Cancer treatment using humanized anti-CD19 chimeric antigen receptors

Abstract

To provide compositions and methods for treating diseases associated with expression of CD19.SOLUTION: In one aspect, the invention pertains to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising an antibody or antibody fragment which includes a humanized anti-CD 19 binding domain, a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain).SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the priority of U.S. Patent Application No. 61 / 802,629, filed on March 16, 2013, and U.S. Patent Application No. 61 / 838,537, filed on June 24, 2013, and the entire contents of each of these applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a sequence listing submitted electronically in ASCII format, the entire contents of which are incorporated by reference. A copy of the ASCII created on March 14, 2014, is named N2067-7002WO_SL.txt and is 228,415 bytes in size.

[0003] The present invention generally relates to the use of T cells engineered to express a chimeric antigen receptor (CAR) for treating diseases associated with the expression of surface antigen classification 19 protein (CD19).

Background Art

[0004] Many patients with B cell malignancies are not treatable with standard therapies. In addition, conventional treatment options often have severe side effects. Attempts have been made to achieve clinical efficacy in cancer immunotherapy, but due to numerous obstacles, it has been an extremely difficult goal. Hundreds of so-called tumor antigens have been identified, but these are generally self-derived and thus have low immunogenicity. Furthermore, tumors use several mechanisms that turn themselves against the initiation and propagation of the immune attack.

[0005] Recent developments using chimeric antigen receptor (CAR)-modified autologous T-cell (CART) therapies, which rely on redirecting T cells to preferred cell surface molecules on cancer cells such as B-cell malignancies, have shown promising results in leveraging the power of the immune system to treat B-cell malignancies and other cancers [see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)]. Clinical outcomes of mouse-derived CART19 (i.e., "CTL019") have shown promise in establishing complete remission in patients with CLL, as well as in pediatric ALL patients [see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)]. Beyond the ability of chimeric antigen receptors on genetically modified T cells to recognize and destroy targeted cells, successful therapeutic T cell therapy requires the ability to proliferate and persist over the long term, as well as the ability to further monitor escaping leukemia cells. While variability in T cell quality, whether as a result of anergy, suppression, or depletion, is expected to affect the performance of CAR-transformed T cells, skilled technicians have only been able to control them to a limited extent at this stage. For CAR-transformed patient T cells to be effective, they must persist and maintain their ability to proliferate in response to CAR antigens. T cells from ALL patients have been shown to achieve this with CART19, including mouse scFv [see, for example, Grupp et al., NEJM 368:1509-1518 (2013)]. [Overview of the project]

[0006] The present invention addresses controlling immune responses in patients by providing optimized, humanized antibody fragments (e.g., scFv) that bind to surface antigen classification 19 protein (CD19) integrated into a chimeric antigen receptor (CAR) construct, which are expected not to induce an immune response in patients, are safe for long-term use, and maintain or possess superior clinical efficacy compared to known CART therapies for treating B-cell derived cancers. The present invention further relates to the use of T cells processed to express a CAR-integrated humanized antibody fragment that binds to CD19 for treating hematological cancers associated with CD19 expression (OMIM accession number 107265, Swiss Prot accession number P15391).

[0007] Accordingly, in one embodiment, the present invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment comprising a humanized anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment comprising a humanized anti-CD19 binding domain as described herein, a transmembrane domain as described herein, and an intracellular signaling domain as described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain).

[0008] In one embodiment, the encoded humanized anti-CD19 binding domain comprises one or more (e.g., all three) light chain complementarity determination regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the humanized anti-CD19 binding domains described herein, and / or one or more (e.g., all three) heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domains described herein, for example, the humanized anti-CD19 binding domain comprises one or more, e.g., all three LC CDRs, and one or more, e.g., all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the encoded humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, the encoded humanized anti-CD19 binding domain has two variable heavy chain regions comprising HC CDR1, HC CDR2, and HC CDR3, respectively, as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the encoded light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the encoded light chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the light chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71 and 87). In one embodiment, the encoded heavy chain variable region includes one, two, three, or all four framework regions of the VH4_4-59 germline sequence.In one embodiment, the encoded heavy chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the heavy chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71, 73, and 78). In one embodiment, the encoded humanized anti-CD19 binding domain includes a humanized light chain variable region and / or a humanized heavy chain variable region described herein (e.g., listed in Table 3). In one embodiment, the encoded humanized anti-CD19 binding domain includes a humanized heavy chain variable region described herein (e.g., listed in Table 3), e.g., at least two humanized heavy chain variable regions described herein (e.g., listed in Table 3). In one embodiment, the encoded anti-CD19 binding domain is an scFv including the light chain and heavy chain of the amino acid sequences in Table 3. In one embodiment, the anti-CD19 binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region shown in Table 3, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 3; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region shown in Table 3, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 3. In one embodiment, the encoded humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, and 72, or a sequence having 95-99% identity with them.In one embodiment, the encoded humanized anti-CD19 binding domain is scFv, and a light chain variable region containing an amino acid sequence as described herein, for example, Table 3, is attached to a heavy chain variable region containing an amino acid sequence as described herein, for example, Table 3, via a linker, for example, a linker as described herein. In one embodiment, the encoded humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of scFv may be, for example, in the following configuration: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0009] In one embodiment, the encoded transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the encoded transmembrane domain includes the sequence of SEQ ID NO: 15. In one embodiment, the encoded transmembrane domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 15, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 15. In one embodiment, the nucleic acid sequence encoding the transmembrane domain includes the sequence of SEQ ID NO: 56, or a sequence having 95-99% identity with it.

[0010] In one embodiment, the encoded anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, for example, a hinge region described herein. In one embodiment, the encoded hinge region includes sequence number 14, sequence number 45, or sequence number 47, or a sequence having 95-99% identity with them. In one embodiment, the nucleic acid sequence encoding the hinge region includes sequence number 55, sequence number 46, or sequence number 48, or a sequence having 95-99% identity with them.

[0011] In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the encoded co-stimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the encoded co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16, but 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the nucleic acid sequence encoding the co-stimulatory domain comprises the sequence of SEQ ID NO: 60, or a sequence having 95-99% identity with it. In one embodiment, the isolated nucleic acid molecule further comprises an intracellular signaling domain, for example, a sequence encoding an intracellular signaling domain as described herein. In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta. In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of CD27 and / or a functional signaling domain of CD3 zeta. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, but having 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43.In one embodiment, the encoded intracellular signaling domain includes the sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, wherein the sequence containing the intracellular signaling domain is expressed as a single polypeptide chain in the same frame. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain includes the sequence of SEQ ID NO: 60, or a sequence having 95-99% identity with them, and / or the sequence of SEQ ID NO: 101 or SEQ ID NO: 44, or a sequence having 95-99% identity with them. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain includes the sequence of SEQ ID NO: 52, or a sequence having 95-99% identity with it, and / or the sequence of SEQ ID NO: 101 or SEQ ID NO: 44, or a sequence having 95-99% identity with them.

[0012] In another embodiment, the present invention relates to an isolated nucleic acid molecule encoding a CAR construct, comprising a leader sequence, e.g., the leader sequence described herein, e.g., the leader sequence of SEQ ID NO: 13; a humanized anti-CD19 binding domain, e.g., a humanized anti-CD19 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3, e.g., the humanized anti-CD19 binding domains listed in Table 3, or sequences having 95-99% identity thereto; a hinge region, e.g., the hinge region of SEQ ID NO: 14 or SEQ ID NO: 45; a transmembrane domain, e.g., a transmembrane domain comprising SEQ ID NO: 15; and an intracellular signaling domain, e.g., an intracellular signaling domain described herein. In one embodiment, the encoded intracellular signaling domain includes a co-stimulatory domain, e.g., a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, and / or a primary signaling domain, e.g., a CD3 zeta-stimulatory domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, e.g., a primary signaling domain, e.g., a CD3 zeta-stimulatory domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the isolated nucleic acid molecule encoding the CAR construct includes a leader sequence encoded by the nucleic acid sequence of SEQ ID NO: 54, or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule encoding the CAR construct includes a humanized anti-CD19 binding domain sequence encoded by the nucleic acid sequences of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule encoding the CAR construct comprises a transmembrane sequence encoded by the nucleic acid sequence of Sequence ID No. 56, or a sequence having 95-99% identity thereto.In one embodiment, the isolated nucleic acid molecule encoding the CAR construct includes an intracellular signaling domain sequence encoded by the nucleic acid sequence of SEQ ID NO: 60, or a sequence having 95-99% identity thereto, and / or the nucleic acid sequence of SEQ ID NO: 101 or SEQ ID NO: 44, or a sequence having 95-99% identity thereto.

[0013] In one embodiment, the isolated nucleic acid molecule comprises a nucleic acid encoding the CAR amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42, or an amino acid sequence having at least one, two, three, four, five, ten, 15, 20 or 30 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42, but with no more than 60, 50 or 40 modifications (e.g., substitutions), or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with them (for example, consisting of).

[0014] In one embodiment, the isolated nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, or SEQ ID NO: 96, or a nucleic acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, or SEQ ID NO: 96 (for example, consisting of).

[0015] In one embodiment, the present invention relates to an isolated nucleic acid molecule encoding a humanized anti-CD19 binding domain, wherein the anti-CD19 binding domain comprises one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the anti-CD19 binding domain described herein, and one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the light chain variable region includes one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse light chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71 and 87). In one embodiment, the heavy chain variable region includes one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse heavy chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71, 73, and 78). In one embodiment, the encoded humanized anti-CD19 binding domain includes a light chain variable region as described herein (e.g., as described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) and / or a heavy chain variable region as described herein (e.g., as described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In one embodiment, the encoded humanized anti-CD19 binding domain is an scFv comprising the light and heavy chains of the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) has at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region provided in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, but with 30, 20, or 10 or fewer modifications (e.g., substitutions), or has 95-99% identity with the amino acid sequence of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. A light chain variable region containing a sequence; and / or a heavy chain variable region containing an amino acid sequence having 30, 20, or 10 or fewer modifications (e.g., substitutions) to at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, the humanized anti-CD19 binding domain contains a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence having 95-99% identity with them. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and 72, or a sequence having 95-99% identity with them.

[0016] In another embodiment, the present invention relates to an isolated polypeptide molecule encoded by the above nucleic acid sequence. In one embodiment, the isolated polypeptide molecule includes a sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. In one embodiment, the isolated polypeptide includes the sequence of SEQ ID NOs: 31. In one embodiment, the isolated polypeptide includes the sequence of SEQ ID NOs: 32. In one embodiment, the isolated polypeptide molecule includes the sequence of SEQ ID NOs: 35. In one embodiment, the isolated polypeptide molecule includes the sequence of SEQ ID NOs: 36. In one embodiment, the isolated polypeptide molecule includes the sequence of SEQ ID NOs: 37.

[0017] In another embodiment, the present invention relates to an isolated chimeric antigen receptor (CAR) molecule comprising a humanized anti-CD19 binding domain (e.g., a humanized antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain including a costimulatory domain and / or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment comprising a humanized anti-CD19 binding domain as described herein (e.g., a humanized antibody or antibody fragment that specifically binds to CD19 as described herein), a transmembrane domain as described herein, and an intracellular signaling domain as described herein (e.g., an intracellular signaling domain including a costimulatory domain and / or a primary signaling domain as described herein).

[0018] In one embodiment, the humanized anti-CD19 binding domain includes one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain includes one or more, for example, all three LC CDRs and one or more, for example, all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain includes at least HC CDR2. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain has two variable heavy chain regions comprising HC CDR1, HC CDR2, and HC CDR3, respectively, as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse light chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71 and 87). In one embodiment, the heavy chain variable region includes one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse heavy chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71, 73, and 78).In one embodiment, the humanized anti-CD19 binding domain includes a light chain variable region and / or a heavy chain variable region as described herein (e.g., Table 3). In one embodiment, the humanized anti-CD19 binding domain is an scFv including the light and heavy chains of the amino acid sequences in Table 3. In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region shown in Table 3, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence in Table 3; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region shown in Table 3, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence in Table 3. In one embodiment, the humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them. In one embodiment, the humanized anti-CD19 binding domain is scFv, and a light chain variable region containing an amino acid sequence as described herein, for example, Table 3, is attached to a heavy chain variable region containing an amino acid sequence as described herein, for example, Table 3, via a linker, for example, a linker as described herein. In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of scFv may be arranged in either of the following configurations, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0019] In one embodiment, the isolated CAR molecule includes a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain includes the sequence of SEQ ID NO: 15. In one embodiment, the transmembrane domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 15, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 15.

[0020] In one embodiment, the humanized anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, such as the hinge region described herein. In one embodiment, the encoded hinge region includes sequence number 14 or sequence number 45, or a sequence having 95-99% identity with them.

[0021] In one embodiment, the isolated CAR molecule further comprises a co-stimulatory domain, for example, a sequence encoding a co-stimulatory domain as described herein. In one embodiment, the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO: 51. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51.

[0022] In one embodiment, the isolated CAR molecule further comprises an intracellular signaling domain, for example, a sequence encoding an intracellular signaling domain as described herein. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 43. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD27 and / or a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 43. In one embodiment, the intracellular signaling domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the intracellular signaling domain includes the sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, wherein the sequence containing the intracellular signaling domain is expressed as a single polypeptide chain in the same frame.

[0023] In one embodiment, the isolated CAR molecule further comprises a leader sequence, for example, a leader sequence described herein. In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 13.

[0024] In another embodiment, the present invention relates to a leader sequence, for example, a leader sequence as described herein, for example, the leader sequence of SEQ ID NO: 13 or a leader sequence having 95-99% identity therewith; a humanized anti-CD19 binding domain as described herein, for example, LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR1 as described herein. The present invention relates to isolated CAR molecules comprising: a humanized anti-CD19 binding domain containing CDR3, e.g., the humanized anti-CD19 binding domains listed in Table 3, or sequences having 95-99% identity thereto; a hinge region, e.g., the hinge region described herein, e.g., the hinge region of SEQ ID NO: 14, or a hinge region having 95-99% identity thereto; a transmembrane domain, e.g., the transmembrane domain described herein, e.g., the transmembrane domain having the sequence of SEQ ID NO: 15, or a transmembrane domain having 95-99% identity thereto; and an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain including a co-stimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain includes a co-stimulatory domain, for example, a 4-1BB co-stimulatory domain having the sequence of the co-stimulatory domain described herein, for example, sequence number 16 or sequence number 51, or a sequence having 95-99% identity thereto, and / or a primary signaling domain, for example, a CD3 zeta-stimulating domain having the sequence of the primary signaling domain described herein, for example, sequence number 17 or sequence number 43, or a sequence having 95-99% identity thereto.

[0025] In one embodiment, the isolated CAR molecule is at least one, two, or three of the amino acid sequences of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, or at least one, two, or three of the amino acid sequences of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. , an amino acid sequence having 4, 5, 10, 15, 20 or 30 modifications (e.g., substitutions) but 60, 50 or 40 or fewer modifications (e.g., substitutions), or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42 (e.g., consisting of ).

[0026] In one embodiment, the present invention relates to a humanized anti-CD19 binding domain comprising one or more (e.g., all three) of the light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the anti-CD19 binding domain described herein, and one or more (e.g., all three) of the heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, a humanized anti-CD19 binding domain comprising one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain has at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the mouse light chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71 and 87). In one embodiment, the heavy chain variable region includes all one, two, three or four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, e.g., substitutions of one or more amino acids found at corresponding positions in the heavy chain variable region of SEQ ID NO: 58, e.g., substitutions at one or more positions 71, 73 and 78). In one embodiment, the humanized anti-CD19 binding domain includes a light chain variable region as described herein (e.g., as described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) and / or a heavy chain variable region as described herein (e.g., as described in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12).In one embodiment, the humanized anti-CD19 binding domain is an scFv comprising the light and heavy chains of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) is an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, but with 30, 20, or 10 or fewer modifications (e.g., substitutions), or having 95-99% identity with the amino acid sequences in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. A light chain variable region containing a sequence; and / or a heavy chain variable region containing an amino acid sequence having 30, 20, or 10 or fewer modifications (e.g., substitutions) to at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a heavy chain variable region containing a sequence having 95-99% identity with the amino acid sequence in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0027] In another embodiment, the present invention relates to a vector comprising a nucleic acid sequence encoding a CAR. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector.

[0028] In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO: 100.

[0029] In one embodiment, the vector is an in vitro transcribed vector, for example, a vector for transcribing RNA of a nucleic acid molecule as described herein. In one embodiment, the nucleic acid sequence in the vector further comprises a poly(A) tail, for example, a polyA tail as described herein, for example, a polyA tail containing about 150 adenosine bases (SEQ ID NO: 104). In one embodiment, the nucleic acid sequence in the vector further comprises a 3'UTR, for example, a 3'UTR as described herein, for example, a 3'UTR containing at least one repeat of a 3'UTR derived from human beta-globulin. In one embodiment, the nucleic acid sequence in the vector further comprises a promoter, for example, a T2A promoter.

[0030] In another embodiment, the present invention relates to cells comprising the above vector. In one embodiment, the cells are human T cells. In one embodiment, the cells are cells described herein, for example, human T cells, for example, human T cells described herein. In one embodiment, the human T cells are CD8+ T cells.

[0031] In another embodiment, the CAR-expressing cells described herein may further express another agent, such as an agent that enhances the activity of the CAR-expressing cells. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide, such as the inhibitory molecule, which associates with a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain described herein. In one embodiment, the drug comprises a first polypeptide of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein [e.g., including a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)]. In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0032] In another embodiment, the present invention relates to a method for producing cells, comprising transducing T cells with a vector comprising a CAR, for example, a nucleic acid encoding a CAR as described herein.

[0033] The present invention also provides a method for generating a population of RNA-processed cells, such as the cell populations described herein, for example, a population of T cells that transiently express exogenous RNA. The method comprises introducing in vitro transcribed or synthetic RNA into cells, wherein the RNA comprises nucleic acids encoding CAR molecules as described herein.

[0034] In another embodiment, the present invention relates to a method for providing antitumor immunity in a mammal, comprising administering an effective amount of cells containing a CAR molecule, for example, CAR molecule-expressing cells as described herein, to the mammal. In one embodiment, the cells are autologous T cells. In one embodiment, the cells are allogeneic T cells. In one embodiment, the mammal is human.

[0035] In another embodiment, the present invention relates to a method for treating a mammal having a disease associated with CD19 expression, comprising administering to the mammal an effective amount of cells containing a CAR molecule, for example, the CAR molecule described herein.

[0036] In one embodiment, the disease associated with CD19 expression is selected from proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal cord malformations, myelodysplastic syndromes or preleukemic states, or non-cancer-related signs associated with CD19 expression. In one embodiment, the disease is a hematological cancer. In one embodiment, the hematological cancer is a leukemia. In one embodiment, cancer includes, but is not limited to, one or more acute leukemias, including B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); but is not limited to, one or more chronic leukemias, including chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL); but is not limited to, B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, and mantle Additional hematological malignancies or hematological conditions, including "preleukemic states," which are a group of hematological conditions common to various hematological conditions of abnormal production (or dysplasia) of blood cells in the bone marrow, such as leukocyte lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and the absence of efficacy in producing (or dysplasia) of blood cells in the bone marrow; and, but not limited to, CD19-related diseases, including atypical and / or atypical CD19-expressing cancers, malignancies, precancerous conditions, or proliferative disorders; and any combination thereof.

[0037] In one embodiment, lymphocyte infusion, for example, allogeneic lymphocyte infusion, is used to treat cancer, wherein the lymphocyte infusion includes at least one type of CD19CAR-expressing cell. In another embodiment, autologous lymphocyte infusion is used to treat cancer, wherein the autologous lymphocyte infusion includes at least one type of CD19-expressing cell.

[0038] In one embodiment, CD19CAR-expressing cells, such as T cells, are administered to a subject who has previously undergone stem cell transplantation, such as autologous stem cell transplantation.

[0039] In one embodiment, CD19CAR-expressing cells, such as T cells, are administered to a subject who has previously received melphalan.

[0040] In one embodiment, CAR molecules, for example, CAR molecule-expressing cells as described herein, are administered in combination with a drug that enhances the efficacy of CAR molecule-expressing cells, for example, a drug as described herein.

[0041] In one embodiment, a CAR molecule, such as a CAR molecule-expressing cell as described herein, is administered in combination with a drug that improves one or more side effects associated with the administration of the CAR molecule-expressing cell, such as a drug as described herein.

[0042] In one embodiment, a CAR molecule, for example, CAR molecule-expressing cells as described herein, is administered in combination with a drug that treats a CD19-related disease, for example, a drug as described herein.

[0043] In one embodiment, a CAR molecule, for example, CAR molecule-expressing cells as described herein, is administered in the dose and / or administration schedule described herein.

[0044] In one embodiment, the CAR molecule is introduced into T cells, for example, by transcription in vitro, and the subject (e.g., human) receives an initial dose of cells containing the CAR molecule, followed by one or more subsequent doses of cells containing the CAR molecule, where the subsequent dose is administered less than 15 days after the previous dose, for example, less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In one embodiment, the subject (e.g., human) receives more than one dose of cells containing the CAR molecule per week, for example, two, three, or four doses of cells containing the CAR molecule per week. In one embodiment, the subject (e.g., human subject) receives more than one dose of cells containing the CAR molecule per week (e.g., two, three, or four doses per week) (also referred to herein as a cycle), followed by a week without administration of cells containing the CAR molecule, and then one or more additional doses of cells containing the CAR molecule (e.g., more than one dose of cells containing the CAR molecule per week) are administered to the subject. In another embodiment, a subject (e.g., a human subject) receives more than one cycle of cells containing the CAR molecule, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells containing the CAR molecule are administered every other day, in three doses per week. In one embodiment, cells containing the CAR molecule are administered for at least 2, 3, 4, 5, 6, 7, 8 weeks or more.

[0045] In one embodiment, a CAR molecule, for example, CAR molecule-expressing cells as described herein, is administered as a first-line treatment for a disease, such as cancer, for example, the cancer as described herein. In another embodiment, a CAR molecule, for example, CAR molecule-expressing cells as described herein, is administered as a second, third, or fourth-line treatment for a disease, such as cancer, for example, the cancer as described herein.

[0046] In one embodiment, a population of cells described herein is administered.

[0047] In another embodiment, the present invention relates to isolated nucleic acid molecules encoding the CAR of the present invention, isolated polypeptide molecules of the CAR of the present invention, vectors containing the CAR of the present invention, and cells containing the CAR of the present invention for use as pharmaceuticals.

[0048] In another embodiment, the present invention relates to an isolated nucleic acid molecule encoding the CAR of the present invention, an isolated polypeptide molecule of the CAR of the present invention, a vector containing the CAR of the present invention, and cells containing the CAR of the present invention, for use in the treatment of diseases expressing CD19.

[0049] In one embodiment, the present invention encompasses a population of autologous cells transfected or transduced with a vector comprising a nucleic acid molecule encoding a CD19-CAR molecule, as described herein, for example. In one embodiment, the vector is a retroviral vector. In one embodiment, the vector is a self-inactivating lentiviral vector, as described elsewhere herein. In one embodiment, the vector is delivered to a cell, for example, a T cell (e.g., by transfection or electroporation), where the vector comprises a nucleic acid molecule encoding a CD19CAR molecule, as described herein, which is transcribed as an mRNA molecule, and the CD19CAR molecule is translated from the RNA molecule and expressed on the cell surface.

[0050] In another embodiment, the present invention provides a population of CAR-expressing cells, for example, CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of different CAR-expressing cells. For example, in one embodiment, the population of CART cells may comprise a first CAR-expressing cell having an anti-CD19 binding domain as described herein, and a second CAR-expressing cell having a different anti-CD19 binding domain, for example, an anti-CD19 binding domain as described herein that is different from the anti-CD19 binding domain in the CAR expressed by the first cell. In another example, the population of CAR-expressing cells may comprise a first CAR-expressing cell containing an anti-CD19 binding domain, for example, as described herein, and a second CAR-expressing cell containing an antigen-binding domain for a target other than CD19 (e.g., CD123). In one embodiment, the population of CAR-expressing cells comprises, for example, a first CAR-expressing cell containing a primary intracellular signaling domain, and a second CAR-expressing cell containing a secondary signaling domain.

[0051] In another embodiment, the present invention provides a population of cells in which at least one type of cell expresses a CAR having an anti-CD19 domain as described herein, and a second cell expresses another agent, such as an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide, such as the inhibitory molecule, which associates with a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain as described herein. In one embodiment, the drug comprises a first polypeptide of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein [e.g., including a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)]. In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0052] In one embodiment, a nucleic acid molecule encoding the CD19CAR molecule, such as that described herein, is expressed as an mRNA molecule. In one embodiment, genetically modified CD19CAR-expressing cells, such as T cells, can be generated by transfecting or electroporating cells with an RNA molecule encoding a desired CAR (e.g., one that does not contain a vector sequence). In one embodiment, once the RNA molecule is taken up, the CD19CAR molecule is translated from the RNA molecule and expressed on the surface of the recombinant cell. [Brief explanation of the drawing]

[0053]

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[0054] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention relates.

[0055] The terms "a" and "an" refer to the grammatical object being one or more (i.e., at least one). For example, "an element" means one or more elements.

[0056] The term "approximately" when referring to measurable values ​​such as quantities, temporary durations, and similar items means to include a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, from the specified value, because such variation is appropriate for performing the disclosed method.

[0057] The term “chimeric antigen receptor” or, instead, “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as the “intracellular signaling domain”) comprising a functional signaling domain derived from a stimulating molecule as defined below. In one embodiment, the stimulating molecule is a zeta chain associated with a T cell receptor complex. In one embodiment, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one embodiment, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulating molecule. In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulator. In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulator. In one embodiment, CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one embodiment, CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence may be cleaved from the antigen recognition domain (e.g., amino acids of scFv) during intracellular processing and localization of CAR to the cell membrane.

[0058] The term "signaling domain" refers to the functional portion of a protein that acts by transmitting information within an cell to regulate cellular activity via a defined signaling pathway, either by generating a second messenger or by functioning as an effector in response to such a messenger.

[0059] The term "CD19," as used herein, refers to the surface antigen classification 19 protein, a detectable antigenic determinant in leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot registry number P15391, and the nucleotide sequence encoding human CD19 can be found under registry number NM_001178098. CD19 is expressed in most B-cell lineage cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells that express CD19 are listed in the definition of "Diseases Associated with CD19 Expression" below. CD19 is also an early marker for B-cell precursors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen-binding site of CART recognizes and binds to the antigen within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed in cancer cells.

[0060] As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multiple or single-chain, or intact immunoglobulins, and may be of natural or recombinant origin. Antibodies may also be tetramers of immunoglobulin molecules.

[0061] The term “antibody fragment” refers to at least one portion of an intact antibody or its recombinant variant, and further refers to an antigen-binding domain, such as the antigen-determining variable region of the intact antibody, to a degree sufficient to result in recognition of the antibody fragment and specific binding to a target such as an antigen. Examples of antibody fragments, but not limited to these, include Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies (either VL or VH) such as sdAb, camel VHH domains, and multispecific antibodies formed from antibody fragments. The term “scFv” refers to the above-mentioned fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are sequentially linked via a short, flexible polypeptide linker, and the fusion protein can be expressed as a single-chain polypeptide, and further, the scFv retains the specificity of the original intact antibody. Unless otherwise specified, when used herein, scFv may have the VL and VH variable regions in either order, and with respect to the N-terminus and C-terminus of the polypeptide, for example, scFv may contain VL-linker-VH or VH-linker-VL.

[0062] A portion of the CAR composition of the present invention, comprising an antibody or an antibody fragment thereof, may exist in various forms, in which case the antigen-binding domain is expressed as part of a continuous polypeptide chain, such as a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0063] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules of their naturally occurring conformations, and this usually determines the class to which the antibody belongs.

[0064] The term "antibody light chain" refers to the smaller of two types of polypeptide chains present in antibody molecules of their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.

[0065] The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as antibodies expressed by bacteriophages or yeast expression systems. This term is also interpreted to mean antibodies produced by the synthesis of a DNA molecule encoding an antibody (which expresses an antibody protein) or an amino acid sequence specifying the antibody, in which case the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technologies available and well known in the industry.

[0066] The terms “antigen” or “Ag” refer to a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immune cells, or both. Those skilled in the art will expect to understand that virtually any macromolecule, encompassing substantially all proteins or peptides, can serve as an antigen. Furthermore, antigens may be recombinant or derived from genomic DNA. Those skilled in the art will expect to understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will consequently encode an “antigen” in the same way as the term “antigen” is used herein. Furthermore, those skilled in the art will expect to understand that antigens are not necessarily encoded only by the full-length nucleotide sequence of a gene. The present invention is not limited to, but will include the use of partial nucleotide sequences of one or more genes, and it will be readily understood that these nucleotide sequences may be arranged in various combinations so as to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will expect to understand that antigens do not necessarily have to be encoded by a “gene.” It will be readily understood that antigens may be generated or synthesized, or derived from biological samples, or may be macromolecules other than polypeptides. Examples of such biological samples include, but are not limited to, tissue samples, tumor samples, and fluids containing cells or other biological components.

[0067] The term "antitumor effect" refers to a biological action that can be demonstrated by various means, but is not limited to, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in expected lifespan, a decrease in tumor cell proliferation, a decrease in tumor cell survival, or an improvement in various physiological symptoms associated with the cancerous state. Furthermore, the "antitumor effect" can also be demonstrated by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the appearance of tumors at their initial site.

[0068] The term "self" refers to all materials originating from the same individual that are intended to be later reintroduced into that individual.

[0069] The term "allogeneic" refers to any material originating from different animals of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic if they do not have identical genes at one or more loci. In some embodiments, allogeneic material from individuals of the same species may be genetically different to a degree sufficient to interact antigenically.

[0070] The term "heterogeneous" refers to a graft derived from an animal of a different species.

[0071] The term "cancer" refers to a disease characterized by rapid, uncontrolled, and abnormal cell growth. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and similar cancers.

[0072] The phrase “diseases associated with CD19 expression” includes, but is not limited to, diseases associated with CD19 expression or conditions associated with CD19-expressing cells, such as proliferative disorders like cancer or malignant tumors, or precancerous conditions like spinal cord malformations, myelodysplastic syndromes or preleukemic states; or non-cancerous signs associated with cells expressing CD19. In one embodiment, cancers associated with CD19 expression are hematolic cancers. In one embodiment, hematolic cancers are leukemias or lymphomas. In one embodiment, cancers associated with CD19 expression include, but is not limited to, cancers and malignant tumors, such as, one or more acute leukemias, such as, but is not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); and, but is not limited to, one or more chronic leukemias, such as, but is not limited to, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Additional cancers or hematological conditions associated with CD19 expression include, but are not limited to, B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative states, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and a group of hematological conditions known as “preleukemic states,” which are common to the unresponsive production (or dysplasia) of blood cells in the bone marrow, as well as similar conditions. Further diseases associated with CD19 expression include, but are not limited to, atypical and / or non-typical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD19 expression. Non-cancer-related signs associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory diseases (allergies and asthma), and transplantation.

[0073] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the industry, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the industry. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CAR of the present invention may be replaced with other amino acid residues of the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0074] The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating signaling events, including, but not limited to, signaling via the TCR / CD3 complex. Stimulation can mediate the downregulation of TGF-β and / or the rearrangement of the cytoskeleton, as well as the altered expression of certain molecules, such as homologs.

[0075] The term “stimulating molecule” refers to a molecule expressed by a T cell that gives rise to a primary cytoplasmic signaling sequence that modulates the primary activation of the TCR complex in the form of a stimulus, with respect to at least some aspects of the T cell signaling pathway. In one embodiment, for example, primary signaling is initiated by the binding of the TCR / CD3 complex to a peptide-presenting MHC molecule, thereby mediating T cell responses such as proliferation, activation, differentiation, and homogeneous, but not limited to these. The primary cytoplasmic signaling sequence that acts in the form of a stimulus (also referred to as the “primary signaling domain”) may contain a signaling motif, which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences that have a particular use in the present invention include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”), and CD66d. In certain CARs of the present invention, the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, the primary signaling sequence of CD3-zeta. In certain CARs of the present invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 17, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their counterparts. In certain CARs of the present invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 43, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their counterparts.

[0076] The term "antigen-presenting cell" or "APC" refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, and allogenes), that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and cause T cells to present them.

[0077] When used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains generate signals that promote the immune effector functions of cells containing CARs, such as CART cells. Examples of immune effector functions in CART cells include cytolytic activity and helper activity, such as cytokine secretion.

[0078] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Typical primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent simulation. In some embodiments, the intracellular signaling domain may include a co-stimulatory intracellular domain. Typical co-stimulatory intracellular signaling domains include those derived from molecules involved in co-stimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0079] The primary intracellular signaling domain may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

[0080] The terms “zeta” or, in lieu of “zeta chain,” “CD3-zeta,” or “TCR-zeta” are defined as the protein provided under GenBan accession number BAG36664.1, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes, and their allies; and the “zeta-stimulating domain” or, in lieu of “CD3-zeta-stimulating domain” or “TCR-zeta-stimulating domain” are defined as amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes, and their allies that are functional orthologs. In one embodiment, the “zeta-stimulating domain” or “CD3-zeta-stimulating domain” is the sequence provided under Sequence ID No. 17. In one embodiment, the "zeta-stimulating domain" or "CD3-zeta-stimulating domain" is the sequence provided as Sequence ID No. 43.

[0081] The term "costimulatory molecule" refers to a co-stimulatory partner on a T cell that mediates a T cell costimulatory response, such as proliferation, by specifically binding to a costimulatory ligand, although these are not limited to costimulatory ligands. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0082] The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. Representative examples of co-stimulatory molecules include the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), and activated NK cell receptors. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83, as well as similar molecules.

[0083] The intracellular signaling domain may include the entire intracellular portion, the entire intrinsic intracellular signaling domain of the molecule from which it is obtained, or functional fragments thereof.

[0084] The term "4-1BB" refers to a member of the TNFR superfamily having the sequence amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their relatives, and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their relatives. In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 16 or equivalent residues from non-human species, such as mice, rodents, monkeys, apes and their relatives.

[0085] The term “coding” refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a specific sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a specific sequence of amino acids, and the biological properties that result from it, serving as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, in cells or other biological systems, when proteins are produced by the transcription and translation of mRNA corresponding to a gene, that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in sequence listings, and the non-coding strand used as a template for the transcription of a gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0086] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate of each other or that encode the same amino acid sequence. Furthermore, the phrase "nucleotide sequence encoding a protein or RNA" may, in some cases, contain introns to the extent that the nucleotide sequence encoding that protein may contain introns.

[0087] The terms “effective dose” or “therapeutic dose” are used synonymously herein and refer to the amount of a compound, preparation, material, or composition, as described herein, that is effective in achieving a particular biological outcome.

[0088] The term "endogenous" refers to any material that originates from an organism, cell, tissue, or system, or any material produced within them.

[0089] The term "exogenous" refers to any material introduced from outside an organism, cell, tissue, or system, or any material produced outside of them.

[0090] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0091] The term "transfer vector" refers to a composition containing isolated nucleic acids that can be used to deliver isolated nucleic acids into cells. Numerous vectors are known in the industry, but are not limited to, linear polynucleotides, polynucleotides conjugated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" encompasses autonomously replicating plasmids or viruses. This term should also be interpreted to further encompass non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and analogues. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and analogues.

[0092] The term “expression vector” refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence ligated to the nucleotide sequence to be expressed in a functional manner. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by the host cell or within the in vitro expression system. Expression vectors encompass everything known in the art, including, for example, cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0093] The term "lentivirus" refers to a genus of retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and because they can deliver a significant amount of genetic information to the host cell's DNA, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0094] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, particularly self-inactivating lentiviral vectors, such as those described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical settings include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX® vector system, and similar products. Non-clinical lentiviral vectors are also available and are expected to be known to those skilled in the art.

[0095] The terms "homologous" or "identical" refer to the identity of subunit sequences between two macromolecules, for example between two nucleic acid molecules, for example between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the positions of subunits in both molecules are occupied by the same monomer subunits, for example, if the positions in each of two DNA molecules are occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a linear function of the number of matched or homologous positions. For example, if half of the positions in the two sequences are homologous (e.g., 5 positions out of 10 subunits in a polymer), then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0096] The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof [e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody] containing the smallest sequence derived from the non-human immunoglobulin. In most cases, the humanized antibody and its antibody fragments are human immunoglobulins (recipient antibodies or antibody fragments) in which residues from the recipient’s complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody / antibody fragment may contain residues not found in either the recipient antibody or the transferred CDR or framework sequence. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or their antibody fragments are expected to contain at least one, typically two, substantially all of variable domains, where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or a substantial portion of the FR region is the FR region of a human immunoglobulin sequence. Humanized antibodies or antibody fragments may also contain at least a portion of the immunoglobulin constant region (Fc), typically the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0097] "Completely human" refers to immunoglobulins, such as antibodies or antibody fragments, where the entire molecule is of human origin or consists of an amino acid sequence identical to that of the human form of an antibody or immunoglobulin.

[0098] The term "isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is "not isolated," but the same nucleic acid or peptide is "isolated" if it has been partially or completely separated from the material that coexists with it in its natural state. Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0099] In the present invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0100] The terms "functionally linked" or "transcriptional regulation" refer to the functional linking of a regulatory sequence to a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, if a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter influences the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Functionally linked DNA sequences may be contiguous with each other, or they may be within the same reading frame, for example, if it is necessary to fused the coding regions of two proteins.

[0101] The term "parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, intratumoral, or infusion techniques.

[0102] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in either single-stranded or double-stranded forms. Unless otherwise specified, the term encompasses nucleic acids containing known analogues of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also effectively encompasses its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences that are explicitly presented. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a hybrid base and / or deoxyinosine residue [Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)].

[0103] The terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide encompasses any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, commonly referred to in the industry as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the industry as proteins, of which many types exist. Examples of “polypeptides” include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Examples of polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0104] The term "promoter" refers to a DNA sequence recognized by a cellular synthetic mechanism, or the introduced synthetic mechanism, that is necessary to initiate the specific transcription of a polynucleotide sequence.

[0105] The term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a gene product that is ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also contain enhancer sequences and other regulatory factors necessary for the expression of the gene product. A promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0106] The term "constitutive" promoter refers to a nucleotide sequence that, when linked to a polynucleotide that codes for or designates a gene product, causes the cell to produce that gene product under most or all physiological conditions.

[0107] The term "inducible" promoter refers to a nucleotide sequence that, when linked to a polynucleotide that codes for or designates a gene product, causes a cell to produce a gene product only when an inducer, essentially corresponding to the promoter, is present in the cell.

[0108] The term "tissue-specific" promoter refers to a nucleotide sequence that, when linked to a polynucleotide that codes for a gene or is specified by a gene, causes the cell to produce a gene product only when the cell is substantially the tissue type corresponding to the promoter.

[0109] The term “flexible polypeptide linker” or “linker,” when used in the context of scFv, refers to a peptide linker consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker comprising the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10 (SEQ ID NO: 105). In one embodiment, examples of flexible polypeptide linkers, but not limited to these, include (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). In another embodiment, the linker comprises multiple iterations of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 108). The linker described in WO2012 / 138475, incorporated herein by reference, is also included within the scope of the present invention.

[0110] 5' cap (RNA cap, RNA7-methylguanosine cap or RNAm 7A 5' cap (also known as a G-cap), as used herein, is a modified guanine nucleotide added to the “prefix” or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of terminal groups that are linked to the first nucleotide to be transcribed. Its presence is important for ribosome recognition and protection from RNases. Capping is performed by coupling with transcription, with each process being transcribed together in a manner that influences the other. Immediately after transcription initiation, the 5' end of the synthesized mRNA is bound to a complex that synthesizes the cap, associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping components may be modified to regulate the functionality of the mRNA, such as its stability or translation efficiency.

[0111] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro, preferably mRNA. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used to produce in vitro transcribed RNA.

[0112] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In a preferred embodiment of the construct for transient expression, the number of poly(A) sequences is between 50 and 5000 (SEQ ID NO: 109), preferably more than 64, more preferably more than 100, and most preferably more than 300 or 400. The poly(A) sequences may be chemically or enzymatically modified to modulate mRNA functionality such as localization, stability, or translation efficiency.

[0113] As used herein, "polyadenylation" refers to the covalent linking of a polyadenylyl component or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' end. The 3' poly(A) tail is a long sequence (often hundreds) of adenine nucleotides added to pre-mRNA via the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to a transcript containing a specific sequence, the polyadenylation signal. The poly(A) tail and the protein it binds to help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA transport out of the cell nucleus, and translation. Polyadenylation occurs in the cell nucleus immediately after DNA transcription to RNA, and can also occur later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved via an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end at the cleavage site.

[0114] As used herein, "transient" refers to the expression of an unintegrated transgene over a period of several hours, days, or weeks, where the expression period is shorter than that of a gene integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0115] The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in signal transmission from one part of a cell to another. The phrase "cell surface receptor" encompasses molecules and molecular complexes that can receive signals and cross the cell membrane to transmit those signals.

[0116] The term "target" is intended to encompass organisms capable of eliciting an immune response (e.g., mammals, humans).

[0117] The term "substantially purified" refers to cells that essentially contain no other cell types. It can also refer to cells isolated from other cell types that normally coexist in their natural environment. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells isolated from cells that naturally coexist in their natural state. In some embodiments, such cells are cultured in vitro. In other embodiments, such cells are not cultured in vitro.

[0118] The term "treatment," as used herein, means a procedure. Therapeutic effects are obtained by recovery, suppression, remission, or eradication of a disease.

[0119] As used herein, the term "prevention" means the prevention or preventive action of a disease or medical condition.

[0120] In the context of the present invention, “tumor antigen,” “antigen of hyperproliferative disorder,” or “antigen associated with hyperproliferative disorder” refers to an antigen common to a particular hyperproliferative disorder. In a given embodiment, the antigens of hyperproliferative disorder of the present invention are, for example, but are not limited to, cancer-derived, such as primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and similar cancers.

[0121] The term "transfection," "transformation," or "transduction" refers to the process of introducing or transferring exogenous nucleic acids into host cells. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids. The cells include the primary cells and their descendants.

[0122] The term "specifically binding" refers to an antibody or ligand that recognizes and binds to a co-originating binding partner protein present in the sample (e.g., a stimulating and / or co-stimulating molecule present on T cells), but these antibodies or ligands do not substantially recognize or bind to other molecules in the sample.

[0123] Scope: Throughout this disclosure, various aspects of the invention may be indicated in the form of scope. It is understood that such indications are for convenience and brevity only and should not be construed as inflexible limitations on the scope of the invention. Accordingly, scope indications shall be deemed to specifically disclose all possible subranges, in addition to the individual numerical values ​​within those ranges. For example, a scope indication such as 1 to 6 is expected to be deemed to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, in addition to the individual numerical values ​​within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity encompasses something that has 95%, 96%, 97%, 98%, or 99% identity, and further encompasses subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

[0124] explanation This specification provides compositions and methods of use of humanized anti-CD19 chimeric antigen receptors (CARs) for treating diseases such as cancer.

[0125] In one embodiment, the present invention provides a number of chimeric antigen receptors (CARs) comprising antibodies or antibody fragments processed to enhance binding to the CD19 protein. In one embodiment, the present invention provides cells (e.g., T cells) processed to express a CAR, where the CART cells ("CART") exhibit antitumor properties. In one embodiment, cells are transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, cells can stably express a CAR. In another embodiment, cells (e.g., T cells) are transfected with a nucleic acid encoding a CAR, such as mRNA, cDNA, or DNA. In some such embodiments, cells can transiently express a CAR.

[0126] In one embodiment, the CAR-binding portion of the anti-CD19 protein is an scFv antibody fragment. In one embodiment, such antibody fragments are functional in that they maintain equivalent binding affinity, for example, they bind to the same antigen with equivalent efficacy to the IgG antibody from which they originated. In one embodiment, such antibody fragments are functional in that they produce biological responses such as activation of an immune response, inhibition of signal initiation from its target antigen, inhibition of kinase activity, and similar responses, which are expected to be understood by those skilled in the art, but are not limited to these. In one embodiment, the anti-CD19 antigen-binding domain of the CAR is a humanized scFv antibody fragment compared to the mouse sequence of its origin. In one embodiment, the parental mouse scFv sequence is the sequence CAR19 construct, provided in PCT Publication WO2012 / 079000 and provided herein as Sequence ID No. 58.

[0127] In several embodiments, the antibodies of the present invention are incorporated into a chimeric antigen receptor (CAR). In one embodiment, the CAR comprises a sequence polypeptide sequence provided as SEQ ID NO: 12 in PCT Publication WO2012 / 079000 and provided herein as SEQ ID NO: 58, where the scFv domain is substituted with one or more sequences selected from SEQ ID NOs: 1-12. In one embodiment, the scFv domains of SEQ ID NOs: 1-12 are humanized variants of the scFv domain of SEQ ID NO: 59, which is a mouse-derived scFv fragment that specifically binds to human CD19. Humanization of this mouse scFv may be desirable for clinical conditions in which mouse-specific residues may induce a human anti-mouse antigen (HAMA) response in patients receiving CART19 treatment, for example, treatment with T cells transduced with a CAR19 construct.

[0128] In one embodiment, the anti-CD19 binding domain, for example, the humanized scFv which is part of the CAR of the present invention, is encoded by a transgene whose codons in its sequence are optimized for expression in mammalian cells. In one embodiment, the entire CAR construct of the present invention is encoded by a transgene whose codons in its entire sequence are optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased across different species. Such codon degeneracy makes it possible to code for the same polypeptide with various nucleotide sequences. Various codon optimization methods are known in the art, including, for example, those disclosed in at least U.S. Patents 5,786,464 and 6,114,148.

[0129] In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 1. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 2. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 3. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 4. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 5. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 6. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 7. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 8. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 9. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 10. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 11. In one embodiment, the humanized CAR19 includes the scFv portion provided in SEQ ID NO: 12.

[0130] In one embodiment, the CAR of the present invention combines a specific antibody-antigen binding domain with an intracellular signaling molecule. For example, in some embodiments, the intracellular signaling molecule may include, but is not limited to, the CD3-zeta chain, 4-1BB and CD28 signaling modules, or combinations thereof. In one embodiment, the antigen-binding domain binds to CD19. In one embodiment, the CD19CAR includes a CAR selected from sequences provided in one or more of SEQ ID NOs: 31-42. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 31. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 32. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 33. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 34. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 35. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 36. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 37. In one embodiment, the CD19CAR includes the sequence provided in SEQ ID NO: 38. In one embodiment, CD19CAR includes the sequence provided in SEQ ID NO: 39. In one embodiment, CD19CAR includes the sequence provided in SEQ ID NO: 40. In one embodiment, CD19CAR includes the sequence provided in SEQ ID NO: 41. In one embodiment, CD19CAR includes the sequence provided in SEQ ID NO: 42.

[0131] Furthermore, the present invention provides CD19CAR compositions and their uses in pharmaceuticals or methods for treating diseases, particularly cancer or any malignant tumor, or autoimmune diseases involving cells or tissues expressing CD19.

[0132] In one embodiment, the CAR of the present invention can be used to eradicate normal cells expressing CD19, thereby making it applicable for use as cellular conditioning therapy before cell transplantation. In one embodiment, the normal cells expressing CD19 are normal stem cells expressing CD19, and the cell transplantation is stem cell transplantation.

[0133] In one embodiment, the present invention provides cells (e.g., T cells) modified to express a chimeric antigen receptor (CAR), where the CART cells ("CART") exhibit antitumor properties. A preferred antigen is CD19. In one embodiment, the antigen-binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment. In another embodiment, the antigen-binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment containing scFv. Thus, the present invention provides CD19-CARs comprising a humanized anti-CD19 binding domain, incorporated into T cells, and methods for using them in adoptive therapy.

[0134] In one embodiment, CD19-CAR comprises at least one intracellular domain selected from the group consisting of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta-signaling domain, and any combination thereof. In one embodiment, CD19-CAR comprises at least one intracellular signaling domain derived from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.

[0135] Chimeric antigen receptor (CAR) The present invention encompasses a recombinant DNA construct comprising a sequence encoding a CAR, wherein the CAR comprises a humanized antibody fragment that specifically binds to CD19, for example, human CD19, and the sequence of the antibody fragment is contiguous with and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain may comprise a co-stimulatory signaling domain and / or a primary signaling domain, such as a zeta chain. The co-stimulatory signaling domain refers to a portion of the CAR that includes at least a portion of the intracellular domain of a co-stimulatory molecule.

[0136] In specific embodiments, the CAR construct of the present invention comprises an scFv domain selected from the group consisting of SEQ ID NO: 1 to 12, where prior to the scFv there may be an arbitrary leader sequence such as that provided by SEQ ID NO: 13, followed by an arbitrary hinge sequence such as that provided by SEQ ID NO: 14, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, a transmembrane region such as that provided by SEQ ID NO: 15, an intracellular signaling domain such as SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence such as SEQ ID NO: 17 or SEQ ID NO: 43, where the domains are contiguous and within the same reading frame, forming a single fusion protein. The present invention also includes nucleotide sequences encoding each polypeptide of an scFv fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. The present invention also encompasses nucleotide sequences encoding each of the scFv fragments selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, as well as each of the polypeptides of the domains of SEQ ID NOs: 13-17, and the encoded CD19CAR fusion protein of the present invention. In one embodiment, a typical CD19CAR construct includes an arbitrary leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulation domain. In one embodiment, a typical CD19CAR construct includes an arbitrary leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular stimulation domain. Specific CD19CAR constructs containing the humanized scFv domain of the present invention are provided as SEQ ID NOs: 31-42.

[0137] Furthermore, the full-length CAR sequence is also provided herein as sequence numbers 31 to 42, as shown in Table 3.

[0138] A typical leader sequence is provided as SEQ ID NO: 13. Typical hinge / spacer sequences are provided as SEQ ID NO: 14, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49. A typical transmembrane domain sequence is provided as SEQ ID NO: 15. A typical sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 16. A typical sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 51. A typical CD3 zeta domain sequence is provided as SEQ ID NO: 17 or SEQ ID NO: 43.

[0139] In one embodiment, the present invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-CD19 binding domain that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain, as described herein, for example. In one embodiment, the anti-CD19 binding domain is selected from one or more of SEQ ID NOs: 1 to 12. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of a sequence provided in one or more of SEQ ID NOs: 61 to 72. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 61. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 62. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 63. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 64. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 65. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 66. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 67. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 68. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 69. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 70. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 71. In one embodiment, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 72.

[0140] In one embodiment, the present invention encompasses a recombinant nucleic acid construct comprising a transgene encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-CD19 binding domain selected from one or more of SEQ ID NOs. 61-72, wherein the sequence is contiguous with and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Typical intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains of the same type, such as CD3-zeta, CD28, 4-1BB, and the like. In some cases, the CAR may comprise any combination of CD3-zeta, CD28, 4-1BB, and the like. In one embodiment, the nucleic acid sequence of the CAR construct of the present invention is selected from one or more of SEQ ID NOs. 85-96. 86. 87. 88. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 89. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 90. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 91. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 92. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 93. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 94. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 95. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 96. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 97. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 98. In one embodiment, the nucleic acid sequence of the CAR construct is sequence number 99.

[0141] Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as screening a library from cells expressing the gene, extracting the gene from a vector known to contain the gene, or directly isolating it from cells and tissues containing the gene using standard techniques. Alternatively, the nucleic acid of interest may be produced synthetically rather than cloned.

[0142] The present invention encompasses retroviral and lentiviral vector constructs that express CARs and can be directly transduced into cells.

[0143] The present invention also encompasses RNA constructs that can be directly transfected into cells. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template using specially designed primers to produce a construct (SEQ ID NO: 118) containing a 3' and 5' untranslated sequence ("UTR") typically 50–2000 nucleotides in length, a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail, followed by poly-A addition. The RNA thus produced can be efficiently transfected into cells of different species. In one embodiment, the template contains a sequence relating to a CAR. In one embodiment, the RNA CAR vector is transduced into T cells by electroporation.

[0144] antigen-binding domain In one embodiment, the CAR of the present invention comprises a target-specific binding element, also referred to as an antigen-binding domain. The selection of components depends on the type and number of ligands that characterize the target cell surface. For example, the antigen-binding domain can be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease condition. Thus, examples of cell surface markers that may act as ligands for the antigen-binding domain in the CAR of the present invention include those associated with viruses, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0145] In one embodiment, the CAR-mediated T cell response may be directed towards the target antigen in order to incorporate an antigen-binding domain that specifically binds to a desired antigen into the CAR.

[0146] In one embodiment, a portion of the CAR containing an antigen-binding domain includes an antigen-binding domain that targets CD19. In one embodiment, the antigen-binding domain targets human CD19. In one embodiment, the antigen-binding domain of the CAR has the same or similar binding specificity as the scFv fragment of FMC63 described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

[0147] The antigen-binding domain may be any domain that binds to an antigen, such as, but is not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and their functional fragments, such as, but is not limited to, single-domain antibodies, such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of a camel-derived nanobody, as well as alternative scaffolds known in the industry to function as antigen-binding domains, such as recombinant fibronectin domains and any similar domains. In some cases, it is beneficial for the antigen-binding domain that the CAR is of the same species from which it is ultimately expected to be used. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to contain human or humanized residues with respect to the antigen-binding domain of the antibody or antibody fragment.

[0148] Therefore, in one embodiment, the antigen-binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain comprises one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs. In one embodiment, the humanized anti-CD19 binding domain includes one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each including HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized anti-CD19 binding domain includes a humanized light chain variable region and / or a humanized heavy chain variable region as described herein (e.g., listed in Table 3). In one embodiment, the humanized anti-CD19 binding domain includes a humanized heavy chain variable region as described herein (e.g., listed in Table 3), for example, at least two humanized heavy chain variable regions as described herein (e.g., listed in Table 3). In one embodiment, the anti-CD19 binding domain is an scFv containing the light and heavy chains of the amino acid sequences shown in Table 3.In one embodiment, the anti-CD19 binding domain (e.g., scFv) includes a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region shown in Table 3, but with 30, 20, or 10 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 3; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region shown in Table 3, but with 30, 20, or 10 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 3. In one embodiment, the humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain includes a sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, and 72, or a sequence having 95-99% identity with them. In one embodiment, the humanized anti-CD19 binding domain is scFv, and a light chain variable region containing an amino acid sequence as described herein, for example, Table 3, is attached to a heavy chain variable region containing an amino acid sequence as described herein, for example, Table 3, via a linker, for example, a linker as described herein. In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of scFv may be arranged in either of the following configurations, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0149] In one embodiment, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NOs: 1 to 12. In one embodiment, the humanized CAR is selected from one or more sequences selected from SEQ ID NOs: 31 to 42. In some embodiments, the non-human antibody is humanized, in which case a specific sequence or region of the antibody is modified to increase its similarity to antibodies or fragments thereof naturally produced in humans. In one embodiment, the antigen-binding domain is humanized.

[0150] Humanized antibodies can be produced using various techniques known in the industry, including, but not limited to, CDR grafting (see, for example, European Patent EP239,400, which is incorporated herein by reference in its entirety; International Patent Publication WO91 / 09967; and U.S. Patents 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (see, for example, European Patents EP592,106 and EP519,596, which are incorporated herein by reference in their entirety; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS). See 91:969-973, chain shuffling (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety), and, for example, U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Patent Publication WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res.Examples include the techniques disclosed in 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). To alter, for example, antigen binding, framework residues within the framework region are often expected to be substituted with corresponding residues from the CDR donor antibody. These framework substitutions are identified by methods well known in the art, such as modeling the interaction between CDRs and framework residues to identify framework residues crucial for antigen binding, and by sequence comparison to identify abnormal framework residues at specific locations (see, for example, U.S. Patent No. 5,585,089 by Queen et al., and Riechmann et al., 1988, Nature, 332:323, which are incorporated in their entirety by reference).

[0151] Humanized antibodies or antibody fragments contain one or more residual amino acid residues from a non-human source. These non-human amino acid residues are often referred to as “implant” residues, which are typically taken from “implant” variable domains. As described herein, humanized antibodies or antibody fragments contain one or more CDRs and framework regions from a non-human immunoglobulin molecule, where the amino acid residues containing the framework are entirely or largely derived from human germline cells. Several techniques for humanizing antibodies or antibody fragments are well known in the industry, essentially the methods of Winter and collaborators [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536]. This can be done by substituting the CDR sequence with respect to the corresponding sequence of a rodent CDR or human antibody, i.e., by CDR grafting (the contents of which are incorporated herein by reference as a whole EP239,400; PCT Publication WO91 / 09967; and U.S. Patents 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640). In such humanized antibodies and antibody fragments, a portion substantially smaller than the intact human variable domain is substituted with the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which several CDR residues and possibly several framework (FR) residues are substituted with residues from similar sites in rodent antibodies.Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing [the contents thereof are incorporated herein by reference as a whole EP592,106;EP519,596;Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498;Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)] or chain shuffling (U.S. Patent No. 5,565,332).

[0152] Selecting both light and heavy chain human variable domains for use in the production of humanized antibodies would reduce antigenicity. The variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences according to a so-called "best-fit" method. The human sequences most closely resembling the rodent sequences are then accepted as the human framework (FR) for humanized antibodies [their contents are incorporated herein by reference as a whole: Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)]. Alternatively, a specific framework derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain is used. The same framework can be used for various different humanized antibodies [see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference as a whole]. In some embodiments, all four framework regions of the framework region, e.g., the heavy chain variable region, are derived from the VH4_4-59 germline sequence. In one embodiment, the framework region may include, for example, one, two, three, four, or five modifications, e.g., substitutions, from amino acids in the corresponding mouse sequence (e.g., SEQ ID NO: 58). In one embodiment, all four framework regions of the framework region, e.g., the light chain variable region, are derived from the VK3_1.25 germline sequence. In one embodiment, the framework region may include, for example, one, two, three, four, or five modifications, such as substitutions, from the amino acids in the corresponding mouse sequence (e.g., sequence number 58).

[0153] In some embodiments, a portion of the CAR composition of the present invention, including an antibody fragment, is humanized while retaining high affinity to a target antigen and other favorable biological properties. According to one aspect of the present invention, humanized antibodies and antibody fragments are prepared by a process of analyzing the parent sequence and various conceptual humanization products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are commonly available and well known to those skilled in the art. Computer programs are available that exemplify and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. By examining these displays, it is possible to analyze the roles of promising residues in the functionalization of the candidate immunoglobulin sequence, for example, the residues that affect the candidate immunoglobulin's ability to bind to a target antigen. In this method, FR residues can be selected and combined from the recipient and transfer sequences so that desired antibody or antibody fragment characteristics, such as high affinity to a target antigen, are achieved. Generally, CDR residues are directly and most substantially involved in the effect on antigen binding.

[0154] Humanized antibodies or antibody fragments may retain antigen specificity similar to the original antibody, for example, the ability to bind to human CD19 in this invention. In some embodiments, humanized antibodies or antibody fragments may have improved affinity and / or specificity for binding to human CD19.

[0155] In one embodiment, the anti-CD19 binding domain is characterized by a specific functional feature or property of the antibody or antibody fragment. For example, in one embodiment, a portion of the CAR composition of the present invention comprising an antigen-binding domain specifically binds to human CD19. In one embodiment, the antigen-binding domain has the same or similar binding specificity to human CD19 as FMC63scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the present invention relates to an antigen-binding domain comprising an antibody or antibody fragment, wherein the antibody-binding domain specifically binds to a CD19 protein or a fragment thereof, and the antibody or antibody fragment comprises a variable light chain and / or variable heavy chain encompassing the amino acid sequences of SEQ ID NOs. 1-12. In one embodiment, the antigen-binding domain comprises the amino acid sequence of an scFv selected from SEQ ID NOs. 1-12. In a given embodiment, the scFv is contiguous with a leader sequence and lies within the same reading frame. In one embodiment, the leader sequence is a polypeptide sequence provided as SEQ ID NO. 13.

[0156] In one embodiment, the anti-CD19 binding domain is a fragment, for example, a single-chain variable fragment (scFv). In one embodiment, the anti-CD19 binding domain is Fv, Fab, a(Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody [e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)]. In one embodiment, the antibodies and their fragments of the present invention bind to the CD19 protein with wild-type or enhanced affinity.

[0157] In some cases, scFv can be prepared according to methods known in the art [see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883]. scFv molecules can be produced by linking the VH and VL regions together using a flexible polypeptide linker. scFv molecules contain a linker with optimized length and / or amino acid composition (e.g., a Ser-Gly linker). The length of the linker can significantly influence how the variable regions of the scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5 and 10 amino acids), intrachain folding is prevented. Interchain folding is also necessary to bridge the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publications WO2006 / 020258 and WO2007 / 024715, which are incorporated herein by reference.

[0158] scFv may contain a linker between its VL and VH regions consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 amino acid residues, or more. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains a series of glycine and serine repeats, for example, (Gly4Ser)n, where n is a positive integer of 1 or more (SEQ ID NO: 18). In one embodiment, the linker may be (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). By varying the length of the linker, it is possible to maintain or enhance activity, thereby providing superior efficacy in activity studies.

[0159] Stability and mutation The stability of the anti-CD19 binding domain, for example, the scFv molecule (e.g., soluble scFv), can be evaluated by referring to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or full-length antibody. In one embodiment, the humanized scFv has a thermal stability of about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 degrees higher than the control binding molecule (e.g., conventional scFv molecule) in the assay described.

[0160] The improved thermal stability of the anti-CD19 binding domain, e.g., scFv, is subsequently conferred to the entire CART19 construct, resulting in improved therapeutic properties of the CART19 construct. The thermal stability of the anti-CD19 binding domain, e.g., scFv, can be improved by at least about 2°C or 3°C compared to conventional antibodies. In one embodiment, the anti-CD19 binding domain, e.g., scFv, has a thermal stability that is 1°C higher than that of conventional antibodies. In another embodiment, the anti-CD19 binding domain, e.g., scFv, has a thermal stability that is 2°C higher than that of conventional antibodies. In yet another embodiment, scFv has a thermal stability that is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15°C higher than that of conventional antibodies. Comparisons can be made, for example, between the scFv molecule disclosed herein and the scFv molecule or Fab fragment of the antibody from which the VH and VL of scFv were obtained. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm may be measured. The following sections provide a more detailed explanation of how to measure Tm and how to determine the stability of other proteins.

[0161] Mutations in scFv (resulting from humanization of soluble scFv or direct mutagenesis) alter the stability of scFv, improving the overall stability of scFv and the CART19 construct. The stability of humanized scFv is compared to mouse scFv using measurements such as Tm, temperature-induced denaturation, and temperature-induced aggregation.

[0162] The binding ability of mutant scFv can be determined using the assay described in the examples.

[0163] In one embodiment, the anti-CD19 binding domain, e.g., scFv, includes at least one mutation resulting from the humanization process, such that the mutated scFv confers improved stability to the CART19 construct. In another embodiment, the anti-CD19 binding domain, e.g., scFv, includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting from the humanization process, such that the mutated scFv confers improved stability to the CART19 construct.

[0164] Methods for evaluating protein stability The stability of the antigen-binding domain may be examined using methods, for example, as described below. Such methods allow for the determination of multiple thermal unfolding transitions, where the least stable domain either does not fold at all or does not fold cooperatively, limiting the overall stability of the multi-domain unit (e.g., a multi-domain protein exhibiting a single unfolding transition). The least stable domain can be identified by numerous additional methods. Mutagenesis can be performed to explore which domain limits overall stability. In addition, protease resistance of multi-domain proteins can be achieved via DSC or other spectroscopic methods under conditions where the least stable domain is known not to fold in essence [Fontana, et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol. 393: 672-692]. Once the least stable domain is identified, the sequence encoding this domain (or a portion thereof) may be used as a test sequence in this method.

[0165] a) Thermal stability The thermal stability of a composition can be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is evaluated by analytical spectroscopic analysis.

[0166] A typical analytical spectroscopic method is differential scanning calorimetry (DSC). DSC employs a calorimeter that is sensitive to the endothermic effects associated with the unfolding of most proteins or protein domains (see, e.g., Sanchez-Ruiz, et al., Biochemistry, 27: 1648-52, 1988). To determine the thermal stability of a protein, a sample of the protein is inserted into the calorimeter and the temperature is raised until the Fab or scFv stops folding. The temperature at which the protein stops folding is an indicator of the overall stability of the protein.

[0167] Another typical analytical spectroscopic method is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures the difference in absorption between left-handed and right-handed polarized light, which arises from structural asymmetry. Disordered or unfolded structures yield CD spectra that are quite different from those of regular or folded structures. CD spectra are an indicator of protein thermal stability because they reflect the protein's sensitivity to denaturation due to increasing temperature [see van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000].

[0168] Another typical analytical spectroscopic method for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma above). Yet another typical analytical spectroscopic method for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma above).

[0169] The thermal stability of a composition can be measured biochemically. A typical biochemical method for examining thermal stability is a thermal challenge assay. In a thermal challenge assay, the composition is exposed to a series of temperature increases over a set period of time. For example, in one embodiment, a test scFv molecule or a molecule containing scFv molecules is exposed to a series of temperature increases over, for example, 1 to 1.5 hours. The activity of the protein is then assayed by an appropriate biochemical assay. For example, if the protein is a binding protein (e.g., scFv or scFv-containing polypeptide), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

[0170] Such assays may be performed in a high-throughput manner, or in the manner disclosed in the examples using E. coli and high-throughput screening. Libraries of anti-CD19 binding domains, e.g., scFv variants, can also be prepared using methods known in the art. Expression of anti-CD19 binding domains, e.g., scFv, may be induced, or the anti-CD19 binding domains, e.g., scFv, may be subjected to thermal induction. The induced test samples may be assayed for binding, and stable anti-CD19 binding domains, e.g., scFv, may be scaled up and further characterized.

[0171] Thermal stability is assessed by measuring the melting temperature (Tm) of the composition using one of the techniques described above (e.g., analytical spectroscopy). The melting temperature is the temperature at the midpoint of the temperature transition curve, where 50% of the composition's molecules are folded [see, for example, Dimasi et al. (2009) J. Mol Biol. 393: 672-692]. In one embodiment, the Tm values ​​of the anti-CD19 binding domain, e.g., scFv, are approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C The temperatures are 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C. In one embodiment, the Tm value of IgG is approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C The temperatures are 69°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C. In one embodiment, the Tm value of the polyvalent antibody is approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 6 The temperatures are 8°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C.

[0172] Thermal stability can also be assessed by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetry techniques (e.g., DSC). The specific heat of a composition is the energy required to raise the temperature of 1 mole of water by 1°C (e.g., expressed in kcal / mol). A higher Cp is a quality indicator that the composition is a denatured or inactive protein. The change in the heat capacity (ΔCp) of a composition is measured by determining the specific heat of the composition before and after the temperature transition. Thermal stability can also be assessed by measuring or determining other parameters of thermodynamic stability, such as the Gibbs free energy of unfolding (ΔG), the enthalpy of unfolding (ΔH), or the entropy of unfolding (ΔS). One or more of the above biochemical assays (e.g., thermal induction assays) can be used to determine the temperature at which 50% of the composition retains its activity (e.g., binding activity) (i.e., T). C The value is determined.

[0173] In addition, mutations in the anti-CD19 binding domain, e.g., scFv, alter the thermal stability of the anti-CD19 binding domain, e.g., scFv, compared to an unmutated anti-CD19 binding domain, e.g., scFv. When a humanized anti-CD19 binding domain, e.g., scFv, is incorporated into the CART19 construct, it confers thermal stability to the entire anti-CD19 CART construct. In one embodiment, the anti-CD19 binding domain, e.g., scFv, includes a single mutation that confers thermal stability to the anti-CD19 binding domain, e.g., scFv. In another embodiment, the anti-CD19 binding domain, e.g., scFv, includes multiple mutations that confer thermal stability to the anti-CD19 binding domain, e.g., scFv. In one embodiment, multiple mutations in the anti-CD19 binding domain, e.g., scFv, have an additive effect on the thermal stability of the anti-CD19 binding domain, e.g., scFv.

[0174] b) % flocculation The stability of a composition can be determined by measuring its aggregation properties. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, aggregation of a composition may be evaluated using chromatography, such as size exclusion chromatography (SEC). SEC separates molecules based on size. The column is packed with semi-solid beads of polymer gel, which are expected to allow ions and small molecules to pass through but not larger ones. When a protein composition is applied to the top of the column, small folded proteins (i.e., non-aggregated proteins) are dispersed in a solvent volume greater than the capacity to accept larger protein aggregates. As a result, larger aggregates move more rapidly through the column, thus making it possible to separate the mixture or fractionate it into its components. Each fraction can be quantified separately when eluted from the gel (e.g., by light scattering). Thus, the aggregation % of a composition can be determined by comparing the concentration of the fraction with the total concentration of the protein applied to the gel. A stable composition will elute from the column as essentially a single fraction and appear as essentially a single peak in the elution profile or chromatogram.

[0175] c) binding affinity The stability of a composition can be investigated by determining its target binding affinity. A wide variety of methods for determining binding affinity are known in the industry. A typical method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using BIAcore systems (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0176] In one embodiment, the antigen-binding domain of the CAR comprises an amino acid sequence homologous to the amino acid sequence of the antigen-binding domain described herein, and the antigen-binding domain retains the desired functional properties of the anti-CD19 antibody fragment described herein. In a specific embodiment, the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the antibody fragment comprises an scFv.

[0177] In various embodiments, the antigen-binding domain of a CAR is modified by altering one or more amino acids within one or both variable regions (e.g., VH and / or VL), for example, within one or more CDR regions and / or one or more framework regions. In one specific embodiment, the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the antibody fragment comprises an scFv.

[0178] Those skilled in the art will understand that the antibodies or antibody fragments of the present invention may be further modified (e.g., from the wild type) to alter their amino acid sequence without altering their desired activity. For example, additional nucleotide substitutions resulting in amino acid substitutions at "non-essential" amino acid residues may be made to the protein. For example, non-essential amino acid residues in the molecule may be replaced with other amino acid residues from the same side-chain family. In another embodiment, amino acid chains may be replaced with structurally similar chains that differ in order and / or composition from members of the side-chain family, for example, conservative substitutions may be made in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

[0179] The family of amino acid residues having similar side chains is defined in the art and includes basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0180] In the context of two or more nucleic acid or polypeptide sequences, "percent identity" means that the two or more sequences are identical. When measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection, two sequences are "substantially identical" if, when compared and aligned to the maximum extent possible across a comparison window or indicated region, they have a certain percentage of identical amino acid residues or nucleotides (for example, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). Identity may exist over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, or 200 or more amino acids) in length.

[0181] For sequence comparison, typically one sequence acts as a reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the coordinates of the subsequences are specified, and the program parameters of the sequence algorithm are specified as needed. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters. The method for aligning sequences for comparison is well known in the industry. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA at Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection [see, for example, Brent et al., (2003) Current Protocols in Molecular Biology].

[0182] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0183] Furthermore, the percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller [(1988) Comput. Appl. Biosci. 4:11-17], which is incorporated into the ALIGN program (version 2.0) and uses the PAM120 weight residue table, a gap elongation penalty of 12, and a gap penalty of 4. In addition, the percentage identity between two amino acid sequences can also be determined using the GAP program (available at www.gcg.com) in the GCG software package, which uses either the Blossom62 matrix or the PAM250 matrix, as well as the algorithm of Needleman and Wunsch [(1970) J. Mol. Biol. 48:444-453], which uses a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0184] In one embodiment, the present invention intends to modify the amino acid sequence of an initial antibody or fragment (e.g., scFv) to generate a functionally equivalent molecule. For example, the anti-CD19 binding domain contained in the CAR, e.g., the VH or VL of scFv, may be modified such that at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the identity of the initial VH or VL framework region of scFv is retained. The present invention intends to modify the entire CAR construct, e.g., modify the amino acid sequence of one or more of the various domains of the CAR construct to generate a functionally equivalent molecule. The CAR construct may be modified so as to retain at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the identity of the initial CAR construct.

[0185] transmembrane domain With respect to the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain attached to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids that associate with the extracellular region of the protein from which transmembrane is induced (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 of the extracellular region) and / or one or more additional amino acids that associate with the intracellular region of the protein from which transmembrane protein is induced (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 of the intracellular region). In one embodiment, the transmembrane domain is a domain that associates with one of the other domains of the CAR used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution so that it does not bind to the transmembrane domain of the same or different surface membrane protein, for example, so that interaction with other members of the receptor complex is minimized. In one embodiment, the transmembrane domain may be homodimerized with another CAR on the surface of the CART cell. In a different embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interaction with the binding domain of a native binding partner present in the same CART.

[0186] The transmembrane domain may be obtained from a natural source or from a recombinant source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one embodiment, the transmembrane domain can signal to the intracellular domain whenever the CAR binds to a target. Transmembrane domains particularly useful in the present invention may include, for example, the transmembrane regions of the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0187] In some cases, the transmembrane domain can attach to the extracellular region of the CAR, for example, the antigen-binding domain of the CAR, via a hinge, for example, a hinge derived from a human protein. For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge, for example, an IgG4 hinge, or a CD8a hinge. In one embodiment, the hinge or spacer comprises the amino acid sequence of SEQ ID NO: 14 (for example, consisting of ). In one embodiment, the transmembrane domain comprises the transmembrane domain of SEQ ID NO: 15 (for example, consisting of ).

[0188] In one embodiment, the hinge or spacer includes an IgG4 hinge. For example, in one embodiment, the hinge or spacer includes a hinge having the amino acid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (Sequence ID 45).In some embodiments, the hinge or spacer includes a hinge encoded by the nucleotide sequence (SEQ ID NO: 46).

[0189] In one embodiment, the hinge or spacer includes an IgD hinge. For example, in one embodiment, the hinge or spacer includes a hinge having the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 47).In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO: 48).

[0190] In one embodiment, the transmembrane domain may be recombinant, in which case it is expected to mainly contain hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

[0191] A short oligolinker or polypeptide linker, between 2 and 10 amino acids in length, may form a linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine pair confers a particularly suitable linker. For example, in one embodiment, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 49). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 50).

[0192] Intracellular domain The cytoplasmic domain or region of a CAR contains an intracellular signaling domain. The intracellular signaling domain is generally involved in the activation of at least one normal effector function of the immune cell into which the CAR has been introduced. The term “effector function” refers to a specialized function of the cell. The effector function of a T cell could be, for example, cytolytic activity or helper activity such as cytokine secretion. Therefore, the term “intracellular signaling domain” refers to the portion of a protein that translates the effector function signal to enable the cell to perform its specialized function. While the entire intracellular signaling domain may be employed, in many cases, the entire chain is not necessarily required. A truncated portion of the intracellular signaling domain can be used in place of the intact chain, as long as it translates the effector function signal to the extent that it is used. Therefore, the term “intracellular signaling domain” means encompassing any truncated portion of an intracellular signaling domain that is sufficient to translate the effector function signal.

[0193] Examples of intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to initiate signal transduction after contact with an antigen receptor, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities.

[0194] It is known that signals generated by TCRs alone are insufficient to fully activate T cells, and that secondary and / or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: a class that initiates antigen-dependent primary activation via TCRs (primary intracellular signaling domains) and a class that acts in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary intracellular domains, e.g., co-stimulatory domains).

[0195] The primary signaling domain modulates the primary activation of the TCR complex either through stimulation or inhibition. Primary intracellular signaling domains acting through stimulation may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as ITAM.

[0196] Examples of primary intracellular signaling domains containing ITAM that are particularly useful in the present invention include the domains of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the CAR of the present invention includes an intracellular signaling domain, for example, the primary signaling domain of CD3-zeta.

[0197] In one embodiment, the primary signaling domain includes a modified ITAM domain, for example, a mutated ITAM domain whose activity is altered (e.g., increased or decreased) compared to a natural ITAM domain. In one embodiment, the primary signaling domain includes a primary intracellular signaling domain containing modified ITAM, for example, a primary intracellular signaling domain containing optimized and / or truncated ITAM. In one embodiment, the primary signaling domain includes one, two, three, four or more ITAM motifs.

[0198] The intracellular signaling domain of the CAR may consist solely of a CD3-zeta signaling domain, or it may be combined with any other desired intracellular signaling domain useful in the context of the CAR of the present invention. For example, the intracellular signaling domain of the CAR may consist of a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand necessary for an efficient lymphocyte response to an antigen. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, as well as similar molecules. For example, CD27 co-stimulation has been shown to enhance the expansion, effector function, and survival of human CART cells in vitro, and to increase the persistence and antitumor activity of human T cells in vivo [Song et al. Blood. 2012; 119(3):696-706].

[0199] The intracellular signaling sequences within the cytoplasmic portion of the CAR of the present invention may be linked to each other in a random or specific order. For example, short oligolinkers or polypeptide linkers with a length between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may form links between the intracellular signaling sequences. In one embodiment, a glycine-serine pair can be used as a suitable linker. In another embodiment, a single amino acid, such as alanine or glycine, can be used as a suitable linker.

[0200] In one embodiment, the intracellular signaling domain is designed to include two or more, for example, 2, 3, 4, 5, or more, co-stimulatory signaling domains. In one embodiment, two or more, for example, 2, 3, 4, 5, or more, co-stimulatory signaling domains are separated by a linker molecule, for example, a linker molecule described herein. In one embodiment, the intracellular signaling domain includes two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0201] In one embodiment, the intracellular signaling domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In one embodiment, the intracellular signaling domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain. In one embodiment, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 16. In one embodiment, the CD3-zeta signaling domain is the signaling domain of SEQ ID NO: 17.

[0202] In one embodiment, the intracellular signaling domain is designed to include a CD3-zeta signaling domain and a CD27 signaling domain. In one embodiment, the CD27 signaling domain includes the amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 51). In one embodiment, the CD27 signaling domain is encoded by the nucleic acid sequence AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 52).

[0203] In one embodiment, the CAR-expressing cells described herein may further include a second CAR, for example, a second CAR containing different antigen-binding domains for the same target (CD19) or different targets (e.g., CD123). In one embodiment, if the CAR-expressing cells include two or more different CARs, the antigen-binding domains of the different CARs may be configured so that they do not interact with each other. For example, cells expressing the first and second CARs may have an antigen-binding domain of the first CAR that does not form association with the antigen-binding domain of the second CAR, for example, as a fragment, e.g., scFv, and the antigen-binding domain of the second CAR is VHH.

[0204] In another embodiment, the CAR-expressing cells described herein may further express another agent, such as an agent that enhances the activity of the CAR-expressing cells. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. In some embodiments, the inhibitory molecule, such as PD1, can reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide, such as the inhibitory molecule, which associates with a second polypeptide, such as an intracellular signaling domain described herein, that provides a positive signal to the cell. In one embodiment, the drug comprises a first polypeptide of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein [e.g., including a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)]. In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein). PD1 is part of the inhibitory CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed in activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75).Two ligands for PD1, PD-L1, and PD-L2 have been shown to downmodulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be reversed by inhibiting the local interaction between PD1 and PD-L1.

[0205] In one embodiment, the drug comprises the extracellular domain (ECD) of an inhibitory molecule, such as Programmed Death 1 (PD1), and can be fused to transmembrane domains and intracellular signaling domains such as 41BB and CD3 zeta (also referred to herein as PD1CAR). In one embodiment, PD1CAR, when used in combination with CD19CAR as described herein, improves T cell persistence. In one embodiment, the CAR is a PD1CAR comprising the extracellular domain of PD1, indicated by the underlined part in SEQ ID NO: 121. In one embodiment, PD1CAR comprises the amino acid sequence of SEQ ID NO: 121.

[0206] Malpvtalllplalllhaarp pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr(Sequence ID 121).

[0207] In one embodiment, PD1CAR includes the following amino acid sequence (SEQ ID NO: 119).

[0208] pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (Sequence ID 119).

[0209] In one embodiment, the drug comprises a nucleic acid sequence encoding PD1CAR, for example, the PD1CAR described herein. In one embodiment, the nucleic acid sequence of PD1CAR is shown below, with PD1 ECD underlined in the following SEQ ID NO: 120.

[0210] atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacca cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc (SEQ ID NO: 120).

[0211] In another embodiment, the present invention provides a population of CAR-expressing cells, for example, CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of different CAR-expressing cells. For example, in one embodiment, the population of CART cells may comprise a first CAR-expressing cell having an anti-CD19 binding domain as described herein, and a second CAR-expressing cell having a different anti-CD19 binding domain, for example, an anti-CD19 binding domain as described herein that is different from the anti-CD19 binding domain in the CAR expressed by the first cell. In another example, the population of CAR-expressing cells may comprise a first CAR-expressing cell containing an anti-CD19 binding domain, for example, as described herein, and a second CAR-expressing cell containing an antigen-binding domain for a target other than CD19 (e.g., CD123). In one embodiment, the population of CAR-expressing cells comprises, for example, a first CAR-expressing cell containing a primary intracellular signaling domain, and a second CAR-expressing cell containing a secondary signaling domain.

[0212] In another embodiment, the present invention provides a population of cells in which at least one type of cell expresses a CAR having an anti-CD19 domain as described herein, and a second cell expresses another agent, such as an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. In some embodiments, the inhibitory molecule can, for example, reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide, such as the inhibitory molecule, which associates with a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain as described herein. In one embodiment, the drug comprises a first polypeptide of an inhibitory molecule such as PD1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein [e.g., including a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein)]. In one embodiment, the drug comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0213] RNA transfection This specification discloses a method for producing RNA CARs transcribed in vitro. The invention also encompasses RNA constructs encoding CARs that can be directly transfected into cells. A method for generating mRNA for use in transfection may involve in vitro transcription (IVT) of a template using specially designed primers to produce a construct (SEQ ID NO: 118) containing a 3' and 5' untranslated sequence ("UTR") typically 50–2000 nucleotides in length, a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail, followed by poly-A addition. The RNA thus produced can be efficiently transfected into cells of different species. In one embodiment, the template comprises a sequence relating to the CAR.

[0214] In one embodiment, anti-CD19CAR is encoded by messenger RNA (mRNA). In one embodiment, the mRNA encoding anti-CD19CAR is introduced into T cells for CART cell production.

[0215] In one embodiment, an RNA CAR transcribed in vitro can be introduced into cells as a form of transient transfection. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Target DNA from any source can be directly converted by PCR into a template for mRNA synthesis using appropriate primers and RNA polymerase in vitro. The DNA source may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source. The desired template for in vitro transcription is the CAR of the present invention. For example, a template for an RNA CAR includes an extracellular region containing a single-chain variable domain of an antitumor antibody; a hinge region, a transmembrane domain (e.g., the CD8a transmembrane domain); and an intracellular region containing an intracellular signaling domain, e.g., an intracellular region containing the CD3-zeta signaling domain and the 4-1BB signaling domain.

[0216] In one embodiment, the DNA intended for use in PCR contains an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid may contain some or all of the 5' and / or 3' untranslated region (UTR). The nucleic acid may contain exons and introns. In one embodiment, the DNA intended for use in PCR is a human nucleic acid sequence. In another embodiment, the DNA intended for use in PCR is a human nucleic acid sequence containing the 5' and 3' UTR. Alternatively, the DNA may be a naturally occurring artificial DNA sequence that is not typically expressed in organisms. A typical artificial DNA sequence is a sequence containing a portion of genes that, together, ligate to form an open reading frame encoding a fusion protein. The portions of DNA ligated together may be from a single organism or from one or more organisms.

[0217] PCR is used to generate templates for in vitro transcription of mRNA used for transfection. The method of performing PCR is well known in the industry. Primers for use in PCR are designed to contain a region substantially complementary to the region of DNA to be used as a template for PCR. "Substantially complementary," as used herein, refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal to or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, a primer can be designed to amplify a portion of nucleic acid (open reading frame) that is normally transcribed in cells and includes the 5' and 3' UTRs. Alternatively, a primer can be designed to amplify a portion of nucleic acid that codes for a specific domain of interest. In one embodiment, a primer is designed to amplify the coding region of human cDNA that includes all or part of the 5' and 3' UTRs. Primers useful for PCR can be produced by synthetic methods well known in the industry. A “forward primer” is a primer containing a region of nucleotides substantially complementary to the nucleotides in the DNA template upstream of the DNA sequence to be amplified. In this specification, “upstream” refers to position 5 of the DNA sequence to be amplified with respect to the coding strand. A “reverse primer” is a primer containing a region of nucleotides substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. In this specification, “downstream” refers to position 3’ of the DNA sequence to be amplified with respect to the coding strand.

[0218] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from numerous suppliers.

[0219] Chemical structures that have the ability to enhance stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between 1 and 3000 nucleotides in length. The lengths of the 5' and 3' UTR sequences to be added to the coding region are not limited to these, but can be modified in various ways, such as by designing primers for PCR that anneal to different regions of the UTR. Using this approach, those skilled in the art can modify the lengths of the 5' and 3' UTRs necessary to achieve optimal translation efficiency after transfection of the transfected RNA.

[0220] The 5' and 3' UTRs may be naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, non-endogenous UTR sequences may be added to the nucleic acid of interest by incorporating the UTR sequences into forward and reverse primers, or by any other modification of the template. The use of non-endogenous UTR sequences for the nucleic acid of interest may be useful in modifying RNA stability and / or translation efficiency. For example, AU-rich elements in the 3' UTR sequence are known to potentially reduce mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of transcribed RNA based on UTR properties well known in the industry.

[0221] In one embodiment, the 5'UTR may contain the Kosack sequence of an endogenous nucleic acid. Alternatively, if a 5'UTR that is not endogenous for the nucleic acid of interest is to be added by PCR as described above, the design of the consensus Kosack sequence may be modified by adding the 5'UTR sequence. While the Kosack sequence can increase the translation efficiency of some RNA transcripts, it is not considered necessary for all RNAs to enable efficient translation. The requirements for the Kosack sequence for many mRNAs are known in the art. In another embodiment, the 5'UTR may be the 5'UTR of an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' or 5'UTR to inhibit the degradation of mRNA by exonucleases.

[0222] To enable RNA synthesis from a DNA template without requiring gene cloning, the transcription promoter is expected to attach to the DNA template upstream of the sequence to be transcribed. When a sequence functioning as an RNA polymerase promoter is added to the 5' end of a forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is the T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0223] In a preferred embodiment, mRNA possesses both a cap at the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. In circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemer-like products that are unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3'UTR end yields mRNA of normal size, which is not effective in eukaryotic transfection even if polyadenylated after transcription.

[0224] In a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript even after passing the last base of the template [(Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)].

[0225] The conventional method for integrating poly(A / T) stretches into DNA templates is molecular cloning. However, poly(A / T) sequences integrated into plasmid DNA can destabilize the plasmid, which explains why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. As a result, cloning methods are not only cumbersome and time-consuming but also unreliable. This is why a method that allows for the construction of DNA templates with poly(A / T) 3' stretches without using cloning is extremely desirable.

[0226] The poly(A) tail of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a poly(T) tail, e.g., a 100T tail (SEQ ID NO: 110) [the size may be 50 to 5000 T (SEQ ID NO: 111)], or after PCR by any other method, but not limited to these, such as DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA, reducing its degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO: 112).

[0227] The poly(A) tail of RNA can be further elongated after transcription in vitro using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 113) results in approximately a twofold increase in RNA translation efficiency. In addition, the attachment of different chemical groups to the 3' end can increase the stability of mRNA. Such attachments may contain modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs may be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase the stability of RNA.

[0228] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method disclosed herein contains the 5' cap. The 5' cap is provided using techniques known in the industry or techniques described herein [Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)].

[0229] RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral sequence, chromosomal sequence, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates translation initiation. It may also contain any solute suitable for cellular electroporation, i.e., solutes that may contain factors that promote cellular permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0230] RNA can be introduced into target cells using a number of different methods, such as commercially available methods, including, but not limited to, electroporation [Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)], [ECM830 (BTX) (Harvard Instruments, Boston, Mass.)] or GenePulser II (BioRad, Denver, Colo.)], multiporators (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or gene gun particle delivery systems, such as "GeneGun" [see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)].

[0231] Nucleic acid constructs encoding CAR The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one embodiment, the nucleic acid molecule is provided as a transcript of messenger RNA. In one embodiment, the nucleic acid molecule is provided as a DNA construct.

[0232] Accordingly, in one embodiment, the present invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 binding domain (e.g., a humanized anti-CD19 binding domain), a transmembrane domain, and an intracellular signaling domain including a stimulatory domain, e.g., a co-stimulatory signaling domain and / or a primary signaling domain, e.g., an intracellular signaling domain including a zeta chain. In one embodiment, the anti-CD19 binding domain is an anti-CD19 binding domain as described herein, for example, an anti-CD19 binding domain comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them. In one embodiment, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain includes the sequence of SEQ ID NO: 15, or a sequence having 95-99% identity thereto. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, for example, a hinge as described herein. In one embodiment, the hinge region includes the sequence of SEQ ID NO: 14, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49, or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further includes a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain includes the sequence of SEQ ID NO: 16, or a sequence having 95-99% identity thereto.In one embodiment, the intracellular signaling domain includes a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain includes the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity with them, and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity with them, wherein the sequences containing the intracellular signaling domain are expressed as a single polypeptide chain in the same frame.

[0233] In another embodiment, the present invention relates to an isolated nucleic acid molecule encoding a CAR construct, comprising: a leader sequence of SEQ ID NO: 13; an scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 (or a sequence having 95-99% identity with them); a hinge region of SEQ ID NO: 14, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49 (or a sequence having 95-99% identity with them); a transmembrane domain having the sequence of SEQ ID NO: 15 (or a sequence having 95-99% identity with them); a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 16 or a CD27 costimulatory domain having the sequence of SEQ ID NO: 51 (or a sequence having 95-99% identity with them); and a CD3 zeta-stimulating domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43 (or a sequence having 95-99% identity with them).

[0234] In another embodiment, the present invention relates to an isolated polypeptide molecule encoded by the nucleic acid molecule described above. In one embodiment, the isolated polypeptide molecule includes a sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42, or a sequence having 95-99% identity with them.

[0235] In another embodiment, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule comprising an anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain including a stimulating domain, wherein the anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a sequence having 95-99% identity with them.

[0236] In one embodiment, the encoded CAR molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 15. In one embodiment, the intracellular signaling domain comprises the functional signaling domain of 4-1BB and the functional signaling domain of zeta. In one embodiment, the intracellular signaling domain includes the sequence of SEQ ID NO: 16 and the sequence of SEQ ID NO: 17, where the sequence containing the intracellular signaling domain is expressed as a single polypeptide chain in the same frame. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region includes SEQ ID NO: 14. In one embodiment, the hinge region includes SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0237] In another embodiment, the present invention relates to an encoded CAR molecule comprising a leader sequence of SEQ ID NO: 13, an scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or a sequence having 95-99% identity with them, a hinge region of SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane domain having the sequence of SEQ ID NO: 15, a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 16 or a CD27 costimulatory domain having the sequence of SEQ ID NO: 51, and a CD3 zeta-stimulating domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the encoded CAR molecule comprises a sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, or a sequence having 95-99% identity with them.

[0238] Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as screening a library from cells expressing the gene, extracting the gene from a vector known to contain it, or directly isolating it from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically rather than cloned.

[0239] The present invention also provides a vector into which the DNA of the present invention is inserted. Retroviral vectors, such as lentiviruses, are suitable as tools for achieving long-term gene transfer because they enable stable integration of the transgene over the long term and its proliferation in daughter cells. Lentiviral vectors have an additional advantage over onchoretrovirus-derived vectors, such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. Lentiviral vectors also have the additional advantage of low immunogenicity.

[0240] In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the present invention is an adenovirus vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as Sleeping Beauty, CRISPR, CAS9, and zinc finger nucleases. See below for June et al. 2009 Nature Reviews Immunology 9.10: 704-716, incorporated herein by reference.

[0241] In short, the expression of native or synthetic nucleic acids encoding CARs is typically achieved by incorporating the construct into an expression vector, by functionally ligating a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter. The vector may be suitable for eukaryotic replication and integration. Typical cloning vectors contain transcriptional and translational terminators, start sequences, and promoters useful for regulating the expression of desired nucleic acid sequences.

[0242] The expression construct of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are publicly known in the industry. See, for example, U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0243] Nucleic acids can be cloned into numerous types of vectors. For example, nucleic acids can be cloned into vectors such as plasmids, phagemids, phage derivatives, animal viruses, and cosmids, though these are not limited to these. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0244] Furthermore, the expression vector may be supplied to the cell in the form of a viral vector. Viral vector technology is well known in the industry and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers in at least one organism (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0245] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes may be inserted into vectors using techniques known in the art and packaged in retroviral particles. Recombinant viruses may then be isolated and delivered to target cells either in vivo or ex vivo. Numerous retroviral systems are known in the art. Adenovirus vectors are used in some embodiments. Numerous adenovirus vectors are known in the art. Lentiviral vectors are used in one embodiment.

[0246] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located in the 30–110 bp region upstream of the initiation site, although numerous promoters have been shown to contain similarly functional elements downstream of the initiation site. The spacing between promoter elements is often flexible to conserve promoter function when elements are reversed or moved relative to one another. In thymidine kinase (TK) promoters, the spacing between promoter elements can be as large as 50 bp, just before activity begins to decline. Individual elements may function either cooperatively or independently, depending on the promoter, to activate transcription.

[0247] An example of a promoter capable of expressing CAR transgenes in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the extension factor-1 complex, which is involved in the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into lentiviral vectors. See, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In one embodiment, the EF1a promoter includes the sequence provided as Sequence ID No. 100.

[0248] Another example of a promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence ligated to function with it. However, other constitutive promoter sequences can also be used, and such constitutive promoter sequences include, but are not limited to, the Simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters, such as, but are not limited to, the actin promoter, myosin promoter, extension factor-1α promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of inductive promoters provides a molecular switch that initiates the expression of a polynucleotide sequence linked to the molecular switch when its expression is desired, or stops its expression when its expression is undesirable. Examples of inductive promoters, but not limited to these, include metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0249] To investigate the expression of CAR polypeptides or parts thereof, the expression vector to be introduced into cells may also contain either or both a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via the viral vector. In other embodiments, the selection marker may be loaded onto another fragment of DNA and used in cotransfection techniques. Both the selection marker and the reporter gene may have appropriate regulatory sequences at their ends to enable expression in host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo and their allogenes.

[0250] Reporter genes can be used to identify cells that may be transfected, and furthermore, to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and furthermore, a gene that encodes a polypeptide whose expression is demonstrated by several readily detectable characteristics, such as enzymatic activity. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into recipient cells. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or may be commercially available. Generally, a construct having the smallest 5' flanking region that exhibits the maximum level of expression of the reporter gene is identified as the promoter. Such promoter regions can be ligated to reporter genes and used to evaluate drugs in terms of their ability to regulate promoter-driven transcription.

[0251] Methods for introducing and expressing genes in cells are well known in the art. In the case of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0252] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, electroporation, and analogues. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the industry. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0253] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are becoming the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and their counterparts. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0254] Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system for use as an in vitro and in vivo delivery medium is liposomes (e.g., artificial membrane vesicles). Other state-of-the-art methods for targeted nucleic acid delivery are available, such as polynucleotide delivery using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0255] In cases where non-viral delivery systems are used, the typical delivery medium is liposomes. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids may be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that associate with both liposomes and oligonucleotides, confined within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained with or complexed with micelles, or otherwise associated with lipids. Compositions of associated lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, the above compositions may exist in a bilayer structure, as micelles, or in a "disintegrated" structure. Furthermore, the above compositions may simply be dispersed in a solution, or they may form aggregates that are not uniform in size or shape. Lipids are either naturally occurring fatty substances or synthetic lipids. For example, lipids include small droplets of fat that occur naturally in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0256] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid vesicles (lipid vehicles) formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized by having a vesicular structure comprising a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. These spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reorganization before a closed structure is formed, trapping water and dissolving solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from the usual vesicular structure in solution are also included. For example, lipids may be presumed to be in a micelle structure, or they may simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0257] Regardless of the method used to introduce exogenous nucleic acid into a host cell or, alternatively, the method of exposing cells to an inhibitor of the present invention, various assays can be performed to confirm the presence of a recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detection of the presence or absence of a specific peptide by immunological means (ELISA and Western blot), or by the assays described herein for identifying agents within the scope of the present invention.

[0258] The present invention further provides a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into cells, such as T cells. In one aspect, the vector is a cloning or expression vector, such as, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs, and the like. In one aspect, the vector can express the CAR construct in mammalian T cells. In one aspect, the mammalian T cells are human T cells.

[0259] T cell source T cell sources are obtained from subjects before expansion and genetic modification. The term “subject” is intended to encompass organisms (e.g., mammals) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural exudate, splenic tissue, and tumors. In certain embodiments of the present invention, numerous T cell lines available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from units of blood collected from a subject using numerous techniques known to those skilled in the art, such as Ficoll® separation. In a preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically include lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction, and the cells may be placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution may be calcium-free, magnesium-free, or contain not all, but many of the divalent cations. The initial activation step in the absence of calcium can increase activation. As is expected to be readily apparent to those skilled in the art, the washing step may be achieved by methods known to those skilled in the art, for example, by using a semi-automatic "run-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, e.g., Ca-free, Mg-free PBS, PlasmaLyte A, etc., or other buffer-containing or buffer-free salines. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be resuspended directly in the culture medium.

[0260] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example, by centrifugation via a PERCOLL® gradient or by countercurrent centrifugation. Specific T cell subpopulations, e.g., CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3×28) conjugated beads, e.g., DYNABEADS® M-450 CD3 / CD28T, for a period sufficient for positive selection of the desired T cells. In one embodiment, the period is approximately 30 minutes. In a further embodiment, the period is in the range of 30 minutes to 36 hours or longer, and any integer value in between. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the duration is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where T cells are few in number compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunodeficient individuals. Furthermore, the use of longer incubation times can increase the capture efficiency of CD8+ T cells. Thus, by simply shortening or lengthening the time, T cells can be bound to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), a subpopulation of T cells can be selectively selected at the start of culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies to the beads or other surfaces, a subpopulation of T cells can be selectively selected at the start of culture or at other desired time points. Those skilled in the art will expect to recognize that multiple selections can also be used in the context of the present invention.In a given embodiment, it may be desirable to use "arbitrarily extracted" cells by performing a selection technique during the activation and expansion process. These "arbitrarily extracted" cells may also be processed in an additional selection round.

[0261] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed at surface markers unique to negatively selected cells. One method is selection via cell sorting and / or negative magnetic immunoadherence, or flow cytometry using a cocktail of monoclonal antibodies directed at cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In a given embodiment, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in a given embodiment, regulatory T cells may be depleted by anti-C25 conjugated beads or other similar selection methods.

[0262] In one embodiment, a population of T cells expressing one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, such as other cytokines, may be selected. A method for screening cell expression can be determined, for example, by the method described in PCT Publication WO2013 / 126712.

[0263] To isolate a desired population of cells by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) may vary. In a given embodiment, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) in order to maximize the contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 1 million cells per ml is used. In a further embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. In a further embodiment, cell concentrations of 75 million or more, 80 million or more, 85 million or more, 90 million or more, 95 million or more, or 100 million cells per ml are used. In a further embodiment, concentrations of 125 million or 150 million cells per ml can be used. The use of higher concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of higher cell concentrations allows for more efficient capture of cells that may have weak expression of the target antigen of interest, such as CD28-negative T cells, or cells from samples containing many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which normally have relatively weak CD28 expression.

[0264] In the relevant embodiments, it may be desirable to use lower cell concentrations. By considerably diluting the mixture of T cells and surface (e.g., particles such as beads), the interaction between particles and cells is minimized. This selects cells that express a large amount of the desired antigen for binding to the particles. For example, CD4+ T cells express higher levels of CD28 but are captured more efficiently than CD8+ T cells at lower concentrations. In one embodiment, the cell concentration used is 5 × 10⁶ e⁶ / ml. In other embodiments, the concentration used is approximately 1 × 10⁶ e⁶5 From / ml to 1 x 10 6 Up to / ml, or any integer value in between.

[0265] In another embodiment, cells can be incubated in a rotating vessel for varying lengths of time, at varying speeds, at either 2–10°C or room temperature.

[0266] T cells for stimulation may be frozen after the washing step. While not bound by theory, the freezing and subsequent thawing steps result in a more homogeneous product by removing granulocytes and some monocytes from the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in the freezing solution. Many freezing solutions and parameters are known in the industry and are expected to be useful in this situation, but one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or a culture medium containing 31.25% Plasmalyte-A, 31.25% 5% dextrose, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, hespan and PlasmaLyte A, then freezing the cells to -80°C at a rate of 1 degree / min and storing them in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used, and uncontrolled freezing may also be performed immediately at -20°C or in liquid nitrogen.

[0267] In a predetermined embodiment, the cryogenically preserved cells are thawed, washed as described herein, and left at room temperature for 1 hour before being activated using the method of the present invention.

[0268] Furthermore, in the context of the present invention, it is also considered to collect blood samples or apheresis products from a subject in a period prior to when the enlarged cells as described herein may be needed. As such, the source of the cells to be enlarged can be collected at any time as needed, and further, desired cells, such as T cells, may be isolated and frozen for later use in T cell therapy for a number of diseases or conditions that are expected to benefit from T cell therapy, such as those described herein. In one embodiment, the blood sample or apheresis is taken from a generally healthy subject. In a given embodiment, the blood sample or apheresis is taken from a generally healthy subject that is at risk of developing the disease but has not yet developed the disease, and further, the cells of the subject may be isolated and frozen for later use. In a given embodiment, T cells may be enlarged, frozen, and used at a later time. In a given embodiment, the sample is collected from the patient immediately after the diagnosis of a particular disease as described herein, but before any treatment. In a further embodiment, but not limited to, cells are isolated from a blood sample or apheresis from a subject before treatment with a number of related procedures, such as drugs including natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228, and radiation.

[0269] In a further embodiment of the present invention, T cells are obtained directly from a patient after a procedure that leaves functional T cells in the subject. In this regard, it has been observed that the quality of T cells obtained immediately after a procedure, specifically after a procedure with a drug that damages the immune system, during the period when the patient is expected to recover from the procedure, may be optimal or improved in terms of their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, in the scope of the present invention, it is considered to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic cell lineage, during this recovery period. Furthermore, in a given embodiment, mobilization (e.g., mobilization with GM-CSF) and a conditioning regimen can be used to create conditions in a subject that are favorable for regrowth, recirculation, regeneration, and / or expansion of specific cell types, particularly during a specified time period after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0270] T cell activation and expansion T cells may generally be activated and expanded using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0271] Generally, the T cells of the present invention can be expanded by contacting a surface to which a drug that stimulates CD3 / TCR complex-related signaling is attached, along with a ligand that stimulates a co-stimulatory molecule on the surface of the T cell. Specifically, as described herein, the T cell population may be stimulated, for example, by contact with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For co-stimulation of accessory molecules on the T cell surface, ligands that bind to the accessory molecules are used. For example, the T cell population may be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Anti-CD3 antibodies and anti-CD28 antibodies are used to stimulate the proliferation of either CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can be used in the same manner as other methods generally known in the art [Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999].

[0272] In a given embodiment, primary stimulatory and co-stimulatory signals to T cells can be supplied by a variety of protocols. For example, the drugs supplying each signal may be lysed or surface-coupled. If surface-coupled, the drugs may be coupled to the same surface (i.e., "cis" formation) or to a different surface (i.e., "trans" formation). Alternatively, one drug may be surface-coupled and the other drug may be lysed. In one embodiment, the drug supplying the co-stimulatory signal may be bound to the cell surface, and the drug supplying the primary activation signal may be lysed or surface-coupled. In a given embodiment, both drugs may be lysed. In one embodiment, the drugs may be in a soluble form and then crosslinked to the surface of cells expressing Fc receptors, or to antibodies or other binders expected to bind to the drug. In this regard, see, for example, U.S. Patent Applications Publications 20040101519 and 20060034810 relating to artificial antigen-presenting cells (aAPCs) intended for use in the present invention to activate and expand T cells.

[0273] In one embodiment, two drugs are immobilized on beads either on the same bead, i.e., "cis," or on different beads, i.e., "trans." For example, the drug supplying the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, and the drug supplying the costimulatory signal is an anti-CD28 antibody or its antigen-binding fragment, and both drugs are immobilized together on the same bead in equal molecular amounts. In one embodiment, each antibody bound to the beads in a 1:1 ratio is used for CD4+ T cell expansion and T cell growth. In a certain embodiment of the present invention, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In a particular embodiment, an increase of about 1 to about 3 times is observed compared to the expansion observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to the beads is in the range of 100:1 to 1:100 and all integer values ​​in between. In one embodiment of the present invention, more anti-CD28 antibody than anti-CD3 antibody is bound to the particle, i.e., the CD3:CD28 ratio is less than 1. In a certain embodiment of the present invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In a particular embodiment, antibodies bound to beads with a CD3:CD28 ratio of 1:100 are used. In one embodiment, antibodies bound to beads with a CD3:CD28 ratio of 1:75 are used. In a further embodiment, antibodies bound to beads with a CD3:CD28 ratio of 1:50 are used. In one embodiment, antibodies bound to beads with a CD3:CD28 ratio of 1:30 are used. In a preferred embodiment, antibodies bound to beads with a CD3:CD28 ratio of 1:10 are used. In one embodiment, antibodies bound to beads with a CD3:CD28 ratio of 1:3 are used. In a further embodiment, antibodies bound to beads with a CD3:CD28 ratio of 3:1 are used.

[0274] To stimulate T cells or other target cells, particle-to-cell ratios ranging from 1:500 to 500:1, and any integer values ​​in between, can be used. As will be readily apparent to those skilled in the art, the particle-to-cell ratio may depend on the particle size relative to the target cells. For example, small beads can bind to only a few cells, while larger beads can bind to many. In a given embodiment, the cell-to-particle ratio is in the range of 1:100 to 100:1, and any integer value in between, and in a further embodiment, this ratio includes 1:9 to 9:1, and any integer value in between, and can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupled particles to T cells that results in T cell stimulation may vary as described above, but some preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, where one preferred ratio is at least 1:1 particles to T cells. In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In a particular embodiment, a preferred particle:cell ratio is 1:5. In a further embodiment, the particle-to-cell ratio can be changed depending on the day of stimulation. For example, in one embodiment, the particle-to-cell ratio is 1:1 to 10:1 on day 1, and then additional particles are added to the cells daily or every other day for up to 10 days, ultimately resulting in a ratio of 1:1 to 1:10 (based on the number of cells added on the day). In a specific embodiment, the particle-to-cell ratio is 1:1 on day 1 of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In one embodiment, particles are added daily or every other day to reach the final 1:1 ratio on day 1 and 1:5 on days 3 and 5 of stimulation. In one embodiment, the particle-to-cell ratio is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In one embodiment, particles are added daily or every other day to reach the final 1:1 ratio on day 1 and 1:10 on days 3 and 5 of stimulation.Those skilled in the art will expect to recognize that various other ratios may be suitable for use in the present invention. Specifically, the ratios are expected to vary depending on particle size and cell size and type. In one embodiment, the most typical ratios for use are around 1:1, 2:1, and 3:1 on day 1.

[0275] In a further embodiment of the present invention, cells such as T cells are combined with drug-coated beads, the beads and cells are then separated, and the cells are subsequently cultured. In an alternative embodiment, the drug-coated beads and cells are cultured together without separation before culturing. In a further embodiment, the beads and cells are first enriched by the application of a force such as magnetism, thereby inducing cell stimulation by increasing the ligation of cell surface markers.

[0276] For example, by bringing paramagnetic beads (3 × 28 beads) with anti-CD3 and anti-CD28 attached to T cells into contact with them, cell surface proteins can be ligated. In one embodiment, cells (e.g., 10 4 from 10 9A number of T cells and beads [e.g., DYNABEADS® M-450CD3 / CD28T paramagnetic beads in a 1:1 ratio] are combined in a buffer, e.g., PBS (which does not contain divalent cations such as calcium and magnesium). Here again, it will be readily apparent to those skilled in the art that any cell concentration can be used. For example, the target cells may be extremely rare in the sample, may constitute only 0.01% of the sample, or the entire sample (i.e., 100%) may consist of the target cells of interest. Thus, any number of cells is within the scope of this invention. In a given embodiment, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) so that the cells and particles are in maximum contact. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In one embodiment, more than 100 million cells per ml is used. In a further embodiment, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. In a further embodiment, concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per ml are used. In a further embodiment, concentrations of 125 million or 150 million cells / ml are available. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may have weak expression of the target antigen of interest, such as CD28-negative T cells. In a given embodiment, such cell populations may have therapeutic value and are expected to be desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which normally have relatively weak CD28 expression.

[0277] In one embodiment of the present invention, the mixture may be cultured for several hours (about 3 hours) to about 14 days, or any integer value in one-hour increments during that period. In one embodiment, the mixture may be cultured for 21 days. In one embodiment of the present invention, the beads and T cells are cultured together for about 8 days. In one embodiment, the beads and T cells are cultured together for 2-3 days. It may also be desirable to stimulate for several cycles so that the T cell culture time is 60 days or longer. Suitable conditions for T cell culture include suitable media [e.g., minimal essential medium or RPMI medium 1640 or X-vivo 15, (Lonza)] which may contain factors necessary for proliferation and viability such as serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Examples of culture media include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate, and vitamins, and are either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokines for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells intended for injection into the target. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air and 5% CO2).

[0278] T cells exposed to various stimulation times may exhibit different characteristics. For example, peripheral blood mononuclear cell products obtained by typical blood or apheresis have a helper T cell population (TH, CD4+) larger than the cytotoxic or suppressor T cell population (TC, CD8+). T cell expansion by stimulating CD3 and CD28 receptors ex vivo produces a T cell population consisting mainly of TH cells before about days 8 - 9, but after about days 8 - 9, the T cell population contains an even greater number of TC cell populations. Thus, depending on the treatment objective, it may be advantageous to inject a T cell population consisting mainly of TH cells. Similarly, if an antigen - specific subset of TC cells is isolated, such a subset may be beneficial for more highly expanding this subset.

[0279] Furthermore, in addition to the CD4 and CD8 markers, during the course of the cell expansion process, other phenotypic markers change significantly, but mostly reproducibly. Thus, such reproducibility enables the ability to tailor activated T cell products to specific purposes.

[0280] Once the CD19CAR is constructed, various assays can be used to evaluate the activity of molecules such as the ability to expand T cells after antigen stimulation and maintain T cell expansion in the absence of restimulation, and anti - cancer activity in appropriate in vitro and animal models. Assays for evaluating the action of CD19CAR are described in further detail below.

[0281] Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, for example, Milone et al., Molecular Therapy 17(8): 1453 - 1464 (2009). To put it very simply, T cells expressing CAR (CD4 + and CD8 +A 1:1 mixture of T cells) is expanded in vitro for more than 10 days and subsequently lysed for SDS-PAGE under reducing conditions. The CAR containing the full-length TCR-ζ cytoplasmic domain and the endogenous TCR-ζ chain is detected by Western blotting using an antibody against the TCR-ζ chain. The same T cell subset is used for SDS-PAGE analysis under non-reducing conditions that allow evaluation of covalent dimer formation.

[0282] CAR+ T cell expansion after antigen stimulation in vitro can be measured by flow cytometry. For example, CD4 + and CD8 + A mixture of T cells is stimulated with αCD3 / αCD28 aAPCs and subsequently transduced with a lentiviral vector expressing GFP under the control of the promoter to be analyzed. Typical promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerate kinase (PGK) promoter. On day 6 from culture, CD4 + and / or CD8 + GFP fluorescence in the T cell subset is evaluated by flow cytometry. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, on day 0, a mixture of CD4 + and CD8 + T cells is stimulated with magnetic beads coated with αCD3 / αCD28 and on day 1 is transduced with a bicistronic lentiviral vector expressing CAR together with eGFP using a 2A ribosomal skipping sequence. After washing, culture is restimulated with either CD19 + K562 cells expressing hCD32 and 4-1BBL (K562-CD19), wild-type K562 cells (K562 wild-type) or K562 cells in the presence of anti-CD3 and anti-CD28 antibodies (K562-BBL-3 / 28). Exogenous IL-2 is added to the culture at 100 IU / ml every other day. GFP +T cells are counted by flow cytometry using bead-based counting. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

[0283] Furthermore, sustained CAR+ T cell expansion in the absence of restimulation can also be measured. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, cells are stimulated with αCD3 / αCD28-coated magnetic beads on day 0, transfected with presented CARs on day 1, and then the average T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter.

[0284] Animal models can also be used to measure CART activity. For example, a xenograft model using human CD19-specific CAR+ T cells can be used to treat primary human pre-B ALL in immunodeficient mice. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). In very simple terms, after ALL is established, mice are randomized as a treatment group. NOD-SCID-γ with B ALL - / - Mice are injected with a large number of T cells processed with different αCD19-ζ and αCD19-BB-ζ vectors in a 1:1 ratio. The copy numbers of αCD19-ζ and αCD19-BB-ζ vectors in the spleen DNA of mice are evaluated at various time points after T cell injection. Animals are examined for leukemia every week. Peripheral blood CD19 in mice injected with αCD19-ζCAR+ T cells or mock transduced T cells + The number of B-ALL blast cells will be measured. Survival curves for each group will be compared using the log-rank test. In addition, NOD-SCID-γ will be measured 4 weeks after T cell injection. - / - Peripheral blood CD4 in mice + and CD8 +The absolute number of T cells will also be analyzed. Leukemia cells will be injected into mice, and three weeks later, T cells modified to express CAR using a bicistronic lentiviral vector encoding CAR linked to eGFP will be injected. By mixing the T cells with mock transduced cells before injection, 45-50% of the T cells will be enriched with eGFP. + The data is normalized to T cells and confirmed by flow cytometry. Animals are examined for leukemia every week. Survival curves for each CAR+ T cell group are compared using the log-rank test.

[0285] Dose-dependent CAR treatment responses can be evaluated. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after leukemia is established in mice injected with CART cells, an equal number of mock transduction T cells, or no T cells on day 21. Peripheral blood CD19 + Blood samples were randomly drawn from mice in each group to determine the number of ALL blast cells, and then killed on days 35 and 49. The remaining animals were evaluated on days 57 and 70.

[0286] The study of cell proliferation and cytokine production has already been described, for example, in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, the study of CAR-mediated proliferation is performed by mixing washed T cells with K562 cells expressing CD19 (K19) or CD32 and CD137 (KT32-BBL) in a microtiter plate at a final T cell:K562 ratio of 2:1. Before use, the K562 cells are irradiated with gamma radiation. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to the culture containing KT32-BBL cells to serve as positive controls for stimulating T cell proliferation, as these signals are ex vivo over a long period of time. +This is to support T cell expansion. During culture, T cells are counted using CountBright® fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR+ T cells are identified by GFP expression using T cells processed with a CAR expression lentiviral vector ligated with eGFP-2A. For CAR+ T cells that do not express GFP, CAR+ T cells are detected using biotinylated recombinant CD19 protein and a secondary avidin-PE conjugate. Also, CD4+ and CD8 in T cells are detected. + Expression is also detected simultaneously using a specific monoclonal antibody (BD Biosciences). A human TH1 / TH2 cytokine cytometry bead array kit (BD Biosciences, San Diego, CA) is used according to the manufacturer's instructions, and cytokine measurements are performed on the supernatant collected 24 hours after restimulation. Fluorescence is examined using a FACScalibur flow cytometer, and the data is analyzed according to the manufacturer's instructions.

[0287] Cytotoxicity can be examined by a standard 51Cr-release assay. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (K562 strain and primary pro-B-ALL cells) are packed with 51Cr (as NaCrO4, New England Nuclear, Boston, MA) with frequent stirring at 37°C for 2 hours, washed twice with complete RPMI, and cultured in microtiter plates. Effector T cells and target cells in the wells are mixed in complete RPMI in various effector cell:target cell (E:T) ratios. Additional wells containing only medium (spontaneous release, SR) or a 1% solution of Triton-X100 washing agent (total release, TR) are also prepared. After incubation at 37°C for 4 hours, the supernatant is collected from each well. Next, the emitted 51Cr is measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition is performed for at least three consecutive times, and the percentage of dissolved material is calculated using the formula: % dissolved = (ER - SR) / (TR - SR), where ER represents the average 51Cr emitted under each experimental condition.

[0288] Imaging techniques can be used to evaluate the specific trafficking and proliferation of CARs in animal models with tumors. Such assays are described, for example, in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). In short, NOD / SCID / γc - / -(NSG) mice are injected IV with Nalm-6 cells, and then 7 days later, 4 hours after electroporation with a CAR construct, T cells are injected. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and the mice are imaged for bioluminescence. Alternatively, the therapeutic efficacy and specificity of a single CAR+T cell injection in a Nalm-6 xenograft model can be measured as follows: Transduced Nalm-6 to stably express firefly luciferase is injected into NSG mice, followed 7 days later by a single tail vein injection of T cells electroporated with CD19CAR. The animals are imaged at various time points after injection, for example, on day 5 (2 days before treatment) and day 8 (CAR + A heat map of photon density in representative mice with firefly luciferase-positive leukemia can be created 24 hours after PBL (Problem-Based Lighting).

[0289] Furthermore, other assays, such as those described in the Examples section of this specification, as well as assays known in the industry, can also be used to evaluate the CD19CAR construct of the present invention.

[0290] Therapeutic applications CD19-related illnesses and / or disorders In one embodiment, the present invention provides a method for treating a disease associated with CD19 expression. In one embodiment, the present invention provides a method for treating a disease in which a portion of the tumor is CD19-negative and a portion of the tumor is CD19-positive. For example, the CAR of the present invention is useful for treating a subject treated for a disease associated with elevated CD19 expression, where the subject treated for elevated CD19 levels exhibits a disease associated with elevated CD19 levels.

[0291] In one embodiment, the present invention relates to a vector comprising a CD19CAR ligated to function as a promoter for expression in mammalian T cells. In one embodiment, the present invention provides recombinant T cells expressing a CD19CAR for use in the treatment of tumors expressing CD19, wherein the recombinant T cells expressing a CD19CAR are named CD19 CART. In one embodiment, the CD19 CART of the present invention allows tumor cells to be brought into contact with at least one CD19CAR of the present invention expressed on their surface, such that the CART targets tumor cells and inhibits tumor growth.

[0292] In one embodiment, the present invention relates to a method for inhibiting the growth of CD19-expressing tumor cells, comprising contacting the tumor cells with the CD19CART cells of the present invention such that CART is activated in response to an antigen and targets cancer cells, thereby inhibiting tumor growth.

[0293] In one embodiment, the present invention relates to a method for treating cancer in a subject. The method comprises administering the CD19CART cells of the present invention to a subject so that cancer is treated in the subject. Examples of cancers treatable by the CD19CART cells of the present invention are cancers associated with CD19 expression. In one embodiment, cancers associated with CD19 expression are hematolic cancers. In one embodiment, hematolic cancers are leukemias or lymphomas. In one embodiment, cancers associated with CD19 expression include, but are not limited to, cancers and malignant tumors, which include, but are not limited to, one or more acute leukemias, including, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); and, but are not limited to, one or more chronic leukemias, including, for example, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and the like. Additional cancers or hematological conditions associated with CD19 expression include, but are not limited to, B-cell prelymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative states, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and a group of hematological conditions known as “preleukemic states,” which are common to the unresponsive production (or dysplasia) of blood cells in the bone marrow, as well as similar conditions. Further diseases associated with CD19 expression include, but are not limited to, atypical and / or non-typical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD19 expression.

[0294] In some embodiments, the cancer that may be treated with CD19CAR, for example the CD19CAR described herein, is multiple myeloma. Multiple myeloma is a blood cancer characterized by the accumulation of plasma cell clones in the bone marrow. Current therapies for multiple myeloma include, but are not limited to, treatment with lenalidomide, an analogue of thalidomide. Lenalidomide has activities such as antitumor activity, angiogenesis inhibition, and immunomodulation. Generally, myeloma cells are considered negative for CD19 expression by flow cytometry. The present invention encompasses the recognition that only a few percent of myeloma tumor cells express CD19, as demonstrated in Example 6. Therefore, in some embodiments, CD19CAR, for example, as described herein, can be used to target myeloma cells. In some embodiments, CD19CAR therapy can be used in combination with one or more additional therapies, such as lenalidomide treatment.

[0295] This invention encompasses a type of cell therapy in which T cells are genetically modified to express chimeric antigen receptors (CARs), and these CART cells are injected into recipients who require them. The injected cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR-modified T cells are capable of replication in vivo, resulting in long-term persistence that leads to sustained tumor control. In various embodiments, the T cells administered to the patient, or their offspring, remain in the patient for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 months, 2, 3, 4, or 5 years after the T cells have been administered.

[0296] The present invention also encompasses a type of cell therapy in which T cells are modified with RNA transcribed in vitro to transiently express, for example, a chimeric antigen receptor (CAR), and the CART cells are injected into recipients who need them. The injected cells can kill tumor cells in the recipient. Thus, in various embodiments, the T cells administered to the patient are present for less than one month, e.g., three weeks, two weeks, or one week, after the T cells have been administered to the patient.

[0297] While we do not wish to be bound by any particular theory, antitumor immune responses induced by CAR-modified T cells may be active or passive immune responses, or alternatively, may be due to direct versus indirect immune responses. In one embodiment, CAR-transduced T cells exhibit specific pro-inflammatory cytokine secretion, and their strong cytolytic activity in response to CD19-expressing human cancer cells is resistant to lytic CD19 inhibition, mediates bystander death, and mediates regression of established human tumors. For example, tumor cells with fewer antigens in a heterogeneous tumor field expressing CD19 are more susceptible to indirect destruction by CD19-redirected T cells that previously responded to adjacent antigen-positive cancer cells.

[0298] In one embodiment, the T cells modified with the fully human CAR of the present invention may be a type of vaccine for ex vivo immunization and / or in vivo therapy in mammals. In one embodiment, the mammal is a human.

[0299] Regarding ex vivo immunization, before administering cells to mammals, at least one of the following is performed in vitro: i) cell enlargement, ii) introduction of CAR-encoding nucleic acids into the cells, or iii) cryopreservation of the cells.

[0300] Ex vivo methods are well-known in the industry and will be discussed in more detail below. Simply put, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transfected or transfected in vitro) with a vector expressing the CAR disclosed herein. Therapeutic benefits can be obtained by administering the CAR-modified cells to a mammalian recipient. The mammalian recipient may be human, and the CAR-modified cells may be self to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or heterogeneic to the recipient.

[0301] A method for expanding hematopoietic stem and progenitor cells ex vivo is described in U.S. Patent No. 5,199,942, incorporated herein by reference, and can be applied to the cells of the present invention. Other preferred methods are known in the art, and therefore the present invention is not limited to any particular method of cell expansion ex vivo. Briefly, the culture and expansion of T cells ex vivo comprises (1) collecting mammalian-derived CD34+ hematopoietic stem and progenitor cells from peripheral blood recapture or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligands can be used for cell culture and expansion.

[0302] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to induce an immune response directed against an antigen in a patient.

[0303] Generally, cells activated and enlarged as described herein can be used for the treatment and prevention of diseases occurring in immunocompromised individuals. Specifically, CAR-modified T cells of the present invention are used for the treatment of diseases, disorders, and conditions associated with CD19 expression. In a given embodiment, the cells of the present invention can be used for the treatment of patients at risk of developing diseases, disorders, and conditions associated with CD19 expression. Accordingly, the present invention provides a method for treating or preventing diseases, disorders, and conditions associated with CD19 expression, comprising administering a therapeutically effective amount of CAR-modified T cells of the present invention to a subject in need.

[0304] In one embodiment, the CART cells of the present invention can be used to treat proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as spinal cord malformations, myelodysplastic syndromes, or preleukemic conditions. In one embodiment, cancer is a hematological cancer. In one embodiment, hematological cancer is leukemia or lymphoma. In one embodiment, the CART cells of the present invention can be used to treat cancer and malignant tumors, and are not limited to, but include, acute leukemias, such as B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); one or more chronic leukemias, such as, but not limited to, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL); and, but not limited to, B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, Additional hematological malignancies or hematological conditions, as well as allogenes, include diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative states, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and “preleukemic states,” a group of hematological conditions common to the undetectable production (or dysplasia) of blood cells in the bone marrow. Further CD19 expression-related diseases, though not limited to these, include, for example, atypical and / or non-typical CD19-expressing cancers, malignancies, precancerous states, or proliferative disorders. Non-cancer-related signs associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory diseases (allergies and asthma), and transplantation.

[0305] The CAR-modified T cells of the present invention may be administered alone or as part of a pharmaceutical composition in combination with a diluent and / or other components or cell populations such as IL-2 or other cytokines.

[0306] Blood cancer Blood cancers include types of cancer such as leukemia, as well as malignant lymphoproliferative conditions that affect the blood, bone marrow, and lymphatic system.

[0307] Leukemia can be classified into acute leukemia and chronic leukemia. Acute leukemia can be further classified into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemic states"), a group of hematological conditions common to impaired production (or dysplasia) of blood cells in the bone marrow, and the risk of conversion to AML.

[0308] The present invention provides compositions and methods for treating cancer. In one embodiment, cancer is a hematological cancer, and hematological cancers are, but are not limited to, leukemia or lymphoma. In one embodiment, the CART cells of the present invention can be used to treat cancer and malignant tumors, and are not limited to, acute leukemias, including, but are not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), and acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including, but are not limited to, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL); and, but are not limited to, B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, Additional hematological malignancies or hematological conditions, as well as allogenes, include diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative states, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and “preleukemic states,” a group of hematological conditions common to the undetectable production (or dysplasia) of blood cells in the bone marrow. Further CD19 expression-related diseases, though not limited to these, include, for example, atypical and / or non-typical CD19-expressing cancers, malignancies, precancerous states, or proliferative disorders.

[0309] The present invention also provides a method for inhibiting or reducing the proliferation of a population of CD19-expressing cells, the method comprising contacting a population of cells containing CD19-expressing cells with anti-CD19CART cells that bind to the CD19-expressing cells of the present invention. In a specific embodiment, the present invention provides a method for inhibiting or reducing the proliferation of a population of cancer cells expressing CD19, the method comprising contacting a population of cancer cells expressing CD19 with anti-CD19CART cells that bind to the CD19-expressing cells of the present invention. In one embodiment, the present invention provides a method for inhibiting or reducing the proliferation of a population of cancer cells expressing CD19, the method comprising contacting a population of cancer cells expressing CD19 with anti-CD19CART cells that bind to the CD19-expressing cells of the present invention. In a given embodiment, the anti-CD19CART cells of the present invention reduce the amount, number, quantity, or percentage of cells and / or cancer cells in subjects having myeloid leukemia or another cancer associated with CD19-expressing cells, or in animal models thereof, by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% compared to a negative control. In one embodiment, the subject is human.

[0310] The present invention also provides methods for preventing, treating and / or managing diseases associated with CD19-expressing cells (e.g., hematological cancers or cancers expressing atypical CD19), the methods comprising administering anti-CD19CART cells bound to the CD19-expressing cells of the present invention to subjects in need. In one embodiment, the subjects are human. Non-limiting examples of disorders associated with CD19-expressing cells include autoimmune disorders (e.g., lupus), inflammatory diseases (e.g., allergies and asthma), and cancers (e.g., hematological cancers or cancers expressing atypical CD19).

[0311] The present invention also provides a method for preventing, treating and / or managing diseases associated with CD19-expressing cells, the method comprising administering anti-CD19CART cells bound to the CD19-expressing cells of the present invention to a subject in need thereof. In one embodiment, the subject is human.

[0312] The present invention provides a method for preventing the recurrence of cancer associated with CD19-expressing cells, the method comprising administering anti-CD19CART cells bound to the CD19-expressing cells of the present invention to a subject in need. In one embodiment, the method comprises administering an effective dose of anti-CD19CART cells bound to the CD19-expressing cells described herein, in combination with an effective dose of another therapy, to a subject in need.

[0313] Combination therapy CAR-expressing cells described herein may be used in combination with other known agents and therapies. “Combined” means, as used herein, that two (or more) different treatments are delivered to a subject while the subject is suffering from a disorder, for example, that two or more treatments are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before the treatment is discontinued for any other reason. In some embodiments, the delivery of one treatment is still ongoing when the delivery of a second treatment begins, with some overlap in administration. This may also be referred to herein as “simultaneous” or “parallel delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of each case, the treatments are more effective by combining the administrations. For example, the second treatment is more effective if it reduces symptoms to the same extent as if the second treatment had not been administered, or to the extent that would be expected if the second treatment had been administered in the absence of the first treatment, or to the extent that a similar situation would be observed using the first treatment. In some embodiments, the delivery is such that the reduction in other parameters relating to symptoms or impairment is greater than the reduction in parameters expected to be observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or more additive. The delivery may be such that the effect of the first treatment delivered is still detectable when the second is delivered.

[0314] The CAR-expressing cells and at least one additional therapeutic agent described herein may be administered simultaneously in the same or different compositions, or sequentially. In the case of sequential administration, the CAR-expressing cells described herein may be administered first, followed by the additional agent, or the order of administration may be reversed.

[0315] In a further embodiment, CAR-expressing cells as described herein can be used in treatment regimens in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunosuppressants such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228, cytokines, as well as irradiation, peptide vaccines, for example, as described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0316] In one embodiment, the CAR-expressing cells described herein can be used in combination with chemotherapeutic agents. Typical chemotherapeutic agents include anthracyclines [e.g., doxorubicin (e.g., liposomal doxorubicin)], vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), and antimetabolites (e.g., folate antagonists). Examples include agonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., acrasinomycin A, gliotoxin, or bortezomib), and immunomodulators, such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0317] Common chemotherapy agents considered for use in combination therapy include anastrozole [Arimidex®], bicalutamide [Casodex®], bleomycin sulfate [Blenoxane®], busulfan [Myleran®], busulfan injection [Busulfex®], capecitabine [Xeloda®], N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin [Paraplatin®], and carmustine [BiCNU®]. ), Chlorambucil [Leukeran®], Cisplatin [Platinol®], Cladribine [Leustatin®], Cyclophosphamide [Cytoxan® or Neosar®], Cytarabine, Cytosine Arabinoside [Cytosar-U®], Cytarabine Liposome Injection [DepoCyt®], Dacarbazine [DTIC-Dome®], Dactinomycin [Actinomycin D, Cosmegan], Daunorubicin Hydrochloride [Cer [ubidine®], daunorubicin citrate liposome injection [DaunoXome®], dexamethasone, docetaxel [Taxotere®], doxorubicin hydrochloride [Adriamycin®, Rubex®], etoposide [Vepesid®], fludarabine phosphate [Fludara®], 5-fluorouracil [Adrucil®, Efudex®], flutamide [Eulexin®], tezacitibine Gemcitabine (difluorodeoxycytidine), hydroxyurea [Hydrea®], idarubicin [Idamycin®], ifosphamide [IFEX®], irinotecan [Camptosar®], L-asparaginase [ELSPAR®], leucovorin calcium, melphalan [Alkeran®], 6-mercaptopurine [Purinethol®], methotrexate [Folex®], mitoxantrone [Novantrone®],Examples include Mylotarg, paclitaxel [Taxol®], Phoenix (yttrium 90 / MX-DTPA), pentostatin, carmustine implant-containing polyfeprosan 20 [Gliadel®], tamoxifen citrate [Nolvadex®], teniposide [Vumon®], 6-thioguanine, thiotepa, tirapazamine [Tirazone®], topotecan hydrochloride for injection [Hycamptin®], vinblastine [Velban®], vincristine [Oncovin®], and vinorelbine [Navelbine®].

[0318] Typical alkylating agents include, but are not limited to, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uracil mustard [Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®], chlormethine [Mustargen®], cyclophosphamide [Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune®], ifosphamide [Mitoxana®], melphalan [Alkeran®], chlorambucil [Leukeran®], Examples include pipobromane [Amedel®, Vercyte®], triethylenemelamine [Hemel®, Hexalen®, Hexastat®], triethylenethiophosphatamine, temozolomide [Temodar®], thiotepa [Thioplex®], busulfan [Busilvex®, Myleran®], carmustine [BiCNU®], lomustine [CeeNU®], streptozocin [Zanosar®], and dacarbazine [DTIC-Dome®].Additional typical alkylating agents include, but are not limited to, oxaliplatin [Eloxatin®]; temozolomide [Temodar® and Temodal®]; dactinomycin [also known as actinomycin-D, Cosmegen®]; melphalan [also known as L-PAM, L-sarcolicin, and phenylalanine mustard, Alkeran®]; altoretamine [also known as hexamethylmelamine (HMM), Hexalen®]. (Trademarks); Carmustine [BiCNU(registered trademark)]; Bendamustine [Treanda(registered trademark)]; Busulfan [Busulfex(registered trademark) and Myleran(registered trademark)]; Carboplatin [Paraplatin(registered trademark)]; Lomustine [also known as CCNU, CeeNU(registered trademark)]; Cisplatin (also known as CDDP, Platinol(registered trademark) and Platinol(registered trademark)-AQ); Chlorambucil [Leukeran(registered trademark)]; Cyclophosphamide [Cytoxan( (Registered Trademark) and Neosar (Registered Trademark); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome (Registered Trademark)); Altretamine (also known as hexamethylmelamine (HMM), Hexalen (Registered Trademark)); Ifosfamide (Ifex (Registered Trademark)); Prednimastine; Procarbazine (Matulane (Registered Trademark)); Mechloretamine (also known as nitrogen mustard, mustine and mechloretamine hydrochloride (mechlor Examples include oethamine (also known as Mustargen®); streptozocin [Zanosar®]; thiotepa [also known as thiophosphoamide, TESPA and TSPA, Thioplex®]; cyclophosphamide [Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®]; and bendamustine HCl [Treanda®].

[0319] Typical mTOR inhibitors include, for example, temsirolimus; ridafololimus (officially deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 [Hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate is known and also known as AP23573 and MK8669, as described in PCT Publication WO03 / 064383); everolimus (Afinitor® or RAD001); rapamycin [AY22989, Sirolimus®]; simapimod (CAS 164301-51- 3); emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholine-4-yl]pyrido[2,3-d]pyrimidine-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3-d]pyrimidine-7(8H)-one (PF04691502, CAS1013101-36-4); and N 2 Examples include -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine-, intramolecular salt (SF1126, CAS936487-67-1), and XL765.

[0320] Typical immunomodulators include, for example, aftuzumab (available from Roche®); pegfilgrastim [Neulasta®]; lenalidomide [CC-5013, Revlimid®]; thalidomide [Thalomid®]; actimide (CC4047); and IRX-2 (a mixture of human cytokines such as interleukin-1, interleukin-2, and interferon-γ, CAS951209-71-5, available from IRX Therapeutics).

[0321] Typical anthracyclines include, for example, doxorubicin [Adriamycin® and Rubex®]; bleomycin [lenoxane®]; daunorubicin [dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®]; daunorubicin liposomes [daunoXome®]; mitoxantrone [DHAD, Novantrone®]; epirubicin [Ellence®]; idarubicin [Idamycin®, Idamycin PFS®]; mitomycin C [Mutamycin®]; geldanamycin; harbimycin; rabidomycin; and desacetylrabidomycin.

[0322] Typical vinca alkaloids include, for example, vinorelbine tartrate [Navelbine®], vincristine [Oncovin®], and vindesine [Eldisine®]; vinblastine [also known as vinblastine sulfate, vincaloicoblastine, and VLB, Alkaban-AQ® and Velban®]; and vinorelbine [Navelbine®].

[0323] Typical proteosome inhibitors include bortezomib [Velcade®]; carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutane) Examples include amide)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxyranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serineamide (ONX-0912).

[0324] Typical GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as the GITR fusion protein described in, for example, U.S. Patent No. 6,111,090, European Patent No. 090505B1, U.S. Patent No. 8,586,023, PCT Publications WO2010 / 003118 and 2011 / 090754, or, for example, U.S. Patent No. 7,025,962, European Patent No. 1947183B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent Examples include anti-GITR antibodies described in EP1866339, PCT Publication WO2011 / 028683, PCT Publication WO2013 / 039954, PCT Publication WO2005 / 007190, PCT Publication WO2007 / 133822, PCT Publication WO2005 / 055808, PCT Publication WO99 / 40196, PCT Publication WO2001 / 03720, PCT Publication WO99 / 20758, PCT Publication WO2006 / 083289, PCT Publication WO2005 / 115451, U.S. Patent No. 7,618,632, and PCT Publication WO2011 / 051726.

[0325] In one embodiment, cells expressing the CAR described herein are administered to a subject in combination with an mTOR inhibitor, such as a rapalog, such as everolimus. In one embodiment, the mTOR inhibitor is administered before the CAR-expressing cells. For example, in one embodiment, the mTOR inhibitor may be administered before cell apheresis. In one embodiment, the subject has CLL.

[0326] In one embodiment, cells expressing the CAR described herein are administered to a subject in combination with a GITR agonist, for example, the GITR agonist described herein. In one embodiment, the GITR agonist is administered before the CAR-expressing cells. For example, in one embodiment, the GITR agonist may be administered before cell apheresis. In one embodiment, the subject has CLL.

[0327] Either a drug that inhibits the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or a drug that inhibits the p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993) may be used. In a further embodiment, the cell composition of the present invention may be administered to a patient together with (e.g., before, concurrently with, or after) bone marrow transplantation, T-cell depletion therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, and / or antibodies such as OKT3 or CAMPATH. In one embodiment, the cell composition of the present invention is administered after B-cell depletion therapy with a drug that reacts with CD20, such as rituxan. For example, in one embodiment, the subject may receive high-dose chemotherapy followed by standard treatment in peripheral blood stem cell transplantation. In a given embodiment, after transplantation, the subject receives an injection of the expanded immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

[0328] In one embodiment, the subject may be administered an agent that reduces or improves side effects associated with the administration of CAR-expressing cells. Side effects associated with the administration of CAR-expressing cells include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and similar symptoms. Therefore, the method described herein may include administering the CAR-expressing cells described herein to the subject and further administering an agent to manage elevated levels of soluble factors resulting from the treatment of the CAR-expressing cells. In one embodiment, the elevated soluble factors in the subject are one or more of IFN-γ, TNFα, IL-2, and IL-6. Therefore, the agent administered to treat this side effect may be an agent that neutralizes one or more of these soluble factors. Such agents include, but are not limited to, steroids, TNFα inhibitors, and IL-6 inhibitors. An example of a TNFα inhibitor is etanercept. An example of an IL-6 inhibitor is tocilizumab (toc).

[0329] In one embodiment, subjects may be administered a drug that enhances the activity of CAR-expressing cells. For example, in one embodiment, the drug may be a drug that inhibits an inhibitory molecule. In some embodiments, an inhibitory molecule, such as programmed death 1 (PD1), can reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. Inhibition of the inhibitory molecule, for example by inhibition at the DNA, RNA, or protein level, can optimize the performance of CAR-expressing cells. In embodiments, inhibitory nucleic acids, such as dsRNA, siRNA, or shRNA, can be used to inhibit the expression of the inhibitory molecule in CAR-expressing cells. In one embodiment, the inhibitor is shRNA. In some embodiments, the inhibitory molecule is inhibited within the CAR-expressing cell. In these embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule is ligated to a component, such as a nucleic acid that encodes all the components of CAR. In one embodiment, the inhibitor of the inhibitory signal may be, for example, an antibody or antibody fragment that binds to the inhibitory molecule. For example, the drug may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 [e.g., ipilimumab (also known as MDX-010 and MDX-101, marketed as Yervoy®); Bristol-Myers Squibb; tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as tisilimubab, CP-675,206)]. In some embodiments, the drug is an antibody or antibody fragment that binds to TIM3. In some embodiments, the drug is an antibody or antibody fragment that binds to LAG3.

[0330] PD1 is part of the CD28 family of receptor inhibitors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed in activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1, and PD-L2 have been shown to downmodulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be restored by inhibiting the local interaction between PD1 and PD-L1. Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1, and PD-L2 are available in the industry and can be used in combination with CD19CAR as described herein. For example, nivolumab (also known as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US8,008,449 and WO2006 / 121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1 pidilizumab, and other humanized anti-PD1 monoclonal antibodies are disclosed in WO2009 / 101611. Lambrolizumab (also known as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD1.Lambrolizumab and other humanized anti-PD1 antibodies are disclosed in US8,354,509 and WO2009 / 114335. MDPL3280A (Genentech / Roche) is an IgG1 monoclonal antibody optimized for human Fc binding to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Patent Application Publication No. 20120039906. Other anti-PD-L1 conjugates include YW243.55.S70 (WO2010 / 077634, with heavy and light chain variable regions shown in SEQ ID NOs. 20 and 21) and MDX-1 105 (also known as BMS-936559, an anti-PD-L1 conjugate disclosed, for example, in WO2007 / 005874). AMP-224 (B7-DCIg; amplimune; e.g., disclosed in WO2010 / 027827 and WO2011 / 066342) is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1. Other anti-PD1 antibodies include AMP514 (amplimune), e.g., the anti-PD1 antibody disclosed in US8,609,089, US2010028330, and / or US20120114649.

[0331] In some embodiments, the agent that enhances the activity of CAR-expressing cells may be, for example, a fusion protein comprising a first domain and a second domain, where the first domain is an inhibitory molecule or a fragment thereof, and the second domain is a polypeptide that associates with a positive signal, for example, an intracellular signaling polypeptide as described herein. In some embodiments, the polypeptide that associates with a positive signal may be the co-stimulatory domains of CD28, CD27, ICOS, for example, the intracellular signaling domains of CD28, CD27, and / or ICOS, and / or the primary signaling domain of CD3 zeta, for example, as described herein. In one embodiment, the fusion protein is expressed by the same cells that express the CAR. In another embodiment, the fusion protein is expressed by cells, for example, T cells that do not express anti-CD19CAR.

[0332] In one embodiment, the agent that enhances the activity of CAR-expressing cells as described herein is miR-17-92.

[0333] Pharmaceutical compositions and treatments The pharmaceutical compositions of the present invention may contain CAR-expressing cells, for example, a plurality of CAR-expressing cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers, such as neutral buffered saline, phosphate-buffered saline, and similar; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one embodiment, the compositions of the present invention are formulated for intravenous administration.

[0334] The pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The dosage and frequency of administration are expected to be determined by factors such as the patient's condition, as well as the type and severity of the patient's disease, although appropriate medication may be determined by clinical trials.

[0335] In one embodiment, the pharmaceutical composition is substantially free of contaminants selected from the group consisting of, for example, endotoxins, mycoplasmas, reproducible lentiviruses (RCLs), p24, VSV-G nucleic acids, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria, and fungi, and for example, their levels are not detectable. In one embodiment, the bacteria is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes A.

[0336] Where an "immunologically effective dose," "antitumor effective dose," "tumor inhibitory effective dose," or "therapeutic dose" is presented, the exact amount of the composition of the present invention to be administered can be determined by a physician considering the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in condition. Generally, the pharmaceutical compositions containing T cells described herein include all integer values ​​within the range of 10 cells. 4 from 109 Dosage per kg of body weight, in some cases 10 cells 5 from 10 6 It can be outlined that the T cell composition may be administered at a dose of cells / kg body weight. Furthermore, the T cell composition may be administered multiple times at these doses. The cells may also be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0337] In a predetermined embodiment, it may be desirable to administer activated T cells to a subject, then subsequently collect blood again (or perform apheresis), activate the T cells therefrom according to the present invention, and reinject these activated and expanded T cells into the patient. This process may be performed multiple times over several weeks. In a predetermined embodiment, T cells can be activated from 10cc to 400cc of collected blood. In a predetermined embodiment, T cells can be activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc of collected blood.

[0338] The composition can be administered by any convenient method, such as aerosol inhalation, injection, uptake, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient via artery, subcutaneous, intradermal, intratumor, lymph node (intranodally), intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In one embodiment, the T cell composition of the present invention is administered by iv injection. The T cell composition may be injected directly into a tumor, lymph node, or site of infection.

[0339] In certain typical embodiments, a subject may receive a leukocyte-depleted blood transfusion, during which leukocytes are collected, concentrated, or depleted ex vivo to select and / or isolate the subject's cells, such as T cells. These T cell isolates can be augmented in methods known in the art and treated to produce the CART cells of the present invention by introducing one or more CAR constructs of the present invention. Subjects requiring them may then receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In a given embodiment, following or in parallel with transplantation, the subject receives an infusion of the augmented CART cells of the present invention. In an additional embodiment, the augmented cells are administered before or after surgery.

[0340] The administration of the above treatments to be administered to patients is expected to vary depending on the exact nature of the condition being treated and the recipient receiving the treatment. Dosage scaling for human administration can be carried out according to industry-accepted practices. For example, the dosage for CAMPATH is generally expected to range from 1 mg to approximately 100 mg for adult patients, and is usually administered daily for a period of 1 to 30 days. The preferred daily dose is 1 mg to 10 mg / day, but in some cases, higher doses of up to 40 mg / day may be used (as described in U.S. Patent No. 6,120,766).

[0341] In one embodiment, the CARs are introduced into T cells, for example, using in vitro transcription, and a subject (e.g., human) receives an initial dose of the CART cells of the present invention, and one or more subsequent doses of the CART cells of the present invention, where the subsequent doses are administered less than 15 days after the previous dose, for example, less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In one embodiment, the subject (e.g., human) receives more than one dose of the CART cells of the present invention per week, for example, two, three, or four doses of the CART cells of the present invention per week. In one embodiment, the subject (e.g., human subject) receives more than one dose of the CART cells per week (e.g., two, three, or four doses per week) (also referred to herein as cycles), followed by a week without CART cell administration, and then one or more additional doses of CART cells (e.g., more than one dose of CART cells per week) are administered to the subject. In another embodiment, a subject (e.g., a human subject) receives more than one cycle of CART cells, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, CART cells are administered every other day, in three doses per week. In one embodiment, the CART cells of the present invention are administered for at least 2, 3, 4, 5, 6, 7, 8 or more weeks.

[0342] In one embodiment, CD19 CART is generated using a lentiviral viral vector, such as a lentivirus. The CART thus generated is expected to have stable CAR expression.

[0343] In one embodiment, CART transiently expresses the CAR vector 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 days after transduction. Transient expression of CAR can be performed by RNA CAR vector delivery. In one embodiment, CAR RNA is transduced into T cells by electroporation.

[0344] A potential problem that may arise in patients treated with transient expression of CART cells (particularly with CART cells bound to mouse scFv) is anaphylaxis after multiple treatments.

[0345] While we do not wish to be bound by this theory, it is thought that such anaphylactic responses may be triggered by the patient developing a humoral anti-CAR response, i.e., anti-CAR antibodies with an anti-IgE isotype. When antigen exposure is interrupted for 10 to 14 days, it is thought that the patient's antibody-producing cells undergo a class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype.

[0346] If a patient is at high risk of developing an anti-CAR antibody response during the course of transient CAR therapy (e.g., those caused by RNA translocation), interruption of CAR infusion should not be prolonged beyond 10 to 14 days. [Examples]

[0347] The present invention will be described in further detail with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Therefore, the present invention shall not be construed as being limited to the following examples, but rather as encompassing all variations that may become apparent as a result of the teachings provided herein.

[0348] Those skilled in the art will likely be able to carry out the claimed method by preparing and utilizing the compounds of the present invention using the examples provided above and below, without further explanation. The following examples specifically illustrate various aspects of the present invention, but should not be construed as limiting other parts of the disclosure. [Examples]

[0349] Humanization of mouse anti-CD19 antibodies Humanization of mouse CD19 antibodies is desirable for clinical conditions in which mouse-specific residues may induce a human anti-mouse antigen (HAMA) response in patients receiving CART19 treatment, i.e., treatment with T cells transduced with the CAR19 construct. The VH and VL sequences of hybridoma-derived mouse CD19 antibodies were obtained from published literature (Nicholson et al, 1997, above). Humanization was achieved by grafting the CDR region from mouse CD19 antibodies onto the human germline acceptor framework VH4_4-59 and VK3_L25 (vBASE database). In addition to the CDR region, five framework residues that are thought to support the structural integrity of the CDR region, namely VH71, 73, 78 and VL71, 87, were retained from the mouse sequence. Furthermore, human J elements JH4 and JK2 were used in the heavy chain and light chain, respectively. The amino acid sequences obtained from the humanized antibodies were named FMC63_VL_hz and FMC63_VH_hz1, respectively, and are shown in Table 1 below. Residue numbering followed Kabat's method (Kabat EA et al, 1991, see above). For the CDR definition, both Kabat and Chothia et al. (1987, see above) were used. Residues derived from mouse CD19 are shown in bold / italic. Position numbers 60 / 61 / 62 enclosed in boxes indicate potential post-translational modification (PTM) sites in CDR H2, also known as HCDR2.

[0350] [Table 1]

[0351] Using these humanized CD19 IgGs, we generated soluble scFvs for expression testing and scFvs for the complete CART CD19 construct (see examples below). Interestingly, during humanization, position 62 in the CDRH2 region selected a serine residue rather than the alanine present in mouse CDRH2. The mouse sequence lacks post-translational modification (PTM) and has asparagine-serine-alanine at positions 60 / 61 / 62, respectively, in CDRH2. This generates a potential PTM motif (presented as a boxed region in CDRH2) during the course of humanization. We tested whether the PTM site generated during the humanization process was actually a "true" PTM site or simply theoretical. We hypothesized that the amino acid motif asparagine followed by serine (NS) might be susceptible to post-translational deamide, but not to an easily discoverable degree. We also hypothesized that all amino acids except asparagine and the subsequent proline, followed by serine (NxS, where x is not P), may be susceptible to post-translational N-glycosylation. To test this hypothesis, we generated two IgG mutants in which asparagine at position 60 (a known glycosylation site) was mutated to serine or glutamine, and named them FMC63_VH_hz2(N60S) and FMC63_VH_hz2(N60Q), respectively. These constructs were generated to remove potential post-translational modification sites (PTMs) and test for retained activity (see Example 2 below).

[0352] Cloning: We obtained DNA sequences encoding mouse and humanized VL and VH domains and optimized the construct codons for expression in human-derived cells.

[0353] Sequences encoding the VL and VH domains were subcloned from the cloning vector into expression vectors suitable for secretion in mammalian cells. The heavy and light chains were cloned into individual expression vectors so that they could be transfected together. The elements of the expression vectors include a promoter (cytomegalovirus (CMV) enhancer-promoter), signal sequences to facilitate secretion, polyadenylation signals and transcriptional terminators (bovine growth hormone (BGH) gene), elements that enable episomal replication and replication in prokaryotes (e.g., derived from SV40 and ColE1 or others known in the art), and elements that enable selection (ampicillin resistance gene and zeosin marker).

[0354] Expression: Chimeric and humanized IgG candidates were expressed in HEK293F mammalian cells on a 1 ml scale. The supernatant was used for FACS-bound studies. More precisely, HEK293F cells were diluted to 5E5 cells / ml in FreeStyle medium supplemented with penicillin / streptomycin, and 1 ml was transferred to a 24-round-bottom deep-well plate. 0.5 μg of light chain and 0.5 μg of heavy chain mammalian expression plasmids were diluted in the same medium with 4 μl of FuGENE HD (Roche REF 04709705001). After incubation at room temperature for 15 minutes, the DNA / FuGENE mix was added dropwise to the cells and incubated in a 5% CO2 incubator at 250 rpm, 37°C for 5 days. The supernatant was then separated from the cells by centrifugation. To measure IgG content, 200 μL aliquots were placed in the wells of a 96-well microtiter plate. All samples and standards were measured in a dual-cycle configuration using a Protein A dip and reading biosensor (Fortebio, catalog no. 18-5010). The plates were placed in an Octet instrument (ForteBio) and equilibrated at 27°C in a thermostat-controlled chamber. Data were processed automatically using Octet user software version 3.0, and concentrations were determined by comparison with the IgG standard curve.

[0355] Combined analysis using FACS: Humanized and chimeric antibodies were evaluated by flow cytometry-bound assay using the cell line 300.19-hsCD19FL. This cell line was generated by transfecting the mouse pre-B cell line 300.19 with a vector encoding the full-length human CD19 sequence and innate promoter, as well as the zeosin resistance gene (hCD19 FL / pEF4-myc-His A). Briefly, 300.19 cells were electroporated with a linearized plasmid, and then cells expressing high levels of hsCD19 were identified using anti-human CD19Ab (clone HIB19555415 from BD) conjugated with APC, and subsequently sorted using a FACS Aria flow cytometer. The sorted hsCD19+ cells were cultured and confirmed to stably express high levels of hsCD19.

[0356] The binding assay could be performed directly using serum-free culture medium containing expressed IgG. All evaluated IgG was normalized to the same concentration (85 nM) and then diluted 3-fold serially to 1.4 pM. Subsequently, 5 × 10⁶ cells were sampled in a 96-well plate. 5 Aliquots of cells / well were incubated with diluted IgG at 4°C for 30 minutes. Cells were washed twice with FACS buffer (0.5% BSA in PBS), and then a specific Fc fragment of goat anti-huIgG (Dianova number 109-136-098) conjugated with the detection antibody APC was added and diluted 1:1000 with FACS buffer. Cells were incubated for a further 30 minutes at 4°C, then washed twice with FACS buffer and assayed using FACS caliber (BD Bioscience). Binding curves were plotted (median fluorescence intensity against IgG concentration) and EC2 were plotted. 50 The decision was made using GraphPad Prism™ 3.0 software, employing nonlinear regression analysis and S-shaped dose-response (variable slope).

[0357] FACS analysis shows that apparent binding for all IgGs evaluated can vary widely, and some constructs exhibit a 5 to 10-fold shift in EC50 as IgG versus scFv. 50 Based on the values, lead candidates with twice or better binding affinity compared to the reference chimera are selected. [Examples]

[0358] Characterization of anti-CD19 soluble scFv fragments derived from humanized CD19IgG antibodies Soluble scFv fragments were generated from humanized CD19IgG as described in Example 1 using standard molecular biology techniques. These soluble scFvs were used in characterization studies to test their stability, cell surface expression, and scFv binding properties. In addition, experiments were conducted to investigate the effects of potential PTMs introduced during the humanization process.

[0359] scFv expression and purification For transfection of each scFv construct, approximately 3e8 293F cells were transfected with 100 μg of plasmid using PEI in a 3:1 (PEI:DNA) ratio as the transfection reagent. The cells were grown in 100 ml of EXPi293 expression medium (Invitrogen) in a shaking flask at 37°C, 125 rpm, and 8% CO2. The cultures were harvested after 6 days and used for protein purification.

[0360] 293F cells were harvested by centrifugation at 3500g for 20 minutes. The supernatant was collected and filtered through a VacuCap90PF filter unit (with a 0.8 / 0.2 μm supermembrane, PALL). Approximately 400 μl of Ni-NTA agarose beads (Qiagen) were added to the supernatant. The mixture was rotated and incubated at 4°C for 4 hours. This was loaded into a purification column and washed with a wash buffer containing 20 mM histidine. Proteins were eluted with 500 μl of elution buffer containing 300 mM histidine. The samples were dialyzed overnight in PBS buffer at 4C. Protein samples were quantified using a NanoDrop 2000c.

[0361] Analysis of the conformation and colloidal stability of scFv The thermal stability of scFv was determined by DSF: 10-20 μl of protein samples in a mix containing cypro orange dye (Invitrogen, catalog no. S6650) at a final dilution of 1:1000 in a total volume of 25 μl in PBS were treated with a BioRad CFX1000 (25°C for 2 minutes, then increasing by 0.5°C every 30 seconds from 25°C to 95°C).

[0362] For analytical SEC experiments, approximately 15-20 μg of scFv protein sample in 20 μl of PBS was injected into a TSKgel Super SW2000 using an Agilent 1100 series at a flow rate of 0.3 ml / min.

[0363] EC50 via FACS coupling Mouse cell line 300.CD19 was grown in RPMI1640 containing 0.5 mg / ml zeocin. Approximately 5e5 cells / well were transferred to a BD Falcon 96-well plate. The cells were centrifuged at 900 rpm (Sorval Legend XT centrifuge) for 3 minutes. The supernatant was removed. Anti-CD19scFv protein samples were diluted in DPBS containing 5% FBS. The samples ...

Claims

1. A pharmaceutical composition comprising cells and a pharmaceutically or physiologically acceptable carrier, diluent or excipient, wherein the cells comprise an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprises an scFv comprising an intracellular signaling domain including a CD19-binding domain, a transmembrane domain and a stimulating domain, and the CD19-binding domain is (i) Light chain complementarity determination region 1 (LC CDR1) containing the amino acid sequence of SEQ ID NO: 25, (ii) Light chain complementarity determination region 2 (LC CDR2) containing the amino acid sequence of SEQ ID NO: 26, (iii) Light chain complementarity determination region 3 (LC CDR3) containing the amino acid sequence of SEQ ID NO: 27, (iv) Heavy chain complementarity determination region 1 (HC CDR1) containing the amino acid sequence of SEQ ID NO: 19, (v) Heavy chain complementarity determination region 2 (HC CDR2) containing the amino acid sequence of any of SEQ ID NOs: 22, 21, or 23, and (vi) Heavy chain complementarity determination region 3 (HC CDR3) containing the amino acid sequence of SEQ ID NO: 24 A pharmaceutical composition containing the above.

2. (a) Light chain variable regions (VLs) shown in Sequence IDs 32, 31, or 33-42; (b) Heavy chain variable region (VH) as shown in Sequence ID No. 32, 31, or 33-42; or (c) VL shown in SEQ ID NOs. 32, 31, or 33-42 and any VH shown in SEQ ID NOs. 32, 31, or 33-42 A pharmaceutical composition according to claim 1, which codes for the pharmaceutical composition described in claim 1.

3. (a) VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NOs. 32, 31, or 33-42, or (b) VH comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NOs. 32, 31, or 33-42. The pharmaceutical composition according to claim 1 or 2.

4. (a) The CD19 binding domain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, SEQ ID NOs: 1, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs: 12, or an amino acid sequence having at least 95% identity with them; (b) The nucleic acid sequence encoding the CD19 binding domain is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 62, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, and 72, or a nucleic acid sequence having at least 95% identity with them; or (c) The cells contain a nucleic acid sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, or a nucleic acid sequence having at least 95% identity with them. A pharmaceutical composition according to any one of claims 1 to 3.

5. The coded CAR is, (a) A transmembrane domain containing a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; (b) A transmembrane domain containing the transmembrane domain of the alpha chain of CD8; or (c) A transmembrane domain encoded with the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

15. A nucleic acid sequence containing or encoding a transmembrane domain includes the nucleic acid sequence of Sequence ID No. 56, or a sequence having at least 95% identity thereto. A pharmaceutical composition according to any one of claims 1 to 4.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the encoded CD19-binding domain is connected to the transmembrane domain by a hinge region.

7. The pharmaceutical composition according to claim 6, wherein the encoded hinge region comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 95% identity thereto, or the nucleic acid sequence encoding the hinge region comprises the nucleic acid sequence of SEQ ID NO: 55, or a nucleic acid sequence having at least 95% identity thereto.

8. The intracellular signaling domain includes a co-stimulatory domain. (a) The co-stimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), or (b) The encoded co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 16, or the nucleic acid sequence encoding the co-stimulatory domain includes the nucleic acid sequence of SEQ ID NO: 60 or a nucleic acid sequence having at least 95% identity with it. A pharmaceutical composition according to any one of claims 1 to 7.

9. The intracellular signaling domain includes the primary signaling domain, (a) The encoded intracellular signaling domain includes the functional signaling domain of 4-1BB and / or the functional signaling domain of CD3 zeta, (b) The encoded intracellular signaling domain comprises (i) the amino acid sequence of SEQ ID NO: 16 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43 or (ii) an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 16 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO:

43. (c) The encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, and the sequence comprising the intracellular signaling domain is expressed as a single polypeptide chain in the same frame, or (d) The nucleic acid sequence encoding the intracellular signaling domain includes the nucleic acid sequence of Sequence ID No. 60, or a nucleic acid sequence having at least 95% identity thereto, and / or the nucleic acid sequence of Sequence ID No. 101 or Sequence ID No. 44, or a nucleic acid sequence having at least 95% identity thereto. A pharmaceutical composition according to any one of claims 1 to 8.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the isolated nucleic acid molecule further comprises a leader sequence.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the CD19 binding domain includes a humanized CD19 binding domain.

12. A pharmaceutical composition comprising cells and a pharmaceutically or physiologically acceptable carrier, diluent or excipient, wherein the cells are (i) Light chain complementarity determination region 1 (LC CDR1) containing the amino acid sequence of SEQ ID NO: 25, (ii) Light chain complementarity determination region 2 (LC CDR2) containing the amino acid sequence of SEQ ID NO: 26, (iii) Light chain complementarity determination region 3 (LC CDR3) containing the amino acid sequence of SEQ ID NO: 27, (iv) Heavy chain complementarity determination region 1 (HC CDR1) containing the amino acid sequence of SEQ ID NO: 19, (v) Heavy chain complementarity determination region 2 (HC CDR2) containing the amino acid sequence of any of SEQ ID NOs: 22, 21, or 23, and (vi) Heavy chain complementarity determination region 3 (HC CDR3) containing the amino acid sequence of SEQ ID NO: 24 A pharmaceutical composition comprising scFv containing a humanized CD19-binding domain.

13. The humanized CD19 binding domain, (i) The VL and VH of any of the amino acid sequences of SEQ ID NOs: 32, 31, or 33-42; (ii) A VL having an amino acid sequence that is at least 95% identical to the amino acid sequence of a VL shown in any of SEQ ID NOs: 32, 31, or 33-42; and / or (iii) A VH having an amino acid sequence that is at least 95% identical to the amino acid sequence of the VH shown in any of SEQ ID NOs: 32, 31, or 33-42. A pharmaceutical composition according to claim 12, comprising:

14. (i) Light chain complementarity determination region 1 (LC CDR1) containing the amino acid sequence of SEQ ID NO: 25, (ii) Light chain complementarity determination region 2 (LC CDR2) containing the amino acid sequence of SEQ ID NO: 26, (iii) Light chain complementarity determination region 3 (LC CDR3) containing the amino acid sequence of SEQ ID NO: 27, (iv) Heavy chain complementarity determination region 1 (HC CDR1) containing the amino acid sequence of SEQ ID NO: 19, (v) Heavy chain complementarity determination region 2 (HC CDR2) containing the amino acid sequence of any of SEQ ID NOs: 22, 21, or 23, and (vi) Heavy chain complementarity determination region 3 (HC CDR3) containing the amino acid sequence of SEQ ID NO: 24 A humanized CD19-binding domain containing, The CD19 binding domain is (i) Maintaining affinity for human CD19 for scFv having the amino acid sequence of SEQ ID NO: 59 Humanized CD19-binding domain.

15. An isolated chimeric antigen receptor (CAR) molecule comprising a humanized CD19-binding domain, a transmembrane domain, and an intracellular signaling domain as defined in claim 14.

16. An isolated nucleic acid molecule encoding a CD19-binding domain as defined in claim 14 or a CAR molecule as defined in claim 15.

17. A vector comprising a nucleic acid molecule encoding a CAR molecule as defined in claim 15.

18. The vector according to claim 17, wherein the vector is selected from the group consisting of DNA, RNA, plasmids, viral vectors, lentiviral vectors, adenovirus vectors, and retroviral vectors.

19. The vector according to claim 17 or 18, further comprising a promoter.

20. (a) The vector is a vector that was transcribed in vitro; (b) The nucleic acid sequence in the vector further includes a poly(A) tail; and / or (c) The nucleic acid sequence in the vector further includes a 3'UTR, The vector according to any one of claims 17 to 19.

21. (a) comprising the nucleic acid molecule of claim 16; (b) comprising the vector according to any one of claims 17 to 20; or (c) expressing the CAR molecule of claim 15, cell.

22. The cell according to claim 21, wherein the cell is a human T cell and / or a CD8+ T cell.

23. An in vitro or ex vivo method for producing cells, comprising transducing T cells in vitro or ex vivo with a vector according to any one of claims 17 to 20 or a nucleic acid molecule according to claim 16.

24. A method for generating a population of RNA-processed cells, comprising introducing RNA transcribed in vitro or synthetic RNA into cells in vitro or ex vivo, wherein the RNA comprises a nucleic acid encoding the CAR molecule described in claim 15, in vitro or ex vivo method.

25. A pharmaceutical composition according to any one of claims 1 to 11, for use in providing antitumor immunity to a subject having a hematological cancer that is positive for CD19, wherein the cells in the pharmaceutical composition are T cells or NK cells.

26. Use of the pharmaceutical composition according to any one of claims 1-11 in the manufacture of a pharmaceutical for providing antitumor immunity to a subject having a hematological cancer that is positive for CD19, wherein the cells in the pharmaceutical composition are T cells or NK cells.

27. (a) The cells are autologous T cells or allogeneic T cells; and / or (b) Antitumor immunity is provided to humans, The pharmaceutical composition according to claim 25.

28. A pharmaceutical composition according to any one of claims 1 to 11, for use in treating a hematological cancer that is positive for CD19, wherein the cells in the pharmaceutical composition are T cells or NK cells.

29. Use of the pharmaceutical composition according to any one of claims 1-11 in the manufacture of a pharmaceutical for the treatment of a hematological cancer that is positive for CD19, wherein the cells in the pharmaceutical composition are T cells or NK cells.

30. The pharmaceutical composition according to claim 25, 27, or 28, wherein the hematological cancer that is positive for CD19 is selected from the group consisting of B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prelymphocytic leukemia, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, Waldenström macroglobulinemia, and combinations thereof.

31. (a) an agent that enhances the efficacy of CAR molecule-expressing cells, an agent that improves one or more side effects associated with the administration of CAR molecule-expressing cells, or an agent that treats hematological cancers that are positive for CD19; and / or (b) Chemotherapy agents, immunosuppressants, treatments for CRS (cytokine release syndrome), or agents that inhibit PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, or 2B4 Administered in combination with, The pharmaceutical composition according to claim 25, 27, 28, or 30.

32. (a) CAR molecule expressing cells, cell 10 ４ from 10 ９ It is administered at a dose of one unit / kg body weight; (b) CAR molecule-expressing cells are administered in a quantity of 1.4 × 10⁷ to 1.1 × 10⁹ cells per dose; (c) Use involves one or more subsequent administrations of cells, nucleic acid molecules or CAR molecules; or (d) Use involves administering cells, nucleic acid molecules or CAR molecules more than once per week, The pharmaceutical composition according to claim 25, 27, 28, 30, or 31.

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