RU2856016C2 - Treatment of cancer using humanised anti-cd19 chimeric antigen receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: there are provided chimeric antigen receptors (CARs) specific for CD19, nucleic acids encoding them and vectors, as well as recombinant T-cells containing the anti-CD19 CAR. The invention also relates to methods for producing a genetically modified T-cell expressing the anti-CD19 CAR, and to the use of said cell. Furthermore, the use of the anti-CD19 CAR and cells according to the invention for generating anti-tumour immunity, as well as for treating diseases associated with CD19 expression, is disclosed.

EFFECT: further application in the therapy of proliferative diseases associated with CD19 expression, in particular cancer.

29 cl, 12 dwg, 6 tbl, 6 ex

Description

This application claims priority from U.S. Provisional Patent Application Ser. No. 61/802,629, filed March 16, 2013, and U.S. Provisional Patent Application Ser. No. 61/838,537, filed June 24, 2013, the entire contents of each of which are incorporated herein by reference.

SEQUENCE LIST

This application contains a sequence listing, which is provided in electronic form in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 14, 2014, is named N2067-7002WO_SL.txt and is 228,415 bytes in size.

FIELD OF TECHNICAL INVENTION

The present invention relates generally to the use of T cells genetically altered to express a chimeric antigen receptor (CAR) for the treatment of a disease associated with expression of cluster of differentiation 19 (CD19) protein.

BACKGROUND OF THE INVENTION

Many patients with B-cell malignancies are resistant to standard therapeutic approaches. Furthermore, traditional treatments often have serious side effects. Attempts have been made to develop cancer immunotherapy, but several factors make achieving clinical efficacy challenging. Although hundreds of so-called tumor antigens have been identified, they are typically derived from the body's own tumors and therefore have weak immunogenicity. Furthermore, tumors have several mechanisms to resist the initiation and spread of immune attack.

Recent developments in chimeric antigen receptor (CAR)-modified autologous T cell therapy (CART), which redirects T cells to relevant molecules on the surface of cancer cells such as B-cell malignancies, are showing promise in harnessing the potential of the immune system to treat B-cell malignancies and other cancers (see, e.g., Sadelain et al., Cancer 1907393 1 Discovery 3: 388–398 (2013)). Clinical results using mouse CART19 (i.e., “CTL019”) have shown promise in achieving complete remission in patients with CLL, as well as in childhood ALL (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)). In addition to the ability of genetically modified T cells bearing a chimeric antigen receptor to recognize and kill target cells, successful treatment with therapeutic T cells requires that they proliferate and persist for a long time, and subsequently control surviving leukemia cells. Variations in T cell quality, resulting from anergy, suppression, or exhaustion, will impact the efficacy of CAR-transformed T cells, which skilled practitioners can currently only control to a limited extent. To be effective, a patient's CAR-transformed T cells must persist and maintain the ability to proliferate in response to the CAR antigen. T cells from an ALL patient have been shown to do this with CART19 containing mouse scFv (see, e.g., Grupp et al., NEJM 368: 1509–1518 (2013)).

ESSENCE OF THE INVENTION

The invention relates to controlling the immune response in patients by providing optimized and humanized antibody fragments (e.g., scFv) that bind cluster of differentiation protein 19 (CD19) integrated into a chimeric antigen receptor (CAR) construct that will not elicit an immune response in patients, is safe for long-term use, and exhibits similar or superior clinical efficacy to a known CAR-based therapeutic for the treatment of B-cell malignancies. The invention further relates to the use of T cells genetically altered to express a humanized antibody fragment that binds CD19 integrated into a CAR for the treatment of blood cancers associated with CD19 expression (OMIM accession no. 107265, Swiss Prot. accession no. P15391).

Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment that comprises 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 basic signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment that comprises 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 basic signaling domain).

In one embodiment, the encoded humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and/or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., 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 comprises at least an HC CDR2. In one embodiment, the encoded humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, the encoded humanized anti-CD19 binding domain has two heavy chain variable regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least an 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 a modification (e.g., a substitution, such as a substitution, of one or more amino acids located at the corresponding position in the light chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71 and 87). In one embodiment, the encoded heavy chain variable region comprises 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 a modification (e.g., a substitution, such as a substitution, of one or more amino acids located at the corresponding position in the heavy chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71, 73, and 78). In one embodiment, the encoded humanized anti-CD19 binding domain comprises a humanized light chain variable region as described herein (e.g., in Table 3) and/or a humanized heavy chain variable region as described herein (e.g., in Table 3). In one embodiment, the encoded humanized anti-CD19 binding domain comprises a humanized heavy chain variable region as described herein (e.g., in Table 3), such as at least two humanized heavy chain variable regions as described herein (e.g., in Table 3). In one embodiment, the encoded anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain with an amino acid sequence as set forth in Table 3. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the light chain variable region amino acid sequence as set forth in Table 3, or a sequence having 95-99% identity to 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), but not more than 30, 20 or 10 modifications (e.g., substitutions), of the amino acid sequence of the heavy chain variable region set forth in Table 3, or a sequence having 95-99% identity to the amino acid sequence in Table 3. In one embodiment, the encoded humanized anti-CD19 binding domain comprises 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 thereto. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting 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: 68, 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 encoded humanized anti-CD19 binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein, for example, in Table 3, is linked to a heavy chain variable region comprising an amino acid sequence described herein, for example, in Table 3, via a linker, for example, a linker described herein. In one embodiment, the encoded humanized anti-CD19 binding domain comprises a linker (Gly ₄ -Ser)n, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region in the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

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 a 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 comprises the sequence of SEQ ID NO: 15. In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 20, 10, or 5 modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 15, 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 comprises the sequence of SEQ ID NO: 56 or a sequence having 95-99% identity thereto.

In one embodiment, the encoded anti-CD19 binding domain is linked to the transmembrane domain via a hinge region, such as a hinge region described herein. In one embodiment, the encoded hinge region comprises SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47, or a sequence having 95-99% identity therewith. In one embodiment, the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO: 55 or SEQ ID NO: 46 or SEQ ID NO: 48, or a sequence having 95-99% identity therewith.

In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain derived 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 costimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the encoded costimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 20, 10, or 5 modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 16, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the nucleic acid sequence encoding the costimulatory domain comprises the sequence of SEQ ID NO: 60, or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding an intracellular signaling domain, such as an intracellular signaling domain as described herein. In one embodiment, the encoded intracellular signaling domain comprises a functional 4-1BB signaling domain and/or a functional CD3-zeta signaling domain. In one embodiment, the encoded intracellular signaling domain comprises a functional CD27 signaling domain and/or a functional CD3-zeta signaling domain. 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), but no more than 20, 10, or 5 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, or a sequence having 95-99% identity to 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 comprises 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 sequences constituting the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO: 60 or a sequence having 95-99% identity thereto, and/or the sequence of SEQ ID NO: 101 or SEQ ID NO: 44, or a sequence having 95-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO: 52 or a sequence having 95-99% identity thereto, and/or the sequence of SEQ ID NO: 101 or SEQ ID NO: 44, or a sequence having 95-99% identity thereto.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence, e.g., a leader sequence as described herein, e.g., SEQ ID NO: 13; a humanized anti-CD19 binding domain as described herein, e.g., a humanized anti-CD19 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 as described herein, e.g., the humanized anti-CD19 binding domain described in Table 3, or a sequence having 95-99% identity thereto; a hinge region as described herein, e.g., SEQ ID NO: 14 or SEQ ID NO: 45; a transmembrane domain described herein, 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 comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, and/or a core signaling domain, e.g., a core signaling domain described herein, e.g., a CD3-zeta signaling domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, an isolated nucleic acid molecule encoding a CAR construct comprises 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, an isolated nucleic acid molecule encoding a CAR construct comprises a humanized anti-CD19 binding domain sequence encoded by the nucleic acid sequence 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, an isolated nucleic acid molecule encoding a CAR construct comprises a transmembrane sequence encoded by the nucleic acid sequence of SEQ ID NO: 56, or a sequence having 95-99% identity thereto. In one embodiment, an isolated nucleic acid molecule encoding a CAR construct comprises 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.

In one embodiment, the isolated nucleic acid molecule comprises (e.g., consists of) a nucleic acid encoding the amino acid sequence of a CAR 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, 10, 15, 20, or 30 modifications (e.g., substitutions), but not more than 60, 50, or 40 modifications (e.g., substitutions), of the amino acid sequence, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to 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.

In one embodiment, the isolated nucleic acid molecule comprises (e.g., consists of) 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 to 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.

In one aspect, the invention provides 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 determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining 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 determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, for example, 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 comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has a modification (e.g., a substitution, such as a substitution of one or more amino acids located at the corresponding position in the mouse light chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has a modification (e.g., a substitution, such as a substitution of one or more amino acids located at the corresponding position in the mouse heavy chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71, 73, and 78). In one embodiment, the encoded humanized anti-CD19 binding domain comprises a light chain variable region as described herein (e.g., in SEQ ID NO: 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., in SEQ ID NO: 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 a light chain and a heavy chain with an amino acid sequence set forth in SEQ ID NO: 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) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence having 95-99% identity to amino acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or a sequence having 95-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting 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: 68, 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 another aspect, the invention relates to an isolated polypeptide molecule encoded by a nucleic acid sequence. In one embodiment, the isolated polypeptide 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. In one embodiment, the isolated polypeptide comprises the sequence of SEQ ID NO: 31. In one embodiment, the isolated polypeptide comprises the sequence of SEQ ID NO: 32. In one embodiment, the isolated polypeptide molecule comprises the sequence of SEQ ID NO: 35. In one embodiment, the isolated polypeptide molecule comprises the sequence of SEQ ID NO: 36. In one embodiment, the isolated polypeptide molecule comprises the sequence of SEQ ID NO: 37.

In another aspect, the invention provides 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 comprising a costimulatory domain and/or a basic signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment comprising a humanized anti-CD19 binding domain described herein (e.g., a humanized antibody or antibody fragment that specifically binds to CD19 as described herein), a transmembrane domain described herein, and an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a basic signaling domain as described herein).

In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining 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 determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., 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 comprises at least an HC CDR2. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., the humanized anti-CD19 binding domain has two heavy chain variable regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least an 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 a modification (e.g., a substitution, such as a substitution, of one or more amino acids located at the corresponding position in the mouse light chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has a modification (e.g., a substitution, such as a substitution, of one or more amino acids located at the corresponding position in the mouse heavy chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71, 73, and 78). In one embodiment, the humanized anti-CD19 binding domain comprises a light chain variable region as described herein (e.g., Table 3) 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 comprising a light chain and a heavy chain with the amino acid sequence set forth in Table 3. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the light chain variable region set forth in Table 3, or a sequence having 95-99% identity to 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), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the heavy chain variable region set forth in Table 3, or a sequence having 95-99% identity to the amino acid sequence in Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises 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 thereto. In one embodiment, the humanized anti-CD19 binding domain is an scFv, and the light chain variable region comprising the amino acid sequence described herein, for example, in Table 3, is linked to the heavy chain variable region comprising the amino acid sequence described herein, for example, in Table 3, via a linker, for example, a linker described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a (Gly ₄ -Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The variable region of the light chain and the variable region of the heavy chain in the scFv may be, for example, in any of the following orientations: variable region of the light chain-linker-variable region of the heavy chain or variable region of the heavy chain-linker-variable region of the light chain.

In one embodiment, the isolated CAR molecule comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD 154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 15. In one embodiment, the transmembrane domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 15, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 15.

In one embodiment, the humanized anti-CD19 binding domain is linked to the transmembrane domain via a hinge region, such as the hinge region described herein. In one embodiment, the encoded hinge region comprises SEQ ID NO: 14 or SEQ ID NO: 45, or a sequence having 95-99% identity thereto.

In one embodiment, the isolated CAR molecule further comprises a sequence encoding a costimulatory domain, such as a costimulatory domain as described herein. In one embodiment, the costimulatory 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), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 16. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 51. In one embodiment, the costimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 20, 10, or 5 modifications (e.g., substitutions), of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51.

In one embodiment, the isolated CAR molecule further comprises a sequence encoding an intracellular signaling domain, such as an intracellular signaling domain as described herein. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and/or a functional CD3-zeta signaling domain. 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 CD27 signaling domain and/or a functional CD3-zeta signaling domain. 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 comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but no more than 20, 10 or 5 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, or a sequence having 95-99% identity to 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 contains 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 sequences constituting the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain.

In one embodiment, the isolated CAR molecule further comprises a leader sequence, such as 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 to the amino acid sequence of SEQ ID NO: 13.

In another aspect, the invention provides an isolated CAR molecule comprising a leader sequence, e.g., a leader sequence described herein, e.g., the leader sequence of SEQ ID NO: 13, or having 95-99% identity thereto; a humanized anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 binding domain comprising the LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, e.g., the humanized anti-CD19 binding domain set forth in Table 3, or having 95-99% identity thereto; a hinge region, e.g., a hinge region described herein, e.g., the hinge region of SEQ ID NO: 14, or having 95-99% identity thereto; a transmembrane domain, such as a transmembrane domain as described herein, such as a transmembrane domain having the sequence of SEQ ID NO: 15 or a sequence having 95-99% identity thereto; an intracellular signaling domain, such as an intracellular signaling domain as described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a basic signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, such as a costimulatory domain as described herein, such as a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or having 95-99% identity thereto, and/or a core signaling domain, such as a core signaling domain as described herein, such as a CD3-zeta signaling domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or having 95-99% identity thereto.

In one embodiment, an isolated CAR molecule comprises (e.g., consists 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, or an amino acid sequence having at least one, two, three, four, five, 10, 15, 20, or 30 modifications (e.g., substitutions), but not more than 60, 50, or 40 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, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to 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.

In one aspect, the invention provides a humanized anti-CD19 binding domain comprising one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining 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 determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., 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 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 a modification (e.g., a substitution, such as a substitution of one or more amino acids located at the corresponding position in the mouse light chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has a modification (e.g., a substitution, such as a substitution of one or more amino acids located at the corresponding position in the heavy chain variable region of SEQ ID NO: 58, such as a substitution at one or more of positions 71, 73, and 78). In one embodiment, the humanized anti-CD19 binding domain comprises a light chain variable region as described herein (e.g., in 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) and/or a heavy chain variable region as described herein (e.g., in 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). In one embodiment, the humanized anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain with the amino acid sequence set forth in 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. In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the light chain variable region set forth in 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 to the amino acid sequence 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; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 30, 20 or 10 modifications (e.g., substitutions), of the amino acid sequence of the heavy chain variable region set forth in 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 to the amino acid sequence 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.

In another aspect, the 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, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

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

In one embodiment, the vector is an in vitro transcribed vector, e.g., a vector that transcribes the RNA of a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the vector further comprises a poly(A) sequence, e.g., a poly(A) sequence described herein, e.g., comprising about 150 adenosine bases (SEQ ID NO: 104). In one embodiment, the nucleic acid sequence in the vector further comprises a 3'UTR, e.g., a 3'UTR described herein, e.g., comprising at least one 3'UTR repeat from human beta globulin. In one embodiment, the nucleic acid sequence in the vector further comprises a promoter, e.g., a T2A promoter.

In another aspect, the invention relates to a cell comprising a vector. In one embodiment, the cell is a human T cell. In one embodiment, the cell is a cell as described herein, such as a human T cell, such as a human T cell as described herein. In one embodiment, the human T cell is a CD8+ T cell.

In another embodiment, the CAR-expressing cell described herein may further express 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 an inhibitory molecule comprises a first polypeptide, such as an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TEVI3, 2B4, and TIGIT, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and/or a basic signaling domain (e.g., the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of a PD1 molecule or fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain, described in this document).

In another aspect, the invention relates to a method of producing a cell comprising transducing a T cell with a vector containing a nucleic acid encoding a CAR, such as a CAR described herein.

The present invention also relates to a method for producing a population of cells with genetically altered RNA, such as cells described herein, such as T cells, transiently expressing exogenous RNA. The method comprises introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid encoding a CAR molecule described herein.

In another aspect, the invention relates to a method for generating anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell comprising a CAR molecule, for example, a cell expressing a CAR molecule as described herein. In one embodiment, the cell is an autologous T cell. In one embodiment, the cell is an allogeneic T cell. In one embodiment, the mammal is a human.

In another aspect, the invention relates to a method of treating a mammal suffering from a disease associated with CD19 expression, comprising administering to the mammal an effective amount of a cell comprising a CAR molecule, for example, a CAR molecule described herein.

In one embodiment, the disease associated with CD19 expression is selected from a proliferative disease such as cancer or a malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome or preleukemia, or it is a non-cancer symptom associated with CD19 expression. In one embodiment, the disease is a blood cancer. In one embodiment, the blood cancer is leukemia. In one embodiment, the cancer is selected from the group consisting of one or more forms of acute leukemia, including but not limited to B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more forms of chronic leukemia, including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); other blood cancers or blood disorders, including, but not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia", which are a diverse collection of blood disorders that have in common inefficient production (or Myeloid dysplasia (dysplasia) of blood myeloid cells, and diseases associated with CD19 expression include, but are not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases in which CD19 is expressed; and any combinations thereof.

In one embodiment, lymphocyte infusion, such as allogeneic lymphocyte infusion, is used in the treatment of cancer, wherein the infused lymphocytes comprise at least one CD19 CAR-expressing cell. In one embodiment, autologous lymphocyte infusion is used in the treatment of cancer, wherein the autologous lymphocytes infused comprise at least one CD19 CAR-expressing cell.

In one embodiment, a CD19 CAR-expressing cell, such as a T cell, is administered to a subject who has previously received a stem cell transplant, such as an autologous stem cell transplant.

In one embodiment, a CD19 CAR-expressing cell, such as a T cell, is administered to a subject who has previously received a dose of melphalan.

In one embodiment, cells expressing a CAR molecule, such as a CAR molecule described herein, are administered in combination with an agent that enhances the efficacy of the cell expressing the CAR molecule, such as an agent described herein.

In one embodiment, cells expressing a CAR molecule, such as a CAR molecule described herein, are administered in combination with an agent that mitigates one or more side effects associated with administration of a cell expressing the CAR molecule, such as an agent described herein.

In one embodiment, cells expressing a CAR molecule, such as a CAR molecule described herein, are administered in combination with an agent that treats a CD19-associated disease, such as an agent described herein.

In one embodiment, cells expressing a CAR molecule, such as a CAR molecule described herein, are administered at a dose and/or dosing regimen described herein.

In one embodiment, a CAR molecule is introduced into T cells, for example, using in vitro transcription, and a subject (e.g., a human) is initially administered cells containing the CAR molecule, with one or more subsequent administrations of cells containing the CAR molecule, wherein one or more subsequent administrations are performed less than 15 days, such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of cells containing the CAR molecule is performed for a subject (e.g., a human) per week, such as 2, 3, or 4 administrations of cells containing the CAR molecule per week. In one embodiment, a subject (e.g., a human subject) receives more than one administration of cells containing a CAR molecule per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle), followed by a week without administration of cells containing a CAR molecule, and then one or more additional administrations of cells containing a CAR molecule (e.g., more than one administration of cells containing a CAR molecule per week) are performed on the subject. In another embodiment, a subject (e.g., a human subject) receives more than one cycle of administration of cells containing a CAR molecule, and the time period between cycles is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the cells containing a CAR molecule are administered every other day, which is 3 administrations per week. In one embodiment, the cells containing a CAR molecule are administered for at least two, three, four, five, six, seven, eight, or more weeks.

In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered as a first-line therapy for a disease, for example, a cancer, for example, a cancer described herein. In another embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered as a second-, third-, or fourth-line therapy for a disease, for example, a cancer, for example, a cancer described herein.

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

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR of the invention, an isolated CAR polypeptide molecule of the invention, a vector comprising a CAR of the invention, and a cell comprising a CAR of the invention for use as a medicament.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR of the invention, an isolated CAR polypeptide molecule of the invention, a vector comprising a CAR of the invention, and a cell comprising a CAR of the invention for use in the treatment of a disease in which CD19 is expressed.

In one aspect, the invention relates to a population of autologous cells that are transfected or transduced with a vector comprising a nucleic acid molecule encoding a CD19 CAR molecule, such as described herein. 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 (e.g., by transfection or electroporation) to a cell, such as a T cell, wherein the vector comprises a nucleic acid molecule encoding a CD19 CAR molecule, such as described herein, which is transcribed as an mRNA molecule, and the CD19 CAR molecule is translated from the RNA molecule and expressed on the cell surface.

In another aspect, the present invention relates to a population of CAR-expressing cells, such as CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CART cells may comprise a first cell expressing a CAR having an anti-CD19 binding domain as described herein, and a second cell expressing a CAR having a different anti-CD19 binding domain, such as an anti-CD19 binding domain as described herein, which differs from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, the population of CAR-expressing cells may comprise a first cell expressing a CAR having an anti-CD19 binding domain, such as described herein, and a second cell expressing a CAR having 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 cell expressing a CAR having a major intracellular signaling domain and a second cell expressing a CAR having an auxiliary signaling domain.

In another aspect, the present invention relates to a population of cells in which at least one cell expresses a CAR having an anti-CD19 domain as described herein, and a second cell expresses another agent, for example, 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 an inhibitory molecule comprises a first polypeptide, for example, an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, for example, an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TEVI3, 2B4, and TIGIT, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them) and a second polypeptide that is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and/or a basic signaling domain (e.g., the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of a PD1 molecule or fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain, described in this document).

In one embodiment, a nucleic acid molecule encoding a CD19 CAR molecule, such as those described herein, is expressed as an mRNA molecule. In one embodiment, genetically modified CD19 CAR-expressing cells, such as T cells, can be produced by transfecting or electroporating an RNA molecule encoding the desired CAR (e.g., without a vector sequence) into the cell. In one embodiment, the CD19 molecule is translated from the RNA molecule after its introduction and expressed on the surface of the recombinant cell.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1A, 1B, and 1C graphically represent the results of a cytotoxicity assay using ND317 T cells (normal donor) transduced with a murine anti-CD19 CAR or a humanized anti-CD19 CAR of the invention and cultured with either control K562 cells that do not express CD19 (K562cc), as shown in Figure 1A, K562 cells transformed with CD19 (K562.CD19), as shown in Figure 1B, or malignant B cells obtained from a patient with CLL (B cell isolate Pt 14), as shown in Figure 1C.

Figures 2A and 2B show graphs demonstrating the proliferative response of cells expressing humanized and murine anti-CD19 CARs to CD19+ cells, with higher numbers of viable CAR+ T cells correlating with populations exhibiting maximal proliferation of CD4+ and CD8+ T cells in response to primary CLL cells.

Figure 3 graphically shows the expanded HPLC mass spectra for scFvs of the invention, with the top row corresponding to the unprocessed scFv and the bottom row corresponding to the related deglycosylated scFv.

Figure 4 graphically depicts conformational stability as measured by differential scanning fluorimetry. The Tm for murine scFv was 57°C (bold line). All humanized scFv variants exhibited higher Tm values of approximately 70°C compared to the parental murine scFv. Residues introduced during humanization resulted in an increase in Tm of more than 10°C.

Figure 5 graphically depicts the proliferation of CD19-transduced CAR T cells, wherein the CART19 cells were directed against either (a) a chronic myelogenous leukemia (“CML”) cell line that is negative for CD19 expression and was therefore used as a negative control; (b) recombinant K562 cells that are positive for CD19 expression and were therefore used as a positive control; or (c) Pt14 B cells derived from a patient with CLL that express CD19 on the cell surface.

Figures 6A and 6B show schematic representations of representative CARs.

Figure 7 shows the progression of primary HALLX5447 ALL disease in NSG mice after treatment with CD19-transduced CAR T cells. Primary human ALL cell expansion in NSG mice after treatment with CD19-specific CAR T cells demonstrated control of disease progression. The mean percentage of human CD19+ ALL cells in peripheral blood of NSG mice served as an indicator of disease burden at day 65 after tumor implantation. Black circles: mice received 100 μL of PBS via the tail vein; red squares: mice received surrogate transduced T cells; blue triangles: mice received murine CD19 CAR T cells; and inverted purple triangles: mice received humanized CD19 CAR T cells. Statistical significance was calculated by ANOVA; * denotes P<0.01.

Figure 8 shows CD19 expression in patient tumor cells. CD138+CD45 ^dim tumor cells were stained for CD19 (x-axis) and CD38 (y-axis).

DETAILED DESCRIPTION

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains.

Singular nouns correspond to the grammatical form "one or more than one" (that is, at least one) objects. For example, "element" means one element or more than one element.

The expression "about" when used in connection with a measurand such as a quantity, a time duration, and the like, covers variations within approximately ±20% or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the stated value, insofar as such variations are appropriate for carrying out the described methods.

The term "chimeric antigen receptor" or, alternatively, "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule described below. In one aspect, the stimulatory molecule is a zeta chain associated with a T-cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule described below. In one aspect, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, and/or CD28. In one aspect, a CAR is 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 stimulatory molecule. In one aspect, a CAR is 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 costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR is 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 costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR is 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 costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity through specific signaling pathways, generating second messengers, or acting as effectors in response to such messengers.

As used herein, the term "CD19" refers to cluster of differentiation protein 19, which is an antigenic determinant expressed on leukemic progenitor cells. Human and mouse amino acid and nucleotide sequences can be found in publicly available databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt/Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. CD19 is expressed on most cancer cells of the B-cell lineage, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells expressing CD19 are listed below, where the definition of "CD19-associated disease" is provided. It is also an early marker of B-cell precursors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the antigen-binding portion of CART recognizes and binds antigen in the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed on a cancer cell.

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, contain multiple or single chains, or be intact immunoglobulins, and may be obtained from natural sources or from recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

The term "antibody fragment" means at least one portion of an intact antibody or recombinant variants thereof, and refers to an antigen-binding domain, such as an antigen-binding variable region of an intact antibody, sufficient to provide recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments. The term "scFv" means a fusion protein that comprises at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are linked sequentially by a short flexible polypeptide linker, and which can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, the scFv described herein may have the VL and VH variable regions in any order, for example, in the direction from the N-terminus to the C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

A portion of a CAR composition of the invention comprising an antibody or fragment thereof may exist in various forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, 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 aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In another aspect, the CAR comprises an antibody fragment that is an scFv.

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their natural conformations and which generally defines the class to which an antibody belongs.

The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their natural conformations. Kappa (κ) and lambda (λ) light chains are the two major isotypes of antibody light chains.

The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed in a bacteriophage or yeast expression system. The term also refers to an antibody produced by synthesizing a DNA molecule encoding the antibody, from which the antibody protein or amino acid sequence defining the antibody is expressed, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequencing technologies that are available and widely known in the art.

The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either the production of antibodies, the activation of specific immunologically competent cells, or both. One skilled in the art understands that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. One skilled in the art understands that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen" within the meaning of this definition. Furthermore, one skilled in the art understands that an antigen need not be encoded exclusively by the full-length nucleotide sequence of a gene. It is clear that the present invention includes, but is not limited to, the use of a partial nucleotide sequence of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response. Furthermore, one skilled in the art understands that an antigen need not be encoded by a "gene." Clearly, an antigen can be synthesized or obtained from a biological sample, or can be another macromolecule in addition to a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a fluid containing other biological components.

The term "antitumor effect" refers to a biological effect that can be manifested in a variety of ways, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell count, a reduction in metastases, an increase in life expectancy, a decrease in tumor cell proliferation, a decrease in tumor cell survival, or an attenuation of various physiological symptoms associated with cancer. An "antitumor effect" can also be manifested through the ability of the peptides, polynucleotides, cells, and antibodies of the invention to initially prevent tumor formation.

The term "autologous" refers to any material obtained from the same individual into which it will subsequently be reintroduced.

The term "allogeneic" refers to any material obtained from another animal of the same species as the individual being administered the material. Two or more individuals are said to be allogeneic with respect to each other when the genes at one or more loci are not identical. In some respects, allogeneic material from individuals of the same species may differ genetically enough to be antigenic.

The term "xenogeneic" refers to a transplant obtained from an animal of another species.

The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various types of cancer are described herein and include, 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 the like.

The definition of "a disease associated with CD19 expression" includes, but is not limited to, a disease associated with CD19 expression or a condition associated with cells that express CD19, including, for example, proliferative diseases such as cancer or malignancy, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or non-cancerous symptoms associated with cells that express CD19. In one aspect, a cancer associated with CD19 expression is a blood cancer. In one aspect, the blood cancer is leukemia or lymphoma. In one aspect, cancer associated with CD19 expression includes forms of cancer and malignancies, including, but not limited to, for example, one or more forms of acute leukemia, including, but not limited to, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more forms of chronic leukemia, including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Other blood cancers or blood conditions associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," representing a diverse collection of blood conditions. which share ineffective production (or dysplasia) of myeloid blood cells, and the like. Other diseases associated with CD19 expression include, but are not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression. Non-cancer symptoms associated with CD19 expression include, but are not limited to, autoimmune disease (e.g., lupus), inflammatory diseases (allergy and asthma), and transplantation.

The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody or antibody fragment comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. 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). Thus, one or more amino acid residues in a CAR of the invention can be replaced with other amino acid residues from a family of residues with the same side chains, and the altered CAR can be tested using the functional assays described herein.

The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., the TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate changes in the expression of certain molecules, such as downregulation of TGF-β, and/or reorganization of cytoskeletal structures, etc.

The term "stimulatory molecule" means a molecule expressed by a T cell that provides a core cytoplasmic signaling sequence(s) that regulates the core activation of the TCR complex in a stimulatory manner for at least some aspects of the T cell signaling pathway. In one aspect, the core signal is initiated, for example, by binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, resulting in the mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The core cytoplasmic signaling sequence (also referred to as the "core signaling domain") that acts in a stimulatory manner may comprise a signaling motif known as an immune receptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing major cytoplasmic signal sequences that are particularly useful for 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 particular CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signal sequence, such as a CD3-zeta major signal sequence. In particular CARs of the invention, the CD3-zeta major signal sequence is the sequence set forth in SEQ ID NO: 17, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, and the like. In a particular CAR of the invention, the CD3-zeta core signal sequence is the sequence set forth in SEQ ID NO: 43, or equivalent residues from a non-human species, such as mice, rodents, monkeys, apes, and the like.

The term "antigen-presenting cell" or "APC" refers to an immune cell, such as a helper cell (e.g., a B cell, dendritic cell, etc.), that displays a foreign antigen complexed with major histocompatibility complexes (MHC) on its surface. T cells can recognize these complexes through their T cell receptors (TCRs). APCs process the antigens and present them to T cells.

As used herein, the term "intracellular signaling domain" refers to the intracellular portion of the molecule. The intracellular signaling domain generates a signal that stimulates the immune effector function of a CAR-containing cell, such as a CART cell. Examples of immune effector function, such as in a CART cell, include cytolytic activity and helper activity, including cytokine secretion.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Illustrative primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Illustrative costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may comprise a cytoplasmic sequence from a T-cell receptor, and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a coreceptor or costimulatory molecule.

The major intracellular signaling domain may contain a signaling motif known as an immune receptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing major cytoplasmic signaling sequences 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.

The term "zeta" or, alternatively, "zeta chain", "CD3 zeta" or "TCR zeta" refers to the protein having GenBan accession number BAG36664.1, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, and the like, and the "zeta stimulatory domain" or, alternatively, the "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the primary signal required for T cell activation. In one aspect, the cytoplasmic zeta domain comprises residues 52 to 164 of the protein with GenBank accession number BAG36664.1 or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, and the like, that are functional orthologs thereof. In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence set forth in SEQ ID NO: 17. In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence set forth in SEQ ID NO: 43.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory T cell response, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are essential for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and the Toll-like receptor, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), and 4-1BB (CD137).

The costimulatory intracellular signaling domain may be the intracellular portion of a costimulatory molecule. The costimulatory molecule may be present in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include 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, CD83-specific binding ligand, and the like.

The intracellular signaling domain may comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

The term "4-1BB" refers to a member of the TNFR superfamily with the amino acid sequence having the GenBank accession number AAA62478.2, or equivalent residues from a non-human species, such as mice, rodents, monkeys, apes, and the like; and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of the protein with the GenBank accession number AAA62478.2, or equivalent residues from a non-human species, such as mice, rodents, monkeys, apes, and the like. In one aspect, the "4-1BB costimulatory domain" is the sequence set forth in SEQ ID NO: 16 or equivalent residues from a non-human species, such as mice, rodents, monkeys, apes, and the like.

The term "coding" refers to the intrinsic property of certain nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes, having either a specific nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a specific amino acid sequence, and the resulting biological properties. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to the gene results in the formation of the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically listed in sequence listings, and the noncoding strand used as a template for transcription of a gene or cDNA may be said to encode a protein or other product of the gene or cDNA.

Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate variants of one another and that encode the same amino acid sequence. The term "nucleotide sequence encoding a protein or RNA" may also include introns if the nucleotide sequence encoding the protein may, in some variants, contain intron(s).

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, drug, material, or composition described herein that is effective in achieving a particular biological result.

The term "endogenous" refers to any material derived from, or produced within, an organism, cell, tissue, or system.

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

The term "expression" means the transcription and/or translation of a specific nucleotide sequence driven by a promoter.

The term "transfer vector" refers to a composition of chemically linked components containing an isolated nucleic acid that can be used to deliver the isolated nucleic acid into a cell. A variety of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or virus. It should be understood that the term also includes non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression vector" means a vector containing a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other expression elements may be provided by the host cell or present in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that contain a recombinant polynucleotide.

The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are capable of delivering significant amounts of genetic information into the host cell's DNA, making them one of the most effective gene delivery vectors. HIV, SIV, and FIV are examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, but not limited to, the self-inactivating lentiviral vector described in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical practice include, but are not limited to, the ^{LENTIVECTOR®} gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Non-clinically used types of lentiviral vectors are also available and known to those skilled in the art.

The term "homologous" or "identity" refers to the identity of subunit sequences between two polymer molecules, such as between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomeric subunit, such as if a position in each of two DNA molecules is occupied by an adenine residue, then they are homologous, or identical, at that position. The homology between two sequences is directly related to the number of positions that match or are homologous; for example, if half of the positions (e.g., five positions in a ten-subunit polymer) in the two sequences are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments are human immunoglobulins (the recipient antibody or antibody fragment) in which residues from the complementarity-determining region (CDR) of the recipient are replaced with residues from the CDR of a non-human species (the donor antibody), such as mouse, rat, or rabbit, that have the desired specificity, affinity, and functionality. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced with corresponding non-human residues. Moreover, a humanized antibody/antibody fragment may contain residues that are not found in either the recipient antibody or the introduced CDR or framework sequences. Such modifications can further refine and optimize the performance of the antibody or antibody fragment. Typically, a humanized antibody or antibody fragment will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions are human immunoglobulin sequences. The humanized antibody or antibody fragment may also contain at least a portion of the constant region (Fc) of an immunoglobulin, typically a human immunoglobulin. For more 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.

The term "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, when the entire molecule is derived from a human or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

The term "isolated" means altered or removed from its natural environment. For example, a nucleic acid or peptide naturally present in an animal's body is not "isolated," but the same nucleic acid or peptide, partially or completely separated from its naturally occurring environment, is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as a host cell.

In the context of the present invention, the following abbreviations for commonly occurring nucleic acid bases are used: "A" stands for adenosine, "C" stands for cytosine, "G" stands for guanosine, "T" stands for thymidine, and "U" stands for uridine.

The terms "functionally linked" or "transcriptional control" refer to the functional relationship between a regulatory sequence and a heterologous nucleotide sequence, resulting in the expression of the latter. For example, a first nucleotide sequence is functionally linked to a second nucleotide sequence if the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is functionally linked to a coding sequence if the promoter influences the transcription or expression of the coding sequence. Functionally linked DNA sequences can be adjacent to each other and, for example, if two protein-coding regions need to be joined, be in the same reading frame.

The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral administration methods, or infusion.

The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), as well as polymers thereof, in both single- and double-stranded forms. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to those of the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly specified. In particular, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (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)).

The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can make up a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, typically referred to in the art as proteins, which come in a wide variety of types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptide includes a natural peptide, a recombinant peptide, or a combination of both.

The term "promoter" means a DNA sequence recognized by the cellular or imported synthetic apparatus, which is necessary for the initiation of specific transcription of a polynucleotide sequence.

The term "promoter/regulatory sequence" refers to a nucleotide sequence required for the expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, this sequence may be the core sequence of the promoter, while in other cases, this sequence may also include an enhancer sequence and other regulatory elements necessary for the expression of the gene product. A promoter/regulatory sequence may, for example, be a sequence that results in tissue-specific expression of the gene product.

The term "constitutive" promoter means a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the cell to produce the gene product under most or all physiological conditions in the cell.

The term "inducible" promoter means a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

The term "tissue-specific" promoter means a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product substantially only if the cell is a cell of the tissue type corresponding to the promoter.

The term "flexible polypeptide linker" or "linker" as used in the context of scFv means a peptide linker consisting of amino acids such as glycine and/or serine residues, used alone or in combination to link together a heavy chain variable region and a light chain variable region. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises 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, flexible polypeptide linkers include, but are not limited to, (Gly ₄ Ser) ₄ (SEQ ID NO: 106) or (Gly ₄ Ser) ₃ (SEQ ID NO: 107). In another embodiment, linkers include multiple repeats of (Gly ₂ Ser), (GlySer) or (Gly ₃ Ser) (SEQ ID NO: 108). The invention also includes linkers described in WO 2012/138475, the contents of which are incorporated herein by reference).

As used herein, the term "5'cap" (also called RNA cap, RNA 7-methylguanosine cap, or RNA ^m7G cap) is a modified guanine nucleotide added "upstream" or at the 5' end of eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group that is linked to the first nucleotide to be transcribed. Its presence is critical for ribosome recognition and protection from RNases. Cap addition is coupled to transcription and occurs cotranscriptionally, such that each event influences the other. Shortly after transcription begins, the 5' end of nascent mRNA is bound by the cap-synthesizing complex, which is associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions required for mRNA capping. Synthesis occurs as a multistep biochemical reaction. The capping moiety can be modified to modulate functional properties of mRNA, such as its stability or translation efficiency.

As used herein, the term " in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro . Typically, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains the template used to create in vitro transcribed RNA.

As used herein, the term "poly(A)" refers to a series of adenosine residues added to mRNA by polyadenylation. In a preferred embodiment of a transient expression construct, the polyA is between 50 and 5000 (SEQ ID NO: 109), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate functional properties of mRNA, such as localization, stability, or translation efficiency.

As used herein, the term "polyadenylation" refers to the covalent attachment of a polyadenyl moiety or a modified version thereof to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) sequence is a long sequence of adenine nucleotides (often several hundred) added to pre-mRNA by the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) sequence is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) sequence and the protein associated with it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA exit from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after DNA is transcribed into RNA, but can also occur later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved by 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 mRNA cleavage, adenosine residues are added to the free 3' end at the cleavage site.

As used herein, the term "transient" refers to the expression of a non-integrated transgene over a period of hours, days, or weeks, wherein the expression time period is shorter than the time period for expression of the gene if it is integrated into the genome or is in a stable plasmid replicon in the host cell.

The term "signal transduction pathway" refers to the biochemical interactions between various signal transduction molecules that play a role in transmitting a signal from one part of a cell to another. The term "cell surface receptor" encompasses molecules and complexes of molecules capable of receiving and transmitting a signal across the cell membrane.

The term "subject" includes living organisms in which an immune response can be induced (e.g. mammals, humans).

The term "substantially purified" cell means a cell that is substantially free of other cell types. A substantially purified cell also means a cell that has been separated from other cell types with which it is normally associated in its natural state. In some cases, a population of substantially purified cells means a homogeneous population of cells. In other cases, the term simply means a cell that has been separated from the cells with which it is naturally associated in its natural state. In some aspects, the cells are cultured in vitro . In other aspects, the cells are not cultured in vitro .

As used in this document, the term "therapeutic" means curative. A therapeutic effect is achieved by alleviating, suppressing, remitting, or eliminating a disease state.

As used herein, the term "prophylaxis" means the prevention or preventive treatment of a disease or condition.

In the context of the present invention, the terms "tumor antigen" or "hyperproliferative disease antigen" or "hyperproliferative disease-associated antigen" refer to antigens that are typically characteristic of certain hyperproliferative diseases. In some aspects, the hyperproliferative disease antigens of the present invention are obtained from malignant tumors, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemias, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

The term "transfection" or "transformation" or "transduction" refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. A cell includes the subject's primary cell and its progeny.

The term "specifically binds" refers to an antibody or ligand that recognizes and binds a corresponding binding partner protein (e.g., a stimulatory and/or costimulatory molecule present on a T cell) present in a sample, but the antibody or ligand does not significantly recognize or bind other molecules in the sample.

Ranges: Throughout this specification, various aspects of the invention may be presented in range format. It should be understood that the use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a range description should be construed as specifically disclosing all possible subranges, as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose such subranges as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and so on, as well as individual numerical values within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98%, or 99% identity, and includes such subranges as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

Description

The present invention relates to compositions and methods of using the same for treating a disease such as cancer using humanized anti-CD19 chimeric antigen receptors (CARs).

In one aspect, the invention provides a series of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment engineered to enhance binding to the CD19 protein. In one aspect, the invention provides a cell (e.g., a T cell) genetically altered to express a CAR, wherein the CAR T cell ("CART") exhibits anti-tumor properties. In one aspect, the cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding the 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, the cell can stably express the CAR. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell can transiently express the CAR.

In one aspect, the binding portion of a CAR directed against the CD19 protein is an scFv antibody fragment. In one aspect, such antibody fragments are functional in that they retain equivalent binding affinity, for example, they bind the same antigen with an efficiency comparable to that of the IgG antibody from which they are derived. In one aspect, such antibody fragments are functional in that they provide a biological response, which may include, but is not limited to, activation of an immune response, inhibition of signal transduction from its target antigen, inhibition of kinase activity, and the like, as will be understood by one of skill in the art. In one aspect, the anti-CD19 antigen-binding domain of a CAR is an scFv antibody fragment that is humanized compared to the murine scFv sequence from which it is derived. In one aspect, the mouse scFv parent sequence is the CAR19 construct described in PCT publication WO 2012/079000 and set forth herein as SEQ ID NO: 58.

In some aspects, the antibodies of the invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises the polypeptide sequence set forth as SEQ ID NO: 12 in PCT publication WO 2012/079000 and set forth herein as SEQ ID NO: 58, wherein the scFv domain is replaced with one or more sequences selected from SEQ ID NOS: 1-12. In one aspect, the scFv domains of SEQ ID NOS: 1-12 are humanized versions of the scFv domain of SEQ ID NO: 59, which is a murine scFv antibody fragment that specifically binds to human CD19. Humanization of this murine scFv may be desirable for clinical application where mouse-specific residues can induce a human immune response to mouse antigen (HAMA) in patients receiving CART19-mediated treatment, such as treatment with T cells transduced with the CAR19 construct.

In one aspect, an anti-CD19 binding domain, e.g., a humanized scFv, part of a CAR of the invention, is encoded by a transgene whose sequence has been codon-optimized for expression in a mammalian cell. In one aspect, the entire CAR construct of the invention is encoded by a transgene whose entire sequence has been codon-optimized for expression in a mammalian cell. Codon optimization relies on the discovery that the frequency of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA varies across species. This codon degeneracy results in the same polypeptide being encoded by different nucleotide sequences. A variety of methods for codon optimization are known in the art and include, for example, those described in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 1.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 2.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 3.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 4.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 5.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 6.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 7.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 8.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 9.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 10.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 11.

In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 12.

In one aspect, the CAR of the invention combines the antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, the intracellular signaling molecule includes, but is not limited to, the CD3 zeta chain, 4-1BB, and CD28 signaling modules, as well as combinations thereof. In one aspect, the antigen-binding domain binds to CD19. In one aspect, the CD19 CAR is a CAR selected from the sequences set forth in one or more of SEQ ID NOS: 31-42. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 31. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 32. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 33. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 34. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 35. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 36. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 37. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 38. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 39. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 40. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 41. In one aspect, the CD19 CAR comprises the sequence set forth in SEQ ID NO: 42.

Furthermore, the present invention relates to CD19 CAR compositions and their use in medicaments or methods for treating, among other diseases, cancer or any malignant neoplasms or autoimmune diseases in which cells or tissues expressing CD19 are involved.

In one aspect, the CAR of the invention can be used to kill CD19-expressing normal cells, making them suitable for cellular therapy prior to cell transplantation. In one aspect, the CD19-expressing normal cell is a CD19-expressing normal stem cell, and the cell transplantation is a stem cell transplant.

In one aspect, the invention relates to a cell (e.g., a T cell) genetically altered to express a chimeric antigen receptor (CAR), wherein the CAR T cell ("CART") exhibits antitumor properties. A preferred antigen is CD19. In one aspect, the antigen-binding domain of the CAR comprises a partially humanized fragment of an anti-CD19 antibody. In one aspect, the antigen-binding domain of the CAR comprises a partially humanized fragment of an anti-CD19 antibody that is an scFv. Accordingly, the invention relates to a CD19 CAR that comprises a humanized anti-CD19 binding domain and is genetically engineered into a T cell, as well as methods for using it for adoptive therapy.

In one aspect, the CD19 CAR comprises at least one intracellular domain selected from the group consisting of the CD137 (4-1BB) signaling domain, the CD28 signaling domain, the CD3-zeta signaling domain, and any combinations thereof. In one aspect, the CD19 CAR comprises at least one intracellular signaling domain of one or more costimulatory molecules other than CD137 (4-1BB) or CD28.

Chimeric antigen receptor (CAR)

The present invention relates to a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises a humanized antibody fragment that specifically binds to CD19, for example, human CD19, wherein the sequence of the antibody fragment is adjacent to and is in the same reading frame as a nucleotide sequence encoding an intracellular signaling domain. The intracellular signaling domain may comprise a costimulatory signaling domain and/or a basic signaling domain, for example, a zeta chain. A costimulatory signaling domain refers to a portion of a CAR that comprises at least a portion of the intracellular domain of a costimulatory molecule.

In particular aspects, a CAR construct of the invention comprises an scFv domain selected from the group consisting of SEQ ID NOS: 1-12, wherein the scFv may optionally be preceded by a leader sequence such as SEQ ID NO: 13, and wherein the scFv may optionally be followed by a hinge sequence such as SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region such as SEQ ID NO: 15, an intracellular signaling domain that includes SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3-zeta sequence that includes SEQ ID NO: 17 or SEQ ID NO: 43, wherein the domains are contiguous and in frame, thereby forming a fusion protein. Also included in the invention is a nucleotide sequence encoding a polypeptide of each of the scFv fragments, 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. Also included in the invention is a nucleotide sequence encoding a polypeptide of each of the scFv fragments, 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, and each of the domains of SEQ ID NOS: 13-17, plus the encoded CD19 CAR fusion protein of the invention. In one aspect, exemplary CD19 CAR constructs comprise an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, an exemplary CD19 CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain. Specific CD19 CAR constructs comprising humanized scFv domains of the invention are set forth as SEQ ID NOS: 31-42.

The full-length CAR sequences are also provided herein at SEQ ID NOS: 31-42, as shown in Table 3.

An exemplary leader sequence is set forth in SEQ ID NO: 13. An exemplary hinge/spacer sequence is set forth in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49. An exemplary transmembrane domain sequence is set forth in SEQ ID NO: 15. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is set forth in SEQ ID NO: 16. An exemplary sequence of the intracellular signaling domain of CD27 is set forth in SEQ ID NO: 51. An exemplary sequence of the CD3-zeta domain is set forth in SEQ ID NO: 17 or SEQ ID NO: 43.

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

In one aspect, the present invention relates to a recombinant nucleic acid construct comprising a transgene encoding a CAR, wherein the nucleic acid molecule comprises a nucleotide sequence encoding an anti-CD19 binding domain selected from one or more of SEQ ID NOS: 61-72, wherein the sequence is adjacent to, and is in the same reading frame with, a nucleotide sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that can be used in a CAR includes, but is not limited to, one or more intracellular signaling domains, such as CD3-zeta, CD28, 4-1BB, and the like. In some cases, a CAR may comprise any combination of CD3-zeta, CD28, 4-1BB, and the like. In one aspect, the nucleotide sequence of the CAR construct of the invention is selected from one or more of SEQ ID NOS: 85-96. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 85. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 86. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 87. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 88. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 89. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 90. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 91. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 92. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 93. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 94. In one aspect, the nucleotide sequence of the CAR construct is is SEQ ID NO: 95. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 96. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 97. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 98. In one aspect, the nucleotide sequence of the CAR construct is SEQ ID NO: 99.

Nucleotide sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the gene, extracting the gene from a vector known to contain it, or isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than by cloning.

The present invention includes retroviral and lentiviral vector constructs expressing CARs that can be directly transduced into a cell.

The present invention also includes an RNA construct that can be directly transfected into a cell. A method for producing mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by the addition of polyA to produce a construct containing the 3' and 5' untranslated sequence ("UTR"), the 5' cap and/or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA sequence, typically 50-2000 bases in length (SEQ ID NO: 118). The RNA thus obtained can efficiently transfect various cell types. In one embodiment, the template includes sequences for a CAR. In one embodiment, the CAR RNA vector is transduced into a T cell by electroporation.

Antigen-binding domain

In one aspect, the CAR of the invention comprises a target-specific binding element, otherwise known as an antigen-binding domain. The choice of moiety depends on the type and number of ligands that define the target cell surface. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a specific disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of the CAR of the invention include markers associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

In one aspect, a CAR-mediated T cell response can be directed to an antigen of interest by engineering an antigen-binding domain that specifically binds the desired antigen to the CAR.

In one aspect, the antigen-binding domain portion of the CAR comprises an antigen-binding domain directed to CD19. In one aspect, the antigen-binding domain is directed to human CD19. In one aspect, the antigen-binding domain of the CAR has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

The antigen-binding domain may be any domain that binds to an antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including, but not limited to, a single-domain antibody such as the variable heavy chain (VH), variable light chain (VL), and variable domain (VHH) of a nanobody derived from camelid antibodies, as well as an alternative scaffold known in the art that acts as an antigen-binding domain, such as a recombinant fibronectin domain, and the like. In some cases, it is beneficial if the antigen-binding domain is derived from an organism of the same species for which the CAR will ultimately be used. For example, in the case of use in humans, it may be beneficial if the antigen-binding domain of the CAR contains human or humanized residues in the antigen-binding domain of an antibody or antibody fragment.

Thus, in one aspect, 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) of the light chain complementarity determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and/or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., 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 comprises one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, e.g., the humanized anti-CD19 binding domain has two heavy chain variable regions, each comprising the HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a humanized light chain variable region described herein (e.g., in Table 3) and/or a humanized heavy chain variable region described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain comprises a humanized heavy chain variable region as described herein (e.g., Table 3), e.g., at least two humanized heavy chain variable regions as described herein (e.g., Table 3). In one embodiment, the anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain with an amino acid sequence as set forth in Table 3. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the light chain variable region as set forth in Table 3, or a sequence having 95-99% identity to 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), but no more than 30, 20, or 10 modifications (e.g., substitutions), of the amino acid sequence of the heavy chain variable region set forth in Table 3, or a sequence having 95-99% identity to the amino acid sequence in Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises 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 thereto. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting 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: 68, 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 humanized anti-CD19 binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein, for example, in Table 3, is linked to a heavy chain variable region comprising an amino acid sequence described herein, for example, in Table 3, via a linker, for example, a linker described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a linker (Gly ₄ -Ser)n, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region in the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In one aspect, the antigen-binding domain region comprises one or more sequences selected from SEQ ID NOS: 1-12. In one aspect, the humanized CAR is selected from one or more sequences selected from SEQ ID NOS: 31-42. In some aspects, the non-human antibody is humanized, wherein certain sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in the human body, or a fragment thereof. In one aspect, the antigen-binding domain is humanized.

A humanized antibody can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (see, e.g., European Patent No. EP 239400, International Patent Publication No. WO 91/09967, and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, the entire contents of each of which are incorporated herein by reference), veneering, or resurfacing (see, e.g., European Patent Nos. EP 592106 and EP 519596, Padlan, 1991, Molecular Immunology, 28(4/5): 489-498; Studnicka et al., 1994, Protein Engineering, 7(6): 805-814; and Roguska et al., 1995). al., 1994, PNAS, 91: 969-973, the entire contents of each of which are incorporated herein by reference), chain shuffling (see, e.g., U.S. Patent No. 5,565,332, the entire contents of which are incorporated herein by reference), as well as the methods disclosed in, e.g., U.S. Patent Application Publication No. US 2005/0042664, U.S. Patent Application Publication No. US 2005/0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Patent Publication No. WO 9317105, 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): 106–78 (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., 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), the entire contents of which are incorporated herein by reference. Frequently, framework residues in framework regions will be replaced by the corresponding residue from a CDR donor antibody to alter, for example, improve, antigen binding. These framework substitutions are determined by methods well known in the art, for example, by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unusual framework residues at specific positions. (See, for example, Queen et al., U.S. Patent No. 5,585,089 and Riechmann et al., 1988, Nature, 332:323, the entire contents of which are incorporated herein by reference.)

A humanized antibody or antibody fragment has one or more amino acid residues retained from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically derived from an "import" variable domain. As described herein, humanized antibodies or antibody fragments comprise one or more CDRs from non-human immunoglobulin molecules and framework regions in which the amino acid residues constituting the framework are derived entirely or substantially from human germline sequences. Numerous methods for humanizing antibodies or antibody fragments are well known in the art, and humanization can essentially be accomplished following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., performing CDR grafting (EP 239400, PCT Publication No. WO 91/09967 and U.S. Patent Nos. 4,816,567, 6,331,415, 5,225,539, 5,530,101, 5,585,089, 6,548,640, the entire contents of which are incorporated herein by reference). In such humanized antibodies and antibody fragments, significantly less than the intact human variable domain is replaced by the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are replaced by residues from analogous regions in rodent antibodies. Humanization of antibodies and antibody fragments can also be accomplished by coating or surface modification (EP 592106, EP 519596, 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), the entire contents of which are incorporated herein by reference.

The selection of human variable domains, both light and heavy chains, for use in the creation of humanized antibodies is aimed at reducing antigenicity. In the so-called "best-fit" method, the variable domain sequence of a rodent antibody is screened against the complete library of known human variable domain sequences. The human sequences that are closest to the rodent sequences are then selected as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993), Chothia et al., J. Mol. Biol., 196: 901 (1987), the entire contents of which are incorporated herein by reference). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (see, e.g., 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 entire contents of which are incorporated herein by reference). In some embodiments, the framework region, e.g., all four framework regions of the variable heavy chain, is derived from the VH4_4-59 germline sequence. In one embodiment, the framework region can comprise one, two, three, four, or five modifications, e.g., substitutions, e.g., from an amino acid in the corresponding mouse sequence (e.g., SEQ ID NO: 58). In one embodiment, the framework region, for example, all four framework regions of the light chain variable region, are derived from the VK3_1.25 germline sequence. In one embodiment, the framework region may comprise one, two, three, four, or five modifications, for example, substitutions, for example, from an amino acid in the corresponding mouse sequence (e.g., SEQ ID NO: 58).

In some aspects, the antibody fragment portion of the CAR composition of the invention is humanized while maintaining high affinity for the target antigen and other beneficial biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are produced by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available and familiar to those skilled in the art. Computer programs that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are also available. Studying these projections allows for analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, for example, by analyzing residues that influence the ability of the candidate immunoglobulin to bind the target antigen. Thus, FR residues can be selected and combined with recipient and imported sequences to achieve a desired characteristic of the antibody or antibody fragment, such as increased affinity for the target antigen. Typically, CDR residues directly and significantly influence antigen binding.

A humanized antibody or antibody fragment may retain the same antigen specificity as the parent antibody, for example, in the present invention, the ability to bind human CD19. In some embodiments, a humanized antibody or antibody fragment may have improved binding affinity and/or specificity for human CD19.

In one aspect, the anti-CD19 binding domain is characterized by particular functional features or properties of an antibody or antibody fragment. For example, in one aspect, a portion of a CAR composition of the invention that comprises the antigen-binding domain specifically binds human CD19. In one aspect, the antigen-binding domain has the same or similar binding specificity for human CD19 as scFv FMC63 described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention relates to an antigen-binding domain comprising an antibody or antibody fragment, wherein the binding domain of the antibody specifically binds to a CD19 protein or a fragment thereof, wherein the antibody or antibody fragment comprises a light chain variable domain and/or a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NOs: 1-12. In one aspect, the antigen-binding domain comprises an scFv amino acid sequence selected from SEQ ID NOs: 1-12. In some aspects, the scFv is adjacent to, and in frame with, a leader sequence. In one aspect, the leader sequence is the polypeptide sequence set forth in SEQ ID NO: 13.

In one aspect, the anti-CD19 binding domain is a fragment, such as a single-chain variable fragment (scFv). In one aspect, the anti-CD19 binding domain is an Fv, Fab, (Fab') ₂ , or a bifunctional (e.g., bispecific) chimeric antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention bind the CD19 protein with comparable or improved affinity to the wild-type.

In some cases, scFv fragments can be prepared by methods known in the art (see, for example, 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 prepared by linking together the VH and VL regions using flexible polypeptide linkers. scFv molecules contain a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The length of the linker can greatly influence how the variable regions of scFv fold and interact. Indeed, by using a short polypeptide linker (e.g., 5–10 amino acids long), intrachain folding is prevented. Interchain folding is also necessary to bring two variable regions together to form a functional epitope-binding site. Examples of linker orientation and dimensions can be found, for example, in Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90: 6444–6448, US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO 2006/020258 and WO 2007/024715, the contents of which are incorporated herein by reference.

ScFv may comprise a linker 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 or more amino acid residues between the VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises repeat groups of glycine and serine, such as (Gly ₄ Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO: 18). In one embodiment, the linker may be (Gly ₄ Ser) ₄ (SEQ ID NO: 106) or (Gly ₄ Ser) ₃ (SEQ ID NO: 107). Variations in linker length may help maintain or enhance activity, resulting in high performance in activity assays.

Stability and mutations

The stability of an anti-CD19 binding domain, such as an scFv molecule (e.g., a soluble scFv), can be assessed by comparison with the biophysical properties (e.g., thermal stability) of a conventional scFv control molecule or a full-length antibody. In one embodiment, the humanized scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than the thermal stability of a control binding molecule (e.g., a conventional scFv molecule) in the assays described.

The increased thermal stability of the anti-CD19 binding domain, e.g., scFv, is subsequently transferred 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 increased by at least about 2°C or 3°C compared to a conventional antibody. In one embodiment, the anti-CD19 binding domain, e.g., scFv, has a thermal stability increased by 1°C compared to a conventional antibody. In another embodiment, the anti-CD19 binding domain, e.g., scFv, has a thermal stability increased by 2°C compared to a conventional antibody. In another embodiment, the scFv has a thermal stability increased by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and the scFv molecules or Fab fragments of the antibody from which the VH and VL of the scFv were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

Mutations in scFv (resulting from humanization or direct mutagenesis of soluble scFv) alter the stability of scFv and increase the overall stability of scFv and the CART19 construct. The stability of humanized scFv is compared with that of murine scFv using parameters such as Tm, denaturation temperature, and aggregation temperature.

The binding capacity of mutant scFvs can be determined using the assays described in the examples.

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

Methods for assessing protein stability

The stability of the antigen-binding domain can be assessed using, for example, the methods described below. Such methods allow the determination of multiple thermally induced unfolding conformational transitions in which the least stable domain either unfolds first or limits the overall stability threshold of the multidomain unit that unfolds concertedly (e.g., a multidomain protein that exhibits a single unfolding conformational transition). The least stable domain can be determined using a variety of additional methods. Mutagenesis can be performed to investigate which domain limits overall stability. In addition, the protease stability of the multidomain protein can be studied under conditions where the least stable domain is known to actually unfold using DSC or other spectroscopic methods (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 part of it) can be used as a test sequence in the methods.

a) Thermal stability

The thermal stability of compositions can be analyzed using a variety of non-limiting biophysical or biochemical methods known in the art. In specific embodiments, thermal stability is assessed using analytical spectroscopy.

An illustrative method of analytical spectroscopy is differential scanning calorimetry (DSC). DSC uses a calorimeter that is sensitive to the heat absorption that accompanies 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 protein sample is inserted into the calorimeter, and the temperature is increased until the Fab or scFv unfolds. The temperature at which the protein unfolds is an indicator of its overall stability.

Another illustrative method of analytical spectroscopy is circular dichroism (CD) spectroscopy. CD spectrometry measures the optical activity of a compound as a function of temperature. Circular dichroism (CD) spectroscopy measures the difference in absorption of left- and right-polarized light, which arises from structural asymmetry. A distorted or unfolded structure results in CD spectra that differ significantly from the spectrum of an ordered or folded structure. The CD spectrum reflects the sensitivity of proteins to the denaturing effect of increasing temperature and is thus an indicator of the thermal stability of the protein (see van Mierlo and Steemsma, J. Biotechnol., 79(3): 281–98, 2000).

Another illustrative analytical spectroscopy method for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, supra). Another illustrative analytical spectroscopy method for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma, supra).

The thermal stability of a composition can be measured using biochemical methods. An exemplary biochemical method for assessing thermal stability is a temperature exposure assay. In a temperature exposure assay, a composition is exposed to a range of elevated temperatures for a specified period of time. For example, in one embodiment, scFv molecules or molecules containing scFv molecules are exposed to a range of increasing temperatures, for example, for 1-1.5 hours. The protein activity is then analyzed in an appropriate biochemical assay. For example, if the protein is a binding protein (e.g., an scFv or scFv-containing polypeptide), the binding activity of the binding protein can be determined using a functional or quantitative ELISA.

Such an assay can be performed in a high-throughput format, and such assays using E. coli and high-throughput screening are described in the Examples section. A library of anti-CD19 binding domains, such as scFv variants, can be generated using methods known in the art. Expression of an anti-CD19 binding domain, such as scFv, can be induced, and the anti-CD19 binding domain, such as scFv, can be exposed to elevated temperatures. The exposed test samples can be assayed for binding, and these stable anti-CD19 binding domains, such as scFv, can be produced in large quantities and further characterized.

Thermal stability is assessed by measuring the melting point (Tm) of the composition using any of the above methods (e.g., analytical spectroscopy methods). The melting point is the temperature at the midpoint of the thermal transition curve at which 50% of the molecules of the composition are in the folded state (see, e.g., Dimasi et al. (2009) J. Mol Biol. 393: 672–692). In one embodiment, the Tm for an anti-CD19 binding domain, e.g., scFv, is about 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, 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, 100°C. In one embodiment, the Tm value for IgG is about 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, 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, 100°C. In one embodiment, the Tm value for the multivalent antibody is about 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, 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, 100°C.

Thermal stability is also assessed by measuring the specific heat capacity or heat capacity (Cp) of the composition using an analytical calorimetry method (e.g., DSC). The specific heat capacity of a composition is the energy (e.g., in kcal/mol) required to raise the temperature of 1 mole of water by 1°C. A high Cp value is indicative of a denatured or inactive protein composition. The change in heat capacity (ΔCp) of a composition is measured by determining the specific heat capacity of the composition before and after its thermal transition. Thermal stability can also be assessed by measuring or determining other thermodynamic stability parameters, including 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., temperature exposure assay) is used to determine the temperature (i.e., the Tc value) at which 50% of the composition retains its activity (e.g., binding activity).

In addition, mutations in the anti-CD19 binding domain, e.g., scFv, result in a change in the thermal stability of the anti-CD19 binding domain, e.g., scFv, compared to a non-mutated anti-CD19 binding domain, e.g., scFv. When a humanized anti-CD19 binding domain, e.g., scFv, is included in a CART19 construct, the anti-CD19 binding domain, e.g., the humanized scFv, confers thermal stability to the entire anti-CD19 CART construct. In one embodiment, the anti-CD19 binding domain, e.g., scFv, comprises one 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, comprises multiple mutations that confers thermal stability to the anti-CD19 binding domain, e.g., scFv. In one embodiment, multiple mutations in an anti-CD19 binding domain, e.g., scFv, have a further impact on the thermal stability of the anti-CD19 binding domain, e.g., scFv.

b) % Aggregation

The stability of a composition can be determined by measuring its tendency to aggregate. Aggregation can be measured using a number of non-limiting biochemical or biophysical methods. For example, aggregation of a composition can be assessed using a chromatographic method, such as size-exclusion chromatography (SEC). In SEC, molecules are separated based on size. A column is filled with semi-solid polymer gel beads that allow ions and small molecules to penetrate, but not large molecules. When a protein composition is loaded onto the column, tightly folded proteins (i.e., non-aggregated proteins) are distributed across a larger volume of solvent than is possible for large protein aggregates. Consequently, large aggregates pass through the column more rapidly, and thus the mixture can be separated or fractionated into its components. Each fraction can be individually quantified (e.g., by light scattering) as it elutes from the gel. Accordingly, the % aggregation in the composition can be determined by comparing the concentration of the fraction to the total concentration of the protein loaded on the gel. Stable compounds elute from the column as a virtually single fraction and appear as virtually a single peak on the elution profile or chromatogram.

c) Binding affinity

The stability of a composition can be assessed by determining the binding affinity of the target. Many different methods for determining binding affinity are known in the art. An illustrative method for determining binding affinity uses surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows real-time analysis of biospecific interactions by detecting changes in protein concentrations in a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For more information, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19–26, Jonsson, U., et al. (1991) Biotechniques 11: 620–627, Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131 and Johnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

In one aspect, the antigen-binding domain of the CAR comprises an amino acid sequence that is 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 fragments described herein. In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In another aspect, the antibody fragment is an scFv.

In various aspects, the antigen-binding domain of a CAR is engineered by modifying one or more amino acids in one or both variable regions (e.g., VH and/or VL), such as one or more CDR regions and/or one or more framework regions. In one particular aspect, a CAR composition of the invention comprises an antibody fragment. In another aspect, the antibody fragment is an scFv.

One skilled in the art will appreciate that an antibody or antibody fragment of the invention can be further modified to differ in amino acid sequence (e.g., from the wild-type) but not in desired activity. For example, additional nucleotide substitutions can be made, resulting in amino acid substitutions at "nonessential" amino acid residues in the protein. For example, a nonessential amino acid residue in the molecule can be replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid sequence can be replaced with a structurally similar sequence that differs in the order and/or composition of side chain family members; for example, a conservative substitution can be made in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains are known in the art, including 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), non-polar 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).

Percent identity in the context of two or more nucleotide or polypeptide sequences is determined for two or more sequences that are the same. Two sequences are "substantially identical" if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 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% identity over a specified region or, if not specifically stated, over the entire sequence), when compared and aligned for maximum correspondence in a comparison window, or a specified region, as quantified using one of the following sequence comparison algorithms or when manual alignment and visual inspection. Optionally, the identity exists 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, 200 or more amino acids) in length.

To compare sequences, one sequence is typically taken as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified, if necessary, and the program parameters for sequence comparison are set. The default program parameters can be used or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequences relative to the reference sequence, based on the program parameters. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the Smith-Waterman local homology algorithm, (1970) Adv. Appl. Math. 2:482c, the Needleman-Wunsch global alignment algorithm, (1970) J. Mol. Biol. 48: 443, and the Pearson-Lipman similarity search method, (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444, using computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or using manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 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 analyses is publicly available through the National Center for Biotechnology Information.

The percentage identity between two amino acid sequences can also be determined using the algorithm described in E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4: 11–17, which is included in the ALIGN program (version 2.0), using the PAM120 residue substitution weight table, a deletion extension penalty of 12, and a deletion introduction penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman–Wunsch algorithm (1970) J. Mol. Biol. 48: 444–453), which is included in the GAP program in the GCG program package (available at www.gcg.com), using either the Blossom 62 or PAM250 template, and a penalty for introducing a deletion of 16, 14, 12, 10, 8, 6, or 4 and a penalty for continuing a deletion of 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention provides modifications of the parent amino acid sequence of an antibody or fragment (e.g., scFv) that produce functionally equivalent molecules. For example, the VH or VL of an anti-CD19 binding domain, e.g., scFv, contained in a CAR can be modified while maintaining 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% identity to the original VH or VL framework region of the anti-CD19 binding domain, e.g., scFv. The present invention provides modifications throughout the CAR construct, such as modifications in one or more amino acid sequences of different domains of the CAR construct, to produce functionally equivalent molecules. The CAR construct can be modified while maintaining 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% identity to the original CAR construct.

Transmembrane domain

With regard to the transmembrane domain, in various embodiments, a CAR can be designed that comprises a transmembrane domain linked to an extracellular domain of the CAR. The transmembrane domain can comprise one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids linked to the extracellular region of the protein from which the transmembrane region is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids from the extracellular region), and/or one or more additional amino acids linked to the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids from the intracellular region). In one aspect, a transmembrane domain is used that is a domain linked to one of the other domains of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or other surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on the surface of a CART cell. In another aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with the binding domains of a natural binding partner present in the same CART.

The transmembrane domain can be obtained from either a natural or a recombinant source. If the source is natural, the domain can be obtained from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of transmitting a signal to the intracellular domain(s) after the CAR has bound to the target. A transmembrane domain particularly useful for the present invention can comprise at least a transmembrane region(s) from, for example, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

In some cases, the transmembrane domain may be attached to the extracellular region of the CAR, such as the antigen-binding domain of the CAR, via a hinge, such as a hinge from a human protein. For example, in one embodiment, the hinge may be a hinge from a human Ig (immunoglobulin), such as an IgG4 hinge or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 14. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 15.

In one aspect, the hinge or spacer is an IgG4 hinge. For example, in one embodiment, the hinge or spacer is a hinge with the amino acid sequence

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 45).

In some embodiments, the hinge or spacer is a hinge encoded by a nucleotide sequence

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCAGCAGCATCGAG AAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACT ACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO: 46).

In one aspect, the hinge or spacer is an IgD hinge. For example, in one embodiment, the hinge or spacer is a hinge with an amino acid sequence

RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLLTLPRS LWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 47).

In some embodiments, the hinge or spacer is a hinge encoded by a nucleotide sequence

AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGG GCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCC CTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGA GGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO: 48).

In one aspect, the transmembrane domain may be recombinant, in which case it will contain predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan, and valine may be present at each end of the recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, 2 to 10 amino acids in length, can serve as a link between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet is a particularly suitable linker. For example, in one aspect, 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).

Cytoplasmic domain

The cytoplasmic domain or region of a CAR includes an intracellular signaling domain. The intracellular signaling domain is typically responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to a specialized function of the cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of the protein that transmits a signal to the effector function and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can typically be used, in many cases the entire chain is not necessary. If a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, provided that it transmits a signal to the effector function. Thus, the term "intracellular signaling domain" should include any truncated portion of the intracellular signaling domain sufficient to transmit a signal to the effector function.

Examples of intracellular signaling domains for use in the CAR of the invention include cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act together to initiate signal transduction upon interaction of an antigen with the receptor, as well as any derivative or variant of these sequences and any recombinant sequence having the same functional capacity.

It is known that signals generated by the TCR alone are insufficient for full T-cell activation and that an accessory and/or costimulatory signal is also required. Thus, it can be argued that T-cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (the primary intracellular signaling domains) and those that act in an antigen-independent manner to provide an accessory or costimulatory signal (the accessory cytoplasmic domain, e.g., the costimulatory domain).

The core signaling domain regulates basal activation of the TCR complex, either stimulatory or inhibitory. Core intracellular signaling domains that act stimulatory may contain signaling motifs known as immune receptor tyrosine-based activation motifs, or ITAMs.

Examples of ITAM-containing core intracellular signaling domains that are particularly useful for the present invention include the TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b, and CD66d domains. In one embodiment, a CAR of the invention comprises an intracellular signaling domain, such as the CD3-zeta core signaling domain.

In one embodiment, the primary signaling domain comprises a modified ITAM domain, for example, a mutant ITAM domain that has altered (e.g., increased or decreased) activity compared to a natural ITAM domain. In one embodiment, the primary signaling domain is a primary intracellular signaling domain comprising a modified ITAM, for example, a primary intracellular signaling domain comprising an optimized and/or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

The intracellular signaling domain of a CAR may be the CD3-zeta signaling domain itself, or it may be combined with any other desired intracellular signaling domain(s) useful in the context of the CAR of the invention. For example, the intracellular signaling domain of a CAR may comprise a portion of the CD3-zeta chain and a costimulatory signaling domain. A costimulatory signaling domain refers to the portion of a CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligands, that is necessary for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, etc. For example, costimulation of CD27 has been shown to enhance the growth, 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).

The intracellular signal sequences in the cytoplasmic portion of the CAR of the invention may be linked to one another in a random or specific order. Optionally, a short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), may serve as a linker between the intracellular signal sequences. In one embodiment, a glycine-serine doublet may be used as a suitable linker. In one embodiment, a single amino acid, for example, alanine, glycine, may be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed to contain two or more, for example, 2, 3, 4, 5, or more, costimulatory signaling domains. In one embodiment, the two or more, for example, 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, for example, a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is engineered to comprise a CD3-zeta signaling domain and a CD28 signaling domain. In one aspect, the intracellular signaling domain is engineered to comprise a CD3-zeta signaling domain and a 4-1BB signaling domain. In one aspect, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 16. In one aspect, the CD3-zeta signaling domain is the signaling domain of SEQ ID NO: 17.

In one aspect, the intracellular signaling domain is engineered to comprise a CD3-zeta signaling domain and a CD27 signaling domain. In one aspect, the CD27 signaling domain comprises the amino acid sequence

QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 51). In one aspect, the CD27 signaling domain is encoded by the nucleotide sequence

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 52).

In one aspect, the CAR-expressing cell described herein may further comprise a second CAR, for example, a second CAR that comprises a different antigen-binding domain, for example, for the same target (CD19) or a different target (e.g., CD123). In one embodiment, if the CAR-expressing cell comprises two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, a cell expressing a first and second CAR may have the antigen-binding domain of the first CAR, for example, in the form of a fragment, such as an scFv, that does not form a bond with the antigen-binding domain of the second CAR, for example, the antigen-binding domain of the second CAR is VHH.

In another aspect, the CAR-expressing cell described herein may further express 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. Inhibitory molecules, such as PD1, may, in some embodiments, reduce the ability of a CAR-expressing cell to mount 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 an inhibitory molecule comprises a first polypeptide, such as an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TEVI3, 2B4, and TIGIT, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, e.g., as described herein) and/or a basic signaling domain (e.g., the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of a PD1 molecule or fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain, described herein). PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on 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 suppress T cell activation when bound to PD1 (Freeman et al. 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 present in high amounts in human malignancies (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). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises an extracellular domain (ECD) of an inhibitory molecule, for example, the programmed cell death 1 (PD1) protein can be fused with a transmembrane domain and intracellular signaling domains, such as 4-1BB and CD3-zeta (also referred to herein as the PD1 CAR). In one embodiment, the PD1 CAR, when used in combination with the CD19 CAR described herein, improves T cell persistence. In one embodiment, the CAR is a PD1 CAR comprising the PD1 extracellular domain indicated by underlining in SEQ ID NO: 121. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 121.

Malpvtalllplalllhaarp pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcel rvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 121).

In one embodiment, the PD1 CAR comprises the amino acid sequence set forth below (SEQ ID NO: 119).

pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcel rvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 119).

In one embodiment, the agent comprises a nucleotide sequence encoding a PD1 CAR, such as the PD1 CAR described herein. In one embodiment, the nucleotide sequence for the PD1 CAR is provided below, with the PD1 ECD underlined, in SEQ ID NO: 120.

atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacca cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctcca acacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttc cgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggccc aaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtc (SEQ ID NO: 120).

In another aspect, the present invention relates to a population of CAR-expressing cells, such as CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CART cells may comprise a first cell expressing a CAR having an anti-CD19 binding domain as described herein, and a second cell expressing a CAR having a different anti-CD19 binding domain, such as an anti-CD19 binding domain as described herein, which differs from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, the population of CART cells may comprise a first cell expressing a CAR that comprises an anti-CD19 binding domain, such as described herein, and a second cell expressing a CAR that comprises 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 cell expressing a CAR that comprises a major intracellular signaling domain and a second cell expressing a CAR that comprises an auxiliary signaling domain.

In another aspect, the present invention relates to a population of cells, wherein at least one cell in the population expresses a CAR having an anti-CD19 domain as described herein, and a second cell expresses another agent, for example, an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, for example, may, in some embodiments, reduce the ability of a CAR-expressing cell to mount 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, an agent that inhibits an inhibitory molecule comprises a first polypeptide, for example, an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, for example, an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, e.g., as described herein) and/or a basic signaling domain (e.g., the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of a PD1 molecule or fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and/or the signaling domain CD3-zeta described in this document).

RNA transfection

Methods for producing in vitro transcribed CAR RNA are described herein. The present invention also includes an RNA construct encoding a CAR that can be directly transfected into a cell. A method for producing mRNA for use in transfection can include in vitro transcription (IVT) of a template with specially designed primers, followed by the addition of polyA, to produce a construct comprising a 3' and 5' untranslated sequence ("UTR"), a 5' cap and/or an internal ribosome entry site (IRES), a nucleic acid to be expressed, and a polyA sequence, typically 50-2000 bases in length (SEQ ID NO: 118). The RNA thus produced can efficiently transfect a variety of cell types. In one aspect, the template comprises sequences for a CAR.

In one aspect, the anti-CD19 CAR is encoded by messenger RNA (mRNA). In one aspect, mRNA encoding the anti-CD19 CAR is introduced into a T cell to generate a CAR T cell.

In one embodiment, in vitro transcribed CAR RNA can be introduced into a cell in the form of transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, a genomic DNA sequence, plasmid DNA, phage DNA, cDNA, synthetic DNA, or any other suitable DNA source. A desirable template for in vitro transcription is a CAR of the present invention. For example, a template for CAR RNA comprises 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 a cytoplasmic region that includes an intracellular signaling domain, such as one containing the CD3-zeta signaling domain and the 4-1BB signaling domain.

In one embodiment, the DNA used for PCR contains an open reading frame. The DNA may be from a natural 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 regions (UTR). The nucleic acid may contain exons and introns. In one embodiment, the DNA used for PCR is a human nucleic acid sequence. In another embodiment, the DNA used for PCR is a human nucleic acid sequence containing 5' and 3' UTR. Alternatively, the DNA may be an artificial DNA sequence that is not typically expressed in natural organisms. An exemplary artificial DNA sequence is one that contains gene portions ligated together to form an open reading frame that encodes a fusion protein. The DNA portions that are ligated together may be from one organism or from more than one organism.

PCR is used to generate a template for in vitro transcription of mRNA used for transfection. PCR techniques are well known in the art. Primers for use in PCR are designed to contain regions that are substantially complementary to the DNA regions used as the PCR template. As used herein, the phrase "substantially complementary" refers to nucleotide sequences in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the intended target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTR. Primers can also be designed to amplify a portion of nucleic acid that encodes a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of the 5' and 3' UTR. Primers useful for PCR can be obtained by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template located upstream of the DNA sequence to be amplified. As used herein, the term "upstream" refers to the 5' direction of the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides that are largely complementary to the double-stranded DNA template located downstream of the DNA sequence to be amplified. In this document, the term "downstream" refers to the 3' direction of the DNA sequence to be amplified relative to the coding strand.

Any DNA polymerase suitable for PCR can be used in the methods disclosed herein. Reagents and polymerase are commercially available from a number of sources.

Chemical structures capable of enhancing translational stability and/or efficiency can also be used. The RNA preferably has a 5' and 3' UTR. In one embodiment, the 5' UTR is from one to 3000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5' and 3' UTR to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs can be the natural, endogenous 5' and 3' UTRs of the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating UTR sequences into the forward and reverse primers or by any other template modification. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or efficiency of RNA translation. For example, it is known that AU-rich elements in 3' UTR sequences can decrease mRNA stability. Therefore, 3' UTRs can be selected or designed to enhance the stability of transcribed RNA based on UTR properties that are well known in the art.

In one embodiment, the 5' UTR may comprise a Kozak sequence of an endogenous nucleic acid. Alternatively, if a 5' UTR that is not endogenous to the nucleic acid of interest is added by PCR as described above, the Kozak consensus sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but do not appear to be necessary for all RNAs to ensure efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR may be the 5' UTR of RNA viruses whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to protect mRNA from degradation by exonucleases.

To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be attached to the DNA template upstream of the sequence to be transcribed. If a sequence that acts as a promoter for RNA polymerase is added to the 5' end of the 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, described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

In a preferred embodiment, the mRNA has both a 5' cap and a 3' poly(A) sequence, which determine ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase forms a long concatemeric product that is unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR results in the formation of normal-sized mRNA that is ineffective for eukaryotic transfection, even if polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase is able to extend the 3' end of the transcript beyond 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).

Molecular cloning is a common method for integrating polyA/T regions into a DNA template. However, the polyA/T sequence integrated into plasmid DNA can lead to plasmid instability, so plasmid DNA templates obtained from bacterial cells are often heavily contaminated with deletions and other aberrations. This makes cloning procedures not only labor-intensive and time-consuming but also often unreliable. Therefore, a method for constructing DNA templates with a 3' polyA/T region without cloning is highly desirable.

The polyA/T segment of the transcriptional DNA template can be produced by PCR using a reverse primer containing a polyT sequence, such as the 100T sequence (SEQ ID NO: 110) (the size can be 50-5000 T (SEQ ID NO: 111)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) sequences also promote the stability of RNA molecules and reduce their degradation. Typically, the length of the poly(A) sequence positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) sequence comprises from 100 to 5000 adenosine residues (SEQ ID NO: 112).

Poly(A) sequences of RNA molecules can be further extended after in vitro transcription using a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) sequence from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 113) results in an approximately twofold increase in RNA translation efficiency. Furthermore, the addition of various chemical groups to the 3' end can improve mRNA stability. Such an addition moiety may contain modified/artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) sequence using poly(A) polymerase. ATP analogs can further improve RNA stability.

The presence of 5' caps also contributes to the stability of RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein contain a 5' cap. The 5' cap is produced using methods known in the art and 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)).

RNA molecules produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially engineered sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solutes suitable for cell electroporation may be added, which may include factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be introduced into target cells using any of a number of different methods, such as commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8): 861-70 (2001).

Nucleic acid constructs encoding CAR

The present invention also relates to nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

Accordingly, in one aspect, the 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 comprising a stimulatory domain, e.g., a costimulatory signaling domain, and/or a basic signaling domain, e.g., a zeta chain. In one embodiment, the anti-CD19 binding domain is an anti-CD19 binding domain as described herein, such as an anti-CD19 binding domain that comprises 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 thereto. 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 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 comprises the sequence of SEQ ID NO: 15 or a sequence having 95-99% identity thereto. In one embodiment, the anti-CD19 binding domain is linked to the transmembrane domain via a hinge region, such as the hinge region described herein. In one embodiment, the encoded hinge region comprises 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 therewith. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory 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 costimulatory domain comprises the sequence of SEQ ID NO: 16 or a sequence having 95-99% identity therewith. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and a functional CD3-zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity therewith, and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity therewith, wherein the sequences constituting the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain.

In another aspect, the 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 therewith), a hinge region 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 therewith), a transmembrane domain having the sequence of SEQ ID NO: 15 (or a sequence having 95-99% identity therewith), the 4-1BB costimulatory domain having the sequence of SEQ ID NO: 16, or the CD27 costimulatory domain having the sequence of SEQ ID NO: 51 (or a sequence having 95-99% identity therewith), and the CD3-zeta stimulatory domain having the sequence of SEQ ID NO: 17 or SEQ ID NO: 43 (or a sequence having 95-99% identity therewith).

In another aspect, the invention relates to an isolated polypeptide molecule encoded by a nucleic acid molecule. In one embodiment, the isolated polypeptide 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 thereto.

In another aspect, the invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that comprises an anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein said anti-CD19 binding domain comprises 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 thereto.

In one embodiment, the encoded CAR molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory 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 costimulatory 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 a functional 4-1BB signaling domain and a functional zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and the sequence of SEQ ID NO: 17, wherein the sequences constituting the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain. In one embodiment, the anti-CD19 binding domain is linked to the transmembrane domain via a hinge region. In one embodiment, the hinge region comprises SEQ ID NO: 14. In one embodiment, the hinge region comprises SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49.

In another aspect, the 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 thereto, 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 stimulatory 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 thereto.

Nucleotide sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the gene, extracting the gene from a vector known to contain it, or isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than by cloning.

The present invention also relates to vectors incorporating the DNA of the present invention. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they enable long-term, stable integration of the transgene and its dissemination to daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they are capable of transducing non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, expression of nucleic acids encoding CARs can be achieved using transposons such as sleeping beauty, CRISPR-Cas9, and zinc finger nucleases. See below the article by June et al. 2009, Nature Reviews Immunology 9,10: 704-716, the contents of which are incorporated herein by reference.

Briefly, expression of natural or synthetic CAR-encoding nucleic acids is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. Vectors can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference. In another embodiment, the invention relates to a gene therapy vector.

Nucleic acid can be cloned into a variety of vectors. For example, nucleic acid can be cloned into vectors including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replicating vectors, probe vectors, and sequencing vectors.

Further, the expression vector can be introduced into the cell in the form of a viral vector. Viral vector technology is well known in the art 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 textbooks on virology and molecular biology. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584, WO 01/29058, and U.S. Patent No. 6,326,193).

A variety of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses are a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells either in vivo or ex vivo . A variety of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A variety of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. They are typically located in the region 30-110 bp upstream of the initiation site, although many promoters have been shown to also contain functional elements downstream of the initiation site. The space between promoter elements is often flexible, so that promoter function is preserved if the elements are inverted or shifted relative to each other. In the thymidine kinase (tk) promoter, the space between promoter elements can increase to 50 bp before activity begins to decline. It appears that, depending on the promoter, individual elements can act either jointly or independently to activate transcription.

An example of a promoter that is capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The naturally occurring EF1a promoter drives expression of the alpha subunit of the elongation factor 1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter is widely used in expression plasmids in mammalian cells and has been shown to be effective in driving CAR expression from transgenes cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453–1464 (2009). In one aspect, the EF1a promoter comprises the sequence set forth in SEQ ID NO: 100.

Another example of a promoter is the cytomegalovirus (CMV) immediate-early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the murine mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor 1α promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the invention is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of inducible promoters provides a molecular switch capable of turning on the expression of a polynucleotide sequence operably linked to it when such expression is desired, or turning it off when expression is undesired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of a CAR polypeptide or portion thereof, the expression vector introduced into the cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells suspected of being transfected or infected with viral vectors. In other aspects, the selectable marker may be located on a separate DNA fragment and used in a co-transfection procedure. Both selectable marker genes and reporter genes may be flanked by appropriate regulatory sequences to allow expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo , and the like.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression results in some readily detectable property, such as enzymatic activity. Reporter gene expression is analyzed at an appropriate time after DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be generated using known methods or purchased commercially. Typically, a construct with a minimal 5'-flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

Methods for introducing and expressing genes in cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method known in the art. For example, an expression vector can be transferred into the host cell by physical, chemical, or biological methods.

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

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most common means for introducing genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Patents 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersed systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle). Other modern nucleic acid delivery methods are also available, such as delivery of polynucleotides with targeted nanoparticles or other suitable submicron-sized delivery systems.

In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. The use of lipid preparations is contemplated for the introduction of nucleic acids into a host cell ( in vitro , ex vivo , or in vivo ). In another aspect, the nucleic acid can be linked to a lipid. The lipid-linked nucleic acid can be encapsulated in the aqueous interior of a liposome, enclosed in the lipid bilayer of a liposome, attached to a liposome via a linker molecule that is linked to both the liposome and an oligonucleotide, enclosed in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in, or complexed with micelles, or otherwise associated with a lipid. Lipid compositions, lipid/DNA mixtures, or lipid/expression vector mixtures are not limited to a specific structure in solution. For example, they can be present in a bilayer structure, as micelles, or have a "distorted" structure. They can also be simply dispersed in solution, possibly forming aggregates of varying size or shape. Lipids are fatty substances that can be natural or synthetic. For example, lipids include fatty droplets that occur naturally in the cytoplasm, as well as a class of compounds that includes long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Suitable lipids can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be purchased from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be purchased from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be purchased from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be purchased from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol mixtures can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the creation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an aqueous medium within. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before forming closed structures and entrapping water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures in solution that differ from normal vesicular structures are also included within the scope of the invention. For example, lipids can adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to the inhibitor of the present invention, various assays can be performed to confirm the presence of the 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 detecting the presence or absence of a particular peptide, for example, by immunological methods (various ELISAs and Western blots), or the assays described herein for identifying agents within the scope of the invention.

Furthermore, the present invention relates to a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into a cell, such as a T cell. In one aspect, the vector is a cloning or expression vector, such as a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

Sources of T cells

Prior to expansion and genetic modification, T cells are obtained from a subject. The term "subject" refers to living organisms in which an immune response can be induced (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic animal species. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In some aspects of the present invention, any number of T cell lines available in the art can be used. In some aspects of the present invention, T cells can be obtained from a blood sample of a subject using various techniques known to those skilled in the art, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected during apheresis can be washed to remove the plasma fraction and transfer the cells to an appropriate buffer or medium for subsequent processing steps. In one aspect of the invention, the cells are washed with phosphate-buffered saline (PBS). Alternatively, the washing solution lacks calcium and may lack magnesium, or may lack many, if not all, divalent cations. The initial steps of activation in the absence of calcium can lead to enhanced activation. As those skilled in the art will readily appreciate, the washing step can be performed using methods familiar to those skilled in the art, such as using a semiautomated continuous-flow centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or another buffered or unbuffered saline solution. Alternatively, unwanted components of the apheresis sample can be removed and the cells can be resuspended directly in the culture medium.

In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, such as by Percoll™ gradient centrifugation or flow-through elutriation centrifugation. Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection methods. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as ^DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient to positively select the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period is in the range of 30 minutes to 36 hours or more, including all integer values within this range. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In another preferred aspect, the time period is from 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times can be used to isolate T cells in any situation where a small number of T cells are present compared to other cell types, for example, when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or in the case of samples obtained from immunocompromised individuals. Furthermore, the use of longer incubation periods can improve the efficiency of CD8+ T cell capture. Thus, simply by shortening or increasing the incubation time for T cells to bind to CD3/CD28 beads, and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected in a positive or negative manner at the start of culturing or at any time points during culturing. Furthermore, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or another surface, subpopulations of T cells can be preferentially selected in a positive or negative manner at the start of culturing or at any time points during culturing. One of skill in the art will appreciate that multiple rounds of selection can also be used in the context of the present invention. In some aspects, it may be desirable to perform the selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies directed against surface markers unique to negatively selected cells. One method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry, which utilizes a cocktail of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically contains antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some aspects, enrichment or positive selection for regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+, may be desirable. Alternatively, in some aspects, regulatory T cells are depleted using anti-C25-conjugated beads or another similar selection method.

In one embodiment, a population of T cells that express 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 relevant molecules, such as other cytokines, can be selected. Cell expression screening methods can be selected, for example, from the methods described in PCT Publication No. WO 2013/126712.

To isolate a desired cell population using positive or negative selection, the cell concentration and surface size (e.g., particles such as beads) can be varied. In some aspects, it may be desirable to significantly decrease the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, in one aspect, a concentration of 2 billion cells/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In a further aspect, a concentration of more than 100 million cells/mL is used. In a further aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In another aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further aspects, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell proliferation. Furthermore, using high cell concentrations allows for more efficient capture of cells that may weakly express the target antigens of interest, such as CD28-negative T cells, or in samples containing many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value, and their recovery may be desirable. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells, which typically exhibit weak CD28 expression.

In a related aspect, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and the binding surface (e.g., particles such as beads), interactions between the particles and the cells are minimized. This allows for the selection of cells that express high amounts of the desired antigens that are expected to bind to the particles. For example, CD4+ T cells express high levels of CD28 and are more efficiently captured than CD8+ T cells at low concentrations. In one aspect, the cell concentration used is 5× ¹⁰⁶ /mL. In other aspects, the concentration used may be from about 1× ¹⁰⁵ /mL to 1× ¹⁰⁶ /mL, including any integer value within this range.

In other aspects, cells can be incubated on a rotator for varying times at different speeds, either at 2-10°C or at room temperature.

T cells for stimulation can also be frozen after the washing step. While not bound by any theory, freezing and subsequent thawing results in a more homogeneous product due to the removal of granulocytes and, to some extent, monocytes from the cell population. After the washing step, which removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, 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 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or another suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, the cells are then frozen at -80°C at a rate of 1° per minute and stored in the vapor phase in a reservoir with Liquid nitrogen. Other controlled freezing methods can also be used, as well as uncontrolled freezing directly at -20°C or in liquid nitrogen.

In some aspects, cryopreserved cells are thawed and washed as described herein and left for one hour at room temperature before activation using the methods of the present invention.

The invention also provides for collecting blood samples or apheresis products from a subject some time before the expanded cells described herein may be needed. Thus, a source of cells to be expanded can be obtained at any desired time, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for a variety of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect, the blood sample or apheresis product is obtained from a generally healthy subject. In some aspects, the blood sample or apheresis product is obtained from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In some aspects, the T cells can be expanded, frozen, and used at a later time. In some aspects, samples are collected from a patient shortly after diagnosis of a particular disease described herein, but prior to any treatment. In a further aspect, cells are isolated from a blood sample or apheresis product obtained from the subject prior to any relevant treatments, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunoablative agents such as campath, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

In other aspects of the present invention, T cells are obtained from a patient immediately following treatment, leaving the subject with functional T cells. In this regard, it has been noted that following certain anti-cancer treatments, particularly treatment with drugs that damage the immune system, shortly after treatment during the period when patients typically recover from treatment, the quality of the obtained T cells may be optimal or improved in terms of their ability to expand ex vivo . Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a favorable state for more efficient engraftment and expansion in vivo . Thus, in the context of the present invention, the collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, is contemplated during this recovery phase. Furthermore, in some aspects, mobilization regimens (e.g., GM-CSF-mediated mobilization) and conditioning can be used to create a state in the subject that promotes the repopulation, recycling, regeneration, and/or proliferation of specific cell types, particularly within a specific time window after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

T cell activation and recruitment

T cells can be activated and expanded, typically using methods described in, for example, U.S. Patents 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.

In general, T cells of the invention can be expanded by contact with a surface to which is attached an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of T cells. In particular, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. To costimulate an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. To stimulate the proliferation of either CD4+ T cells or CD8+ T cells, anti-CD3 antibody and anti-CD28 antibody are used. Examples of anti-CD28 antibodies that can be used include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), and other methods well known in the art (Berg et al., Transplant Proc. 30(8): 3975–3977, 1998, Haanen et al., J. Exp. Med. 190(9): 1319–1328, 1999, Garland et al., J. Immunol Meth. 227(1–2): 53–63, 1999).

In some aspects, the primary stimulatory signal and the costimulatory signal for a T cell can be provided according to different protocols. For example, the agents providing each signal can be in solution or surface-bound. If surface-bound, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be surface-bound and the other agent can be in solution. In one aspect, the agent providing the costimulatory signal is cell-bound and the agent providing the primary activation signal is in solution or surface-bound. In some aspects, both agents can be in solution. In one aspect, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents. In this regard, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for information on artificial antigen-presenting cells (iAPCs) contemplated for use in activating and expanding T cells of the present invention.

In one aspect, two agents are immobilized on beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same bead in equivalent molecular amounts. In one aspect, a 1:1 ratio of each of the antibodies bound to the beads is used for the expansion of CD4+ T cells and the growth of T cells. In some aspects of the present invention, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increased expansion of T cells is observed compared to the expansion observed using a 1:1 ratio. In one specific aspect, an increase of from about 1 to about 3 times is observed compared to the expansion observed using a 1:1 ratio. In one aspect, the ratio of anti-CD3:CD28 antibodies bound to the beads is in the range of 100:1 to 1:100, including all integer values within this range. In one aspect of the present invention, a greater amount of anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than one. In some aspects of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In one specific aspect, the ratio of CD3:CD28 antibodies bound to the beads is 1:100. In one aspect, the ratio of CD3:CD28 antibodies bound to the beads is 1:75. In a further aspect, the ratio of CD3:CD28 antibodies bound to the beads is 1:50. In one aspect, the ratio of CD3:CD28 antibodies bound to the beads is 1:30. In one preferred aspect, a ratio of CD3:CD28 antibodies bound to the beads of 1:10 is used. In one aspect, a ratio of CD3:CD28 antibodies bound to the beads of 1:3 is used. In another aspect, a ratio of CD3:CD28 antibodies bound to the beads of 3:1 is used.

Particles and cells can be used in a ratio of 1:500 to 500:1, including any integer values within this range, to stimulate T cells or other target cells. As those skilled in the art will readily appreciate, the particle-to-cell ratio can depend on the particle size relative to the target cell. For example, small beads may bind only a few cells, while larger beads can bind many cells. In some aspects, the cell-to-particle ratio is in the range of 1:100 to 100:1, including any integer values within this range, and in other aspects, a ratio of 1:9 to 9:1, including any integer values within this range, can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells that results in stimulation of T cells may vary as indicated above, however, 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, with one preferred particle to T cell ratio of at least 1:1. In one aspect, a particle to cell ratio of 1:1 or less is used. In one specific aspect, the preferred particle:cell ratio is 1:5. In further aspects, the particle:cell ratio may vary depending on the day of stimulation. For example, in one aspect, the particle:cell ratio is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or every other day thereafter for up to 10 days, with a final ratio of from 1:1 to 1:10 (based on the number of cells on the day of addition). In one specific aspect, the particle:cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, particles are added daily or every other day to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In one aspect, the particle:cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, particles are added daily or every other day to a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. Those skilled in the art will appreciate that various other ratios may be suitable for use in the present invention. In particular, the ratio will depend on the particle size and the cell size and type. In one aspect, the most typical ratios used are approximately 1:1, 2:1, and 3:1 on the first day.

In other aspects of the present invention, cells, such as T cells, are combined with agent-coated beads, the beads and cells are then separated, and the cells are then cultured. In an alternative aspect, the agent-coated beads and cells are not separated before culturing, but are instead cultured together. In a further aspect, the beads and cells are first concentrated by applying a force, such as magnetic force, resulting in enhanced binding of cell surface markers, causing cell stimulation.

As an example, cell surface proteins can be bound by contacting paramagnetic beads to which anti-CD3 and anti-CD28 (3x28 beads) are bound with T cells. In one aspect, cells (e.g., ^104-109 T cells) and beads (e.g., ^DYNABEADS® M- ⁴⁵⁰ CD3/CD28 T paramagnetic beads at a 1:1 ratio) are combined in a buffer, such as PBS (without divalent cations such as calcium and magnesium). Again, those skilled in the art will readily appreciate that cells of any concentration can be used. For example, a target cell may be very rare in a sample, comprising only 0.01% of the sample, or the entire sample (i.e., 100%) may represent the target cell of interest. Accordingly, any number of cells are within the scope of the present invention. In some aspects, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and the particles. For example, in one aspect, a concentration of about 2 billion cells/mL is used. In one aspect, a concentration of more than 100 million cells/mL is used. In another aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In another aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further aspects, concentrations of 125 or 150 million cells/mL can be used. The use of high concentrations can lead to increased cell yield, cell activation, and cell proliferation. Furthermore, using high cell concentrations allows for more efficient capture of cells that may weakly express the target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value, and in some contexts, their production may be desirable. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells, which typically exhibit weak CD28 expression.

In one aspect of the present invention, the mixture can be cultured for a period ranging from several hours (approximately 3 hours) to approximately 14 days, including any integer number of hours within this range. In one aspect, the mixture can be cultured for 21 days. In one aspect, the beads and T cells are cultured together for approximately eight days. In one aspect, the beads and T cells are cultured together for 2-3 days. It may also be desirable to use multiple stimulation cycles, such that the T cell culture time can be 60 days or more. Suitable conditions for culturing T cells include an appropriate medium (e.g., minimal essential medium or RPMI 1640 medium, or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum 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 cell growth supplements known to those skilled in the art. Other cell growth supplements include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and/or a certain amount of cytokine(s) sufficient to support T-cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures to be administered to the subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% _CO2 ).

T cells stimulated for different periods of time may have different characteristics. For example, typical blood or peripheral blood mononuclear cells obtained by apheresis contain a population of helper T cells (Th, CD4+) that is larger than the population of cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors results in a T cell population that is predominantly Th cells until approximately day 8-9, with the T cell population increasingly containing Th cells after approximately day 8-9. Accordingly, depending on the treatment goal, infusing a subject with a T cell population containing primarily Th cells may have certain advantages. Similarly, if a subset of antigen-specific T cells has been isolated, expanding this subset to a greater extent may be advantageous.

Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but are largely reproducible, during cell proliferation. This reproducibility therefore enables the precise selection of activated T cells for specific purposes.

Once the CD19 CAR is engineered, various assays can be used to evaluate the molecule's activity, including, but not limited to, the ability of T cells to expand after antigen stimulation, the maintenance of T cell expansion in the absence of re-stimulation, and anti-cancer activity in relevant in vitro and animal models. Assays for assessing the effects of CD19 CARs are described in more detail below.

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). Briefly, T cells (1:1 mixture of CD4+ and CD8+ T cells) expressing CAR are expanded in vitro for over 10 days, followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-length TCR-ζ cytoplasmic domain and endogenous TCR-ζ chain are detected by Western blotting using an antibody to the TCR-ζ chain. The same T cell sets are analyzed by non-reducing SDS-PAGE to assess covalently linked dimer formation.

In vitro , CAR+ T cell expansion following antigen stimulation can be quantified by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells is stimulated with αCD3/αCD28 iAPCs, followed by transduction with lentiviral vectors expressing GFP under the control of promoters to be analyzed. Illustrative promoters include the CMV IE, EF-1, ubiquitin C, or phosphoglyceride kinase (PGK) gene promoters. GFP fluorescence is assessed at day 6 of culture in CD4+ and/or CD8+ T cell subsets by flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8): 1453–1464 (2009). Alternatively, a mixture of CD4+ and CD8+ T cells are stimulated with αCD3/αCD28-coated magnetic beads on day 0 and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using the 2A ribosome skipping sequence. Cultures are restimulated with either CD19+ K562 cells (K562-CD19), wild-type K562 cells (WT K562), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibodies (K562-BBL-3/28) after washing. Exogenous IL-2 is added to cultures every other day at a concentration of 100 IU/ml. GFP+ T cells are enumerated by flow cytometry using a bead-based count. See, for example, Milone et al., Molecular Therapy 17(8): 1453–1464 (2009).

Sustained expansion of CAR+ T cells in the absence of re-stimulation can also be measured. See, for example, Milone et al., Molecular Therapy 17(8): 1453–1464 (2009). Briefly, the mean T-cell volume (fL) is measured on day 8 of culture using a Coulter Multisizer III particle counter, following stimulation with αCD3/αCD28-coated magnetic beads on day 0 and transduction with the indicated CAR on day 1.

Animal models can also be used to measure cART activity. For example, a xenograft model in immunodeficient mice can be used to treat primary human pre-B-ALL using human CD19-specific CAR+ T cells. See, for example, Milone et al., Molecular Therapy 17(8): 1453–1464 (2009). Briefly, after ALL development, mice were randomly assigned to treatment groups. Varying numbers of αCD19-ζ and αCD19-ΒΒ-ζ engineered T cells were coinjected at a 1:1 ratio into NOD-SCID-γ ^-/- mice bearing B-ALL. The number of αCD19-ζ and αCD19-ΒΒ-ζ vector copies in mouse spleen DNA was assessed at various time points after T cell injection. Animals were assessed for signs of leukemia at weekly intervals. The number of CD19+ B-ALL blast cells in the peripheral blood of mice injected with αCD19-ζ CAR+ T cells or surrogate transduced T cells was measured. Survival curves for groups were compared using the log-rank test. In addition, absolute numbers of CD4+ and CD8+ T cells in the peripheral blood can also be analyzed 4 weeks after T cell injection in NOD-SCID-γ ^-/- mice. Mice were injected with leukemia cells and 3 weeks later injected with T cells engineered to express CAR using a bicistronic lentiviral vector encoding a CAR linked to eGFP. T cells were normalized to 45-50% of the administered GFP+ T cell content by mixing with surrogate transduced cells before injection, which was confirmed by flow cytometry. Animals were assessed for signs of leukemia at 1-week intervals. Survival curves for the groups receiving CAR+ T cells were compared using the log-rank test.

Dose-dependent responses to CAR treatment can be assessed. See, for example, Milone et al., Molecular Therapy 17(8): 1453–1464 (2009). For example, peripheral blood was collected 35–70 days after leukemia development from mice that received an injection of CAR T cells on day 21, an equivalent number of surrogate transduced T cells, or no T cells. Mice in each group were randomly bled to determine the number of CD19+ ALL blast cells in peripheral blood and then sacrificed on days 35 and 49. The remaining animals were assessed on days 57 and 70.

Methods for assessing cell proliferation and cytokine production have been described previously, for example, in Milone et al., Molecular Therapy 17(8): 1453–1464 (2009). Briefly, CAR-mediated proliferation was assessed in microtiter plates by mixing washed T cells with K562 cells expressing CD19 (K19) or CD32 and CD137 (KT32-BBL) to a final T cell:562 cell ratio of 2:1. K562 cells were gamma-irradiated prior to use. Monoclonal anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) antibodies were added to KT32-BBL cell cultures as a positive control to stimulate T cell proliferation, as these signals support long-term expansion of CD8+ T cells ex vivo . T cells were counted in cultures by flow cytometry using CountBright™ fluorescent beads (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. CAR+ T cells were identified by GFP expression using T cells engineered with lentiviral vectors expressing eGFP-2A-linked CAR. For CAR+ T cells that did not express GFP, CAR+ T cells were detected using biotinylated recombinant CD19 protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells was also simultaneously determined using specific monoclonal antibodies (BD Biosciences). Cytokine quantification was performed in supernatants collected 24 hours after restimulation using a polystyrene bead kit with human monoclonal antibodies against TH1/TH2 cytokines (BD Biosciences, San Diego, CA), according to the manufacturer's instructions. Fluorescence was assessed using a FACScalibur flow cytometer, and data were analyzed according to the manufacturer's instructions.

Cytotoxicity can be assessed using a standard ^51Cr release assay. See, for example, Milone et al., Molecular Therapy 17(8): 1453–1464 (2009). Briefly, target cells (K562 and primary pro-B-ALL cells) were loaded with ^51Cr (as _NaCrO4 , New England Nuclear, Boston, MA) at 37°C for 2 h with frequent agitation, washed twice with complete RPMI medium, and seeded in microtiter plates. Effector T cells were mixed with target cells in wells in complete RPMI medium at various effector:target cell (E:T) ratios. Additional wells containing medium alone (spontaneous release, SR) or 1% Triton-X 100 detergent solution (total release, TR) were also prepared. After 4 h of incubation at 37°C, the supernatant from each well was collected. The released ^51Cr was then quantified using a gamma counter (Packard Instrument Co., Waltham, MA). The experiment for each condition was performed at least in triplicate, and the percent lysis was calculated using the formula: % lysis = (ER - SR) / (TR - SR), where ER corresponds to the average value of ^51Cr released for each experimental condition.

Imaging technologies can be used to assess the specific targeting of CAR cells in animal models of tumors. Such assays are described, for example, in Barrett et al., Human Gene Therapy 22: 1575–1586 (2011). Briefly, NOD/SCID/γc ^-/- (NSG) mice were intravenously injected with Nalm-6 cells, followed by T cell administration 7 days later, 4 hours after electroporation of CAR constructs. The T cells were stably transfected with a lentiviral construct expressing firefly luciferase, and the mice were imaged for bioluminescence. Alternatively, the therapeutic efficacy and specificity of a single injection of CAR+ T cells in a Nalm-6 xenograft model could be measured as follows: NSG mice were injected with Nalm-6 cells transduced to stably express firefly luciferase, followed 7 days later by a single tail vein injection of T cells electroporated with CD19 CAR. Animals were imaged at various time points post-injection. For example, heat maps of the photon density of firefly luciferase-positive leukemia cells could be obtained in representative mice on day 5 (2 hours before treatment) and day 8 (24 hours after CAR+ LPC administration).

Other assays, including those described in the Examples section herein, as well as those known in the art, can also be used to evaluate the CD19 CAR construct of the invention.

Therapeutic use

CD19-associated diseases and/or disorders

In one aspect, the invention relates to methods for treating a disease associated with CD19 expression. In one aspect, the invention relates to methods for treating a disease in which a portion of a tumor is negative for CD19 and a portion of the tumor is positive for CD19. For example, a CAR of the invention is useful for treating subjects who have been treated for a disease associated with increased CD19 expression, wherein the subject who has received treatment for increased CD19 levels has a disease associated with increased CD19 levels.

In one aspect, the invention relates to a vector comprising a CD19 CAR operably linked to a promoter for expression in mammalian T cells. In one aspect, the invention relates to a recombinant T cell expressing the CD19 CAR for use in the treatment of CD19-expressing tumors, wherein the recombinant T cell expressing the CD19 CAR is referred to as a CD19 CART. In one aspect, the CD19 CART of the invention is capable of contacting a tumor cell via at least one CD19 CAR of the invention expressed on its surface, such that the CART targets the tumor cell and tumor growth is inhibited.

In one aspect, the invention relates to a method for inhibiting the growth of a CD19-expressing tumor cell, comprising contacting the tumor cell with a CD19 CAR T cell of the present invention such that the CAR T cell is activated in response to an antigen and targets the cancer cell, wherein tumor growth is inhibited.

In one aspect, the invention provides a method of treating cancer in a subject. The method comprises administering to the subject a CD19 CAR T cell of the present invention such that the cancer in the subject is treated. An example of a cancer that can be treated with a CD19 CAR T cell of the invention is a cancer associated with CD19 expression. In one aspect, the cancer associated with CD19 expression is a blood cancer. In one aspect, the blood cancer is leukemia or lymphoma. In one aspect, the cancer associated with CD19 expression includes forms of cancer and malignancy, including, but not limited to, for example, one or more forms of acute leukemia, including, but not limited to, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more forms of chronic leukemia, including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Other blood cancers or blood conditions associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," representing a diverse collection of blood conditions. which share ineffective production (or dysplasia) of myeloid blood cells, etc. Furthermore, diseases associated with CD19 expression include, but are not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression.

In some embodiments, a cancer that can be treated with a CD19 CAR, such as that described herein, is multiple myeloma. Multiple myeloma is a blood cancer characterized by the accumulation of a plasma cell clone in the bone marrow. Current treatments for multiple myeloma include, but are not limited to, treatment with lenalidomide, which is an analog of thalidomide. Lenalidomide has activities that include antitumor activity, angiogenesis inhibition, and immunomodulation. Myeloma cells are generally considered to be negative for CD19 expression, as determined by flow cytometry. The present invention also includes evidence that a small percentage of myeloma tumor cells express CD19, as demonstrated in Example 6. Thus, in some embodiments, a C19 CAR, such as that described herein, can be used to target myeloma cells. In some embodiments, treatment with a CD19 CAR may be used in combination with one or more additional treatments, such as treatment with lenalidomide.

The invention involves a type of cell therapy in which T cells are genetically modified to express a chimeric antigen receptor (CAR), and the CAR T cell is infused into a recipient in need. The infused cell is capable of killing tumor cells in the recipient. Unlike antibody therapy, CAR-modified T cells are capable of replicating in vivo , resulting in long-term persistence, potentially leading to sustained tumor control. In various aspects, the T cells administered to the patient, or their progeny, persist in the patient's body for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after the T cell is administered to the patient.

The invention also includes a type of cell therapy in which T cells are modified, for example, with in vitro transcribed RNA to transiently express a chimeric antigen receptor (CAR), and the CAR T cell is infused into a recipient in need thereof. The infused cell is capable of killing tumor cells in the recipient. Thus, in various aspects, the T cells infused into the patient are present for less than one month, for example, three weeks, two weeks, or one week, after the T cell is infused into the patient.

Without being bound by any particular theory, the antitumor immune response elicited by CAR-modified T cells may be an active or passive immune response, or alternatively, may be associated with a direct rather than an indirect immune response. In one aspect, transduced CAR T cells exhibit specific secretion of proinflammatory cytokines and potent cytolytic activity in response to human cancer cells expressing CD19, are resistant to inhibition by soluble CD19, mediate nonspecific cytolysis, and mediate regression of established human tumors. For example, antigen-depleted tumor cells in a heterogeneous zone of CD19-expressing tumors may be susceptible to indirect killing by CD19-redirected T cells that previously responded to neighboring antigen-positive cancer cells.

In one aspect, the fully human CAR-modified T cells of the invention can be a type of vaccine for ex vivo immunization and/or in vivo therapy of a mammal. In one aspect, the mammal is a human.

With respect to ex vivo immunization, at least one of the following events occurs in vitro prior to administration of the cell to the mammal: i) expansion of the cells, ii) introduction of a nucleic acid encoding a CAR into the cells, or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are obtained from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro ) with a vector expressing the CAR disclosed herein. The CAR-modified cell can be administered to a recipient mammal to achieve a therapeutic effect. The recipient mammal can be a human, and the CAR-modified cell can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

The ex vivo method for expanding hematopoietic stem and progenitor cells described in U.S. Patent No. 5,199,942, the contents of which are incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art; therefore, the present invention is not limited to any particular ex vivo method for expanding cells. Briefly, ex vivo culturing and expanding T cells comprises: (1) obtaining CD34+ hematopoietic stem and progenitor cells from a mammal from collected peripheral blood or bone marrow explants and (2) expanding such cells ex vivo . In addition to the cellular growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used to cultivate and expand the cells.

In addition to the use of a cell-based vaccine in an ex vivo immunization setting, the present invention also relates to compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

In general, cells activated and expanded as described herein can be used to treat and prevent diseases that occur in individuals with immune deficiencies. In particular, CAR-modified T cells of the invention are used to treat diseases, disorders, and conditions associated with CD19 expression. In some aspects, the cells of the invention are used to treat patients at risk of developing diseases, disorders, and conditions associated with CD19 expression. Thus, the present invention relates to methods for treating or preventing diseases, disorders, and conditions associated with CD19 expression, comprising administering to a subject in need thereof a therapeutically effective amount of CAR-modified T cells of the invention.

In one aspect, the CART cells of the invention can be used to treat a proliferative disease such as a cancer or malignancy, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia. In one aspect, the cancer is a blood cancer. In one aspect, the blood cancer is a leukemia or lymphoma. In one aspect, the CART cells of the invention can be used to treat various forms of cancer and malignancies, such as, but not limited to, for example, acute leukemias, including, but not limited to, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more forms of chronic leukemia, including, but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); other blood cancers or blood disorders, including, but not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," which are a diverse collection of blood disorders that share the common feature of ineffective production (or dysplasia) of myeloid blood cells, and the like. In addition, diseases associated with CD19 expression include, but are not limited to, for example, atypical and/or non-classical forms of cancer, malignant neoplasms, precancerous conditions, or proliferative diseases associated with CD19 expression. Non-cancer conditions associated with CD19 expression include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation.

The CAR-modified T cells of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations.

Blood cancer

Blood cancers are forms of cancer such as leukemia and malignant lymphoproliferative disorders that affect the blood, bone marrow, and lymphatic system.

Leukemia can be divided into acute leukemia and chronic leukemia. Acute leukemia, in turn, can be subdivided into acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, previously known as "preleukemia"), a diverse collection of blood disorders characterized by ineffective production (or dysplasia) of myeloid blood cells and the risk of progression to AML.

The present invention relates to compositions and methods for treating cancer. In one aspect, the cancer is a blood cancer, including, but not limited to, leukemia or lymphoma. In one aspect, the CART cells of the invention can be used to treat various forms of cancer and malignancies, such as, but not limited to, for example, acute leukemias including, but not limited to, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more forms of chronic leukemia, including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); other blood cancers or blood disorders, including, but not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," which are a diverse collection of blood disorders that share the common feature of ineffective production (or dysplasia) of myeloid blood cells, etc. In addition, diseases associated with CD19 expression include, but are not limited to, for example, atypical and/or non-classical forms of cancer, malignant neoplasms, precancerous conditions, or proliferative diseases associated with CD19 expression.

The present invention also relates to methods of inhibiting proliferation or reducing a population of CD19-expressing cells, comprising contacting a population of cells comprising a CD19-expressing cell with an anti-CD19 CART cell of the invention that binds to the CD19-expressing cell. In a particular aspect, the present invention relates to methods of inhibiting proliferation or reducing a population of CD19-expressing cancer cells, comprising contacting a population of CD19-expressing cancer cells with an anti-CD19 CART cell of the invention that binds to the CD19-expressing cell. In one aspect, the present invention relates to methods of inhibiting proliferation or reducing a population of CD19-expressing cancer cells, comprising contacting a population of CD19-expressing cancer cells with an anti-CD19 CART cell of the invention that binds to the CD19-expressing cell. In some aspects, an anti-CD19 CART cell of the invention reduces the number, abundance, volume, or percentage of cells and/or cancer cells 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% in a subject suffering from, or in an animal model with, myeloid leukemia or another form of cancer associated with CD19-expressing cells, compared to a negative control. In one aspect, the subject is a human.

The present invention also relates to methods for preventing, treating, and/or managing a disease associated with CD19-expressing cells (e.g., blood cancer or atypical cancer associated with CD19 expression), comprising administering to a subject in need thereof an anti-CD19 CART cell of the invention that binds to a CD19-expressing cell. In one aspect, the subject is a human. Non-limiting examples of diseases associated with CD19-expressing cells include autoimmune diseases (such as lupus), inflammatory diseases (such as allergies and asthma), and cancer (such as various forms of blood cancer or atypical cancers associated with CD19 expression).

The present invention also relates to methods for preventing, treating, and/or managing a disease associated with CD19-expressing cells, comprising administering to a subject in need thereof an anti-CD19 CART cell of the invention that binds to a CD19-expressing cell. In one aspect, the subject is a human.

The present invention relates to methods for preventing the recurrence of cancer associated with CD19-expressing cells, comprising administering to a subject in need thereof an anti-CD19 CART cell of the invention that binds to a CD19-expressing cell. In one aspect, the methods comprise administering to a subject in need thereof an effective amount of an anti-CD19 CART cell described herein that binds to a CD19-expressing cell, in combination with an effective amount of another therapeutic agent.

Combination treatment methods

The CAR-expressing cell described herein can be used in combination with other known agents and therapies. As used herein, the term "in combination" means that a subject receives two (or more) different therapeutic agents while the subject is suffering from a disease, for example, two or more therapeutic agents are administered after the subject has been diagnosed with the disease and before the disease has been cured or eliminated, or the treatment has been discontinued for other reasons. In some embodiments, administration of one therapeutic agent is still ongoing when administration of the other begins, such that their administration overlaps. This is sometimes referred to herein as "simultaneous" or "co-administration." In other embodiments, administration of one therapeutic agent is stopped before administration of the other therapeutic agent begins. In some embodiments of either case, the treatment is more effective due to the combined administration. For example, the second therapeutic agent is more effective, e.g., an equivalent effect is observed with a smaller amount, or the second therapeutic agent reduces symptoms to a greater extent than would be the case if the second therapeutic agent were administered in the absence of the first therapeutic agent, or a similar situation occurs for the first therapeutic agent. In some embodiments, administration results in a reduction in a symptom or other disease-related parameter to a greater extent than would be the case if one drug were administered in the absence of the other. The effect of the two drugs may be partially additive, fully additive, or more than additive. Administration may result in the effect of the first drug being still noticeable when the second drug is administered.

The CAR-expressing cell described herein and at least one additional therapeutic agent may be administered simultaneously, in the same or separate compositions, or sequentially. If administered sequentially, the CAR-expressing cell described herein may be administered first, and the additional agent may be administered second, or the order of administration may be reversed.

In other aspects, the CAR-expressing cell described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as campath, anti-CD3 antibodies or other therapeutic antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate acid, steroids, FR901228, cytokines and radiation, a peptide vaccine such as that described in Izumoto et al. 2008 J Neurosurg 108: 963-971.

In one embodiment, a CAR-expressing cell described herein can be used in combination with a chemotherapeutic agent. Illustrative chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozololmide), an immune cell antibody (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), an antimetabolite (including, e.g., folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a glucocorticoid-inducible protein TNFR (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin, or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (eg, lenalidomide).

The main chemotherapeutic agents being 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® ), carmustine ( ^BiCNU® ), chlorambucil ( ^Leukeran® ), cisplatin ( ^Platinol® ), cladribine ( ^Leustatin® ), cyclophosphamide ( ^Cytoxan® or ^Neosar® ), cytarabine, cytosine arabinoside (Cytosar- ^U® ), injection cytarabine liposomes ( ^DepoCyt® ), dacarbazine (DTIC- ^Dome® ), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride ( ^Cerubidine® ), daunorubicin citrate liposome injections ( ^DaunoXome® ), dexamethasone, docetaxel ( ^Taxotere® ), doxorubicin hydrochloride ( ^Adriamycin® , ^Rubex® ), etoposide ( ^Vepesid® ), fludarabine phosphate ( ^Fludara® ), 5-fluorouracil ( ^Adrucil® , ^Efudex® ), flutamide ( ^Eulexin® ), tezacitabine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea ^® ), idarubicin (Idamycin ^® ), ifosfamide (IFEX ^® ), irinotecan (Camptosar ^® ), L-asparaginase (ELSPAR ^® ), leucovorin calcium, melphalan (Alkeran ^® ), 6-mercaptopurine (Purinethol ^® ), methotrexate (Folex ^® ), mitoxantrone (Novantrone ^® ), mylotarg, paclitaxel (Taxol ^® ), phoenix (Yttrium90/MX-DTPA), pentostatin, polyfeprosan 20 implant with carmustine (Gliadel ^® ), tamoxifen citrate (Nolvadex ^® ), teniposide (Vumon ^® ), 6-thioguanine, thiotepa, tirapazamine (Tirazone ^® ), topotecan hydrochloride injection (Hycamptin ^® ), vinblastine (Velban ^® ), vincristine (Oncovin ^® ) and vinorelbine (Navelbine ^® ).

Illustrative alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uramustine (Aminouracil ^Mustard® , ^{Chlorethaminecil®} , ^{Demethyldopan®} , ^{Desmethyldopan®} , ^{Haemanthamine®} , ^Nordopan® , Uracil nitrogen ^mustard® , Uracillost®, ^{Uracilmostaza®} , ^Uramustin® , ^Uramustine® ), chlormethine ( ^Mustargen® ), cyclophosphamide ( ^Cytoxan® , Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide ( ^Mitoxana® , ^Neosar® , ^Clafen® , ^Endoxan® , ^Procytox® , Revimmune™), and cyclophosphamide (Mitoxana®). ^® ), melphalan (Alkeran ^® ), chlorambucil (Leukeran ^® ), pipobroman (Amedel ^® , Vercyte ^® ), triethylenemelamine (Hemel ^® , Hexalen ^® , Hexastat ^® ), triethylenethiophosphoramine, temozolomide (Temodar ^® ), thiotepa (Thioplex ^® ), busulfan (Busilvex ^® , Myleran ^® ), carmustine (BiCNU ^® ), lomustine (CeeNU ^® ), streptozocin (Zanosar ^® ), and dacarbazine (DTIC-Dome ^® ). Additional illustrative 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-sarcolysin, and phenylalanine mustard, Alkeran ^® ), altretamine (also known as hexamethylmelamine (HMM), Hexalen ^® ), carmustine (BiCNU ^® ), bendamustine (Treanda ^® ), busulfan (Busulfex ^® and Myleran ^® ), carboplatin (Paraplatin ^® ), lomustine (also known as CCNU, CeeNU ^® ), cisplatin (also known as CDDP, Platinol ^® and Platinol ^® -AQ), chlorambucil (Leukeran ^® ), cyclophosphamide (Cytoxan ^® and Neosar ^® ), dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome ^® ), altretamine (also known as hexamethylmelamine (HMM), Hexalen ^® ), ifosfamide (Ifex ^® ); prednimustine, procarbazine ( ^Matulane® ), mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, ^Mustargen® ), streptozocin ( ^Zanosar® ), thiotepa (also known as thiophosphoamide, TESPA, and TSPA, ^Thioplex® ), cyclophosphamide ( ^Endoxan® , ^Cytoxan® , ^Neosar® , ^Procytox® , ^Revimmune® ), and bendamustine HCl ( ^Treanda® ).

Illustrative mTOR inhibitors include, for example, temsirolimus, ridaforolimus (formerly known as deferolimus, (1 R ,2 R ,4 S )-4-[(2 R )-2[(1 R ,9 S ,12 S ,15 R ,16 E ,18 R ,19 R ,21 R ,23 S ,24 E ,26 E ,28 Z ,30 S ,32 S ,35 R )-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, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383), everolimus (Afinitor ^® or RAD001), rapamycin (AY22989, Sirolimus ^® ), semapimod (CAS 164301-51-3), emsirolimus, (5-{2,4-bis[( 3S ,)-3-methylmorpholin-4-yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055), 2-amino-8-[ trans -4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3- d ]pyrimidin-7(8 H )-one (PF04691502, CAS 1013101-36-4) and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4 H -1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine-, inner salt (SF1126, CAS 936487-67-1) and XL765.

Illustrative immunomodulators include, for example, afutuzumab (manufactured by Roche ^® ), pegfilgrastim (Neulasta ^® ), lenalidomide (CC-5013, Revlimid ^® ), thalidomide (Thalomid ^® ), actimid (CC4047), and IRX-2 (a human cytokine mixture containing interleukin 1, interleukin 2, and interferon γ, CAS 951209-71-5, manufactured by IRX Therapeutics).

Illustrative anthracyclines include, for example, doxorubicin (Adriamycin ^® and Rubex ^® ), bleomycin (Lenoxane ^® ), daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine ^® ), liposomal daunorubicin (daunorubicin citrate liposomes, DaunoXome ^® ), mitoxantrone (DHAD, Novantrone ^® ), epirubicin (Ellence™), idarubicin (Idamycin ^® , Idamycin PFS ^® ), mitomycin C (Mutamycin ^® ), geldanamycin, herbimycin, ravidomycin, and desacetylravidomycin.

Illustrative vinca alkaloids include, for example, vinorelbine tartrate (Navelbine ^® ), vincristine (Oncovin ^® ), and vindesine (Eldisine ^® ), vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ ^® and Velban ^® ), and vinorelbine (Navelbine ^® ).

Illustrative proteasome 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-phenylpropan-2-yl)-2-(( S )-2-(2-morpholinoacetamido)-4-phenylbutanamido)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-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl] -L- serinamide (ONX-0912).

Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., divalent anti-GITR antibodies), such as, for example, the GITR fusion protein described in U.S. Patent No. 6,111,090, European Patent No. 090505B1, U.S. Patent No. 8,586,023, PCT Publication Nos. WO 2010/003118 and 2011/090754, or the anti-GITR antibody described in, for example, U.S. Patent No. 7,025,962, European Patent No. 1,947,183B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent No. EP 1,866,339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO 2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO 2005/055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCT Publication No. WO 99/20758, PCT Publication No. WO 2006/083289, PCT Publication No. WO 2005/115451, U.S. Patent No. 7618632 and PCT Publication No. WO 2011/051726.

In one embodiment, a CAR-expressing cell described herein is administered to a subject in combination with an mTOR inhibitor, e.g., an mTOR inhibitor described herein, e.g., a rapalog such as everolimus. In one embodiment, the mTOR inhibitor is administered prior to administration of the CAR-expressing cell. For example, in one embodiment, the mTOR inhibitor can be administered prior to cell apheresis. In one embodiment, the subject has CLL.

In one embodiment, a CAR-expressing cell described herein is administered to a subject in combination with a GITR agonist, such as a GITR agonist described herein. In one embodiment, the GITR agonist is administered prior to administration of the CAR-expressing cell. For example, in one embodiment, the GITR agonist can be administered prior to cell apheresis. In one embodiment, the subject has CLL.

Drugs that inhibit either the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase, which is important for growth factor-induced signaling (rapamycin), can also be used (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). In a further aspect, the cellular compositions of the present invention can be administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, T-cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, and/or antibodies such as OKT3 or Campath. In one aspect, the cellular compositions of the present invention are administered after B-cell ablative therapy, for example, using agents that react with CD20, such as Rituxan. For example, in one embodiment, subjects may receive standard treatment with a high dose of a chemotherapeutic agent, followed by a peripheral blood stem cell transplant. In particular embodiments, after transplantation, subjects receive an infusion of the expanded immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

In one embodiment, a subject may be administered an agent that reduces or ameliorates a side effect associated with the administration of a CAR-expressing cell. Side effects associated with the administration of a CAR-expressing cell include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Symptoms of CRS include fever, nausea, transient hypotension, hypoxia, and the like. Accordingly, the methods described herein may comprise administering a CAR-expressing cell described herein to a subject and further administering an agent to control elevated levels of a soluble factor resulting from treatment with the CAR-expressing cell. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-γ, TNFα, IL-2, and IL-6. Therefore, the agent administered to treat this side effect may be one 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).

In one embodiment, a subject may be administered an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, such as programmed cell death protein 1 (PD1), may, in some embodiments, reduce the ability of a CAR-expressing cell to mount 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 an inhibitory molecule, such as inhibition at the DNA, RNA, or protein level, may optimize the efficacy of the CAR-expressing cell. In some embodiments, an inhibitory nucleic acid, such as an inhibitory nucleic acid, such as a dsRNA, such as an siRNA or shRNA, can be used to inhibit the expression of an inhibitory molecule in a CAR-expressing cell. In one embodiment, the inhibitor is an shRNA. In one embodiment, the inhibitory molecule is inhibited in a CAR-expressing cell. In these embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule is linked to a nucleic acid encoding a component, such as all components, of the CAR. In one embodiment, the inhibitor of the inhibitory signal can be, for example, an antibody or antibody fragment that binds the inhibitory molecule. For example, the agent 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, and marketed as ^Yervoy® , Bristol-Myers Squibb), tremelimumab (an IgG2 monoclonal antibody manufactured by Pfizer, formerly known as ticilimumab, CP-675,206)). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on 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 suppress T cell activation when bound to PD1 (Freeman et al. 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 present in high amounts in human malignancies (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). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1, and PD-L2 are available in the art and can be used in combination with the CD19 CAR 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 described in US 8,008,449 and WO 2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are described in WO 2009/101611. Lambrolizumab (also called MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD1. Lambrolizumab and other humanized anti-PD1 antibodies are described in US 8,354,509 and WO 2009/114335. MDPL3280A (Genentech/Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human anti-PD-L1 monoclonal antibodies are described in U.S. Patent No. 7,943,743 and U.S. Patent Publication No. 20120039906. Other anti-PD-L1 binding agents include YW243.55.S70 (heavy and light chain variable regions are provided in SEQ ID NOs 20 and 21 in WO 2010/077634) and MDX-1105 (also referred to as BMS-936559, and, for example, the anti-PD-L1 binding agents described in WO 2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., described in WO 2010/027827 and WO 2011/066342) is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1. Other anti-PD1 antibodies include AMP 514 (Amplimmune), among others, e.g., the anti-PD1 antibodies described in US 8,609,089, US 2010028330, and/or US 20120114649.

In some embodiments, an agent that enhances the activity of a CAR-expressing cell may be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule or a fragment thereof, and the second domain is a polypeptide associated with a positive signal, for example, a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide associated with a positive signal may include a costimulatory domain of CD28, CD27, ICOS, for example, an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a basic signaling domain, for example, CD3-zeta, for example, as described herein. In one embodiment, the fusion protein is expressed by the same cell that expresses the CAR. In another embodiment, the fusion protein is expressed by a cell, for example, a T cell, that does not express the anti-CD19 CAR.

In one embodiment, the agent that enhances the activity of a CAR-expressing cell described herein is miR-17-92.

Pharmaceutical compositions and methods of treatment

Pharmaceutical compositions of the present invention may comprise a CAR-expressing cell, such as 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 comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like, carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelators such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present invention are, in one aspect, formulated for intravenous administration.

The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease being treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the disease, although appropriate dosages can be determined through clinical trials.

In one embodiment, the pharmaceutical composition is substantially free, e.g., absent detectable levels, of an impurity, e.g., selected from the group consisting of endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, 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. In one embodiment, the bacterium is at least a bacterium selected from the group consisting of Alcaligenes faecalis , Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae , and Streptococcus pyogenes group A.

When an "immunologically effective amount,""antitumor effective amount,""tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician, taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the health condition of the patient (subject). Generally, it can be stated that a pharmaceutical composition containing T cells described herein can be administered at a dose of 10 ⁴ -10 ⁹ cells/kg body weight, in some cases 10 ⁵ -10 ⁶ cells/kg body weight, including all integer values in these ranges. T-cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly used in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

In some aspects, it may be desirable to administer activated T cells to a subject, followed by blood collection (or apheresis), activation of T cells therefrom in accordance with the present invention, and reinfusion of these activated and expanded T cells into the patient. This procedure can be performed several times every few weeks. In some aspects, T cells can be activated from a volume of collected blood of from 10 ^cm3 to 400 ^cm3 . In some aspects, T cells are activated from a volume of collected blood of ^{20 cm3} , 30 ^cm3 , 40 ^cm3 , 50 ^cm3 , 60 ^cm3 , 70 cm3, 80 ^cm3 , 90 ^cm3 , or 100 ^cm3 .

Administration of the compositions to a subject can be accomplished by any convenient route, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous (IV) injection, or intraperitoneal. In one aspect, the T-cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T-cell compositions of the present invention are administered by IV injection. The T-cell compositions can be injected directly into a tumor, lymph node, or site of infection.

In a specific illustrative aspect, subjects may undergo leukapheresis, whereby leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate cells of interest, such as T cells. These T cell isolates may be expanded using methods known in the art and processed to allow administration of one or more CAR constructs of the invention, thereby creating a CAR T cell of the invention. Subjects in need thereof may subsequently receive standard treatment with a high dose of a chemotherapeutic agent, followed by a peripheral blood stem cell transplant. In some aspects, after or concurrently with the transplant, subjects receive an infusion of expanded CAR T cells of the present invention. In a further aspect, the expanded cells are administered before or after surgery.

The dosage of the above-mentioned therapeutic agents for administration to a patient will vary depending on the specific condition being treated and the individual recipient of the therapeutic agent. Dose scaling for human administration can be accomplished in accordance with accepted practice in the art. The dose for campath, for example, will typically range from 1 to approximately 100 mg for an adult patient, administered daily for a period of 1 to 30 days. The preferred daily dose is 1 to 10 mg per day, although higher doses of up to 40 mg per day can be used in some cases (described in U.S. Patent No. 6,120,766).

In one embodiment, the CAR molecule is introduced into T cells, such as using in vitro transcription, and the subject (e.g., a human) is initially administered the CAR T cells of the invention, followed by one or more administrations of the CAR T cells of the invention, wherein the one or more subsequent administrations are performed less than 15 days, such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the CAR T cells of the invention is performed to the subject (e.g., a human) per week, such as 2, 3, or 4 administrations of the CAR T cells of the invention per week. In one embodiment, a subject (e.g., a human subject) receives more than one administration of CAR T cells per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle), followed by a week without administration of CAR T cells, and then one or more additional administrations of CAR T cells (e.g., more than one administration of CAR T cells per week) are performed on the subject. In another embodiment, a subject (e.g., a human subject) receives more than one cycle of administration of CAR T cells, and the time period between cycles is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, CAR T cells are administered every other day, which is 3 administrations per week. In one embodiment, CAR T cells of the invention are administered for at least two, three, four, five, six, seven, eight, or more weeks.

In one aspect, CD19 CART is produced using lentiviral vectors, such as lentivirus. CART cells produced in this manner will have stable CAR expression.

In one aspect, CART cells transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 days after transduction. Transient CAR expression can be achieved by vector delivery of CAR RNA. In one aspect, CAR RNA is transduced into a T cell using electroporation.

A potential problem that may arise in patients receiving treatment with transiently expressing CAR T cells (particularly CAR T cells carrying murine scFvs) is anaphylaxis after multiple treatments.

Without being bound by any theory, it is believed that such an anaphylactic response can be triggered in a patient by the development of a humoral anti-CAR response, that is, the production of anti-CAR antibodies of the anti-IgE isotype. It is believed that the patient's antibody-producing cells undergo a class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype after a ten- to fourteen-day gap in antigen exposure.

If a patient is at high risk of developing an anti-CAR antibody response during therapy in the presence of transient CAR expression (which occurs as a result of RNA transduction), interruptions in CAR infusion should not last longer than ten to fourteen days.

EXAMPLES

The invention is now described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise stated. Therefore, the invention should in no way be considered limited to the following examples, but, on the contrary, should be construed as encompassing any and all variations that become apparent from the teachings presented herein.

Without further description, it is believed that one of ordinary skill in the art, using the preceding description and the following illustrative examples, can prepare and use the compounds of the present invention and practice the claimed methods. The following working examples specifically illustrate various aspects of the present invention and are not to be construed as limiting the remainder of the invention in any way.

Example 1: Humanized mouse anti-CD19 antibody

Humanization of the mouse anti-CD19 antibody is needed for clinical practice because mouse-specific residues can induce a human immune response to mouse antigen (HAMA) in patients receiving CART19 therapy, i.e., treatment with T cells transduced with the CAR19 construct. The VH and VL sequences of the hybridoma-derived mouse anti-CD19 antibody were obtained from the literature (Nicholson et al., 1997, supra). Humanization was achieved by grafting the CDR regions from the mouse anti-CD19 antibody onto the acceptor scaffolds of the human germline antibody VH4_4-59 and VK3_L25 (vBASE database). In addition to the CDR regions, five framework residues, i.e., VH #71, #73, #78 and VL #71, #87, which are thought to maintain the structural integrity of the CDR regions, were retained from the mouse sequence. In addition, human J elements JH4 and JK2 were used for the heavy and light chains, respectively. The resulting amino acid sequences of the humanized antibody are designated FMC63_VL_hz and FMC63_VH_hz1, respectively, and are listed below in Table 1. Residue numbering follows the Kabat system (Kabat EA et al., 1991, supra). Both the Kabat system and the system of Chothia et al., 1987, supra, were used to define the CDRs. Residues derived from mouse anti-CD19 are shown in bold/italics. Boxed positions 60/61/62 correspond to potential post-translational modification (PTM) sites in CDR H2, also called HCDR2.

Table 1
Amino acid sequences of the variable domains of the humanized anti-CD19 antibody (SEQ ID NOs: 114-117, respectively, in order of appearance).

These humanized anti-CD19 IgGs were used to generate soluble scFvs to test expression and scFvs for full CD19 CART constructs (see examples below). Interestingly, the humanization process favors a serine residue at position 62 in CDRH2 over the alanine residue present in the mouse CDRH2. The mouse sequence lacks a post-translational modification (PTM) and contains asparagine-serine-alanine residues at positions 60/61/62, respectively, in CDRH2. This creates potential PTM motifs (indicated by the boxed region in CDRH2) during humanization. It was tested whether the PTM site generated during humanization is a "true" PTM site or simply a theoretical one. It was hypothesized that an amino acid motif of an asparagine residue followed by a serine residue (NS) might be susceptible to post-translational deamidation, but this was not readily apparent. It was also hypothesized that the combination of an asparagine residue followed by any amino acid except proline and followed by a serine residue (NxS, x≠P) might be susceptible to post-translational N-glycosylation. To test this hypothesis, two IgG variants were generated in which the asparagine at position 60 (known as the glycosylation site) was mutated to serine or glutamine, and the variants were designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q), respectively. These constructs were generated to eliminate a potential post-translational modification (PTM) site and to test for retention of activity (see Example 2 below).

Cloning:

DNA sequences encoding murine and humanized VL and VH domains were obtained, and the constructs were codon optimized for expression in Homo sapiens cells.

Sequences encoding the VL and VH domains were subcloned from cloning vectors into expression vectors suitable for secretion in mammalian cells. The heavy and light chains were cloned into separate expression vectors to allow for cotransfection. Expression vector elements included a promoter (the cytomegalovirus (CMV) enhancer-promoter), a signal sequence to facilitate secretion, a polyadenylation signal, and a transcription terminator (the bovine growth hormone (BGH) gene), an element enabling episomal replication and prokaryotic replication (e.g., the SV40 and ColE1 origins of replication or others known in the art), and elements enabling selection (an ampicillin resistance gene and a zeocin resistance marker).

Expression:

Chimeric and humanized IgG candidates were expressed in mammalian HEK293F cells at 1-ml scale. Clarified supernatants were used for FACS binding studies. Specifically, HEK293F cells were diluted to 5E5 cells/ml in FreeStyle medium supplemented with pen/strep, and 1 ml was transferred to a 24-well round-bottom deep-well plate. 0.5 μg of light chain plasmid and 0.5 μg of heavy chain plasmid, suitable for expression in mammalian cells, were diluted in the same medium along with 4 μl of FuGENE HD (Roche REF 04709705001). After 15 min of incubation at RT, the DNA/Fugene mixture was added dropwise to the cells and incubated in a 5% _CO2 atmosphere with shaking at 250 rpm at 37°C for five days. The supernatant was then separated from the cells by centrifugation. For IgG measurement, 200 µl aliquots were placed into the wells of 96-well microtiter plates. All samples and standards were measured in duplicate using a Protein A Dip and biosensor readers (Fortebio Cat. No. 18-5010). The plate was placed in an Octet instrument (ForteBio) and equilibrated at 27°C in a thermostatted chamber. Data were processed automatically using Octet User Software version 3.0, and the concentration was determined by comparison with an IgG standard curve.

FACS binding analysis:

Humanized and chimeric antibodies were assessed in a flow cytometric binding assay using the 300.19-hsCD19FL cell line. This cell line was generated by transfecting the murine preB cell line 300.19 with a vector (hCD19FL/pEF4-myc-His A) encoding the full-length human CD19 coding sequence and natural promoter, as well as the zeocin resistance gene. Briefly, the linearized plasmid was electroporated into 300.19 cells, and cells expressing high levels of hsCD19 were identified using APC-conjugated anti-human CD19 Ab (clone HIB 19 from BD 555415) and subsequently sorted using a FACS Aria flow cytometer. Sorted hsCD19+ cells were cultured and stable expression of high levels of hsCD19 was confirmed.

Binding assays can be performed directly in serum-free culture medium containing expressed IgG. All IgG concentrations assessed were normalized to the same concentration (85 nM) before dilution with 3-fold serial dilutions up to 1.4 pM. Aliquots of 5 × ¹⁰⁵ cells/well were then incubated in a 96-well plate for 30 min at 4°C with diluted IgG. Cells were washed twice with FACS buffer (0.5% BSA in PBS) before addition of detection antibody, APC-conjugated goat anti-human IgG Fc-specific antibody (Dianova #109-136-098), diluted 1:1000 in FACS buffer. Cells were incubated for an additional 30 min at 4°C, then washed twice in FACS buffer and analyzed on a FACS Calibur (BD Bioscience). Binding curves (mean fluorescence intensity versus IgG concentration) and _EC50 determination were plotted using GraphPad Prism™ 3.0 software with nonlinear regression analysis of the sigmoidal dose-response curve (with variable slope).

FACS analyses demonstrated that apparent binding for all IgGs evaluated can vary widely, with some constructs exhibiting a 5- to 10-fold shift in _EC50 when comparing IgG to scFv. Based on _EC50 values, the best candidates were selected, those with a binding affinity increased by 2-fold or more compared to the chimeric reference.

Example 2: Characterization of anti-CD19 soluble scFv fragments derived from humanized IgG antibodies to CD19

Soluble scFv fragments were generated from the humanized anti-CD19 IgGs described in Example 1 using standard molecular biology techniques. These soluble scFvs were used in studies to investigate characteristics such as stability, cell surface expression, and binding properties of the scFvs. Experiments were also conducted to examine the impact of a potential PTM introduced during the humanization process.

Expression and purification of scFv

To transfect each scFv construct, approximately 3e8 293F cells were transfected with 100 μg of plasmid using PEI as the transfection reagent at a 3:1 (PEI:DNA) ratio. Cells were grown in 100 ml of EXPi293 expression medium (Invitrogen) at 37°C in a shaking flask at 125 rpm under an 8% _CO2 atmosphere. The culture was harvested after six days and used for protein purification.

293F cells were harvested by centrifugation at 3500 g for 20 min. The supernatant was collected and filtered through a VacuCap90 PF filter cell (w/0.8/0.2 μm Super Membrane, PALL). Approximately 400 μl of Ni-NTA agarose beads (Qiagen) were added to the supernatant. The mixture was vortexed and incubated for 4 h at 4°C. It was loaded onto a purification column and washed with wash buffer containing 20 mM histidine. Protein was eluted with 500 μl of elution buffer containing 300 mM histidine. Samples were dialyzed against PBS buffer at 4°C overnight. Protein samples were quantified using a nanodrop 2000c.

scFv conformation and colloidal stability analysis

Thermostability of scFv was determined using DSF: 10-20 µl of protein sample was mixed with Sypro Orange dye (Invitrogen Cat # S6650) at a final dilution of 1:1000 in a total volume of 25 µl in PBS and analyzed on a BioRad CFX1000 instrument (25°C for 2 minutes, then 0.5°C increments over 30 seconds, from 25 to 95°C).

For the analytical SEC experiment, approximately 15–20 µg of scFv protein sample in 20 µl PBS was injected onto a TSKgel Super SW2000 column at a flow rate of 0.3 ml/min on an Agilent 1100 Series instrument.

Definition of EC ₅₀ FACS binding

The murine 300.CD19 cell line was grown in RPMI 1640 medium with 0.5 mg/mL zeocin. Approximately 5e5 cells/well were transferred to a 96-well BD Falcon plate. The cells were then centrifuged at 900 rpm (Sorval Legend XT centrifuge) for 3 min. The supernatant was discarded. Anti-CD19 scFv protein samples were diluted in DPBS with 5% FBS. Samples were added to wells, mixed thoroughly with the cells, and incubated for 1 hour. Cells were washed twice in DPBS with 5% FBS. Cells were incubated with anti-poly His PE (R&D) for 1 hour and washed twice before FACS analysis (LSRII, BD Biosciences).

Kinetic analysis using Proteon

Kinetics were determined using Bio-Rad Proteon. Immobilization was performed by standard coupling via amine groups on a GLC sensor chip. scFv samples were diluted to 0.03 mg/mL in acetate, pH 4.5, and applied to the chip at a flow rate of 30 μL/min for 300 seconds. CD19 ligand was then serially diluted in PBS-Tween and injected at a flow rate of 50 μL/min for 120 seconds with a dissociation time of 480 seconds. The chip surface was regenerated with glycine, pH 2.5. Curves were constructed from the data using a 1:1 Langmuir model.

Surface expression of the CART19 construct and FACS staining

HEK293F suspension cells transiently transfected with different anti-hCD19 CARTs were harvested 2 days post-transfection. Approximately 1e6 cells were placed in each well of a 96-well V-well plate (Greiner Bio-One, Germany) and washed three times with 0.2 ml FACS buffer (1x PBS containing 4% bovine serum albumin (BSA) (BSA fraction V, Roche Diagnostics, Indianapolis, IN). Cells were resuspended in 0.2 ml FACS buffer with either 0.2 μg biotinylated protein L (GenScript, Piscataway, NJ) or 100 nM hCD19(AA 1-291)-hIgG1 Fc (obtained from NIBRI) and incubated at 4°C for 30 min. Cells were then washed three times with 0.2 ml FACS buffer and incubated with 1 μl Alexa Fluor-labeled streptavidin. 488 (Life Technologies, Grand Island, NY) in 0.2 mL of FACS sample buffer with protein L, or 2 μL of PE anti-human Fcγ (Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.2 mL of FACS sample buffer with hCD19-hIgG1 Fc for 30 min at 4°C in the dark. After three washes with 0.2 mL of FACS buffer, cells were analyzed on an LSRII instrument (BD Biosciences, San Jose, CA) using FACSDiva software (BD Biosciences, San Jose, CA). Immunofluorescence staining was analyzed as the relative log of live cell fluorescence, and the percentage of Alexa Fluor 488-positive or PE-positive cells was measured.

Analysis of potential PMTs obtained during the humanization process

Interestingly, the humanization process favors a serine residue for position 62 in the CDRH2 region over the alanine residue present in the mouse CDRH2 region, as described in Example 1. It was tested whether the PTM site generated during the humanization process was a "true" PTM site or merely a theoretical one. Two IgG variants were generated in which the asparagine at position 60 (known as a glycosylation site) was mutated to serine or glutamine, and the variants were designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q), respectively. These constructs were generated to eliminate a potential post-translational modification (PTM) site and test for retained activity.

Results

Anti-CD19 humanized scFv and murine scFv were expressed in 293F cells and purified using a His-tag. Expression and yield of all humanized scFvs were significantly higher than those of the parental murine scFv (data not shown).

To confirm identity and assess construct integrity, scFv were analyzed with and without incubation with N-glycanase F (PNGaseF), followed by both high-performance liquid chromatography-mass spectrometry (HPLC-MS) (see Figure 3) and SDS-PAGE (data not shown). PNGaseF is an enzyme that specifically removes N-linked glycan structures from the consensus sequence N-X-S/T/C, where X is any amino acid except proline. Briefly, samples were diluted in water to a concentration of 0.1 μg/μL and either left untreated or incubated with PNGaseF at a PNGaseF:scFv ratio of 1:2 (w/w) for 3 hours at 37°C.

SDS-PAGE analysis was performed using NuPAGE 4-12% Bis-Tris gel (Novex). Approximately 2 μg of scFv was loaded into each well, and electrophoresis was performed at a constant voltage of 200 V for 40 minutes. After electrophoresis, the gel was stained with PhastGel Blue R 250 (Amersham Pharmacia) and destained with a mixture of 10% acetic acid and 30% methanol.

LC-MS analysis was performed on a Waters Acquity UPLC system coupled to a Xevo-Tof mass spectrometer. Approximately 1 μg of each sample was loaded onto a POROS R 1/102.1 x 100 mm 10 μm column (Applied Biosciences) equilibrated at 60°C with a flow rate of 0.5 mL/min. The mobile phases were 0.1% formic acid (A) and 0.1% formic acid, 75% isopropanol, 25% acetonitrile (B). Protein was eluted from the column using a reversed-phase gradient of 25% to 90% B over 12 minutes. Data were collected using positive electrospray ionization in the mass scanning range m/z 600–4000 Da with a source cone voltage of 20–50 V. The resulting spectra were unwrapped using MaxEnt1.

The glycosylation site was introduced during the humanization process. Non-PTM variants (VH: N60S or N60Q) lacked this additional form. There was only one construct with a consensus N-linked glycosylation site in HC CDR2. Upon SDS-PAGE analysis, untreated samples migrated as single bands consistent with the approximate molecular masses of the sequences for all constructs except 103101-WT (S/N), for which a doublet was observed. This construct was the only one with a consensus N-linked glycosylation site in HC CDR2. Upon treatment with PNGaseF, the higher molecular mass doublet band was no longer present, indicating partial occupancy of the site. Similarly, the observed molecular masses from the deconvoluted mass spectra were consistent with those predicted based on the amino acid sequences. However, while other constructs exhibited single major molecular species, 103101-WT (S/N) also exhibited molecules 1217 daltons larger than predicted from the sequence, which were no longer present after PNGaseF treatment. This is consistent with the presence of a single predominant N-linked glycoform, most likely oligomannose 5, based on mass. The presence of the glycosylated form was confirmed by MS analysis, as shown in Figure 3.

Conformational stability was measured using differential scanning fluorimetry (DSF). As shown in Figure 4, the Tm of mouse scFvs was 57°C, while the human variants exhibited a higher Tm of approximately 70°C. The Tm for all humanized scFvs exceeded that of mouse scFvs, clearly demonstrating that all humanized scFvs are more stable than mouse scFvs. This stability is likely to be transferred to the CART19 construct, likely leading to improved therapeutic properties.

The activity of purified scFvs was measured by binding to hCD19-expressing cells and by binding to the hCD19 antigen using an SPR-based detection method. Murine cell line 300 was used to determine scFv binding. The _EC50 value of murine scFvs for hCD19 was approximately 0.6–1.6 nM. Humanized variants had _EC50 values in the same range, in the low- or sub-nM _EC50 range.

Example 3: CD19 CAR constructs

ScFvs intended for use in the final CAR construct were generated from the humanized IgGs described in Example 1. To generate the complete domain scFv, the order in which the VL and VH domains appear in the scFv (i.e., VL-VH or VH-VL orientation) and whether three or four copies of the “G4S” subunit (SEQ ID NO: 18), with each subunit containing the sequence GGGGS (SEQ ID NO: 18) (e.g., (G4S) ₃ (SEQ ID NO: 107) or (G4S) ₄ (SEQ ID NO: 106)) connect the variable domains were varied, as shown in Table 2.

Table 2
Humanized scFv constructs for CD19, for which the VH and VL orientation and linker length are shown (“3G4S” corresponds to SEQ ID NO: 107 and “4G4S” corresponds to SEQ ID NO: 106). Name of the design Length, ak Annotation Change in Vh mscFvCTL019 486 VL-VH, 3G4S 104879 491 VL-VH, 4G4S N/S 104880 491 VL-VH, 4G4S N/Q 104881 491 VH-VL, 4G4S N/S 104882 491 VH-VL, 4G4S N/Q 104875 486 VL-VH, 3G4S N/S 104876 486 VL-VH, 3G4S N/Q 104877 486 VH-VL, 3G4S N/S 104878 486 VH-VL, 3G4S N/Q 105974 491 VL-VH, 4G4S S/N 105975 491 VH-VL, 4G4S S/N 105976 486 VL-VH, 3G4S S/N 105977 486 VH-VL, 3G4S S/N

The sequences of the humanized scFv fragments (SEQ ID NOs: 1-12) are given below in Table 3. Full-length CAR constructs were generated using SEQ ID NOs: 1-12 with additional sequences, SEQ ID NOs: 13-17, given below, to generate full-length CAR constructs with SEQ ID NOs: 31-42.

• leader (amino acid sequence) (SEQ ID NO: 13)

MALPVTALLLPLALLLHAARP

• leader (nucleotide sequence) (SEQ ID NO: 54)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC

• CD8 hinge (amino acid sequence) (SEQ ID NO: 14)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

• CD8 hinge (nucleotide sequence) (SEQ ID NO: 55)

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

• transmembrane fragment of CD8 (amino acid sequence) (SEQ ID NO: 15)

IYIWAPLAGTCGVLLLSLVITLYC

• transmembrane fragment (nucleotide sequence) (SEQ ID NO: 56)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCAC CCTTTACTGC

• intracellular domain 4-1BB (amino acid sequence) (SEQ ID NO: 16)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

• intracellular domain 4-1BB (nucleotide sequence) (SEQ ID NO: 60)

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

• CD3-zeta domain (amino acid sequence) (SEQ ID NO: 17)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

• CD3-zeta (nucleotide sequence) (SEQ ID NO: 101)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

• CD3-zeta domain (amino acid sequence; NCBI reference sequence NM_000734.3) (SEQ ID NO: 43)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

• CD3-zeta (nucleotide sequence ; NCBI reference sequence NM_000734.3) (SEQ ID NO: 44)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

• IgG4 hinge (amino acid sequence) (SEQ ID NO: 102)

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

• IgG4 hinge (nucleotide sequence) (SEQ ID NO: 103)

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCAGCAGCATCGAG AAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACT ACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG

All of these clones had an alteration of the Q/K residue in the signaling domain of the 4-1BB-derived costimulatory domain.

The sequences of the humanized scFv CDR domain sequences are shown in Table 4 for the heavy chain variable domains and in Table 5 for the light chain variable domains. "ID" denotes the corresponding SEQ ID NO for each CDR.

Table 4
CDRs of the variable domains of the heavy chain (Kabat) Candidate FW HCDR1 ID HCDR2 ID HCDR3 ID mouse_CART19 GVSLPDYGVS 19 VIWGSETTYYNSALKS 20 HYYYGGSYAMDY 24 humanized_CART19 a VH4 GVSLPDYGVS 19 VIWGSETTYYSSSLKS 21 HYYYGGSYAMDY 24 humanized_CART19 b VH4 GVSLPDYGVS 19 VIWGSETTYYQSSLKS 22 HYYYGGSYAMDY 24 humanized_CART19 c VH4 GVSLPDYGVS 19 VIWGSETTYYNSSLKS 23 HYYYGGSYAMDY 24

Table 5
CDRs of the light chain variable domains Candidate FW LCDR1 ID LCDR2 ID LCDR3 ID mouse_CART19 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27 humanized_CART19 a VK3 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27 humanized_CART19 b VK3 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27 humanized_CART19 c VK3 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27

Table 6 is an identification key for mapping the CD19 construct number designations to a specific orientation of the scFv light and heavy chains, the number of linker units (i.e., (G4S) ₃ (SEQ ID NO: 107) or (G4S) ₄ (SEQ ID NO: 106)) separating the heavy and light chains, and the differing amino acid sequences in the heavy chain CDR2.

Table 6
Designations CD19 CAR. Clone ID/
CAR No. Alternate clone ID Chain orientation Linkers Mutation site in CDR2 of the heavy chain SEQ ID NO 104875 (CAR1) C2136 L2H 3x YSSSL 28 104876
(CAR2) C2137 L2H 3x YQSSL 29 104877
(CAR3) C2138 H2L 3x YSSSL 28 104878
(CAR4) C2139 H2L 3x YQSSL 29 104879
(CAR5) C2140 L2H 4x YSSSL 28 104880
(CAR6) C2141 L2H 4x YQSSL 29 104881
(CAR7) C2142 H2L 4x YSSSL 28 104882
(CAR8) C2143 H2L 4x YQSSL 29 105974
(CAR9) C2144 L2H 4x YNSSL 30 105975
(CAR10) C2145 H2L 4x YNSSL 30 105976
(CAR11) C2146 L2H 3x YNSSL 30 105977
(CAR12) C2147 H2L 3x YNSSL 30 CTL019 muCART19 L2H 3x YNSAL 57

The scFv CAR fragments were then cloned into lentiviral vectors to generate the full-length CAR construct in a single coding reading frame and using the EF1 alpha promoter for expression (SEQ ID NO: 100).

EF1 alpha promoter

CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT TTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTG CCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCC GCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC GGTTTTTGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCG TGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGTGAGTCACCCACACAAA GGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCA CACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA (SEQ ID NO: 100).

Analysis of humanized CAR constructs was performed as described in Example 4.

Example 4: Analysis of humanized constructs in CART for CD19

To evaluate the feasibility of targeting CD19 using CAR technology, single-chain variable fragments of anti-CD19 antibodies were cloned into a lentiviral expression vector for CAR with the CD3 zeta chain and the costimulatory molecule 4-1BB in four different configurations, and the optimal construct was selected based on the quantity and quality of effector T cell responses of CD19 CAR-transduced T cells ("CART19" or "CART19 T cells") to CD19+ targets. Effector T cell responses include, but are not limited to, cell expansion, proliferation, duplication, cytokine production, and target cell killing or cytolytic activity (degranulation).

Materials and methods

Generation of redirected T cells with humanized CART19

Lentiviral transfer vectors for humanized CART19 were used to generate genomic material packaged into pseudotyped lentiviral particles (VSVg). The lentiviral DNA transfer vector was mixed with the three packaging components (VSVg, gag/pol, and rev) in combination with the lipofectamine reagent for cotransfection into 293T cells. After 24 and 48 hours, the medium was collected, filtered, and concentrated by ultracentrifugation. The resulting viral preparation was stored at -80°C. The number of transducing units was determined by titration on SupT1 cells. Redirected CART19 T cells were generated by activating fresh naive T cells with CD3x28 beads for 24 hours and then adding the appropriate number of transducing units to obtain the desired percentage of transduced T cells. These modified T cells were allowed to expand until they became quiescent and shrank in size, after which they were cryopreserved for further analysis. Cell number and size were measured using a Coulter Multisizer™ III. Before cryopreservation, the percentage of transduced cells (expressing CART19 on the cell surface) and the relative fluorescence intensity of this expression were determined by flow cytometry analysis using LSRII. Histograms can be used to determine relative CAR expression levels by comparing the percentage of transduced cells with their relative fluorescence intensity.

Evaluation of cytolytic activity, proliferative capacity and cytokine secretion of redirected T cells with humanized CART19.

To assess the functional abilities of humanized CAR19 T cells to kill, proliferate, and secrete cytokines, cells were thawed and allowed to recover overnight. In addition to humanized CART19 cells, mouse CART19 cells were used for comparative purposes, while SS1-BBz served as a non-targeted, expressed CAR to study the background effects of CAR/T cells. A gold standard (GS) CART19 was used in all assays to compare assay variations. It is important to note that GS CART19 cells represent cells produced under production conditions suitable for research purposes (i.e., not for clinical use), and IL-2 was added to the growing culture. This likely impacts the overall viability and functionality of these cells and should not be directly compared with the production of other research-grade transduced T cell populations. T cells were directed to kill K562, a chronic myelogenous leukemia cell line expressing or lacking CD19, or Pt14, B cells obtained from a patient with CLL. For this flow cytometry-based cytotoxicity assay, target cells were stained with CSFE to quantify their presence. Target cells were stained for CD19 expression to confirm similar levels of target antigens. The cytolytic activity of CAR19 T cells was measured by titration at effector:target cell ratios of 10:1, 3:1, 1:1, 0.3:1, and 0:1, where effectors were defined as T cells expressing the anti-CD19 chimeric receptor. Assays were initiated by mixing the appropriate number of T cells with a constant number of target cells. After 16 hours, each mixture was completely removed and each well was thoroughly washed, combining as appropriate. T cells were stained for CD2, and all cells were stained with the live/dead cell marker 7AAD. After the final wash, the pelleted cells were resuspended in a predetermined volume with a predetermined number of beads for counting. Cell staining data were collected by flow cytometry on an LSRII and analyzed using FloJo software, using beads for quantitative results.

To measure cell proliferation and cytokine production by humanized CAR19 T cells, cells were thawed and allowed to recover overnight. In addition to humanized CART19 cells, mouse CART19 cells were used for comparative purposes, while SS1-BBz served as a nontargeted, expressed CAR to study the background effects of CAR/T cells. The gold standard (GS) CART19 was used in all assays to compare assay variations. T cells were directed against either K562, a chronic myelogenous leukemia cell line that either expresses or lacks CD19, or Pt14, B cells derived from a patient with CLL. Additionally, CD3x28 beads were used to assess the potential ability of T cells to respond to endogenous immunological signals. For proliferation analysis, T cells were stained with CSFE. Proliferation is evident as a dilution of the CSFE dye, reflecting the partitioning of the parental labeling into two daughter cells. The assay was performed only at effector:target ratios of 1:1 and 1:0, with effectors defined as T cells expressing the anti-CD19 chimeric receptor. The assay was performed in duplicate, and 24 hours after cell mixing, 50% of the medium was removed/replaced for cytokine analysis using the Luminex 10-plex human cytokine panel. After 5 days, T cells were stained for CAR expression, phenotyped as either CD4 or CD8 cells, and stained with 7AAD to determine live/dead cells. After a final wash, the pelleted cells were resuspended in a defined volume with a predetermined number of BD beads for counting. Cell staining data were collected by flow cytometry on LSRII and analyzed using FloJo software, using beads to quantify results. Total cell counts were determined as the number of cells counted per bead count, multiplied by the fraction of beads remaining to be counted.

To assess the potential ability of humanized CART19 cells to function similarly to modern, efficient mouse CART19 cells, the authors decided to evaluate their ability to kill target cells, proliferate in response to a target antigen, and demonstrate persistence in vitro . By packaging each of the humanized CART19 lentiviral constructs and titrating them on SupT1 cells, the authors determined that the amount of virus required to normalize transduction was approximately 50%. This allowed for more direct comparisons of activity, starting from a similar average number of integration sites per cell.

Therapeutic CAR19 T cells were obtained from normal donor blood after apheresis, with naive T cells obtained by negative selection for T cells, CD4+, and CD8+ lymphocytes. These cells were activated with CD3x28 beads in 10% RPMI at 37°C, 5% _CO2 .

After 24 hours, the T cells were disrupted and a standardized amount of virus was added. T cells began dividing in a logarithmic growth pattern, which was monitored by measuring cell counts per mL and cell size. As the T cells entered a quiescent state, logarithmic growth weakened and cell size decreased. The combination of a decrease in growth rate and T cell size approaching ~300 fL determines the state at which T cells should be cryopreserved or re-stimulated.

A very similar trend exists for resting T cells, as judged by their sizes. The nearly overlapping patterns between humanized CAR19 cells, conventional mouse CART19 cells, and the UTD population suggest no unusual effect of humanized CAR19 on conventional T cell expansion after activation. SS1-BBz was used as a control to detect unwanted antigen-independent CAR activity. The expansion profile of total cell numbers shows that differences in actual numbers in individual expanding populations appear to arise primarily from different starting cell numbers. When normalized to starting T cell numbers, a tight cluster of all CART19 cells is observed. Furthermore, the unwanted effect of antigen-independent CAR activation was detected in a line with lower values and an outlier in the group.

The surface expression level for each of these CAR19-expressing cells was determined. The titrated virus, normalized by transduction, demonstrated comparable expression levels, correlating with transduction efficiency and the percentage of transduced cells. Titers for some CARs were extrapolated from earlier packaging, and although their percentage of transduced cells was lower, their MFI values also decreased, as expected. The results indicate that there are no apparent negative effects of humanized CAR19 on the ability of cells to proliferate normally compared to UTD and mouse CAR19-expressing T cells.

The ability of cells with humanized CART19 to selectively recognize and kill a specific cell surface epitope expressed on cells was studied. Wild-type K562 cells do not express CD19 but can be transduced to express CD19. When comparing these killing curves, titration of the number of effector cells revealed that these CD19-expressing cells were killed. Redirected T cells from the same donor modified with either humanized CART19 or modern clinical mouse CART19 did not differ in their killing capacity. Killing curves showed that very similar killing capacities were found among cells with humanized CART19 directed against CD19+ CLL cells from patient 14. Interestingly, a decrease in overall cytolytic activity, particularly GS CART19, was observed, suggesting that these cells may have specific inhibitory properties. Similar levels of CD19 expressed on target cells indicate that expression level is not responsible for differences in cell killing.

The requisite ability of humanized CART19 cells to proliferate upon encountering target cells was observed for all constructs after stimulation with control CD3x28 beads and CD19-expressing targets. Pt14-targeted CLL cells appeared to exhibit a slightly higher proliferation rate with the light-to-heavy chain scFv, while no difference was observed with the 3x or 4x GGGGS binder (SEQ ID NOS 107 and 106, respectively). Proliferation assay results reflect the total number of cells accumulated over 5 days, indicating that humanized CART19, 2146, 2144, 2136, 2141, and 2137 provide an enhanced proliferative signal to T cells. Strikingly, this was found in cells with humanized CART19 targeting Pt14 CLL cells.

Overall, humanized CART19 constructs exhibited very similar characteristics to currently used CART19 in terms of cytolytic activity, proliferative response, and cytokine secretion in response to antigen-specific targets. The potential ability of humanized CART19 (2146, 2144, 2136, 2141, and 2137) to impart a stronger proliferative signal to T cells upon target activation is likely an additional advantage of these new constructs for potential enhancement of the therapeutic response.

Results

Using degranulation and cytokine production assays, it was demonstrated that CART19-engineered T cells specifically target CD19+ cells.

ND317 cells transduced with the inventive humanized CD19 CAR constructs (also referred to as "huCART19") were analyzed. There was a significant similarity in T cell size during expansion following CD3x28 activation and transduction with humanized CART19 candidates compared to transduced murine CART19 and unmodified (UTD) T cells.

Experiments showed little difference in the number of T cells that accumulate during their expansion following CD3x28 activation and transduction with various humanized CART19 candidates compared to transduced murine CART19 and unmodified (UTD) T cells.

Cell surface expression levels of humanized CART19 were comparable, and these levels were very similar to those of mouse CART19. Histograms of cell surface expression staining results were constructed for each T cell transduced with humanized CART19, and the mean fluorescence intensity (MFI) calculated from these profiles correlated well with the percentage of transduced cells.

Furthermore, humanized CART19 cells had similar specific cytotoxic activities when directed at CD19-expressing target cells and were comparable to those of mouse CART19 cells. Flow cytometric killing assays were plotted for 16 hours using titrating effector-to-target (E:T) ratios, in which humanized CART19 effector cells were directed at CSFE-labeled K562cc (Figure 1A, non-CD19-expressing controls), K562.CD19 (Figure 1B, K562 cells transduced to express CD19), or Pt14 (Figure 1C, B cells from a patient with CLL). The cytolytic activities of all humanized CART19 cells were similar and comparable to those of mouse CART19 cells. Differences in cytolytic activity between the different targets were similar and comparable, indicating that the activity of cells with murine CART19 is retained in cells with the humanized form of CART19.

Histograms plotted for CFSE-labeled cells with humanized CART19 6 days after mixing with target cells demonstrated their proliferative capacity (Figure 5). The proliferative response induced by CAR19 is necessary after target cell engagement and killing to achieve a positive clinical response. Dilution of SS1-BBz CSFE staining, an indicator of the presence of daughter cells in which parental cell staining is diluted, is a consequence of non-resting T cells continuing to divide via a target-independent mechanism.

The overall proliferative capacity of cell populations was assessed using CD3x28 beads, which mimic endogenous TCR and costimulator CD28 binding. The data indicate that all cell populations have comparable proliferative potential. All humanized and murine CART19 cells proliferated strongly and comparably when exposed to CD19-expressing K562 cells. Humanized CART19 cells also responded well to CLL patient-derived B cells, although some appeared to respond to a slightly lesser extent. As shown in Figures 2A and 2B, humanized CART19 cells 2136, 2137, 2140, 2141, 2144, and 2146 demonstrated slightly greater proliferation, as evidenced by higher dilution of the CSFE dye. All of these constructs had the same orientation of the variable chain, from light chain to heavy chain, indicating that this orientation is preferred. A closer examination of the amino acid changes at the heavy chain CDR2 site (Table 1) revealed that each of the three variations, YSSSL, YQSSL, and YNSSL (SEQ ID NOS: 28, 29, and 30, respectively), was present in constructs that appeared to exhibit greater proliferation upon contact with targets. Furthermore, these constructs had both a G4S linker containing 3 copies of the subunit (3 G4S) (SEQ ID NO: 107) and a G4S linker containing 4 copies of the subunit (4 G4S) (SEQ ID NO: 106), indicating that linker size does not affect function.

Based on the proliferation results described above, the total cell number was determined 5 days after tumor exposure. The cell number was lower than initially seeded, indicating that activation is necessary to maintain viability. Endogenous activation control was analyzed, and the total cell number was similar after 6 days. A study of humanized CART19 cells targeting CD19-expressing K562 cells revealed that both mouse CART19 cell lines ultimately had higher cell numbers, with 2146 cells having slightly higher numbers than all other constructs with similar values. Total cell counts were also analyzed 6 days after exposure to B cells from patient 14 (pt14), and it is interesting to note that previously selected humanized CART19 constructs 2146, 2144, 2136, 2141, and 2137, all of which had a light-to-heavy chain orientation and represented three amino acid variations YSSSL, YQSSL, and YNSSL (SEQ ID NOS: 28, 29, and 30, respectively), had higher total cell counts than those with mouse CART19. This unexpected difference between different clones with humanized anti-CD19 CARs may lead to higher clinical efficacy of CART cells transduced with these constructs.

Baseline levels of cytokines produced by humanized CART19 cells were analyzed after exposure to control K562 cells, which do not express CD19. Supernatants obtained after 24 hours were analyzed using a 30-plex Luminex panel. Potential cytokine profiles following stimulation of the endogenous immune system with CD3x28 beads indicated that all cell populations had comparable cytokine profiles.

The data also showed that cells with humanized CART19 and mouse CART19 produced similar cytokine profiles at similar levels when exposed to the same targets. The cytokine profile was lower but similar when exposed to Pt14 target cells.

Example 5: Treatment with humanized CD19 CAR T cells in in vivo ALL models.

Primary human ALL cells were grown in immunodeficient mice without the need for in vitro culture. These mice can be used to test the efficacy of chimeric antigen receptor (CAR) T cells in a model representative of the clinical patient population. In the model used here, HALLX5447 cells were passaged twice in NOD.Cg- Prkdc ^scid Il2rg ^tmlWjl /SzJ (NSG) mice before use in CAR T cell efficacy studies.

T cells expressing a murine CD19 CAR have previously been shown to target and kill leukemia cells in a NSG mouse model of primary human ALL. Anti-CD19 scFvs (single-chain variable Fc fragments) were humanized, and in this study, the ability of T cells expressing the humanized CD19 CAR (CAR 2) to eliminate ALL tumor cells in vivo was compared with that of T cells expressing a murine CD19 CAR. In this study, the efficacy of these cells was directly compared in mice bearing established primary human ALL, as assessed by FACS analysis of human CD19+ cells in peripheral blood. Following intravenous implantation of 1.5 x ¹⁰⁶ primary ALL cells, a disease burden of 2.5-4% human CD19+ cells in the blood was achieved 2 weeks after tumor implantation. This was the percentage of CD19+ cells among all cells in the blood of mice. All human cells in the mice before CAR T cell treatment were tumor cells. A percentage of CD19+ human cells greater than 2% in peripheral blood is considered a hallmark of advanced human ALL disease in this model. Mice with leukemia were treated with CAR T cells after the mice developed leukemia, approximately two to three weeks after tumor implantation. Mice in each group were treated with a total of 5 × ¹⁰⁶ human T cells. The transduction efficiency of donor human T cells by CAR-expressing lentivirus was 40-60%. After T cell treatment, mice had weekly blood draws to analyze the percentage of CD19+ human cells in the blood as a biomarker of disease progression.

Materials and methods:

Primary human ALL cells: Primary cells were not cultured.in vitrobefore implantation. These cells were obtained from a patient with ALL and then transferred into mice for cell development and expansion. After the tumor cells were expanded in mice, bone marrow and splenocytes were collected and viably frozen in separate batches for reimplantation. Cells were frozen in 90% DMSO and 10% FBS at a minimum concentration of 5×10⁶cells per milliliter. For reimplantation, frozen ALL cells were thawed and then intravenously injected into NSG mice to generate ALL mice that will be used to compare the antitumor efficacy of humanized CD19 CAR T cells and mouse CD19 CAR T cells.

Mice : 6-week-old NSG mice (NOD.Cg-Prkdc ^scid Il2rg ^tmlWjl /SzJ) were obtained from Jackson Laboratory (article 005557). Animals were acclimatized in the Novartis NIBRI animal facility for at least 3 days prior to experiments. Animals were handled in accordance with Novartis ACUC guidelines and principles.

Tumor implantation :Serially passivatedin vivoPrimary human ALL cells, model HALLX5447, were thawed in a 37°C water bath. The cells were then transferred to a 15-mL conical tube and washed twice with cold sterile PBS. Following this, primary ALL cells were counted and resuspended at a concentration of 15×10⁶cells per milliliter of PBS. The cells were placed on ice and immediately (within one hour) implanted into mice. ALL cells were injected intravenously into the tail vein in a volume of 100 μl, for a total of 1.5 x 10⁶cells per mouse.

CAR T cell dosing: Mice were injected with 5×10⁶T cells 16 days after tumor implantation. Cells were partially thawed in a 37°C water bath and then completely thawed by adding 1 ml of cold sterile PBS to the tube containing the cells. Thawed cells were transferred to 15-ml Falcon™ tubes and brought to a final volume of 10 ml with PBS buffer. Cells were washed twice by centrifugation at 1000 rpm for 10 minutes each time and then counted on a hemocytometer. T cells were then resuspended at a concentration of 50 × 10⁶cells per ml of cold PBS and kept on ice until administration to mice. Mice were injected intravenously into the tail vein with 100 μl of CAR T cells, corresponding to a dose of 5×10⁶T cells per mouse. Five mice in each group received 100 μl of either PBS (PBS), untransduced T cells (surrogate), murine CD19 CAR T cells (muCTL019), or humanized CD19 CAR T cells (huCTL019). Untransduced T cells, muCTL019 T cells, and huCTL019 T cells were obtained from the same human donor and prepared in parallel.

Animal Monitoring: The mice's health was monitored daily, including twice-weekly body weight measurements. The percentage change in body weight was calculated using the formula (BW_current- MT_original)/(MT_original)×100%. Tumor burden was monitored weekly by FACS analysis of peripheral blood. Mice were bled weekly via the tail vein into EDTA-coated tubes that were stored on ice. 10–20 μl of blood were transferred from the tubes to 96-well plates on ice. Red blood cells were lysed using ACK red blood cell lysis buffer (Life Technologies, catalog no. A 10492-01) and then washed twice with cold PBS. Cells were incubated with an Fc blocking mixture of human and mouse Fc blockers (Miltenyi Biotec, catalog nos. 130-059-901 and 130-092-575) for 30 min and then incubated with anti-human CD19 antibody for 30 min. Cells were fixed with 2% paraformaldehyde for 20 minutes, washed, and stored in PBS + 2% FBS overnight before analysis on a BD Canto or Fortessa, followed by analysis using FlowJo FACS software. Cells were analyzed to determine the percentage of human CD19+ cells in the blood of NSG mice bearing HALLX5447 human ALL tumor cells. The percentage of CD19+ cells in the blood was expressed as the mean ± standard error of the mean (SEM).

Treatment/control (T/C) percentages were calculated using the following formula:

% T/C=100×ΔΤ/ΔC, if ΔΤ≥0;

% regression = 100×ΔΤ/T _initial , if ΔΤ<0;

where T=mean percentage of CD19+ cells in the peripheral blood of animals in the drug-treated group on the last day of the study; _Tbaseline =percentage of CD19+ cells in the peripheral blood of animals in the drug-treated group on the first day of dosing; ΔT=mean percentage of CD19+ cells in the peripheral blood of animals in the drug-treated group on the last day of the study - the mean percentage of CD19+ cells in the peripheral blood of animals in the drug-treated group on the first day of dosing; C=mean percentage of CD19+ cells in the peripheral blood of animals in the control group on the last day of the study; and ΔC=mean percentage of CD19+ cells in the peripheral blood of animals in the control group on the last day of the study - the mean percentage of CD19+ cells in the peripheral blood of animals in the control group on the first day of dosing.

T/C values between 100% and 42% are interpreted as the absence or presence of minimal antitumor activity; T/C values between ≤ 42% and > 10% are interpreted as the presence of antitumor activity or tumor growth inhibition. T/C values ≤ 10% or regression values ≥ -10% are interpreted as tumor growth delay. Regression values < -10% are interpreted as regression.

Results:

The antitumor activity of murine and humanized CD19 CAR T cells was assessed and directly compared using a primary human ALL model. Following tumor implantation on day 0, mice were randomly assigned to treatment groups and treated intravenously with 5 × ¹⁰⁶ T cells on day 16. ALL disease burden and health status were monitored until the animals reached the endpoint. Mice in all groups were euthanized on day 65 post-tumor implantation, when disease burden in the control groups exceeded 80% human CD19+ cells in peripheral blood.

A clear difference in disease burden was observed between the control groups and those receiving either murine or humanized CD19 CAR T cells, with P < 0.01, beginning at day 24 after tumor implantation and continuing until the end of the study at day 65. Murine and human CD19 CAR T cells demonstrated similar abilities to control the growth of HALLX5447 human ALL tumor cells in NSG mice. Both groups showed a peak in peripheral blood disease, corresponding to 12-15% human CD19+ cells, at day 21 after HALLX5447 implantation. By 42 days after tumor cell implantation, human CD19+ cells were not detected in the huCTL019 group, while the percentage of human CD19+ cells in the muCTL019 group dropped to approximately 1%. Both murine and humanized CD19 CAR T cells exhibited comparable ability to control primary human ALL cell proliferation in this model (P>0.05). The % T/C value for the surrogate transduced T cell group was 94.40%, indicating that the surrogate transduced T cells did not have antitumor activity. The regression rate in the muCTL019 group was -89.75% and in the huCTL019 group was -90.46%, indicating that both treatments were capable of inducing regression in the HALLX5447 tumor model. The percentage of human CD19+ cells in peripheral blood as a measure of disease burden in these mice is shown in Figure 7. In the PBS treatment group, in which animals did not receive any T cells, the baseline growth kinetics of primary ALL tumor cells implanted intravenously in NSG mice were observed. Animals in the surrogate treatment group received untransduced T cells subjected to the same in vitro expansion process as CAR T cells. These cells served as a T-cell control to demonstrate a non-specific response to T cells in this tumor model. Continuous tumor progression was observed throughout the experiment in both the PBS and surrogate transduced T cell treatment groups. Both murine and humanized CD19 CAR T cells controlled disease progression within one week of injection of ^5x106 T cells and demonstrated consistent ability to maintain disease control throughout the 65-day study.

The antitumor activity of murine and humanized CD19 CAR T cells transduced with CAR T cells was assessed in efficacy studies in NSG mice bearing HALLX5447 cells of primary human ALL. This study demonstrated that both murine and humanized CD19 CAR T cells (muCTL019 and huCTL019) were capable of generating an antitumor response in the primary human ALL model. Furthermore, this response, as measured by disease burden in peripheral blood, was consistent between muCTL019 and huCTL019 cells. Both murine and humanized CD19 CAR T cells controlled primary ALL growth one week after mice were injected with the T cells. Initially after treatment, disease burden continued to increase before decreasing to a virtually undetectable level. A single administration of either murine or humanized CAR T cells resulted in a sustained antitumor response for 65 days, during which time disease progression occurred in mice receiving control drugs. Humanized CD19 CAR T cells demonstrated the ability to mount an effective response against CD19+ tumors and control ALL disease burden similar to that observed with murine CD19 CAR T cells.

Example 6: CD19-containing CAR T cells for use in the treatment of multiple myeloma.

Even with modern chemotherapy, targeted therapy, and autologous stem cell transplantation, myeloma is considered an incurable disease. This case report describes the treatment of multiple myeloma (MM) with autologous T cells targeting CD19 using a chimeric antigen receptor (lentivirus/CD19:4-1BB:CD3-zeta; also known as "CART19" or CTL019). This case report demonstrates that treatment with a CD19-targeted CAR has the potential to induce large-scale, durable, and reliable remissions by targeting myeloma stem cells and/or tumor cells that express CD19 at very low levels (undetectable by most assays).

In the treatment of a patient with aggressive secondary plasma cell leukemia, the authors found that administering CART19 two days after life-saving autologous stem cell transplantation resulted in rapid eradication of the plasma cell leukemia and a very good partial response in the patient, who had undergone multiple lines of chemotherapy. This patient had been transfusion-dependent for several months prior to treatment; after two months, her white blood cell count had recovered (with normal platelet and white blood cell counts) and she did not require transfusions, as she was discharged from the hospital after completing treatment.

Because myeloma cells do not naturally express CD19, the fact that treatment with CART19 induced a rapid and significant antitumor response in this tumor was surprising. Without being tied to any particular theory, it was logical to hypothesize that CART19 could be used to treat myeloma because: (1) while myeloma cells are traditionally thought to be negative for CD19 expression by flow cytometry, there is evidence that myeloma cells can express very small amounts of CD19, such that expression is detectable at the RNA level but not by flow cytometry or immunohistochemistry; and (2) there is the concept of targeting clonotypic B cells, which are thought to be the cancer stem cells that give rise to multiple myeloma and are particularly resistant to chemotherapy. There is a clonal relationship between B cells and myeloma tumor cells, but traditional myeloma treatment targets malignant plasma cells rather than B cells. Therefore, CART19-based myeloma treatment targets different cell populations than most melanoma treatments.

In this single case, the patient had circulating plasma cells, and the inventors were able to test her tumor cells for CD19 expression. Approximately 1-2% of her tumor cells expressed the CD19 antigen (Figure 8). Thus, it was reasonable to hypothesize that CART19 might have a direct effect on a very small population of the patient's tumor cells; however, a very good partial response would not be expected based on targeting only a very small population of CD19+ tumor cells.

In this case, CART19 was administered following autologous stem cell transplantation as a salvage transplant following high-dose melphalan treatment. Although this is standard therapy for myeloma, it is not curative. Furthermore, this patient had previously undergone tandem autologous stem cell transplants and relapsed shortly (<6 months) after transplantation. Without being bound by any particular theory, the use of CART19 cells, as described in this case, may employ an entirely separate mechanism for treating myeloma in combination with salvage autologous stem cell transplantation.

Ten more patients with multiple myeloma will be treated with CART19 in a phase I clinical trial.

Dose justification and risk/benefit ratio

For this protocol, the inventors selected a base dose using the intravenous route of administration. The primary objective of this protocol was to test the safety and feasibility of administering CART19 cells to patients with multiple myeloma. The main anticipated toxicities were (1) cytokine release when CAR cells encounter their surrogate antigen CD19 on malignant or normal B cells; (2) depletion of normal B cells, similar to what occurs with rituximab therapy; (3) skin reactions to steroids and gastrointestinal syndromes resembling graft-versus-host disease (GVHD), which have been observed previously when expanded/costimulated autologous T cells were used in combination with ASCT to treat MM. There was a theoretical concern that transformation or uncontrolled proliferation of CART19 T cells might occur in response to high levels of CD19. There was less concern in this case compared to another study for CLL patients, as the clonotypic B cell count in MM is expected to be much lower than the malignant B cell count in the refractory CLL patients treated in this study.

Justification of doses

In the first 3 patients, we observed clinical activity at doses ranging from 1.4 x 10 ⁷ to 1.1 x 10 ⁹ CART19 cells. This result demonstrates that, at least in the first 3 patients treated, there is no apparent dose-response relationship. Complete responses were observed in patients who received doses with a log2 difference. Thus, unlike standard drugs, which are metabolized, CAR T cells have a wide dose-response range. This is likely due to the extensive proliferative capacity of CAR T cells in patients. Therefore, we determined a dose range of 1-5 x 10 ⁸ CART19 cells for infusion. In the described single-patient study, which was offered for compassionate reasons, the patient was offered up to 5 x 10 ⁸ CART19 cells, with no lower dose limit. For the ten-patient trial, they will be offered 1-5× ¹⁰⁷ CART19 cells.

General design

A single-patient study was conducted on a compassionate basis; it was modeled after a phase I clinical trial to determine the safety of infusion of autologous T cells transduced to express CART19. The primary objectives of the study were to determine the safety, tolerability, and engraftment potential of CART19 T cells in patients undergoing salvage ASCT following early relapse following a first ASCT. A protocol for an open-label pilot study was developed.

Subjects enrolled in the study will undergo bone marrow biopsy and evaluation of their MM using routine laboratory and imaging methods. Eligible subjects will undergo inpatient apheresis to obtain large numbers of peripheral blood mononuclear cells (PBMCs) for CART19 production. T cells will be isolated from PBMCs, transduced with the TCRζ/4-1BB lentiviral vector, expanded in vitro , and then frozen for subsequent administration. The number of patients with inadequate T cell collection, expansion, or production results will be compared with the number of patients whose T cells were successfully produced; drug production is not expected to be problematic in this patient population.

Typically, subjects will have enough peripheral blood stem cells stored from the time of mobilization/collection in preparation for their first ASCT to undergo two additional ASCTs. Those who do not have this will undergo a second mobilization/collection procedure before or after their inpatient apheresis, using the regimen chosen by their treating physician. Approximately two weeks after the initial leukapheresis, subjects will be admitted to the hospital and receive a high-dose melphalan (day -2), followed by an autologous stem cell infusion two days later (day 0), and all subjects will receive an infusion of CART19 cells four days later (day +2). The study will enroll up to 10 patients.

All subjects will undergo blood tests to assess safety, engraftment, and persistence of CART19 cells at regular intervals throughout week 4 of the study. On days +42 and +100, subjects will undergo a bone marrow aspiration/biopsy to assess bone marrow plasma cell counts and CART19 cell delivery to the bone marrow. Formal response assessment will be performed at day 100 according to the International Myeloma Working Group (IMWG) 136 criteria, and time to disease progression (TTP) will be monitored according to routine clinical practice for patients with multiple myeloma. The primary efficacy endpoint measured in this study will be the comparison of TTP after the patient's first ASCT with TTP after ASCT in this study.

Because the primary outcome in this study is the safety and feasibility of infusing CART19 cells with ASCT, the study will utilize an early stopping rule. Briefly, if fewer than two severe unexpected adverse events occur in the first five treated patients, five additional subjects will be enrolled to bring the study enrollment to the planned 10 subjects. Subjects will be observed for 40 days after CART19 infusion (i.e., until the first formal response assessment at day 42) before enrolling the next subject, until five subjects have been enrolled and will be observed. No waiting periods will be required between each subsequent subject for treatment of the second group of five patients.

After six months of intensive follow-up, subjects will be assessed at least quarterly for two years, with medical histories, physical examinations, and blood tests. Following this assessment, subjects will be enrolled in an additional study, which will include annual telephone interviews and questionnaires for up to thirteen years to assess long-term health issues, such as the development of new malignancies.

Main outcomes assessed

This pilot study is designed to test the safety and feasibility of using autologous CD19 TCRζ/4-1BB-transduced T cells in patients receiving salvage ASCT for MM after early relapse following a first ASCT.

The main safety and usability outcomes assessed include:

The occurrence of study-related adverse events, defined as NCJ CTC 2: Grade 3 signs/symptoms, laboratory toxicities, and clinical events possibly or definitely related to study drug at any time from the time of infusion to week 24. This includes infusion toxicities as well as any toxicity possibly related to CART19 cells, including, but not limited to:

a. fever

b. rash

c. neutropenia, thrombocytopenia, anemia, bone marrow aplasia

d. liver dysfunction

e. pulmonary infiltrates or other pulmonary toxicity

f. GVHD-like syndromes involving the gastrointestinal tract or skin.

The possibility of generating CART19 cells from patient apheresis products. The number of products obtained that do not meet the final product release criteria in terms of vector transduction efficiency, purity, viability, T-cell sterility, and tumor contaminants will be determined.

The depth and duration of response after CART19-specific autologous stem cell transplantation will be compared with the depth and duration of response achieved in each patient after standard autologous stem cell transplantation.

Selection and exclusion of subjects from the study

Inclusion criteria

Subjects must have previously undergone ASCT for MM and have disease progression within 365 days of stem cell infusion. Subjects who have undergone two prior ASCTs as part of a planned consolidated tandem ASCT regimen are eligible for the study. Disease progression will be defined according to the IMWG criteria for progressive disease or, for patients who achieved a CR or sCR after the initial ASCT, the criteria for relapse after CR (Durie et al. Leukemia 2006; 20(9): 1467-1473). Note: Patients are not required to be enrolled within 365 days of their first ASCT, and patients may have received other treatments, including experimental agents, after relapse/progression following a previous ASCT before enrollment in this study.

Subjects must provide a signed informed consent form.

Subjects must have sufficient vital organ function to receive the high dose of melphalan, as defined by the following, measured within 12 weeks prior to the date of melphalan infusion: a. serum creatinine ≤ 2.5 or estimated creatinine clearance ≥ 30 mL/min and not dependent on dialysis, b. SGOT ≤ 3x the upper limit of normal and total bilirubin ≤ 2.0 mg/dL (except for patients whose hyperbilirubinemia is explained by Gilbert's syndrome), c. left ventricular ejection fraction (LVEF) ≥ 45% or, if LVEF < 45%, formal assessment by a cardiologist certifying the absence of clinically significant impairment of cardiovascular function. LVEF assessment must be performed within six weeks prior to study entry, d. adequate pulmonary function with FEV1, FVC, TLC, and DLCO (after appropriate adjustment for lung volume and hemoglobin concentration) ≥ 40% of the expected values. Pulmonary function testing should be performed within six weeks prior to study inclusion.

Subjects must have an ECOG performance status of 0-2, unless the higher performance status is solely due to bone pain.

Exclusion criteria Subjects must not:

Have any active and uncontrolled infection.

Have active hepatitis B, hepatitis C, or HIV infection.

Any uncontrolled medical condition that precludes participation as described.

Treatment regimen

Therapy for relapsed/progressive multiple myeloma

Patients may receive treatment for relapsed/progressive multiple myeloma prior to study enrollment, as determined by their treating physician. Treatment may continue after study enrollment.

Patients should discontinue all medications two weeks prior to apheresis and two weeks prior to receiving high-dose melphalan. If the expected interval between apheresis and high-dose melphalan is more than two weeks, patients may resume treatment after apheresis at the discretion of their treating physician.

High dose melphalan (day -2)

Patients will be admitted to the hospital on day -3 or -2 and will undergo an examination by the treating physician and routine laboratory testing, including monitoring of tumor lysis syndrome parameters, before starting treatment according to the protocol. Blood for laboratory tests to monitor MM (SPEP, immunoglobulin quantification, and serum free light chain analysis) will be drawn before therapy begins, unless such tests have been performed within 7 days prior to hospital admission.

High-dose therapy will include intravenous melphalan at a dose of 200 mg/ ^m2 over approximately 20 minutes on day -2. The melphalan dose will be reduced to 140 mg/ ^m2 for patients over 70 years of age or for patients of any age who, in the opinion of the treating physician, may not tolerate a dose of 200 mg/ ^m2 . All patients will receive standard antiemetic medications prophylactically, which may include dexamethasone, and a standard antibiotic prophylactically.

Stem cell re-infusion (day 0)

Stem cell infusion will be administered on day 0, at least 18 hours after high-dose melphalan administration. Stem cells will be administered by intravenous infusion over approximately 20-60 minutes after premedication, according to standard institutional practice. At least 2 x ¹⁰⁶ CD34+ progenitors/kg body weight should be administered. In addition, at least 1 x ¹⁰⁶ CD34+ progenitors/kg body weight should be available as reserve stem cells for infusion in case of delayed engraftment or late graft rejection. G-CSF should be administered subcutaneously beginning on day +5, at doses consistent with standard institutional practice. Other supportive care, such as maintenance transfusion, will be administered according to standard institutional guidelines.

CART19 cell infusion (day +2)

A single dose of CART19-transduced T cells, comprising up to 5× ¹⁰ CART19 cells, will be administered. In this protocol, there will be no minimum acceptable dose of infused cells transduced with the CD19 TCRζ/4-1BB vector per patient. CART19 cells will be administered as a single dose by rapid intravenous infusion on day +2 after the stem cell infusion.

Maintenance lenalidomide

Subjects who received and tolerated lenalidomide maintenance therapy after their first ASCT will restart lenalidomide maintenance therapy at approximately day +100, unless contraindicated in the opinion of their treating physician.

Obtaining and administering the study drug

CART19 T cells will be generated at the CVPF and will remain there until they meet FDA-approved product release criteria for infusion cells (e.g., cell dose, cell purity, sterility, average vector copy number/cell, etc.). After release, the cells will be transported to the hospital for administration.

Thawing cells. Frozen cells will be transported on dry ice to the patient's bedside. The cells will be thawed at the patient's bedside using a water bath maintained at 36°C to 38°C. The package should be gently massaged until the cells are completely thawed. No frozen clumps should remain in the container. If the CART19 cell preparation appears spoiled, the package leaks, or any other abnormalities are visible, the preparation should not be administered and should be returned to the CVPF as described below.

Premedication. Adverse effects following T-cell infusion include transient fever, chills, and/or nausea; for review, see Cruz et al. (Cytotherapy 2010; 12(6): 743-749). Premedication of the subject with acetaminophen and diphenhydramine hydrochloride prior to CART19 cell infusion is recommended. These medications can be repeated every six hours as needed. A course of nonsteroidal anti-inflammatory drugs may be prescribed if the patient continues to have a fever despite acetaminophen. It is recommended that patients not receive systemic corticosteroids such as hydrocortisone, prednisone, methylprednisolone, or dexamethasone except in life-threatening emergencies, as these drugs may have adverse effects on T cells. If corticosteroids are needed for an acute infusion reaction, an initial dose of 100 mg of hydrocortisone is recommended.

Pyretic reaction. In the unlikely event that a subject develops sepsis or systemic bacteremia following CAR T-cell infusion, appropriate cultures should be obtained and appropriate medical measures should be taken. If contamination of the CART19 T-cell product is suspected, the product should be retested for sterility using archived samples stored in the CVPF.

Administration. The infusion will be performed in an isolated room at Rhoads, with precautions taken for immunocompromised patients. Transduced T cells will be administered by rapid intravenous infusion at a flow rate of approximately 10 ml to 20 ml per minute through a latex-free Y-shaped infusion set with an 18G needle and a 3-way stopcock. The infusion will last approximately 2-20 minutes. Each infusion bag will be labeled with the following: "FOR AUTOLOGOUS USE ONLY." In addition to the label, at least two unique identifiers will be present, such as the subject's initials, date of birth, and study number. Before the infusion, two individuals will independently verify all this information in the presence of the subject, confirming that this information accurately corresponds to the study participant.

Emergency medical equipment (i.e., an ambulance) will be nearby during the infusion in case the subject develops an allergic reaction, severe hypotensive crisis, or any other reaction to the infusion. Vital signs (temperature, respiratory rate, pulse, and blood pressure) will be monitored before and after the infusion, then every 15 minutes for at least one hour and until these signs are stable and satisfactory. The subject will be asked not to leave the hospital until the physician deems it safe for the patient.

Package

The infusion will consist of a single dose of 1-5 x 10 ⁸ transduced CAR19 T cells, with a minimum acceptable dose of 1 x 10 ⁷ CART19 cells per infusion. Each bag will contain an aliquot (volume depends on the dose) of cryomedia containing the following infusion-grade reagents (% by volume): 31.25% plasmalyte-A, 31.25% dextrose (5%), 0.45% NaCl, up to 7.5% DMSO, 1% dextran 40, 5% human serum albumin.

Apheresis

A large-volume apheresis procedure (12-15 liters or 4-6 blood volumes) is performed at an apheresis center. This procedure yields PBMCs for CART19. A single leukapheresis session is expected to yield at least 50 x 10 ⁹ leukocytes for CART19 T cell generation. Baseline leukocytes for FDA-compliant retrospective studies and research are also obtained and cryopreserved. The cell preparation is expected to be ready for release in approximately 2-4 weeks. Lymphocyte subsets are enumerated by flow cytometry, including CD19 and CD20 B cell counts. Baseline levels are assessed for human anti-VSV-G and anti-mouse antigen (HAMA) antibodies. If a subject has previously had an adequate apheresis cell collection, stored in accordance with current good manufacturing practices at the Clinical Cell and Vaccine Production Facility, these cells could be used as a cell source for CART19 production. Using the stored apheresis product would reduce the cost, time, and risk to the subject should they undergo additional apheresis.

Cytoreductive chemotherapy

Anti-lymphoma chemotherapy will consist of high dose melphalan as described in this document.

CART19 infusion

The infusion will be started on day +2 after the stem cell re-infusion.

On day +2 before the first infusion, patients will have a complete blood count (CBC) with differential and CD3, CD4, and CD8 quantification, as chemotherapy is given in part to induce lymphopenia.

The first dose will be administered as a single dose. Cells will be thawed at the patient's bedside. Thawed cells will be infused at the maximum tolerated infusion rate, resulting in an infusion duration of approximately 10-15 minutes. To facilitate mixing, cells will be administered simultaneously using a Y-shaped adapter. Subjects will receive the infusion and premedication as described in this document. Subjects' vital signs will be assessed, and pulse oximetry will be performed before the dose, after the end of the infusion, and every 15 minutes thereafter for 1 hour and until stable and satisfactory. A blood sample for baseline CART19 levels will be collected at any time before the first infusion and between 20 minutes and 4 hours after each infusion (and sent to TCSL).

For patients experiencing signs of toxicity associated with high-dose melphalan administration, the infusion will be delayed until their condition improves. Specific toxicities that require T-cell infusion to be delayed include: 1) lungs: need for supplemental oxygen to maintain saturation above 95% or presence of radiographic abnormalities on chest x-rays that are progressive; 2) heart: new cardiac arrhythmia refractory to conservative management; 3) hypotension requiring vasopressors; 4) active infection: positive blood culture for bacteria, fungi, or viruses within 48 hours of T-cell infusion.

Monitoring toxicity manifestations

Uncontrolled T-cell proliferation. Toxicity associated with allogeneic or autologous T-cell infusion was managed with pharmacologic immunosuppression. T-cell-related toxicity has been reported to be responsive to systemic corticosteroids. In cases of uncontrolled T-cell proliferation (grade 3 or 4 toxicity associated with CART19 cells), subjects may receive corticosteroids. Subjects will receive methylprednisolone pulse therapy (2 mg/kg IV, divided doses every 8 hours × 2 days), followed by rapid tapering.

Additionally, based on observations in subjects treated with a different protocol, there is some concern about macrophage activation syndrome (MAS), although the CD19+ tumor burden is expected to be much lower in myeloma patients than in CLL patients. The decision regarding treatment and timing of treatment for this toxicity will be made by the patient's treating physician and the trial investigator. Suggested treatment includes: if a subject has a fever greater than 101°F for more than two consecutive days and there is no evidence of infection (negative blood cultures, chest X-ray (CXR), or other tests), tocilizumab at a dose of 4 mg/kg body weight may be considered. Corticosteroids and anti-TNF therapy may be used at the physician's discretion.

B-cell depletion. B-cell depletion and hypogammaglobulinemia are likely to occur. This is a common occurrence with anti-CD20 targeted therapy. In cases of clinically significant hypogammaglobulinemia (i.e., systemic infections), subjects will receive intravenous immunoglobulin (IVIG) according to established clinical dosing guidelines to restore normal serum immunoglobulin levels, as was the case with rituximab.

Rejection of the primary graft. Rejection of the primary graft (i.e., failure to engraft) may be more common after a second ASCT than after the first ASCT. Eligibility criteria for the study stipulate that sufficient stem cells must be available for a life-saving re-infusion, at the discretion of the treating physician, in the event of primary graft rejection.

EQUIVALENTS

The entire contents of each and every patent, patent application, and publication cited herein are incorporated herein by reference. While the invention has been described with reference to specific aspects, it is understood that other aspects and variations of the invention may be devised by those skilled in the art without departing from the spirit and scope of the invention. The appended claims are intended to include all such aspects and equivalent variations.

--->

SEQUENCE LIST

<110> NOVARTIS AG

TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA

<120> CANCER TREATMENT WITH HUMANIZED ANTI-CD19 CHIMERIC

ANTIGEN RECEPTOR

<130> N2067-7002WO

<140> PCT/US2014/029943

<141> 2014-03-15

<150> 61/838,537

<151> 2013-06-24

<150> 61/802,629

<151> 2013-03-16

<160> 121

<170> PatentIn version 3.5

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Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

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Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

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Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

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Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

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Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

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Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

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Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

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Thr Leu Val Thr Val Ser Ser

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Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

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Thr Leu Val Thr Val Ser Ser

245

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<212> PRT

<213> Artificial Sequence

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

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Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

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Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

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Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

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Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

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Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 8

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

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Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

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Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

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Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

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Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

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Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

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Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 9

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 9

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 10

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 11

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 11

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 12

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 13

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 14

<211> 45

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 14

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 15

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 15

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 16

<211> 42

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 16

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 17

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 17

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 18

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 18

Gly Gly Gly Gly Ser

1 5

<210> 19

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 19

Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

1 5 10

<210> 20

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 20

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 21

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 21

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

1 5 10 15

<210> 22

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 22

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

1 5 10 15

<210> 23

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 23

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

1 5 10 15

<210> 24

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 24

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 25

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 25

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 26

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 26

His Thr Ser Arg Leu His Ser

1 5

<210> 27

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 27

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 28

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 28

Tyr Ser Ser Ser Leu

1 5

<210> 29

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 29

Tyr Gln Ser Ser Leu

1 5

<210> 30

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic peptide"

<400> 30

Tyr Asn Ser Ser Leu

1 5

<210> 31

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 31

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 32

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 32

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 33

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 33

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 34

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 34

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 35

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 35

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 36

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 36

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 37

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 37

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 38

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 38

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 39

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 39

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 40

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 40

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 41

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note = "Description of the artificial sequence:

Synthetic polypeptide"

<400> 41

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 42

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 42

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 43

<211> 112

<212> PRT

<213> Homo sapiens

<400> 43

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 44

<211> 336

<212> DNA

<213> Homo sapiens

<400> 44

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc

60

tataacgagc tcaatctagg acgaagagg gagtacgatg ttttggacaa gagacgtggc

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat

180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc

240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc

300

tacgacgccc ttcacatgca ggccctgccc cctcgc

336

<210> 45

<211> 230

<212> PRT

<213> Homo sapiens

<400> 45

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 46

<211> 690

<212> DNA

<213> Homo sapiens

<400> 46

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc

60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag

120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac

180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc

240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa

300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag

360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg

420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc

480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg

540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag

600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag

660

aagagcctga gcctgtccct gggcaagatg

690

<210> 47

<211> 282

<212> PRT

<213> Homo sapiens

<400> 47

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 48

<211> 847

<212> DNA

<213> Homo sapiens

<400> 48

aggtggcccg aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca

60

gaaggcagcc tagccaaagc tactactgca cctgccacta cgcgcaatac tggccgtggc

120

ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga gaccaagacc

180

cctgaatgtc catcccatac ccagccgctg ggcgtctatc tcttgactcc cgcagtacag

240

gacttgtggc ttagagataa ggccaccttt acatgtttcg tcgtgggctc tgacctgaag

300

gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg

360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga

420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca

480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat

540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc

600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc

660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt

720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc

780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact

840

gaccatt

847

<210> 49

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic peptide"

<400> 49

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 50

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic oligonucleotide"

<400> 50

ggtggcggag gttctggagg tggaggttcc

30

<210> 51

<211> 48

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 51

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 52

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 52

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc

60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc

120

tcc

123

<210> 53

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<220>

<221> VARIANT

<222> (6)..(30)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (6)..(30)

<223> //note = "Variant residues given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<220>

<221> MISC_FEATURE

<222> (1)..(30)

<223> /note="This sequence may encompass 1-6 "Gly Gly Gly Gly Ser"

repeating units"

<220>

<221> MISC_FEATURE

<222> (1)..(30)

<223> note = "This sequence may include 1-6

repeating units of "Gly Gly Gly Gly Ser"

<400> 53

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 54

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic oligonucleotide"

<400> 54

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga

60

ccc

63

<210> 55

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 55

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg

60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg

120

gacttcgcct gtgat

135

<210> 56

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic oligonucleotide"

<400> 56

atctacatct gggcgccctt ggccggggact tgtggggtcc ttctcctgtc actggttatc

60

accctttact gc

72

<210> 57

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic peptide"

<400> 57

Tyr Asn Ser Ala Leu

1 5

<210> 58

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 58

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 59

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 59

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 60

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 60

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa

60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt

120

gaactg

126

<210> 61

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 61

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagcc accaccatca tcaccatcac cat

813

<210> 62

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 62

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagcc accaccatca tcaccatcac cat

813

<210> 63

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 63

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

tcatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaac atcaccacca tcatcaccat cac

813

<210> 64

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 64

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

caatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaac atcaccacca tcatcaccat cac

813

<210> 65

<211> 828

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 65

atggccctcc cagtgaccgc tctgctgctg cctctcgcac ttcttctcca tgccgctcgg

60

cctgagatcg tcatgaccca aagccccgct accctgtccc tgtcacccgg cgagagggca

120

accctttcat gcagggccag ccaggacatt tctaagtacc tcaactggta tcagcagaag

180

ccagggcagg ctcctcgcct gctgatctac cacaccagcc gcctccacag cggtatcccc

240

gccagatttt ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag

300

cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta caccttcggt

360

caaggtacca agctggaaat caagggaggc ggagatcag gcggtggcgg aagcggagga

420

ggtggctccg gaggaggagg ttcccaagtg cagcttcaag aatcaggacc cggacttgtg

480

aagccatcag aaaccctctc cctgacttgt accgtgtccg gtgtgagcct ccccgactac

540

ggagtctctt ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg

600

ggatcagaga ctacttacta ctcttcatca cttaagtcac gggtcaccat cagcaaagat

660

aatagcaaga accaagtgtc acttaagctg tcatctgtga ccgccgctga caccgccgtg

720

tactattgtg ccaaacatta ctattacgga gggtcttatg ctatggacta ctggggacag

780

gggaccctgg tgactgtctc tagccatcac catcaccacc atcatcac

828

<210> 66

<211> 828

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 66

atggccctcc cagtgaccgc tctgctgctg cctctcgcac ttcttctcca tgccgctcgg

60

cctgagatcg tcatgaccca aagccccgct accctgtccc tgtcacccgg cgagagggca

120

accctttcat gcagggccag ccaggacatt tctaagtacc tcaactggta tcagcagaag

180

ccagggcagg ctcctcgcct gctgatctac cacaccagcc gcctccacag cggtatcccc

240

gccagatttt ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag

300

cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta caccttcggt

360

caaggtacca agctggaaat caagggaggc ggagatcag gcggtggcgg aagcggagga

420

ggtggctccg gaggaggagg ttcccaagtg cagcttcaag aatcaggacc cggacttgtg

480

aagccatcag aaaccctctc cctgacttgt accgtgtccg gtgtgagcct ccccgactac

540

ggagtctctt ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg

600

ggatcagaga ctacttacta ccagtcatca cttaagtcac gggtcaccat cagcaaagat

660

aatagcaaga accaagtgtc acttaagctg tcatctgtga ccgccgctga caccgccgtg

720

tactattgtg ccaaacatta ctattacgga gggtcttatg ctatggacta ctggggacag

780

gggaccctgg tgactgtctc tagccatcac catcaccacc atcatcac

828

<210> 67

<211> 828

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 67

atggcactgc ctgtcactgc cctcctgctg cctctggccc tccttctgca tgccgccagg

60

ccccaagtcc agctgcaaga gtcaggaccc ggactggtga agccgtctga gactctctca

120

ctgacttgta ccgtcagcgg cgtgtccctc cccgactacg gagtgtcatg gatccgccaa

180

cctcccggga aagggcttga atggattggt gtcatctggg gttctgaaac cacctactac

240

tcatcttccc tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc

300

cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc caagcactac

360

tattacggag gaagctacgc tatggactat tggggacagg gcactctcgt gactgtgagc

420

agcggcggtg gagggtctgg aggtggagga tccggtggtg gtgggtcagg cggaggaggg

480

agcgagattg tgatgactca gtcaccagcc accctttctc tttcacccgg cgagagagca

540

accctgagct gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa

600

ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc tggtatcccc

660

gctcggttta gcggatcagg atctggtacc gactacactc tgaccatttc cagcctgcag

720

ccagaagatt tcgcagtgta tttctgccag cagggcaata cccttcctta caccttcggt

780

cagggaacca agctcgaaat caagcaccat caccatcatc accaccat

828

<210> 68

<211> 828

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 68

atggcactgc ctgtcactgc cctcctgctg cctctggccc tccttctgca tgccgccagg

60

ccccaagtcc agctgcaaga gtcaggaccc ggactggtga agccgtctga gactctctca

120

ctgacttgta ccgtcagcgg cgtgtccctc cccgactacg gagtgtcatg gatccgccaa

180

cctcccggga aagggcttga atggattggt gtcatctggg gttctgaaac cacctactac

240

cagtcttccc tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc

300

cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc caagcactac

360

tattacggag gaagctacgc tatggactat tggggacagg gcactctcgt gactgtgagc

420

agcggcggtg gagggtctgg aggtggagga tccggtggtg gtgggtcagg cggaggaggg

480

agcgagattg tgatgactca gtcaccagcc accctttctc tttcacccgg cgagagagca

540

accctgagct gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa

600

ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc tggtatcccc

660

gctcggttta gcggatcagg atctggtacc gactacactc tgaccatttc cagcctgcag

720

ccagaagatt tcgcagtgta tttctgccag cagggcaata cccttcctta caccttcggt

780

cagggaacca agctcgaaat caagcaccat caccatcatc atcaccac

828

<210> 69

<211> 828

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 69

atggccctcc cagtgaccgc tctgctgctg cctctcgcac ttcttctcca tgccgctcgg

60

cctgagatcg tcatgaccca aagccccgct accctgtccc tgtcacccgg cgagagggca

120

accctttcat gcagggccag ccaggacatt tctaagtacc tcaactggta tcagcagaag

180

ccagggcagg ctcctcgcct gctgatctac cacaccagcc gcctccacag cggtatcccc

240

gccagatttt ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag

300

cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta caccttcggt

360

caaggtacca agctggaaat caagggaggc ggagatcag gcggtggcgg aagcggagga

420

ggtggctccg gaggaggagg ttcccaagtg cagcttcaag aatcaggacc cggacttgtg

480

aagccatcag aaaccctctc cctgacttgt accgtgtccg gtgtgagcct ccccgactac

540

ggagtctctt ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg

600

ggatcagaga ctacttacta caattcatca cttaagtcac gggtcaccat cagcaaagat

660

aatagcaaga accaagtgtc acttaagctg tcatctgtga ccgccgctga caccgccgtg

720

tactattgtg ccaaacatta ctattacgga gggtcttatg ctatggacta ctggggacag

780

gggaccctgg tgactgtctc tagccatcac catcaccacc atcatcac

828

<210> 70

<211> 828

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 70

atggcactgc ctgtcactgc cctcctgctg cctctggccc tccttctgca tgccgccagg

60

ccccaagtcc agctgcaaga gtcaggaccc ggactggtga agccgtctga gactctctca

120

ctgacttgta ccgtcagcgg cgtgtccctc cccgactacg gagtgtcatg gatccgccaa

180

cctcccggga aagggcttga atggattggt gtcatctggg gttctgaaac cacctactac

240

aactcttccc tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc

300

cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc caagcactac

360

tattacggag gaagctacgc tatggactat tggggacagg gcactctcgt gactgtgagc

420

agcggcggtg gagggtctgg aggtggagga tccggtggtg gtgggtcagg cggaggaggg

480

agcgagattg tgatgactca gtcaccagcc accctttctc tttcacccgg cgagagagca

540

accctgagct gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa

600

ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc tggtatcccc

660

gctcggttta gcggatcagg atctggtacc gactacactc tgaccatttc cagcctgcag

720

ccagaagatt tcgcagtgta tttctgccag cagggcaata cccttcctta caccttcggt

780

cagggaacca agctcgaaat caagcaccat caccatcatc accaccat

828

<210> 71

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 71

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactacaatt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagcc accaccatca tcaccatcac cat

813

<210> 72

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 72

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

aactcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaac atcaccacca tcatcaccat cac

813

<210> 73

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 73

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 74

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 74

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 75

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 75

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys His His His His His His His

260 265 270

<210> 76

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 76

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys His His His His His His His

260 265 270

<210> 77

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 77

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

260 265 270

His His His His

275

<210> 78

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 78

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

260 265 270

His His His His

275

<210> 79

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 79

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His

260 265 270

His His His His

275

<210> 80

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 80

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His

260 265 270

His His His His

275

<210> 81

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 81

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

260 265 270

His His His His

275

<210> 82

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 82

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His

260 265 270

His His His His

275

<210> 83

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 83

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 84

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 84

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys His His His His His His His

260 265 270

<210> 85

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 85

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg

1458

<210> 86

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 86

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg

1458

<210> 87

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 87

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

tcatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaaa ccactactcc cgctccaagg ccacccaccc ctgccccgac catcgcctct

840

cagccgcttt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg

1458

<210> 88

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 88

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

caatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaaa ccactactcc cgctccaagg ccacccaccc ctgccccgac catcgcctct

840

cagccgcttt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg

1458

<210> 89

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 89

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gcggaggcgg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta ctcttcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 90

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 90

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gaggcggagg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta ccaatcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 91

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 91

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

tcatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg aggtggcgga

480

agcgaaatcg tgatgaccca gagccctgca accctgtccc tttctcccgg ggaacgggct

540

accctttctt gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag

600

ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag cgggattccc

660

gcacgcttta gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag

720

cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta caccttcggc

780

cagggcacca agcttgagat caaaaccact actcccgctc caaggccacc cacccctgcc

840

ccgaccatcg cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 92

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 92

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

caatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg aggcggtggg

480

tcagaaatcg tgatgaccca gagccctgca accctgtccc tttctcccgg ggaacgggct

540

accctttctt gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag

600

ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag cgggattccc

660

gcacgcttta gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag

720

cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta caccttcggc

780

cagggcacca agcttgagat caaaaccact actcccgctc caaggccacc cacccctgcc

840

ccgaccatcg cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 93

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 93

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gaggcggtgg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta caactcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 94

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 94

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gaggcggtgg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta caactcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 95

<211> 1473

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 95

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

aactcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg aggtggcgga

480

agcgaaatcg tgatgaccca gagccctgca accctgtccc tttctcccgg ggaacgggct

540

accctttctt gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag

600

ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag cgggattccc

660

gcacgcttta gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag

720

cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta caccttcggc

780

cagggcacca agcttgagat caaaaccact actcccgctc caaggccacc cacccctgcc

840

ccgaccatcg cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg

1473

<210> 96

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 96

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactacaact catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg

1458

<210> 97

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 97

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg

60

ccggacatcc agatgaccca aaccacctca tccctctctg cctctcttgg agacaggtg

120

accatttctt gtcgcgccag caggacatc agcaagtatc tgaactggta tcagcagaag

180

ccggacggaa ccgtgaagct cctgatctac catacctctc gcctgcatag cggcgtgccc

240

tcacgcttct ctggaagcgg atcaggaacc gattattctc tcactatttc aaatcttgag

300

caggaagata ttgccaccta tttctgccag cagggtaata ccctgcccta caccttcgga

360

ggagggacca agctcgaaat caccggtgga ggaggcagcg gcggtggagg gtctggtgga

420

ggtggttctg aggtgaagct gcaagaatca ggccctggac ttgtggcccc ttcacagtcc

480

ctgagcgtga cttgcaccgt gtccggagtc tccctgcccg actacggagt gtcatggatc

540

agacaacctc cacggaaagg actggaatgg ctcggtgtca tctggggtag cgaaactact

600

tactacaatt cagccctcaa aagcaggctg actattatca aggacaacag caagtcccaa

660

gtctttctta agatgaactc actccagact gacgacaccg caatctacta ttgtgctaag

720

cactactact acggaggatc ctacgctatg gattactggg gacaaggtac ttccgtcact

780

gtctcttcac accatcatca ccatcaccat cac

813

<210> 98

<211> 271

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 98

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 99

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 99

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg

60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc

120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa

180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca

240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag

300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga

360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc

420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc

480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt

540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca

600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa

660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa

720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc

780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg

840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg

900

aggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg

960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg

1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt

1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg

1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta

1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg

1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag

1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac

1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg

1440

caggccctgc cccctcgc

1458

<210> 100

<211> 1184

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 100

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt

60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg

120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tggggggagaa ccgtatataa

180

gtgcagtagt cgccgtgaac gttctttttc gcaacggggtt tgccgccaga acacaggtaa

240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt

300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg

360

ggtggggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg

420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg

480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt

540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg

600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc

660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg

720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg

780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat

840

ggagaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct

900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc

960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg

1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga

1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc

1140

agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga

1184

<210> 101

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 101

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc

60

tataacgagc tcaatctagg acgaagagg gagtacgatg ttttggacaa gagacgtggc

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat

180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc

240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc

300

tacgacgccc ttcacatgca ggccctgccc cctcgc

336

<210> 102

<211> 230

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 102

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 103

<211> 690

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 103

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc

60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag

120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac

180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc

240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa

300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag

360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg

420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc

480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg

540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag

600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag

660

aagagcctga gcctgtccct gggcaagatg

690

<210> 104

<211> 150

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 104

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

150

<210> 105

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<220>

<221> VARIANT

<222> (5)..(40)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (5)..(40)

<223> /note = "Variant residues given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<220>

<221> MISC_FEATURE

<222> (1)..(40)

<223> /note = "This sequence may include 1-10

repeating units of "Gly Gly Gly Ser"

<400> 105

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser

35 40

<210> 106

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic peptide"

<400> 106

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 107

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic peptide"

<400> 107

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 108

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic peptide"

<400> 108

Gly Gly Gly Ser

1

<210> 109

<211> 5000

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<220>

<221> Variation

<222> (51)..(5000)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (51)..(5000)

<223> /note = "The alternative grounds given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<400> 109

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4980

aaaaaaaaaa aaaaaaaaaa

5000

<210> 110

<211> 100

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 110

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

60

tttttttttt tttttttttt tttttttttt tttttttttt

100

<210> 111

<211> 5000

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<220>

<221> Variation

<222> (51)..(5000)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (51)..(5000)

<223> /note = "The alternative grounds given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<400> 111

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

60

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1980

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2040

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2100

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2160

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2220

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2280

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2340

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2400

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2460

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2520

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2580

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2640

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2700

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2760

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2820

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2880

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2940

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3000

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3060

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4980

tttttttttt ttttttttttt

5000

<210> 112

<211> 5000

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<220>

<221> Variation

<222> (101)..(5000)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (101)..(5000)

<223> /note = "The alternative grounds given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<400> 112

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4980

aaaaaaaaaa aaaaaaaaaa

5000

<210> 113

<211> 400

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<220>

<221> Variation

<222> (101)..(400)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (101)..(400)

<223> /note = "The alternative grounds given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<220>

<221> MISC_FEATURE

<222> (1)..(400)

<223> /note= "See the submitted specification for details

descriptions of replacements and preferred embodiments"

<400> 113

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

400

<210> 114

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 114

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 115

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 115

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 116

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 116

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 117

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 117

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 118

<211> 2000

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<220>

<221> Variation

<222> (51)..(2000)

<223> /replacement=« «

<220>

<221> MISC_FEATURE

<222> (51)..(2000)

<223> /note = "The alternative grounds given in

sequences have no preference over those that

are given in the annotations for the variant provisions"

<400> 118

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1980

aaaaaaaaaa aaaaaaaaaa

2000

<210> 119

<211> 373

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 119

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 120

<211> 1182

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polynucleotide"

<400> 120

atggccctcc ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga

60

ccacccggat ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg

120

gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt ctccaacacc

180

tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt caaaccagac cgacaagctc

240

gccgcgtttc cggaagatcg gtcgcaaccg ggacaggatt gtcggttccg cgtgactcaa

300

ctgccgaatg gcagagactt ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg

360

acctacctgt gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg

420

gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc atccccatcg

480

cctcggcctg cggggcagtt tcagaccctg gtcacgacca ctccggcgcc gcgcccaccg

540

actccggccc caactatcgc gagccagccc ctgtcgctga ggccggaagc atgccgccct

600

gccgccggag gtgctgtgca tacccgggga ttggacttcg catgcgacat ctacatttgg

660

gctcctctcg ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc

720

aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag gcccgtgcaa

780

accacccagg aggaggacgg ttgctcctgc cggttccccg aagaggaaga aggaggttgc

840

gagctgcgcg tgaagttctc ccggagcgcc gacgcccccg cctataagca gggccagaac

900

cagctgtaca acgaactgaa cctgggacgg cgggaagagt acgatgtgct ggacaagcgg

960

cgcggccggg accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg

1020

tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg gatgaaggga

1080

gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag gactgtccac cgccaccaag

1140

gacacatacg atgccctgca catgcaggcc cttccccctc gc

1182

<210> 121

<211> 394

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> //note = "Description of artificial sequence:

Synthetic polypeptide"

<400> 121

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

20 25 30

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

35 40 45

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

50 55 60

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

65 70 75 80

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

85 90 95

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

100 105 110

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

115 120 125

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

130 135 140

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

145 150 155 160

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

165 170 175

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

180 185 190

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

195 200 205

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

210 215 220

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

225 230 235 240

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

245 250 255

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

260 265 270

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

275 280 285

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

290 295 300

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

305 310 315 320

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

325 330 335

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

340 345 350

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

355 360 365

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<---

Claims (81)

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment that comprises: (a) an anti-CD19 binding domain comprising the amino acid sequence of SEQ ID NO: 2, (b) transmembrane domain, (c) a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, ICOS (CD278) and 4-1BB; and (d) a major intracellular signaling domain containing a functional CD3-zeta or FcR-gamma signaling domain. 2. An isolated nucleic acid molecule according to claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. 3. An isolated nucleic acid molecule according to claim 1 or 2, wherein: (a) the transmembrane domain contains the transmembrane domain of the CD8 alpha chain; (b) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 15; (c) the transmembrane domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 15; or (d) the nucleic acid sequence encoding the transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 56, or a nucleic acid sequence having at least 95% identity thereto. 4. An isolated nucleic acid molecule according to any one of claims 1-3, wherein the anti-CD19 binding domain is linked to the transmembrane domain via a hinge region. 5. An isolated nucleic acid molecule according to claim 4, in which: (a) The hinge region is the alpha hinge of CD8; (b) the hinge region comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 95% identity thereto, and/or (c) 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. 6. An isolated nucleic acid molecule according to any one of paragraphs 1-5, in which: (a) the encoded costimulatory domain comprises the amino acid sequence of SEQ ID NO: 16; (b) the encoded costimulatory domain comprises an amino acid sequence that is at least 95% similar to the amino acid sequence of SEQ ID NO: 16; or (c) the nucleic acid sequence encoding the costimulatory domain comprises the nucleic acid sequence of SEQ ID NO: 60, or a nucleic acid sequence having at least 95% identity thereto. 7. An isolated nucleic acid molecule according to any one of claims 1-6, in which: (a) the encoded intracellular signaling domain comprises 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; (b) the encoded intracellular signaling domain comprises an amino acid sequence with at least 95% identity to 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, wherein the sequences containing the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain; and/or (d) the nucleic acid sequence encoding the intracellular signaling domain comprises the nucleic acid sequence of SEQ ID NO: 60 or a nucleic acid sequence having at least 95% identity therewith, and/or the nucleic acid sequence of SEQ ID NO: 101 or SEQ ID NO: 44, or a nucleic acid sequence having at least 95% identity therewith. 8. An isolated nucleic acid molecule according to any one of claims 1-7, further encoding a leader sequence comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with at least 95% identity to SEQ ID NO: 13. 9. An isolated nucleic acid molecule encoding a CAR, wherein the CAR comprises, from N-terminus to C-terminus: (a) an anti-CD19 binding domain comprising the amino acid sequence of SEQ ID NO: 2, a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 15, a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 16, and a major intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43. 10. An isolated chimeric antigen receptor (CAR) molecule that binds to CD19, encoded by a nucleic acid molecule according to any one of claims 1-9. 11. The isolated CAR molecule of claim 10, wherein the anti-CD19 binding domain is an scFv. 12. An isolated chimeric antigen receptor (CAR) molecule that binds to CD19, wherein the CAR molecule comprises, from N-terminus to C-terminus: (a) an anti-CD19 binding domain comprising the amino acid sequence of SEQ ID NO: 2, (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 15, (c) a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 16, and (d) a major intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 43. 13. An expression vector comprising a nucleic acid molecule according to any one of claims 1-9 or a nucleic acid molecule encoding a CAR molecule according to any one of claims 10-12. 14. The expression vector of claim 13, selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. 15. An expression vector according to claim 13 or 14, where: (a) the virus further comprises an EF-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 100; (b) is an in vitro transcribed vector; (c) the nucleic acid sequence in the vector further comprises a poly(A) sequence; and/or (d) the nucleic acid sequence in the vector additionally contains a 3'UTR. 16. A cell expressing the CAR molecule of any one of claims 10-12, comprising the expression vector of any one of claims 13-15. 17. The cell according to item 16, where: (a) the cell is a human T cell; (b) the cell is a CD8+ T cell; (c) the cell further expresses an inhibitory molecule that comprises a first polypeptide comprising at least a portion of the inhibitory molecule linked to a second polypeptide that provides a positive signal from the intracellular signaling domain; and/or (d) the cell further expresses an inhibitory molecule that comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and a basic signaling domain. 18. A method for producing a cell comprising transducing a T cell with an expression vector according to any one of claims 13-15 or a nucleic acid molecule according to any one of claims 1-9. 19. A method for producing a population of cells with genetically modified RNA, comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA contains a nucleic acid encoding a CAR molecule according to any one of claims 10-12. 20. A method for producing in vitro transcribed RNA encoding a CD19 CAR, comprising performing in vitro transcription of a DNA sequence encoding a CD19 CAR, wherein the DNA sequence comprises a nucleic acid molecule according to any one of claims 1-9. 21. A pharmaceutical composition for the treatment of a disease associated with the expression of the cluster of differentiation 19 (CD19) protein, comprising a cell according to claim 16 or 17. 22. Use of a cell expressing a CAR molecule according to any one of claims 10-12, or a pharmaceutical composition according to claim 21 for the production of a medicinal product for creating antitumor immunity in a mammal. 23. Use of a cell according to claim 16 or 17, a nucleic acid molecule according to any of claims 1-9, a CAR molecule according to any of claims 10-12, or a pharmaceutical composition according to claim 21 for the production of a medicament for the treatment of a disease associated with CD19 expression. 24. Use of a cell according to claim 16 or 17 or a pharmaceutical composition according to claim 21 for the production of a medicinal product for cell conditioning therapy before cell transplantation. 25. The use according to any of paragraphs 22-24, in which: (a) the cell is an autologous T cell; (b) the cell is an allogeneic T cell; and/or (c) the mammal is a human. 26. The application of paragraph 23, in which: (a) the disease associated with CD19 expression is selected from the group consisting of cancer or a malignant disease, precancerous condition, or non-cancerous symptom associated with CD19 expression; or (b) the disease associated with CD19 expression is a blood cancer selected from the group consisting of B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and preleukemia. 27. The use according to any of paragraphs 22-26, wherein the medicinal product is administered in combination with: (a) an agent that increases the efficiency of the cell, (b) an agent that mitigates one or more side effects associated with the administration of the cell, (c) an agent that treats a disease associated with CD19, or (d) one or more of: (i) PD-1 CAR, comprising the PD1 extracellular domain, transmembrane domain and intracellular signaling domain; (ii) GITR agonist; (iii) an agent that inhibits an inhibitory molecule selected from PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, or 2B4; (iv) an agent that ameliorates one or more side effects associated with the administration of a cell expressing a CAR molecule; (v) an IL-6 inhibitor; or (vi) mTOR inhibitor. 28. The use according to claim 27, wherein the CD19 CAR and the PD-1 CAR are expressed in the same cell. 29. Use according to any of paragraphs 22-26, where: (a) the cell is administered at a dose of 10 ⁴ - 10 ⁹ cells/kg body weight; (b) the subject receives one or more subsequent administrations of the cell; and/or (c) the subject receives more than one administration (e.g., 2, 3, or 4 administrations) per cage week.