-first aspect of the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0235] By delivering one CAR targeting CD19 and one targeting CD22, each of these markers can be targeted, thereby reducing the problem of cancer escape.

[0236] Because CAR is expressed as a separate molecule on the cell surface, this approach overcomes the space and accessibility issues associated with TanCAR. It also improves the efficiency of cell activation. If each CAR has its own spacer, the spacer can be adjusted for a specific target antigen and thereby adjust the distance the binding domain extends from the cell surface and its flexibility, etc. This choice is not affected by the design considerations of the accompanying TanCAR, i.e., one CAR needs to be juxtaposed to the T cell membrane and one CAR needs to be at the distal end, positioned in tandem with the first CAR.

[0237] By providing a single nucleic acid encoding two CARs separated by a cleavage site, cells can be engineered to co-express both CARs using a simple single transduction procedure. A double transfection procedure with the CAR encoding sequences in separate constructs can be used, but this will be more complex and expensive and require more integration sites for the nucleic acids. The double transfection procedure will also be accompanied by uncertainty as to whether the two CAR encoding nucleic acids have been transduced and effectively expressed.

[0238] CAR will have high homology, for example, transmembrane and / or intracellular signaling domains may be highly homologous. If the same or similar linkers are used for two CARs, then they will also be highly homologous. This suggests that a method in which two CARs are provided on a single nucleic acid sequence will not be suitable because of the possibility of homologous recombination between sequences. However, the inventors have found that by "codon wobbling" of the sequence coding region portion with high homology, two CARs can be efficiently expressed from a single construct. Codon wobbling involves the use of alternative codons in sequence regions encoding identical or similar amino acid sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0240] Figure 1 : a) Schematic diagram illustrating a classic CAR. (b) to (d) Different generations and arrangements of CAR intracellular domains: (b) Initial designs delivered only ITAM signals via the FcεR1-γ or CD3ζ intracellular domains, while later designs delivered additional (c) one or (d) multiple costimulatory signals within the same composite intracellular domain.

[0241] Figure 2 B cell maturation pathway / B cell ontogeny. DR = HLA-DR; cCD79 = cytoplasmic CD79; cCD22 = cytoplasmic CD22. Both CD19 and CD22 antigens are expressed early in B cell maturation. It is these cells that develop into B cell acute leukemia. Simultaneous targeting of both CD19 and CD22 is optimal for targeting B cell acute leukemia.

[0242] Figure 3 : Strategy for designing anti-CD19 OR CD22 CAR gene cassettes. Select a binder that recognizes CD19 and an adaptor that recognizes CD22. Select the optimal spacer domain and signaling domain for each CAR. (a) Construct the OR gate gene cassette so that both CARs are co-expressed using the FMD-2A peptide. Codon wobble any homologous sequences to avoid recombination. (c) Express both CARs as separate proteins on the T cell surface.

[0243] Figure 4 : An example of codon wobbling to allow co-expression of identical peptide sequences in a retroviral vector while avoiding homologous recombination. Here, wild-type HCH2CH3-CD28tmZeta is aligned with codon-wobbled HCH2CH3-CD28tmZeta.

[0244] Figure 5: Demonstration of functionality of anti-CD19 OR CD22 gates. (a) Sketch of the construct: S1-signal peptide 1; HA-hemagglutinin tag; HCH2CH3-hinge, CH2CH3 of the IgG1 wild-type sequence; CD28tmZ-CD28 transmembrane domain and CD3Zeta wobble sequence; 2A-FMD 2A peptide; S2-signal peptide 2; V5-v5 epitope tag; aCD22-anti-CD22 scFv; HCH2CH3'-hinge, CH2CH3 of the IgG1 wobble sequence; CD28tmZ-CD28 transmembrane domain and CD3Zeta wobble sequence; (b) Co-expression of two receptors from a single vector. Peripheral blood T cells were transduced using a bicistronic vector after stimulation with OKT2 and anti-CD28. Cells were analyzed five days after transduction by staining with anti-V5-FITC (invitrogen) and anti-HA-PE (abCam). Both CARs can be detected simultaneously on the T cell surface. (c) Target cells expressing neither CD19 nor CD22, either CD19 or CD22 alone, or both antigens were used to stimulate non-transduced T cells, T cells expressing only anti-CD19 CAR, T cells expressing only anti-CD22 CAR, and T cells expressing either anti-CD19 or CD22 CAR. T cells expressing either anti-CD19 or CD22 CAR can kill target cells even in the absence of one antigen.

[0245] Figure 6 : Biacore affinity determination of murine CD22ALAb scFv, humanized CD22ALAb scFv and M971 scFv.

[0246] Figure 7 : Biacore affinity determination of murine CD19ALAb scFv and humanized CD19ALAb.

[0247] Figure 8 : Comparison of binding kinetics between CD19ALAb scFv and fmc63scFv binding to soluble scFv-CD19.

[0248] Figure 9 : Schematic diagram illustrating the CD19ALAb CAR, fmc63 CAR, CD22ALAb CAR, and M971 CAR used in the comparative study.

[0249] Figure 10 : Killing of CD19-positive target cells was compared between a CAR with a CD19ALAb antigen-binding domain and an equivalent CAR with an fmc63 binding domain.

[0250] Figure 11A) Comparison of killing of CD22-positive target cells by a CAR with the CD22 ALAb antigen binding domain and an equivalent CAR with the M971 binding domain. B) Comparison of IFNγ release after 1:1 co-culture with CD22-positive Sup T1 cells.

[0251] Figure 12 : CD19 structure and exons.

[0252] Figure 13: Schematic diagram and construct map illustrating four constructs tested in Example 5. In the construct map, the parts marked with ' are codon wobble.

[0253] A: Both CD19 and CD22 CARs have a 41BB-CD3zeta complex intracellular domain; B: Both CD19 and CD22 CARs have an OX40-CD3zeta complex intracellular domain; C: CD19 CAR has a 41BB-CD3zeta complex intracellular domain and CD22 CAR has a CD28-CD3zeta complex intracellular domain; and D: CD19 CAR has an OX40-CD3zeta complex intracellular domain and CD22 CAR has a CD28-CD3zeta complex intracellular domain.

[0254] Figure 14 : Killing of target cells by cells expressing the constructs shown in FIG. 13 . Detailed Description of the Invention

[0256] Chimeric Antigen Receptor (CAR)

[0257] CAR, which is schematically shown in Figure 1 In the present invention, it is a chimeric type I transmembrane protein that connects the extracellular antigen recognition domain (binding subdomain) to the intracellular signaling domain (intracellular domain). The binding subdomain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms that contain antibody-like antigen binding sites. A spacer domain is usually required to separate the binding subdomain from the membrane and allow it to be oriented appropriately. The commonly used spacer domain is the Fc of IgG1. Depending on the antigen, a tighter spacer may suffice, such as a stem from CD8α or only a single IgG1 hinge. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the intracellular domain.

[0258] Early CAR designs had intracellular domains derived from the intracellular portion of the γ chain of FcεR1 or CD3ζ. Therefore, these first-generation receptors transmit immune signal 1, which is sufficient to trigger T cells to kill cognate target cells, but cannot fully activate T cells to proliferate or survive. To overcome this limitation, a composite intracellular domain was constructed: the intracellular portion of the T cell co-stimulatory molecule was fused to the intracellular portion of CD3ζ, resulting in a second-generation receptor that can simultaneously transmit activation and co-stimulatory signals after antigen recognition. The most commonly used co-stimulatory domain is the co-stimulatory domain of CD28. This provides the most powerful co-stimulatory signal - called immune signal 2, which triggers T cell proliferation. Some receptors have been described, including TNF receptor family intracellular domains, such as the closely related OX40 and 41BB, which transmit survival signals. Even more powerful third-generation CARs have now been described, which have intracellular domains that can transmit activation, proliferation and survival signals.

[0259] The nucleic acid encoding the CAR can be transferred to T cells using, for example, a retroviral vector. A lentivirus can be used. In this way, a large number of cancer-specific T cells can be generated for adoptive cell transfer. When the CAR binds to the target antigen, this results in the activation signal being transmitted to the T cells on which it is expressed. Therefore, the CAR directs the specificity and cytotoxicity of the T cells to tumor cells expressing the targeted antigen.

[0260] A first aspect of the invention relates to cells that co-express a first CAR and a second CAR, wherein one CAR binds CD19 and the other CAR binds CD22, such that T cells can recognize target cells expressing either of these markers.

[0261] Therefore, the antigen binding domains of the first and second CARs of the present invention bind to different antigens, and the two CARs may include an activation intracellular domain. The two CARs may include a spacer domain, which may be identical, or sufficiently different to prevent cross-pairing between two different receptors. Therefore, cells may be engineered to activate when either or both of CD19 and CD22 are recognized. This is useful in the field of oncology, as described in the Goldie-Coldman hypothesis: individual targeting of a single antigen may result in tumor escape, which is achieved by adjusting the antigen due to the inherent high mutation rate in most cancers. By simultaneously targeting two antigens, the possibility of such escape is greatly reduced.

[0262] Importantly, the two CARs do not heterodimerize.

[0263] The first and second CARs of the T cells of the present invention can be produced as a polypeptide comprising both CARs together with a cleavage site.

[0264] signal peptide

[0265] The CAR of the cells of the invention may comprise a signal peptide such that when the CAR is expressed inside a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell membrane, where it is expressed.

[0266] The core of a signal peptide may contain a long stretch of hydrophobic amino acids that tends to form a single alpha helix. The signal peptide may begin with a short, positively charged stretch of amino acids that helps enforce the correct topological conformation of the polypeptide during translocation. At the end of the signal peptide is typically an amino acid stretch recognized and cleaved by a signal peptidase. The signal peptide can be cleaved during or after translocation to generate a free signal peptide and the mature protein. The free signal peptide is then digested by a specific protease.

[0267] The signal peptide may be at the amino terminus of the molecule.

[0268] The signal peptide may comprise SEQ ID No. 1, 2 or 3, or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions), as long as the signal peptide still functions to cause cell surface expression of CAR.

[0269] SEQ ID No.1:MGTSLLCWMALCLLGADHADG

[0270] The signal peptide of SEQ ID No. 1 is compact and efficient. It is predicted to produce approximately 95% cleavage after the terminal glycine, resulting in efficient removal by signal peptidase.

[0271] SEQ ID No.2:MSLPVTALLLPLALLLHAARP

[0272] The signal peptide of SEQ ID No. 2 is derived from IgG1.

[0273] SEQ ID No.3:MAVPTQVLGLLLLWLTDARC

[0274] The signal peptide of SEQ ID No. 3 is derived from CD8.

[0275] The signal peptide of the first CAR may have a different sequence than the signal peptide of the second CAR.

[0276] CD19

[0277] The human CD19 antigen is a 95 kD transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is classified as a type I transmembrane protein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. The general structure of CD19 is shown in Figure 12 middle.

[0278] CD19 is a biomarker for normal and tumor B cells, as well as follicular dendritic cells. In fact, during development, it is present on B cells from the earliest recognizable B lineage cells to B cell blasts, but is lost when maturing into plasma cells. It primarily acts as a B cell co-receptor together with CD21 and CD81. When activated, the cytoplasmic tail of CD19 becomes phosphorylated, which causes the binding of Src family kinases and the recruitment of PI-3 kinases. CD19 is expressed very early in B cell differentiation and is lost only when B cells differentiate into plasma cells in the late stages. Therefore, CD19 is expressed on B cell malignancies except multiple myeloma.

[0279] Different designs of CARs targeting CD19 have been tested in different centers, as summarized in the table below:

[0280] Table 1

[0281]

[0282] As shown above, most studies to date have used scFv derived from the hybridoma fmc63 as part of the binding domain to recognize CD19.

[0283] like Figure 12 As shown in , the gene encoding CD19 contains ten exons: exons 1 to 4 encode the extracellular domain; exon 5 encodes the transmembrane domain; and exons 6 to 10 encode the cytoplasmic domain;

[0284] In the CD19 / CD22 OR gate of the present invention, the antigen binding domain of the anti-CD19 CAR can bind to the epitope of CD19 encoded by exon 1 of the CD19 gene.

[0285] In the CD19 / CD22 OR gate of the present invention, the antigen binding domain of the anti-CD19 CAR can bind to the epitope of CD19 encoded by exon 3 of the CD19 gene.

[0286] In the CD19 / CD22 OR gate of the present invention, the antigen binding domain of the anti-CD19 CAR can bind to the epitope of CD19 encoded by exon 4 of the CD19 gene.

[0287] CD19ALAb

[0288] The present inventors have developed a novel anti-CD19 CAR with improved properties compared to the known anti-CD19 CAR containing the binder fmc63. The antigen binding domain of the CAR is based on the CD19 binder CD19 ALAb, which has the CDRs and VH / VL regions identified below.

[0289] Thus, the present invention also provides a CAR comprising a CD19 binding domain comprising a) a heavy chain variable region (VH) having a complementarity determining region (CDR), wherein the CDR has the following sequence:

[0290] CDR1–SYWMN (SEQ ID No. 15);

[0291] CDR2–QIWPGDGDTNYNGKFK (SEQ ID No. 16);

[0292] CDR3–RETTTVGRYYYAMDY (SEQ ID No. 17); and

[0293] b) a light chain variable region (VL) having CDRs having the following sequences:

[0294] CDR1–KASQSVDYDGDSYLN(SEQ ID No.18);

[0295] CDR2–DASNLVS(SEQ ID No.19);

[0296] CDR3-QQSTEDPWT (SEQ ID No. 20).

[0297] One or more mutations (substitutions, additions or deletions) can be introduced into each CDR without negatively affecting CD19 binding activity. Each CDR can, for example, have one, two or three amino acid mutations.

[0298] The CAR of the present invention may comprise one of the following amino acid sequences:

[0299] SEQ ID No. 21 (mouse CD19ALAbscFv sequence)

[0300] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQG TTVTVSSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK

[0301] SEQ ID No. 22 (humanized CD19AL AbscFv sequence – Heavy 19, Kappa 16)

[0302] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGT LVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR

[0303] SEQ ID No. 39 (humanized CD19 ALAb scFv sequence – Heavy 19, Kappa 7)

[0304] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGT LVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

[0305] The scFv can be in the VH-VL orientation (as shown in SEQ ID No. 21, 22 and 39) or in the VL-VH orientation.

[0306] The CAR of the present invention may comprise one of the following VH sequences:

[0307] SEQ ID No. 23 (murine CD19 ALAb VH sequence)

[0308] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS

[0309] SEQ ID No. 24 (humanized CD19 ALAb VH sequence)

[0310] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGTLVTVSS

[0311] The CAR of the present invention may comprise one of the following VL sequences:

[0312] SEQ ID No. 25 (murine CD19 ALAb VL sequence)

[0313] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK

[0314] SEQ ID No. 26 (humanized CD19 ALAb VL sequence, Kappa 16)

[0315] DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR

[0316] SEQ ID No.40 (humanized CD19 ALAb VL sequence, Kappa 7)

[0317] DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

[0318] The CAR of the present invention may comprise a variant of the sequence shown in SEQ ID No. 21, 22, 23, 24, 25, 26, 39 or 40 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the ability to bind to CD19 (if applicable, when taken together with complementary VL and VH domains).

[0319] The percent identity between two polypeptide sequences can be readily determined by programs such as BLAST (which is freely available at http: / / blast.ncbi.nlm.nih.gov).

[0320] CD22

[0321] The human CD22 antigen is a molecule belonging to the SIGLEC family of lectins. It is found on the surface of mature B cells and some immature B cells. Generally speaking, CD22 is a regulatory molecule that prevents overactivation of the immune system and the development of autoimmune diseases.

[0322] CD22 is a carbohydrate-binding transmembrane protein that specifically recognizes sialic acid via an immunoglobulin (Ig) domain located at its N-terminus. The presence of the Ig domain makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0323] CD22 is an IgSF molecule that can exist in two isoforms: one with seven domains and an intracytoplasmic tail containing three ITIMs (immunoreceptor tyrosine-based inhibitory motifs) and one ITAM; and a splice variant that instead contains five extracellular domains and an intracytoplasmic tail carrying one ITIM. CD22 is considered an inhibitory receptor involved in controlling the response of B cells to antigens. Similar to CD19, CD22 is widely considered a pan-B antigen, although expression on some non-lymphoid tissues has been described. Targeting CD22 using therapeutic monoclonal antibodies and immunoconjugates has entered clinical testing.

[0324] Haso et al. (Blood; 2013; 121(7)) described an example of an anti-CD22 CAR. Specifically, an anti-CD22 CAR having an antigen binding domain derived from m971, HA22, and BL22 scFv was described.

[0325] The antigen binding domain of the anti-CD22 CAR can be expressed with a K in the range of 30-50 nM, such as 30-40 nM. D Binds to CD22. K D It may be about 32 nM.

[0326] CD22 has seven extracellular IgG-like domains, which are generally identified as Ig domain 1 to Ig domain 7, with Ig domain 7 being the most proximal to the B cell membrane and Ig domain 7 being the most distal to the Ig cell membrane (see Haso et al 2013, supra). Figure 2 B).

[0327] The following table summarizes the positions of the Ig domains according to the amino acid sequence of CD22 (http: / / www.uniprot.org / uniprot / P20273).

[0328] Ig domain amino acids 1 20-138 2 143-235 3 242-326 4 331-416 5 419-500 6 505-582 7 593-676

[0329] The antigen binding domain of the second CAR can bind to a membrane distal epitope on CD22. The antigen binding domain of the second CAR can bind to an epitope on Ig domain 1, 2, 3 or 4 of CD22, such as an epitope on Ig domain 3 of CD22. The antigen binding domain of the second CAR can bind to an epitope located between amino acids 22-416 of CD22, such as an epitope between amino acids 242-326 of CD22.

[0330] The anti-CD22 antibodies HA22 and BL22 (Haso et al 2013, supra) and CD22 ALAb, described below, bind to an epitope on Ig domain 3 of CD22.

[0331] The antigen binding domain of the second CAR may not bind to a membrane proximal epitope on CD22. The antigen binding domain of the second CAR may not bind to an epitope on Ig domain 5, 6, or 7 of CD22. The antigen binding domain of the second CAR may not bind to an epitope located between amino acids 419-676 of CD22, such as an epitope between 505-676 of CD22.

[0332] CD22ALAb

[0333] The present inventors have developed a new anti-CD22 CAR that has improved properties compared to the known anti-CD22 CAR containing the binder m971 (see Examples 2 and 3 and Haso et al (2013), supra). The antigen binding domain of the CAR is based on the CD22 binder CD22ALAb, which has the CDRs and VH / VK regions identified below.

[0334] Therefore, the present invention also provides a CAR comprising a CD22 binding domain, which comprises

[0335] a) a heavy chain variable region (VH) having a complementarity determining region (CDR) having the following sequence:

[0336] CDR1–NYWIN (SEQ ID No. 27);

[0337] CDR2–NIYPSDSFTNYNQKFKD(SEQ ID No.28)

[0338] CDR3–DTQERSWYFDV (SEQ ID No. 29); and

[0339] b) a light chain variable region (VL) having CDRs having the following sequences:

[0340] CDR1–RSSQSLVHSNGNTYLH(SEQ ID No.30);

[0341] CDR2–KVSNRFS (SEQ ID No. 31)

[0342] CDR3-SQSTHVPWT (SEQ ID No. 32).

[0343] One or more mutations (substitutions, additions or deletions) may be introduced into the or each CDR without negatively affecting CD22 binding activity. Each CDR may, for example, have one, two or three amino acid mutations.

[0344] The CAR of the present invention may comprise one of the following amino acid sequences:

[0345] SEQ ID No. 33 (mouse CD22ALAbscFv sequence)

[0346] QVQLQQPGAELVRPGASVKLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIYPSDSFTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRDTQERSWYFDVWGAGTTV TVSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPWTFGGGTKLEIK

[0347] SEQ ID No. 34 (humanized CD22ALAbscFv sequence)

[0348] EVQLVESGAEVKKPGSSVKVSCKASGYTFTNYWINWVRQAPGQGLEWIGNIYPSDSFTNYNQKFKDRATLTVDKSTSTAYLELRNLRSDDTAVYYCTRDTQERSWYFDVWGQGTLV TVSSDIVMTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPARFSGSGSGVEFTLTISSLQSEDFAVYYCSQSTHVPWTFGQGTRLEIK

[0349] The scFv can be in the VH-VL orientation (as shown in SEQ ID Nos 33 and 34) or in the VL-VH orientation.

[0350] The CAR of the present invention may comprise one of the following VH sequences:

[0351] SEQ ID No. 35 (murine CD22ALAb VH sequence)

[0352] QVQLQQPGAELVRPGASVKLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIYPSDSFTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRDTQERSWYFDVWGAGTTVTVSS

[0353] SEQ ID No.36 (humanized CD22ALAb VH sequence)

[0354] EVQLVESGAEVKKPGSSVKVSCKASGYTFTNYWINWVRQAPGQGLEWIGNIYPSDSFTNYNQKFKDRATLTVDKSTSTAYLELRNLRSDDTAVYYCTRDTQERSWYFDVWGQGTLVTVSS

[0355] The CAR of the present invention may comprise one of the following VL sequences:

[0356] SEQ ID No. 37 (murine CD22ALAb VL sequence)

[0357] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPWTFGGGTKLEIK

[0358] SEQ ID No. 38 (humanized CD22ALAb VL sequence)

[0359] DIVMTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPARFSGSGSGVEFTLTISSLQSEDFAVYYCSQSTHVPWTFGQGTRLEIK

[0360] The CAR of the present invention may comprise a variant of the sequence shown in SEQ ID No. 33, 34, 35, 36, 37 or 38 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the ability to bind to CD22 (if applicable, when taken together with complementary VL and VH domains).

[0361] B cell antigen expression during B cell ontogeny and subsequent neoplasia

[0362] CD19 is widely considered to be a pan-B antigen, although occasionally it can also show some lineage infidelity. The CD19 molecule contains two extracellular IgSF domains separated by a smaller domain, and a long intracytoplasmic tail that is almost as large as the extracellular part of the molecule and carries an ITAM. CD19 is a key molecule in B cell development and activation. CD22 is an IgSF molecule that can exist in two subtypes, one with seven domains and an intracytoplasmic tail containing three ITIMs (immunoreceptor tyrosine-based inhibitory motifs) and an ITAM; and a splice variant that instead contains five extracellular domains and an intracytoplasmic tail carrying an ITIM. CD22 is believed to be an inhibitory receptor involved in controlling the response of B cells to antigens. Similar to CD19, CD22 is widely considered to be a pan-B antigen, although expression on some non-lymphoid tissues has been described (Wen et al. (2012) J. Immunol. Baltim. Md 1950 188, 1075–1082). Targeting CD22 using therapeutic monoclonal antibodies and immunoconjugates has entered clinical testing. Generation of CD22-specific CARs has been described (Haso et al, 2013, Blood: Volume 121; 7: 1165-74, and James et al 2008, Journal of immunology, Volume 180; Issue 10; Pages 7028-38).

[0363] Detailed immunophenotyping studies of B-cell leukemias have shown that, while surface CD19 is always present, surface CD22 is almost always present. For example, Raponi et al. (2011, supra) studied the surface antigen phenotype of 427 B-ALL cases and found CD22 present in 341 of the cases studied.

[0364] The possibility of CD19 downregulation following the above-mentioned CAR19 targeting can be explained by the Goldie-Coldman hypothesis. The Goldie-Coldman hypothesis predicts that tumor cells mutate to a resistant phenotype at a rate that depends on their inherent genetic instability, and that the probability that a cancer will contain a resistant clone depends on the mutation rate and the size of the tumor. Although it may be difficult for cancer cells to become inherently resistant to direct killing by cytotoxic T cells, antigen loss is still possible. In fact, this phenomenon has been reported previously with targeting of melanoma antigens and EBV-driven lymphomas. According to the Goldie-Coldman hypothesis, the best chance for a cure would be to simultaneously attack non-cross-resistant targets. Given that CD22 is expressed in almost all cases of B-ALL, simultaneous CAR targeting of CD19 together with CD22 could reduce the emergence of anti-CD19-negative clones.

[0365] Antigen binding domain

[0366] The antigen binding domain is the part of CAR that recognizes the antigen. Many antigen binding domains are known in the art, including those based on the antigen binding sites of antibodies, antibody mimics, and T cell receptors. For example, the antigen binding domain can include: a single chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as Darpin (designed ankyrin repeat protein); or a single chain derived from a T cell receptor.

[0367] The antigen binding domain of a CAR that binds to CD19 can be any domain that can bind to CD 19. For example, the antigen binding domain can comprise a CD19 binder as described in Table 1.

[0368] The antigen binding domain of the CAR that binds CD19 may comprise a sequence derived from one of the CD19s shown in Table 2.

[0369] Table 2

[0370]

[0371] The antigen binding domain of the CAR that binds to CD22 can be any domain that can bind to CD22. For example, the antigen binding domain can comprise a CD22 binder as described in Table 3.

[0372] Table 3

[0373]

[0374] spacer domain

[0375] CARs contain a spacer sequence to connect the antigen-binding domain and the transmembrane domain and to spatially separate the antigen-binding domain and the intracellular domain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding.

[0376] In the cells of the present invention, the first and second CARs can comprise different spacer molecules. For example, the spacer sequence can comprise, for example, an IgG1 Fc region, an IgG1 hinge, or a human CD8 stem or a mouse CD8 stem. Alternatively, the spacer can comprise an alternative linker sequence having a length and / or domain spacing properties similar to that of the IgG1 Fc region, IgG1 hinge, or CD8 stem. The human IgG1 spacer can be altered to remove the Fc binding motif.

[0377] The spacer of the anti-CD19 CAR may comprise a CD8 stem spacer, or have a spacer of the same length as the CD8 stem spacer. The spacer of the anti-CD19 CAR may have at least 30 amino acids or at least 40 amino acids. It may have 35-55 amino acids, such as 40-50 amino acids. It may have about 46 amino acids.

[0378] The spacer of the anti-CD22 CAR may comprise an IgG1 hinge spacer, or have a spacer equivalent in length to an IgG1 hinge spacer. The spacer of the anti-CD22 CAR may have less than 30 amino acids or less than 25 amino acids. It may have 15-25 amino acids, such as 18-22 amino acids. It may have about 20 amino acids.

[0379] Examples of amino acid sequences of these spacers are given below:

[0380] SEQ ID No. 4 (hinge-CH2CH3 of human IgG1)

[0381] AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD

[0382] SEQ ID No.5 (Human CD8 Stem):

[0383] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0384] SEQ ID No.6 (Human IgG1 Hinge):

[0385] AEPKSPDKTHTCPPCPKDPK

[0386] SEQ ID No.7 (CD2 Extracellular Domain)

[0387] KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKE KETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCP

[0388] EKGLD

[0389] SEQ ID No.8 (CD34 Extracellular Domain)

[0390] SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTTSLATSPTKP YTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT

[0391] Since CAR is usually a homodimer (see Figure 1 a), cross-pairing may produce heterodimeric chimeric antigen receptors. This is undesirable for a number of reasons, such as: (1) the epitopes on the target cell may not be at the same "level" so that the cross-paired CAR may only be able to bind to one antigen; (2) the VH and VL from different scFvs may be exchanged so that either cannot recognize the target or, worse, recognize an unexpected and unpredictable antigen. The spacer of the first CAR can be sufficiently different from the spacer of the second CAR to avoid cross-pairing. The amino acid sequence of the first spacer can share less than 50%, 40%, 30% or 20% identity with the second spacer at the amino acid level.

[0392] transmembrane domain

[0393] The transmembrane domain is the sequence that allows the CAR to span the membrane.

[0394] The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. It is typically an alpha helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the present invention. Those skilled in the art can use the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ) to determine the presence and span of the transmembrane domain of a protein. In addition, considering that the transmembrane domain of a protein is a relatively simple structure, that is, a polypeptide sequence that is predicted to form a hydrophobic alpha helix of sufficient length to span the membrane can also be an artificially designed TM domain (US7052906B1 describes a synthetic transmembrane component).

[0395] The transmembrane domain may be derived from CD28, which results in good receptor stability.

[0396] The transmembrane domain may be derived from human type 1. The type 1 transmembrane sequence is shown as SEQ ID No. 45.

[0397] SEQ ID No.45

[0398] IIAIAVVGALLLVALIFGTASYLI

[0399] Activation intracellular domain

[0400] The activation intracellular domain is the signaling portion of the CAR. After antigen recognition, the receptor cluster, native CD45 and CD148, are removed from the synapse and the signal is transmitted to the cell. The most commonly used intracellular domain component is CD3-zeta, which contains three ITAMs. It transmits activation signals to T cells after antigen binding. CD3-zeta may not provide all the complete activation signals, and additional co-stimulatory signals may be required. For example, chimeric CD28 and OX40 can be used together with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0401] The cells of the invention comprise two CARs, each having an intracellular domain.

[0402] The intracellular domain of the first CAR and the intracellular domain of the second CAR may comprise:

[0403] (i) an ITAM-containing intracellular domain, such as that from CD3zeta; and / or

[0404] (ii) a co-stimulatory domain, such as the intracellular domain from CD28; and / or

[0405] (iii) Domains that transmit survival signals, such as intracellular domains of the TNF receptor family such as OX-40 or 4-1BB.

[0406] In one arrangement, the costimulatory and survival signaling domains are "shared" between two (or more) CARs in an OR gate. For example, in an OR gate with two CARs (CAR A and CAR B), CAR A can contain a costimulatory domain (e.g., a CD28 intracellular domain) and CAR B can contain a TNF receptor family intracellular domain, such as OX-40 or 4-1BB.

[0407] Intracellular domains containing ITAM motifs can serve as activation intracellular domains in the present invention. Several proteins are known to contain intracellular domains with one or more ITAM motifs. Examples of such proteins include the CD3 epsilon chain, the CD3 gamma chain, and the CD3 delta chain. The ITAM motif can be easily identified because a tyrosine is separated from a leucine or isoleucine by any two other amino acids, resulting in the signature YxxL / I. Typically, but not always, two of these motifs are separated by 6 to 8 amino acids in the tail of the molecule (YxxL / Ix(6-8)YxxL / I). Therefore, those skilled in the art can easily identify proteins containing one or more ITAMs to transmit activation signals. Furthermore, because the motif is simple and does not require complex secondary structures, those skilled in the art can design polypeptides containing artificial ITAMs to transmit activation signals (see WO2000 / 063372, which relates to synthetic signaling molecules).

[0408] The transmembrane and intracellular T cell signaling domain (intracellular domain) of the CAR having an activation intracellular domain may comprise a sequence as shown in SEQ ID No. 9, 10 or 11, or a variant thereof having at least 80% sequence identity.

[0409] SEQ ID No.9 contains the CD28 transmembrane domain and the CD3Z intracellular domain

[0410] FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0411] SEQ ID No.10 contains the CD28 transmembrane domain and the CD28 and CD3 Zeta intracellular domains

[0412] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0413] SEQ ID No.11 contains the CD28 transmembrane domain and the intracellular domains of CD28, OX40 and CD3 Zeta

[0414] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0415] The variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 9, 10 or 11, provided that the sequence provides an effective transmembrane domain and an effective intracellular T cell signaling domain.

[0416] “Split” OR gate intracellular domain

[0417] The present invention provides an OR gate in which the costimulatory / survival signaling domain is "split" between two CARs.

[0418] In this aspect, the present invention provides a cell that co-expresses a first chimeric antigen receptor (CAR) and a second CAR on the cell surface, each CAR comprising an intracellular signaling domain, wherein the intracellular signaling domain of the first CAR comprises a costimulatory domain; and the intracellular signaling domain of the second CAR comprises a TNF receptor family intracellular domain.

[0419] The first and second CARs can bind to different antigens. For example, the first CAR can bind to CD19 and the second CAR can bind to CD22; or the first CAR can bind to CD22 and the second CAR can bind to CD19.

[0420] The intracellular signaling domain of the first CAR may include a costimulatory domain and may not include a domain that transmits a survival signal (e.g., a TNF receptor family intracellular domain). The intracellular signaling domain of the second CAR may include a TNF receptor family intracellular domain and may not include a costimulatory domain (e.g., a CD28 intracellular domain).

[0421] The costimulatory domain may be a CD28 costimulatory domain. The CD28 costimulatory domain may have a sequence as shown in SEQ ID No. 41.

[0422] SEQ ID No.41 (CD28 co-stimulatory intracellular domain)

[0423] SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0424] The CAR of the present invention may comprise a variant of the sequence shown in SEQ ID No. 41 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the ability to co-stimulate T cells upon antigen recognition, i.e., provide signal 2 to T cells.

[0425] The TNF receptor family intracellular domain can be an OX40 or 4-1BB intracellular domain. The OX40 intracellular domain can have the sequence shown in SEQ ID No. 42. The 4-1BB intracellular domain can have the sequence shown in SEQ ID No. 43.

[0426] SEQ ID No.42 (OX40 intracellular domain)

[0427] RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0428] SEQ ID No.43 (4-1BB intracellular domain)

[0429] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0430] The CAR of the present invention may comprise a variant of the sequence shown in SEQ ID No. 42 or 43 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the ability to transmit a survival signal to T cells upon antigen recognition.

[0431] The intracellular signaling domain of the first and / or second CAR may further comprise an ITAM-containing domain, such as a CD3 zeta domain. The CD3 zeta domain may have a sequence as shown in SEQ ID No. 44.

[0432] SEQ ID No.44 (CD3zeta intracellular domain)

[0433] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0434] The CAR of the present invention may comprise a variant of the sequence shown in SEQ ID No. 44 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the ability to induce T cell signaling upon antigen recognition, i.e., provide signal 1 to T cells.

[0435] The first CAR may have the following structure:

[0436] AgB1-spacer1-TM1-costim-ITAM

[0437] in:

[0438] AgB1 is the antigen-binding domain of the first CAR;

[0439] Spacer 1 is the spacer of the first CAR;

[0440] TM1 is the transmembrane domain of the first CAR;

[0441] Costim is the costimulatory domain; and

[0442] ITAM is an intracellular domain containing ITAM.

[0443] "Costim" may be the CD28 costimulatory domain.

[0444] "ITAM" may be the CD3zeta intracellular domain.

[0445] The second CAR may have the following structure:

[0446] AgB2-Spacer2-TM2-TNF-ITAM

[0447] in:

[0448] AgB2 is the antigen-binding domain of the second CAR;

[0449] Spacer 2 is the spacer for the second CAR;

[0450] TM2 is the transmembrane domain of the second CAR;

[0451] TNF is the intracellular domain of the TNF receptor; and

[0452] ITAM is an intracellular domain containing ITAM.

[0453] "TNF" can be the intracellular domain of a TNF receptor such as the intracellular domain of OX40 or 4-1BB.

[0454] Also provided are nucleic acids encoding both a first and a second chimeric antigen receptor (CAR) having a "split" intracellular domain; and kits comprising two nucleic acids, one encoding the first CAR and one encoding the second CAR, comprising a split intracellular domain as defined above.

[0455] Co-expression sites

[0456] A second aspect of the invention relates to nucleic acids encoding a first and a second CAR.

[0457] The nucleic acid can produce a protein comprising two CAR molecules connected by a cleavage site. The cleavage site can be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the first and second CARs without the need for any external cleavage activity.

[0458] A variety of self-cleavage sites are known, including the foot-and-mouth disease virus (FMDV) 2A peptide and similar sequences (Donnelly et al, Journal of General Virology (2001), 82, 1027-1041), for example the 2A-like peptide from Thoseaasigna virus, which has the sequence shown as SEQ ID No. 12.

[0459] SEQ ID No.12

[0460] RAEGRGSLLTCGDVEENPGP

[0461] The co-expression sequence may be an internal ribosome entry sequence (IRES).The co-expression sequence may be an internal promoter.

[0462] cell

[0463] The present invention relates to cells that co-express a first CAR and a second CAR on the cell surface, wherein one CAR binds to CD19 and the other CAR binds to CD22.

[0464] The cell can be any eukaryotic cell capable of expressing a CAR on its cell surface, such as an immune cell.

[0465] In particular, the cell may be an immune effector cell such as a T cell or a natural killer (NK) cell.

[0466] T cells or T lymphocytes are lymphocyte types that play a central role in cell-mediated immunity. Through the presence of cell surface T cell receptors (TCRs), they can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells). There are various types of T cells, as summarized below.

[0467] Helper T helper cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when they present peptide antigens through MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0468] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. IL-10, adenosine, and other molecules secreted by regulatory T cells can inactivate CD8+ cells to an incompetent state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0469] Memory T cells are a subset of antigen-specific T cells that persist for a long time after the infection has resolved. They rapidly expand into a large number of effector T cells after being exposed to their cognate antigens again, thereby providing the immune system with "memory" for past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0470] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity toward the end of the immune response and to suppress autoreactive T cells that escape the thymic negative selection process.

[0471] Two major types of CD4+ Treg cells have been described - naturally occurring Treg cells and adaptive Treg cells.

[0472] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) appear in the thymus and are associated with interactions between developing T cells and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, leading to the fatal autoimmune disease IPEX.

[0473] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can be generated during a normal immune response.

[0474] The T cells of the present invention may be any of the T cell types described above, in particular CTLs.

[0475] Natural killer (NK) cells are a type of cytolytic cell that forms part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0476] NK cells (belonging to the innate lymphoid cell group) are defined as large granular lymphocytes (LGLs) and constitute the third type of cell that differentiates from the common lymphoid progenitor cells that give rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before they enter the circulation.

[0477] The CAR cells of the present invention can be any of the cell types described above.

[0478] CAR-expressing cells, such as CAR-expressing T cells or NK cells, can be generated ex vivo from the patient's own peripheral blood (first party), or in the setting of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from the peripheral blood of an unrelated donor (third party).

[0479] The present invention also provides a cell composition comprising a T cell expressing CAR according to the present invention and / or a NK cell expressing CAR. The cell composition can be prepared by transducing a blood sample in vitro using the nucleic acid according to the present invention.

[0480] Alternatively, cells expressing CARs can be derived from induced progenitor cells or embryonic progenitor cells that are differentiated in vitro into relevant cell types, such as T cells. Alternatively, immortalized cell lines, such as T cell lines, that retain their lytic function and can be used as therapeutic agents can be used.

[0481] In all of these embodiments, CAR cells are generated by introducing CAR-encoding DNA or RNA by one of a number of means including transduction using a viral vector, transfection using DNA or RNA.

[0482] The CAR T cells of the present invention can be ex vivo T cells from a subject. T cells can be from a peripheral blood mononuclear cell (PBMC) sample. T cells can be activated and / or expanded before being transduced with a nucleic acid encoding CAR, for example, by treatment with an anti-CD3 monoclonal antibody.

[0483] The CAR T cells of the present invention can be prepared as follows:

[0484] (i) isolating a sample containing T cells from a subject or other sources listed above; and

[0485] (ii) transducing or transfecting T cells with one or more nucleic acid sequences encoding the first and second CARs.

[0486] T cells can then be selected by purification, for example, based on co-expression of the first and second CARs.

[0487] Nucleic acid sequence

[0488] The second aspect of the present invention relates to one or more nucleic acid sequences encoding the first CAR and the second CAR as defined in the first aspect of the present invention.

[0489] The nucleic acid sequence may be, for example, an RNA, DNA or cDNA sequence.

[0490] The nucleic acid sequence can encode one chimeric antigen receptor (CAR) that binds CD19 and another CAR that binds CD22.

[0491] The nucleic acid sequence can have the following structure:

[0492] AgB1-Spacer1-TM1-coexpr-AbB2-Spacer2-TM2

[0493] in

[0494] AgB1 is the nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0495] Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

[0496] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0497] coexpr is a nucleic acid sequence that enables co-expression of two CARs;

[0498] AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0499] Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

[0500] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0501] The nucleic acid sequence, when expressed in a T cell, encodes a polypeptide that is cleaved at the cleavage site such that the first and second CARs are co-expressed on the surface of the T cell.

[0502] The first CAR can bind to CD19 and the second CAR can bind to CD22. Alternatively, the first CAR can bind to CD22 and the second CAR can bind to CD19.

[0503] To avoid homologous recombination, alternative codons can be used in sequence regions encoding the same or similar amino acid sequences.

[0504] Due to the degeneracy of codons, it is possible to use alternative codons that encode the same amino acid sequence. For example, the codons "ccg" and "cca" both encode the amino acid proline, so the used "ccg" can be exchanged for "cca" without affecting the amino acid at that position in the translated protein sequence.

[0505] Alternative codons that can be used to encode each amino acid are summarized in Table 3.

[0506] Table 3

[0507]

[0508] Alternative codons may be used in the portion of the nucleic acid sequence encoding the spacer of the first CAR and the spacer of the second CAR, particularly if the same or similar spacer is used in the first and second CARs. Figure 4 Two sequences encoding the spacer region HCH2CH3-hinge are shown, one of which has used alternative codons.

[0509] Alternative codons can be used in the nucleic acid sequence encoding the transmembrane domain of the first CAR and the transmembrane portion of the second CAR, particularly if the same or similar transmembrane domains are used in the first and second CARs. Figure 4 Two sequences encoding the CD28 transmembrane domain are shown, one of which has used alternative codons.

[0510] Alternative codons can be used in the portion of the nucleic acid sequence encoding all or part of the intracellular domain of the first CAR and all or part of the intracellular domain of the second CAR. Alternative codons can be used for the CD3 zeta intracellular domain. Figure 4 Two sequences encoding the CD3zeta intracellular domain are shown, one of which has used alternative codons.

[0511] Alternative codons may be used in one or more costimulatory domains, such as the CD28 intracellular domain.

[0512] Alternative codons can be used to transmit one or more domains of survival signals, such as the OX40 and 41BB intracellular domains.

[0513] Alternative codons may be used in portions of the nucleic acid sequence encoding the intracellular domain of CD3 zeta and / or portions of the nucleic acid sequence encoding one or more costimulatory domains and / or portions of the nucleic acid sequence encoding one or more domains that transmit a survival signal.

[0514] carrier

[0515] The present invention also provides a vector, or a kit of vectors, comprising one or more nucleic acid sequences encoding CARs. Such vectors can be used to introduce nucleic acid sequences into host cells so that they can express the first and second CARs.

[0516] The vector may be, for example, a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

[0517] The vector may be capable of transfecting or transducing T cells.

[0518] Pharmaceutical composition

[0519] The present invention also relates to a pharmaceutical composition comprising a plurality of CAR-expressing cells according to the first aspect of the present invention, such as T cells or NK cells. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds. Such dosage forms may be, for example, in a form suitable for intravenous infusion.

[0520] Treatment

[0521] The cells of the present invention are capable of killing cancer cells, such as B cell lymphoma cells.CAR cells, such as expression T cells, can be produced in vitro from the patient's own peripheral blood (first party), or produced in the setting of hematopoietic stem cell transplantation (second party) from donor peripheral blood, or from the peripheral blood (third party) of an unrelated donor. Alternatively, CAR T cells can be derived from inducible progenitor cells or embryonic progenitor cells to differentiate into T cells in vitro. In these cases, CAR T cells are generated by including using viral vector transduction, using one of the many ways of DNA or RNA transfection to introduce DNA or RNA encoding CAR.

[0522] The cells of the present invention may be capable of killing target cells, such as cancer cells. Target cells may be identified by the expression of CD19 or CD22.

[0523] Table 4. Expression of lymphoid antigens on lymphoid leukemias

[0524]

[0525] Adapted from Campana et al., (Immunophenotyping of leukemia. J. Immunol. Methods 243, 59-75 (2000)). cIgμ - cytoplasmic immunoglobulin heavy chain; sIgμ - surface immunoglobulin heavy chain.

[0526] The expression of commonly studied lymphoid antigens on different types of B cell leukemias is closely related to B cell ontogeny (see Figure 2 ).

[0527] The T cells of the present invention can be used to treat cancer, particularly B cell malignancies.

[0528] Examples of cancers that express CD19 or CD22 are B-cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; and B-cell leukemias.

[0529] For example, the B-cell lymphoma may be diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma (MZL), or mucosa-associated lymphoid tissue lymphoma (MALT), small cell lymphocytic lymphoma (overlapping with chronic lymphocytic leukemia), Mantle cell lymphoma (MCL), Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma (which may present as macroglobulinemia), nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, or primary central nervous system lymphoma.

[0530] B-cell leukemia may be acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, precursor B-lymphocytic leukemia, or hairy cell leukemia.

[0531] The B-cell leukemia may be acute lymphoblastic leukemia.

[0532] Treatment with the T cells of the invention can help prevent the escape or release of tumor cells that typically occurs using standard approaches.

[0533] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to practice the present invention and are not intended to limit the scope of the present invention in any way. Example

[0534] Example 1 - Proof of Concept of CD19 / CD22 Logic 'OR' Gate

[0535] The CD19'OR'CD22 CAR was constructed by co-expressing CD19 and CD22 in the same vector. The anti-CD19 binder is an scFv derived from the surface-remodeled B4 antibody (Roguska et al. (1996) Protein Eng. 9, 895–904), while the anti-CD22 binder is an scFv derived from the humanized RFB4 antibody. The human IgG1 hinge-CH2-CH3 spacer is used for both CARs, and its coding sequence is codon-wobbled to avoid homologous recombination through the integration vector. The TM domain in both CARs is derived from the TM domain of CD28, while the intracellular domain of both CARs is composed of CD3-Zeta. Once again, these homologous sequences are codon-wobbled. Co-expression is achieved by in-frame cloning of two CARs separated by the FMD-2A peptide. The nucleic acid sequence and amino acid sequence of the CD19 / CD22'OR' gate construct are shown as SEQ ID NOs: 13 and 14, respectively.

[0536] SEQ ID NO:13

[0537]

[0538] SEQ ID NO:14

[0539]

[0540] In order to prove the co-expression of two CARs, epitope tags (HA or V5, respectively) were used to mark the scFv of each CAR. This subsequent single open reading frame was cloned into the SFG retroviral vector. T cells were transduced using this vector and expressed by using anti-HA and anti-V5 staining and flow cytometry. Two CARs can be detected on the surface of T cells expressing the gene cassette.

[0541] Next, we challenged T cells expressing the CD19 OR CD22 CAR gate with target cells that expressed neither antigen, both antigens, or one antigen, along with control T cells that expressed no CAR, anti-CD19 CAR alone, or anti-CD22 CAR alone. We found that OR gated CAR T cells were able to kill target cells expressing either or both target antigens ( Figure 5 ).

[0542] Example 2 - Identification and Characterization of CD19 ALAb and CD22 ALAb

[0543] CD19 binders (CD19 ALAbs) were identified, humanized, and the affinities of murine and humanized IgGs and scFvs were determined and compared to the "gold standard" anti-CD19 binder fmc63. In parallel, CD22 binders (CD22 ALAbs) were identified, humanized, and the affinities of murine and humanized IgGs and scFvs were determined and compared to the "gold standard" anti-CD22 binder M971.

[0544] Experiments were performed on a Biacore T200 using HBS-P as running and dilution buffer. BIAevaluation software version 2.0 was used for data processing. For binding kinetics, mouse anti-human IgG / goat anti-mouse IgG were covalently coupled to a CM5 sensor chip. IgG or scFv-Fc protein was captured and various concentrations of the interacting paired protein were injected on the flow cell at a flow rate of 30 μl / min. Kinetic rate constants were obtained by curve fitting according to a 1:1 Langmuir binding model. Bulk refractive index differences were subtracted using a blank control flow cell in which the capture antibody had been immobilized at the same level as the active surface. Double reference subtraction was performed using buffer only.

[0545] The results are shown in Figures 6 to 8 .

[0546] The data showed that humanized CD22ALAb has similar binding affinity to CD22 as murine CD22ALAb ( Figure 6) and similar binding kinetics. The scFv forms of mouse and humanized CD22 ALAbs have significantly higher binding affinity for CD22 than the gold standard CD22 binding antibody M971 ( Figure 6 ).

[0547] Although the binding affinity of murine and humanized CD19ALAb in IgG format was found to be similar (data not shown), surprisingly, the binding affinity of humanized CD19ALAb in scFv format was found to be higher than that of murine CD19ALAb ( Figure 7 The binding affinity of CD19ALAb is comparable (and possibly slightly better) to that of the gold standard anti-CD19Ab, fmc63 ( Figure 8 ).

[0548] Example 3 - Comparative functional assays using CD19ALAb / fmc63 CAR and CD22ALAb / M971 CAR

[0549] The antigen-binding domain of a CAR can influence its function. In this study, CARs containing CD19ALAb and CD22ALAb were created, and their function was compared with that of equivalent CARs with antigen-binding domains based on fmc63 or M971.

[0550] CARs containing scFvs based on fmc63 (anti-CD19) and M971 (anti-CD22) can be considered gold standard antibodies, as both CARs are in clinical development.

[0551] CARs were constructed and expressed based on CD19ALAb, fmc63, CD22ALAb, and M971. Their structures are shown in Figure 2. Figure 9 The CARs differ only in their antigen-binding domains. In all constructs, the binding domain is linked to the membrane using a CD8 stalk spacer and contains intracellular activation motifs from 41BB and CD3-zeta.

[0552] Retrovirus was produced by transiently transfecting 293T cells with plasmids encoding CAR, gag / pol, and envelope protein RD114. The supernatant was harvested 3 days later and used to transduce PHA / IL2-activated PBMCs using the same titer of retrovirus on fibronectin-coated plates. CAR expression was confirmed by flow cytometry 6 days after transduction, and PBMCs were co-cultured with CD19+BFPSupT1 cells (fmc63 and CD19ALAb CAR) or CD22+BFP SupT1 cells (M971 and CD22ALAb CAR) at a 1:1 ratio. Target cell killing was determined one and three days later. After one and three days, the supernatant was also removed and interferon-γ levels were determined by ELISA.

[0553] The results are as follows Figure 10 and11 shown.

[0554] like Figure 10 As shown, CARs with CD19ALAb antigen binding domains gave more killing of CD11+ve target cells on days 1 and 3 than the equivalent CARs with fmc63 binding domains ( Figure 10 ).

[0555] Regarding CD22, the CAR with the CD22ALAb antigen binding domain gave more killing of CD22+ve target cells after three days than the equivalent CAR with the M971 binding domain ( Figure 11 a). After the same time period, the IFNγ release of CD22ALAb CAR was significantly higher than that of M971CAR.

[0556] Thus, CARs with antigen-binding domains based on CD19 ALAb and CD22 ALAb have improved properties in terms of target cell killing compared to equivalent CARs based on fmc63 and M971.

[0557] The CD22 ALAb results are particularly surprising given the results reported by Haso et al. (2013) as described above. In that study, different anti-CD22 CARs were prepared and tested that had binding domains based on the anti-CD22 antibodies HA22, BL22, and m971. The HA22 and BL22 scFvs bind to Ig domain 3 of CD22, while m971 binds to Ig domains 5-7 of CD22 (see Haso et al. (2013)). Figure 2 B). It was reported that m971-derived CARs showed superior target cell killing activity compared to HA22-derived CARs, a finding attributed to the importance of the CD22 epitope targeted by the CAR (Haso et al. (2013) p. 1168, last paragraph). It was concluded that targeting the membrane proximal domain of CD22 was the “key factor” for developing highly active anti-CD22 CARs (Discussion, last paragraph). In contrast to this finding, this paper Figure 11 The data presented indicate that CD22 ALAb, which targets an epitope in Ig domain 3 of CD22—a “membrane distal” epitope compared to Ig domains 5-7 targeted by m9712—has superior target cell killing ability than the m971-based anti-CD22 CAR.

[0558] Example 4 - Study of OR gate constructs with different intracellular domain combinations

[0559] Four OR gate constructs were developed as shown in Figure 13. They all encode a CD19 / CD22 OR gate with the same antigen binding domain, spacer domain and transmembrane domain: the only difference between the constructs is in the intracellular domain, which is shown in the following table:

[0560] Construct CD19 CAR intracellular domain CD22 CAR intracellular domain A 41BB-CD3ζ 41BB-CD3ζ B OX40-CD3ζ OX40-CD3ζ C 41BB-CD3ζ CD28-CD3ζ D OX40-CD3ζ CD28-CD3ζ

[0561] The ability of cells expressing each CD19 / CD22 OR gate to kill Raji cells in vitro was determined as described above. Transduced PBMCs expressing various OR gate combinations were co-cultured with CD19+ / CD22+ Raji target cells at effector:target ratios of 1:1 and 1:10 for 72 hours.

[0562] The results are as follows Figure 14 All four OR gates were found to kill target cells significantly better than the fmc63 and M971 CARs. Using a 1:10 effector:target ratio, the "split" intracellular domain OR gate, which had 4-1BBzeta / OX40zeta on one CAR and CD28zeta on the other, was shown to have the best killing activity.

[0563] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to these specific embodiments. Indeed, various modifications of the modes for carrying out the present invention that are apparent to those skilled in molecular biology, cell biology, or related fields are intended to be within the scope of the appended claims. Sequence Listing <110> UCL Business plc Autorus Co., Ltd. (Autorus Co., Ltd.) <120> cell <130> P106469PCT <150> GB 1423172.4 <151> 2014-12-24 <160> 49 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> signal peptide <400> 1 Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Asp His Ala Asp Gly 20 <210> 2 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> signal peptide <400> 2 Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 3 <211> 20 <212> PRT <213> Artificial sequence <220> <223> signal peptide <400> 3 Met Ala Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys 20 <210> 4 <211> 234 <212> PRT <213> Artificial sequence <220> <223> Spacer sequence, hinge-CH2CH3 of human IgG1 <400> 4 Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp 225 230 <210> 5 <211> 46 <212> PRT <213> Artificial sequence <220> <223> Spacer sequence, human CD8 stem <400> 5 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 35 40 45 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Spacer sequence, human IgG1 hinge <400> 6 Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Lys Asp Pro Lys 20 <210> 7 <211> 185 <212> PRT <213> Artificial sequence <220> <223> Spacer sequence, CD2 extracellular domain <400> 7 Lys Glu Ile Thr Asn Ala Leu Glu Thr Trp Gly Ala Leu Gly Gln Asp 1 5 10 15 Ile Asn Leu Asp Ile Pro Ser Phe Gln Met Ser Asp Asp Ile Asp Asp 20 25 30 Ile Lys Trp Glu Lys Thr Ser Asp Lys Lys Lys Ile Ala Gln Phe Arg 35 40 45 Lys Glu Lys Glu Thr Phe Lys Glu Lys Asp Thr Tyr Lys Leu Phe Lys 50 55 60 Asn Gly Thr Leu Lys Ile Lys His Leu Lys Thr Asp Asp Gln Asp Ile 65 70 75 80 Tyr Lys Val Ser Ile Tyr Asp Thr Lys Gly Lys Asn Val Leu Glu Lys 85 90 95 Ile Phe Asp Leu Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser 100 105 110 Trp Thr Cys Ile Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly Thr 115 120 125 Asp Pro Glu Leu Asn Leu Tyr Gln Asp Gly Lys His Leu Lys Leu Ser 130 135 140 Gln Arg Val Ile Thr His Lys Trp Thr Thr Ser Leu Ser Ala Lys Phe 145 150 155 160 Lys Cys Thr Ala Gly Asn Lys Val Ser Lys Glu Ser Ser Val Glu Pro 165 170 175 Val Ser Cys Pro Glu Lys Gly Leu Asp 180 185 <210> 8 <211> 259 <212> PRT <213> Synthetic Sequence <220> <223> Spacer sequence, CD34 extracellular domain <400> 8 Ser Leu Asp Asn Asn Gly Thr Ala Thr Pro Glu Leu Pro Thr Gln Gly 1 5 10 15 Thr Phe Ser Asn Val Ser Thr Asn Val Ser Tyr Gln Glu Thr Thr Thr 20 25 30 Pro Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly 35 40 45 Asn Glu Ala Thr Thr Asn Ile Thr Glu Thr Thr Val Lys Phe Thr Ser 50 55 60 Thr Ser Val Ile Thr Ser Val Tyr Gly Asn Thr Asn Ser Ser Val Gln 65 70 75 80 Ser Gln Thr Ser Val Ile Ser Thr Val Phe Thr Thr Pro Ala Asn Val 85 90 95 Ser Thr Pro Glu Thr Thr Leu Lys Pro Ser Leu Ser Pro Gly Asn Val 100 105 110 Ser Asp Leu Ser Thr Thr Ser Thr Ser Leu Ala Thr Ser Pro Thr Lys 115 120 125 Pro Tyr Thr Ser Ser Ser Pro Ile Leu Ser Asp Ile Lys Ala Glu Ile 130 135 140 Lys Cys Ser Gly Ile Arg Glu Val Lys Leu Thr Gln Gly Ile Cys Leu 145 150 155 160 Glu Gln Asn Lys Thr Ser Ser Cys Ala Glu Phe Lys Lys Asp Arg Gly 165 170 175 Glu Gly Leu Ala Arg Val Leu Cys Gly Glu Glu Gln Ala Asp Ala Asp 180 185 190 Ala Gly Ala Gln Val Cys Ser Leu Leu Leu Ala Gln Ser Glu Val Arg 195 200 205 Pro Gln Cys Leu Leu Leu Val Leu Ala Asn Arg Thr Glu Ile Ser Ser 210 215 220 Lys Leu Gln Leu Met Lys Lys His Gln Ser Asp Leu Lys Lys Leu Gly 225 230 235 240 Ile Leu Asp Phe Thr Glu Gln Asp Val Ala Ser His Gln Ser Tyr Ser 245 250 255 Gln Lys Thr <210> 9 <211> 140 <212> PRT <213> Artificial sequence <220> <223> Contains the CD28 transmembrane domain and the CD3 Z intracellular domain <400> 9 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe 20 25 30 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 35 40 45 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 50 55 60 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 65 70 75 80 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 85 90 95 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 100 105 110 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 115 120 125 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 130 135 140 <210> 10 <211> 180 <212> PRT <213> Artificial sequence <220> <223> Contains the CD28 transmembrane domain and the CD28 and CD3 Zeta intracellular domains <400> 10 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 65 70 75 80 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 85 90 95 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 100 105 110 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 115 120 125 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 130 135 140 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 145 150 155 160 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 165 170 175 Leu Pro Pro Arg 180 <210> 11 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Containing the CD28 transmembrane domain and the intracellular domains of CD28, OX40 and CD3 Zeta <400> 11 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro 65 70 75 80 Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 85 90 95 Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala 100 105 110 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 115 120 125 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 130 135 140 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 145 150 155 160 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 165 170 175 Glu Ile Gly Met Light Gly Glu Arg Arg Arg Gly Light Gly His Asp Gly 180 185 190 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu Free Mp3 Download 195 200 205 His Met Gln Ala Leu Pro Pro Arg 210 215 <210> 12 <211> 20 <212> PRT <213> The snowstorm <220> <223> 2A snowstorm <400> 12 Arg Ala Gly Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Gly 1 5 10 15 Asn Pro Gly Pro 20 <210> 13 <211> 3402 <212> DNA <213> The snowstorm <220> <223> CD19 / CD22 'OR' sludge <400> 13 atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga ccatacccct acgacgtgcc cgactacgcc agcctgagcg gaggcggcgg cagccaggtg 120 cagctggtgc agagcggagc cgaggtgaag aagcctggcg ccagcgtgaa ggtgtcctgt 180 aaggccagcg gctacacctt caccagcaac tggatgcact gggtgaggca ggcccctgga 240 cagggactgg agtggatggg cgagatcgac cccagcgaca gctacaccaa ctacaaccag 300 aagttcaagg gccgggtgac catcaccgtg gataagagcg ccagcaccgc ctacatggag 360 ctgtccagcc tgagaagcga ggataccgcc gtgtactact gtgccagagg cagcaacccc 420 tactactacg ctatggacta ctggggccag ggcaccctgg tgaccgtgtc cagcggcgga 480 ggaggaagcg gagggggcgg atctggcggc ggagggagcg agatcgtgct gacccagagc 540 cccgccaccc tgagcctgag ccctggcgag agagccaccc tgtcctgtag cgccagcagc 600 ggcgtgaatt acatgcactg gtatcagcag aagcccggcc aggcccccag aagatggatc 660 tacgacacca gcaagctggc cagcggcgtg cccgccagat tcagcggcag cggctccggc 720 accagctaca gcctgaccat cagcagcctg gagcctgagg atttcgccgt gtattattgc 780 caccagaggg gcagctacac ctttggcggc ggaacaaagc tggagatcaa gcgctcagat 840 cccaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 900 ctgtccctgc gcccagaggc gtgccggcca gcggcgggg gcgcagtgca cacgagggg 960 ctggacttcg cctgtgatat cttttgggtg ctggtggtgg ttggtggagt cctggcttgc 1020 tatagcttgc tagtaacagt ggcctttatt atttctggg tgaggagagt gaagttcagc 1080 aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1140 ctaggagagagagagagaga cgatgttttg ggagagagga gtggccggga ccctgagatg 1200 gggggaaagc cgagaaggaa gaaccctcag gaagcctgt acaatgaact cgagaaagat 1260 aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1320 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1380 atgcaggccc tgcctcctcg cagagccgag ggcagggggaa gtcttctaac atgcggggac 1440 gtgggaaa atcccgggcc catggagttt gggctgagct ggctttttct tgtggctatt 1500 ttaaaaggtg tccagtgcga ggtgcagctg gtggagtctg ggggaggctt ggtccagcca 1560 ggggggtccc tgcgcctctc ctgtgcagcc tctggattcg ctttcagtat ctatgacatg 1620 tcttgggtcc gccaggttcc ggggaagggg ctggagtggg tctcatatat tagtagtggt 1680 ggtggtacca cctattaccc ggacactgtg aagggccgct tcaccatctc ccgtgacaat 1740 tcccgcaaca ctctggatct tcaaatgaac agtctgcgcg tcgaggacac ggctgtctat 1800 tattgtgcgc gtcatagtgg ctacggtagt agctacgggg ttttgtttgc ttactggggc 1860 caaggaaccc tggtcaccgt ctcctcaggt ggaggcggtt caggcggagg tggctctggc 1920 ggtggcggat cggacatcca gatgactcag tctccgtcct ccctgtctgc atctgtagga 1980 gaccgcgtca ccatcacctg ccgtgcaagt caggacatta gcaattattt aaactggctt 2040 caacagaaac cggggaaagc cccgaagctc ctgatttact acacatcaat cttacactca 2100 ggagtcccgt cacgcttcag cggcagtgga tctgggacag aattcactct cacaatcagc 2160 agcctgcagc cggaagattt tgcaacttat tactgtcaac agggtaatac gcttccgtgg 2220 acgtttggcc aggggaccaa actggaaatc aaacgttcgg atccagccga accaaagagc 2280 cccgataaga cccacacctg tcccccctgc ccagccccag agctgctggg aggccccagc 2340 gtgtttctgt ttccacccaa gccaaaggat acctgatga ttagtagaac acccgaagtg 2400 acctgtgtgg tggtggatgt gtctcacgag gaccccgagg tgaaatttaa ttggtatgtt 2460 gatggtgttg aagtgcacaa cgccaaaacc aaacccagag aggagcagta caattctacc 2520 tatagagtcg tgtctgtgct gacagtgctg catcaggatt ggctgaacgg aaaagaatac 2580 aaatgtaaag tgagcaataa ggccctgccc gctccaattg agaagacaat tagcaaggcc 2640 aagggccagc caagggagcc ccaggtgtat acactgccac ccagtagaga cgaactgaca 2700 aagaatcagg tgtctctgac atgtctggtg aagggatttt acccatctga tatcgccgtg 2760 gaatgggaat ctaacggcca gcccgagaat aactataaga caaccccacc agtgctggat 2820 agcgatggca gcttttttct gtattctaag ctgacagtgg ataagtcccg gtggcagcag 2880 ggaaatgtgt ttagctgtag tgtcatgcat gaggccctgc acaatcacta tacccagaaa 2940 tctctgagtc tgagcccagg caagaaggac cccaagttct gggtcctggt ggtggtggga 3000 ggcgtgctgg cctgttactc tctcctggtg accgtggcct tcatcatctt ttgggtgcgc 3060 tcccgggtga agttttctcg ctctgccgat gccccagcct atcagcaggg ccagaatcag 3120 ctgtacaatg aactgaacct gggcaggcgg gaggagtacg acgtgctgga taagcggaga 3180 ggcagagacc ccgagatggg cggcaaacca cggcgcaaaa atccccagga gggactctat 3240 aacgagctgc agaaggacaa aatggccgag gcctattccg agatcggcat gaagggagag 3300 agaagacgcg gaaagggcca cgacggcctg tatcagggat tgtccaccgc tacaaaagat 3360 acatatgatg ccctgcacat gcaggccctg ccacccagat ga 3402 <210> 14 <211> 11,33 <212> PRT <213> Artificial Sequence <220> <223> CD19 / CD22 'OR' Gate Construct <400> 14 Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu 20 25 30 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 35 40 45 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 50 55 60 Tyr Thr Phe Thr Ser Asn Trp Met His Trp Val Arg Gln Ala Pro Gly 65 70 75 80 Gln Gly Leu Glu Trp Met Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr 85 90 95 Asn Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys 100 105 110 Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 115 120 125 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala 130 135 140 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 165 170 175 Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 180 185 190 Thr Leu Ser Cys Ser Ala Ser Ser Gly Val Asn Tyr Met His Trp Tyr 195 200 205 Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr Asp Thr Ser 210 215 220 Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 225 230 235 240 Thr Ser Tyr Ser Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala 245 250 255 Val Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly Gly Gly Thr 260 265 270 Lys Leu Glu Ile Lys Arg Ser Asp Pro Thr Thr Thr Pro Ala Pro Arg 275 280 285 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 290 295 300 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 305 310 315 320 Leu Asp Phe Ala Cys Asp Ile Phe Trp Val Leu Val Val Val Gly Gly 325 330 335 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 340 345 350 Trp Val Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 355 360 365 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 370 375 380 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 385 390 395 400 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 405 410 415 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 420 425 430 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 435 440 445 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 450 455 460 Pro Pro Arg Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 465 470 475 480 Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser Trp Leu Phe 485 490 495 Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu 500 505 510 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 515 520 525 Ala Ala Ser Gly Phe Ala Phe Ser Ile Tyr Asp Met Ser Trp Val Arg 530 535 540 Gln Val Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Gly 545 550 555 560 Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile 565 570 575 Ser Arg Asp Asn Ser Arg Asn Thr Leu Asp Leu Gln Met Asn Ser Leu 580 585 590 Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Ser Gly Tyr 595 600 605 Gly Ser Ser Tyr Gly Val Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu 610 615 620 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 625 630 635 640 Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 645 650 655 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 660 665 670 Ile Ser Asn Tyr Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro 675 680 685 Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser 690 695 700 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 705 710 715 720 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn 725 730 735 Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 740 745 750 Ser Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro 755 760 765 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 770 775 780 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 785 790 795 800 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 805 810 815 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 820 825 830 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 835 840 845 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 850 855 860 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 865 870 875 880 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 885 890 895 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 900 905 910 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 915 920 925 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 930 935 940 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 945 950 955 960 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 965 970 975 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys 980 985 990 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 995 1000 1005 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Arg Val 1010 1015 1020 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 1025 1030 1035 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 1040 1045 1050 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 1055 1060 1065 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 1070 1075 1080 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 1085 1090 1095 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 1100 1105 1110 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 1115 1120 1125 Ala Leu Pro Pro Arg 1130 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Complementarity determining region (CDR) variable heavy chain (VH) CDR1 <400> 15 Ser Tyr Trp Met Asn 1 5 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 <400> 16 Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 <400> 17 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Variable light chain (VL) CDR1 <400> 18 Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn 1 5 10 15 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 <400> 19 Asp Ala Ser Asn Leu Val Ser 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 <400> 20 Gln Gln Ser Thr Glu Asp Pro Trp Thr 1 5 <210> twenty one <211> 235 <212> PRT <213> Artificial sequence <220> <223> Chimeric Antigen Receptor (CAR), mouse CD19ALAb scFv sequence <400> twenty one Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Asp Ile Gln Leu 115 120 125 Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr 130 135 140 Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr 145 150 155 160 Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile 165 170 175 Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly 180 185 190 Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Lys 195 200 205 Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp 210 215 220 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 225 230 235 <210> 22 <211> 236 <212> PRT <213> Artificial sequence <220> <223> CAR, humanized CD19ALAb scFv sequence - Heavy 19, Kappa 16 <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Glu Ser Ala Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln Leu 115 120 125 Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr 130 135 140 Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr 145 150 155 160 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 165 170 175 Tyr Asp Ala Ser Asn Leu Val Ser Gly Val Pro Asp Arg Phe Ser Gly 180 185 190 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 195 200 205 Ala Asp Val Ala Val Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp 210 215 220 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 225 230 235 <210> 2 <211> 124 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> CAR, murine CD19ALAb VH sequence <400> 23 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 24 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> CAR, Humanized CD19ALAb VH Sequence <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Glu Ser Ala Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 25 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> CAR, murine CD19ALAb VL sequence <400> 25 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CAR, Humanized CD19ALAb VL Sequence, Kappa16 <400> 26 Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Ala Asp Val Ala Val Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 27 Asn Tyr Trp Ile Asn 1 5 <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 28 Asn Ile Tyr Pro Ser Asp Ser Phe Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 29 <211> 11 <212> PRT <213> Artificial Sequence <220> \><223> VH CDR3 <400> 29 Asp Thr Gln Glu Arg Ser Trp Tyr Phe Asp Val 1 5 10 <210> 30 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 <400> 30 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 <400> 31 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 <400> 32 Ser Gln Ser Thr His Val Pro Trp Thr 1 5 <210> 33 <211> 232 <212> PRT <213> Artificial sequence <220> <223> CAR, mouse CD22ALAb scFv sequence <400> 33 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Phe Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Gln Glu Arg Ser Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Asp Val Val Met Thr Gln Thr Pro 115 120 125 Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg 130 135 140 Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp 145 150 155 160 Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val 165 170 175 Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu 195 200 205 Gly Leu Tyr Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly 210 215 220 Gly Gly Thr Lys Leu Glu Ile Lys 225 230 <210> 34 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> CAR, Humanized CD22ALAb scFv Sequence <400> 34 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Phe Thr Asn Tyr Asn Gln Lys Phe 50 55 60 [[ID=A]]<00... Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Gln Glu Arg Ser Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Asp Ile Val Met Thr Gln Ser Pro 115 120 125 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 130 135 140 Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val 165 170 175 Ser Asn Arg Phe Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Val Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe 195 200 205 Ala Val Tyr Tyr Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly 210 215 220 Gln Gly Thr Arg Leu Glu Ile Lys 225 230 <210> 35 <211> 120 <212> PRT <213> Artificial sequence <220> <223> CAR, murine CD22ALAb VH sequence <400> 35 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Phe Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 ۷۰ ۷۵ ۸۰ Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Gln Glu Arg Ser Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 36 <211> 120 <212> PRT <213> Artificial sequence <220> <223> CAR, Humanized CD22ALAb VH Sequence <400> 36 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Phe Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Gln Glu Arg Ser Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CAR, Mouse CD22ALAb VL Sequence <400> 37 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 38 <211> 112 ​​​​​​​​​​​​​​Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ala Arg Phe Ser Gly Ser Gly Ser Gly Val Glu Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 39 <211> 236 <212> PRT <213> Artificial sequence <220> <223> CAR, humanized CD19ALAb scFv sequence - Heavy 19, Kappa 7 <400> 39 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Glu Ser Ala Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln Leu 115 120 125 Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr 130 135 140 Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr 145 150 155 160 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile 165 170 175 Tyr Asp Ala Ser Asn Leu Val Ser Gly Val Pro Asp Arg Phe Ser Gly 180 185 190 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 195 200 205 Ala Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Thr Glu Asp Pro Trp 210 215 220 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 225 230 235 <210> 40 <211> 112 <212> PRT <213> Artificial sequence <220> <223> CAR, humanized CD19ALAb VL sequence, Kappa 7 <400> 40 Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Val Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Val Pro Asp 50 55 60<00​​​Ser Leu Gln Ala Ala Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 41 <211> 40 <212> PRT <213> Artificial sequence <220> <223> CD28 costimulatory intracellular domain <400> 41 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 1 5 10 15 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 20 25 30 Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 42 <211> 36 <212> PRT <213> Artificial sequence <220> <223> OX40 intracellular domain <400> 427] Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 1 5 10 15 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 20 25 30 Leu Ala Lys Ile 35 <210> 43 <211> 42 <212> PRT <213> Artificial sequence <220> <223> 4-1BB intracellular domain <400> 43 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 44 <211> 112 <212> PRT <213> Artificial sequence <220> <223> CD3zeta intracellular domain <400> 44 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 45 <211> 24 <212> PRT <213> artificial sequence <220> <223> Tyrp‑1 transmembrane sequence <400> 45 Ile Ile Ala Ile Ala Val Val Gly Ala Leu Leu Leu Val Ala Leu Ile 1 5 10 15 Phe Gly Thr Ala Ser Tyr Leu Ile 20 <210> 46 <211> 258 <212> PRT <213> humans <400> 46 Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala Val Leu 1 5 10 15 Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu Thr Trp 20 25 30 Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu Gly Leu 35 40 45 Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu Phe Ile 50 55 60 Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln Pro Gly 65 70 75 80 Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val Glu 85 90 95 Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly Leu 100 105 110 Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro Ser 115 120 125 Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg Pro 130 135 140 Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp Ser Leu 145 150 155 160 Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser Thr Leu 165 170 175 Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly Pro Leu 180 185 190 Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu Ser Leu 195 200 205 Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu Thr 210 215 220 Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr Tyr 225 230 235 240 Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr Ala 245 250 255 Arg Pro <210> 47 <211> 4 <212> PRT <213> Artificial sequence <220> <223> ITAM motif <220> <221> misc_feature <222> (2)..(3) <223> Xaa can be a recognized naturally occurring amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa can be leucine or isoleucine <400> 47 Tyr Xaa Xaa Xaa 1 <210> 48 <211> 1139 <212> DNA <213> Human <400> 48 atcccgccga gcccaaatct cctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60 aactcctggg gggaccgtca gtttctctct tccccccaaa acccaaggac accctcatga 120 tctccccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atcccagcga catcgccgtg gagtgggaga gcaatgggca accggagaac aactacaaga 540 ccacgctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600 acaagacag gtggcagcag gggaacgtct tctcatgctc cgtgatgcac gaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccggg taaaaaagat cccaaatttt 720 gggtgctggt ggtggttggt ggagtcctgg cttgcttag cttgctagta acagtggcct 780 ttattatttt ctgggtgagg agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc 840 agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg 900 ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc 960 ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga 1020 ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca 第1080行 gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgcct cctcgcgag 1139 <210> 49 <211> 1139 <212> DNA <213> Artificial sequence <220> <223> Codon wobble HCH2CH3-CD28tmZeta <400> 49 atccagccga accaaagagc cccgataaga cccacacctg tcccccctgc ccagccccag 60 agctgctggg aggccccagc gtgtttctgt ttccacccaa gccaaaggat accctgatga 120 ttagtagaac acccgaagtg acctgtgtgg tggtggatgt gtctcacgag gaccccgagg 180 tgaaatttaa ttggtatgtt gatggtgttg aagtgcacaa cgccaaaacc aaacccagag 240 Note: There was an error in the original text where "第1080行" was added in the translation of line 8, which is incorrect. It should be "1080" as in the original text. The corrected translation is as follows: ttattatttt ctgggtgagg agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc 840 agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg 900 ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc 960 [[ID=aggagcagta caattctacc tatagagtcg tgtctgtgct gacagtgctg catcaggatt 300 ggctgaacgg aaaagaatac aaatgtaaag tgagcaataa ggccctgccc gctccaattg 360 agaagacaat tagcaaggcc aagggccagc caagggagcc ccaggtgtat acactgccac 420 ccagtagaga cgaactgaca aagaatcagg tgtctctgac atgtctggtg aagggatttt 480 acccatctga tatcgccgtg gaatgggaat ctaacggcca gcccgagaat aactataaga 540 caaccccacc agtgctggat agcgatggca gctttttct gtattctaag ctgacagtgg 600 ataagtcccg gtggcagcag ggaaatgtgt ttagctgtag tgtcatgcat gaggccctgc 660 acaatcacta tacccagaaa tctctgagtc tgagcccagg caagaaggac cccaagttct 720 gggtcctggt ggtggtggga ggcgtgctgg cctgttactc tctcctggtg accgtggcct 780 tcatcatct ttgggtgcgc tcccgggtga agttttctcg ctctgccgat gccccagcct 840 atcagcaggg ccagaatcag ctgtacaatg aactgaacct gggcaggcgg gaggagtacg 900 acgtgctgga taagcggaga ggcagagacc ccgagatggg cggcaaacca cggcgcaaaa 960 atccccagga gggactctat aacgagctgc agaaggacaa aatggccgag gcctattccg 1020 agatcggcat gaagggagag agaagacgcg gaaagggcca cgacggcctg tatcagggat 1080 tgtccaccgc tacaaaagat acatatgatg ccctgcacat gcaggccctg ccacccaga 1139

Claims

1. A cell that co-expresses a first chimeric antigen receptor (CAR) and a second CAR at the cell surface, each CAR comprising an antigen binding domain, wherein the antigen binding domain of the first CAR binds CD19 and the antigen binding domain of the second CAR binds CD22, wherein each CAR comprises a CD3 zeta endodomain; wherein the cell is a T cell; and The intracellular domain of one CAR comprises a costimulatory domain and the intracellular domain of the other CAR comprises a TNF receptor family domain, and wherein the costimulatory domain is a CD28 costimulatory domain and the TNF receptor family domain is an OX-40 or 4-1BB intracellular domain.

2. The cell according to claim 1, wherein each CAR comprises: (i) antigen-binding domain; (ii) a spacer; and (iii) transmembrane domain; The spacer of the first CAR is different from the spacer of the second CAR.

3. The cell according to claim 2, wherein the antigen binding domain of the second CAR binds to an epitope on Ig domain 1, 2, 3 or 4 of CD22.

4. The cell according to claim 1, wherein the antigen binding domain of the first CAR comprises a) a heavy chain variable region (VH) having a complementarity determining region (CDR) consisting of the following sequence: CDR1 – SYWMN (SEQ ID No. 15); CDR2 – QIWPGDGDTNYNGKFK (SEQ ID No. 16); CDR3 – RETTTVGRYYYAMDY (SEQ ID No. 17); and b) a light chain variable region (VL) having CDRs consisting of the following sequences: CDR1 – KASQSVDYDGDSYLN (SEQ ID No. 18); CDR2 – DASNLVS (SEQ ID No. 19); CDR3 – QQSTEDPWT (SEQ ID No. 20).

5. The cell according to claim 4, wherein the antigen binding domain of the first CAR comprises a VH domain consisting of a sequence as shown in SEQ ID No. 23 or SEQ ID No. 24; or a VL domain consisting of a sequence as shown in SEQ ID No. 25, SEQ ID No. 26 or SEQ ID No.

40.

6. The cell according to claim 4, wherein the antigen binding domain of the first CAR comprises the sequence shown in SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No.

39.

7. The cell according to claim 1, wherein the antigen binding domain of the second CAR comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) consisting of the following sequence: CDR1 – NYWIN (SEQ ID No. 27); CDR2 – NIYPSDSFTNYNQKFKD (SEQ ID No. 28) CDR3 – DTQERSWYFDV (SEQ ID No. 29); and b) a light chain variable region (VL) having CDRs consisting of the following sequences: CDR1 – RSSQSLVHSNGNTYLH (SEQ ID No. 30); CDR2 – KVSNRFS (SEQ ID No. 31) CDR3 – SQSTHVPWT (SEQ ID No. 32).

8. The cell according to claim 7, wherein the antigen binding domain of the second CAR comprises a VH domain consisting of a sequence as shown in SEQ ID No. 35 or SEQ ID No. 36; or a VL domain consisting of a sequence as shown in SEQ ID No. 37 or SEQ ID No.

38.

9. The cell according to claim 7, wherein the antigen binding domain of the second CAR comprises the sequence shown in SEQ ID No. 33 or SEQ ID No.

34.

10. A nucleic acid sequence encoding both a first and a second chimeric antigen receptor (CAR) as defined in any one of claims 1 to 9, which, when expressed in a cell, encodes a polypeptide that is cleaved at a cleavage site such that the first and second CARs are co-expressed at the cell surface, the cleavage site being located between the nucleic acid sequence encoding the first CAR and the nucleic acid sequence encoding the second CAR.

11. The nucleic acid sequence according to claim 10, having the following structure: AgB1-Spacer1-TM1-endo1-coexpr-AbB2-Spacer2-TM2-endo2 in AgB1 is a nucleic acid sequence encoding the antigen binding domain of the first CAR; Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; endo1 is a nucleic acid sequence encoding the intracellular domain of the first CAR; coexpr is a nucleic acid sequence that enables co-expression of two CARs; AgB2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR; Spacer 2 is a nucleic acid sequence encoding the spacer of the second CAR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; endo2 is a nucleic acid sequence encoding the intracellular domain of the second CAR; The nucleic acid sequence, when expressed in a T cell, encodes a polypeptide that is cleaved at the cleavage site such that the first and second CARs are co-expressed at the surface of the T cell.

12. The nucleic acid sequence according to claim 11, wherein coexpr encodes a sequence comprising a self-cleaving peptide.

13. Nucleic acid sequence according to claim 11 or 12, wherein in order to avoid homologous recombination, alternative codons are used in the sequence regions encoding the same or similar amino acid sequence.

14. A vector comprising a nucleic acid sequence according to any one of claims 10 to 13.

15. The vector according to claim 14, wherein the vector is a retroviral vector or a lentiviral vector or a transposon.

16. A method for preparing a cell according to any one of claims 1 to 9, comprising the step of introducing into the cell: a nucleic acid sequence according to any one of claims 10 to 13 or a vector according to claim 14 or 15.

17. The method according to claim 16, wherein the cells are from a sample isolated from a subject.

18. A pharmaceutical composition comprising a plurality of cells according to any one of claims 1 to 9.

19. The pharmaceutical composition according to claim 18, for use in the treatment and / or prevention of a disease.

20. Use of a cell according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment and / or prevention of a disease.

21. Use of a nucleic acid sequence according to any one of claims 10 to 13 or a vector according to claim 14 or 15 for the manufacture of a medicament for the treatment and / or prevention of a disease.

22. Use according to claim 20 or 21, wherein the disease is cancer.

23. Use according to claim 22, wherein the cancer is a B-cell malignancy.

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