EA048570B1 - CHIMERIC RECEPTORS TO STEAP1 AND METHODS OF THEIR APPLICATION - Google Patents

Abstract

The present invention discloses antigen-binding molecules, chimeric receptors, and engineered immune cells that bind to STEAP1. The present invention also relates to vectors, compositions, and methods for treatment and/or detection using antigen-binding molecules and engineered immune cells that bind STEAP1.

Description

Background of the invention

Prostate cancer is the most commonly diagnosed cancer in men other than skin cancer. With an estimated 28,170 deaths in 2012, prostate cancer is the second leading cause of cancer death in men. Hormonal therapy, chemotherapy, radiation therapy, or a combination of these treatments are used to treat more advanced disease. Despite the above advances in prostate cancer therapy, there is a great need for additional therapies that can effectively inhibit the progression of prostate cancer, including prostate cancer that has not been treated with an androgen receptor inhibitor.

Engineered immune cells have been shown to have desirable properties in therapeutic treatment, particularly in oncology. The two main types of engineered immune cells are those that contain chimeric antigen receptors (called CARs or CAR-Ts) and T cell receptors (TCRs). These engineered cells are designed to provide antigen specificity while maintaining or enhancing their ability to recognize and kill a target cell. Chimeric antigen receptors may contain, for example, (i) an antigen-specific component (an antigen-binding molecule), (ii) one or more costimulatory domains, and (iii) one or more activating domains. Each domain may be heterogeneous, i.e., composed of sequences derived from different protein chains. Immune cells expressing a chimeric antigen receptor (such as T cells) may be used in a variety of therapies, including cancer therapies. It should be understood that the costimulatory polypeptides defined herein can be used to enhance the activation of CAR-expressing cells against target antigens and thereby increase the efficacy of adoptive immunotherapy.

T cells can be engineered to have activity against one or more desired targets. For example, T cells can be transduced with DNA or other genetic material encoding an antigen-binding molecule, such as one or more single-chain variable fragments (scFv) of an antibody, in combination with one or more signaling molecules and/or one or more activation domains, such as CD3zeta.

In addition to the ability of CAR-T cells to recognize and kill target cells, successful T cell therapy benefits from the ability of CAR-T cells to persist and maintain the ability to proliferate in response to antigen.

There is a need to identify new and improved therapies for STEAP1-associated diseases and disorders.

Brief description of the invention

The present invention relates to engineered immune cells (such as CAR or TCR), antigen-binding molecules (including but not limited to antibodies, scFv, heavy and/or light chains and CDRs of these antigen-binding molecules) with specificity for STEAP1.

The chimeric antigen receptors of the present invention typically comprise: (i) a STEAP1-specific antigen-binding molecule, (ii) one or more costimulatory domains, and (iii) one or more activating domains. It should be understood that each domain may be heterogeneous and thus comprise sequences derived from different protein chains.

In some embodiments, the present invention provides a chimeric antigen receptor comprising an antigen-binding molecule that specifically binds to STEAP1, wherein the antigen-binding molecule comprises at least one of: (a) a heavy chain variable region CDR1 comprising an amino acid sequence that differs from the sequences of SEQ ID NO: 89, 99, 109, 119, 129, or 139 by no more than 3, 2, 1, or 0 amino acid residues; (b) a heavy chain variable region CDR2 comprising an amino acid sequence that differs from the sequences of SEQ ID NO: 90, 100, 110, 120, 130, or 140 by no more than 3, 2, 1, or 0 amino acid residues; (c) a CDR3 of the variable heavy chain comprising an amino acid sequence that differs from the sequences of SEQ ID NO: 91, 101, 111, 121, 131 or 141 by no more than 3, 2, 1 or 0 amino acid residues; (d) a CDR1 of the variable light chain comprising an amino acid sequence that differs from the sequences of SEQ ID NO: 94, 104, 114, 124, 134 or 14 by no more than 3, 2, 1 or 0 amino acid residues; (e) a CDR2 of the variable light chain comprising an amino acid sequence that differs from the sequences of SEQ ID NO: 95, 105, 115, 125, 135 or 145 by no more than 3, 2, 1 or 0 amino acid residues; (f) a light chain variable region CDR3 comprising an amino acid sequence that differs from the sequence of SEQ ID:96, 106, 116, 126, 136 or 146 by no more than 3, 2, 1 or 0 amino acid residues.

In other embodiments, the chimeric antigen receptor further comprises at least one costimulatory domain. In further embodiments, the chimeric antigen receptor further comprises at least one activating domain.

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In some embodiments, the costimulatory domain is a signaling region of CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte functional association antigen 1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating receptors NK cells, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, LL-2R-gamma, LL-2R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

In some embodiments, the costimulatory domain is derived from 4-1BB. In other embodiments, the costimulatory domain is derived from OX40. See also Hombach et al., Oncoimmunology. 2012 Jul. 1; 1(4): 458-466. In yet other embodiments, the costimulatory domain comprises ICOS as described by Guedan et al., August 14, 2014; Blood: 124 (7) and Shen et al., Journal of Hematology & Oncology (2013) 6:33. In yet other embodiments, the costimulatory domain comprises CD27 as described by Song et al., Oncoimmunology. 2012 Jul. 1; 1(4): 547-549.

In certain embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In further embodiments, the CD8 costimulatory domain comprises SEQ ID NO: 14. In further embodiments, the activating domain comprises CD3, CD3-zeta, or CD3-zeta having the sequence set forth in SEQ ID NO: 10.

In other embodiments, the present invention relates to a chimeric antigen receptor, wherein the costimulatory domain comprises SEQ ID NO: 2 and the activating domain comprises SEQ ID NO: 10.

The present invention further relates to polynucleotides encoding chimeric antigen receptors and vectors comprising the polynucleotides. The vector may be, for example, a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated virus vector, a lentiviral vector, or any combination thereof. The present invention further relates to immune system cells comprising the vectors. In some embodiments, the lentiviral vector is a pGAR vector.

Exemplary immune system cells include, but are not limited to, T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR expressing cells, dendritic cells, or NKT cells. T cells may be autologous, allogeneic, or heterologous. In other embodiments, the present invention relates to pharmaceutical compositions comprising the immune cells described herein.

In some embodiments, the present invention relates to antigen-binding molecules (and chimeric antigen receptors comprising these molecules) comprising at least one of:

(a) a VH region that differs from the amino acid sequence of the VH region of 2F3 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region of 2F3 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(b) a VH region that differs from the amino acid sequence of the VH region of 11C2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region of 11C2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(c) a VH region that differs from the amino acid sequence of the VH region 1A1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region 1A1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(d) a VH region that differs from the amino acid sequence of VH region 7A4 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues, and a VL region that differs from the amino acid sequence of VL region 7A4 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues; and (e) a VH region that differs from the amino acid sequence of VH region 7A5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues, and a VL region that differs from the amino acid sequence of VL region 7A5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues, and

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(f) a VH region that differs from the amino acid sequence of the 14C1 VH region by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the 14C1 VL region by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

and wherein either the VH and VL region or regions are linked by at least one linker.

In other embodiments, the present invention relates to antigen-binding molecules (and chimeric antigen receptors comprising these molecules), wherein the linker comprises at least one of a G4S scFv linker and a Whitlow scFv linker.

In other embodiments, the present invention relates to vectors encoding the polypeptides of the present invention and immune cells comprising these polypeptides. Preferred immune system cells include T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells. T cells may be autologous, allogeneic, or heterologous.

In other embodiments, the present invention relates to isolated polynucleotides encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), comprising an antigen binding molecule that specifically binds to STEAP1, wherein the antigen binding molecule comprises a CDR3 of a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 27. The polynucleotides may further comprise an activating domain. In preferred embodiments, the activating domain is CD3, more preferably CD3-zeta, more preferably the amino acid sequence presented under SEQ ID NO: 9.

In other embodiments, the present invention comprises a costimulatory domain such as Cd28, CD28T, OX40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte functional-associated antigen-1 (LFA-1 (CD1 la/CD 18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig-alpha (CD79a), DAP-10, Fc-gamma receptor, MHC class I molecule, TNF, TNFr, integrin, lymphocyte activation signaling molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, III-beta, III-2K-gamma, 1b-7K-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 ligand, or fragments or combinations thereof. Preferred costimulatory domains are listed below.

The present invention also relates to methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject antigen-binding molecules, CARs, TCRs, polynucleotides, vectors, cells or compositions according to the present invention. Diseases suitable for treatment include, but are not limited to, prostate cancer, including metastatic castration-resistant prostate cancer.

Brief description of graphic materials

Fig. 1 shows a flow cytometric analysis of cell surface expression of STEAP1 in human cell lines.

Fig. 2 shows CAR expression in primary human T cells electroporated with mRNA encoding different CARs.

Fig. 3 shows the cytolytic activity of electroporated CAR T cells against multiple cell lines.

Fig. 4, consisting of Figs. 3A and 3B, shows the production of IFNy, IL-2, and TNFa by electroporated CAR T cells.

Fig. 5 shows CAR expression in lentivirus-transduced primary human T cells from two healthy donors.

Fig. 6 shows a map of the pGAR vector.

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Detailed description of the invention

It should be understood that chimeric antigen receptors (CAR or CAR-T) and T-cell receptors (TCR) are receptors created by genetic engineering. These engineered receptors can be easily inserted and expressed by immune cells, including T cells, according to techniques known in the art. With CAR, one receptor can be programmed to both recognize a specific antigen and, if it binds to that antigen, to activate an immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses CAR can target and destroy the tumor cell.

CARs can be engineered to bind an antigen (e.g., a cell surface antigen) by incorporating an antigen-binding molecule that will interact with the target antigen. Preferably, the antigen-binding molecule is an antibody or fragment thereof, and more preferably one or more single-chain antibody fragments (scFv). An scFv is a single-chain antibody fragment comprising the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494 and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFv retains the ability of the original antibody to specifically interact with the target antigen. scFvs are preferred for use in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain along with other CAR components. Id. See also Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797. It will be appreciated that the antigen-binding molecule is typically contained in the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs are within the scope of the present invention with specificity for more than one target of interest.

Costimulatory domains. Chimeric antigen receptors may include costimulatory (signaling) domains to enhance their activity. See U.S. Patent Nos. 7,741,465 and 6,319,494 and Krause et al. and Finney et al. (supra), Song et al., Blood 119:696–706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725–33 (2011); and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:5983 (2016). For example, CD28 is a costimulatory protein that is naturally found on T cells. The complete native amino acid sequence of CD28 is described in NCBI Accession No. NP_006130.1. The complete native nucleic acid sequence of CD28 is described in NCBI Accession No. NM_006139.1.

Certain domains of CD28 are used in chimeric antigen receptors. In one embodiment, a novel extracellular domain of CD28, called CD28T, can be used and has surprisingly been found to provide certain advantages when used in a CAR design.

The nucleotide sequence of the CD28T molecule, including the CD28T extracellular domain, the CD28 transmembrane domain and the CD28 intracellular domain, is provided under SEQ ID NO: 1:

CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTG TCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGTTGGTCGTAGTGGGT GGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGGTTA GATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGC CCTGGCCCCACAAGGAAACACTACCAGCTTACGCACCACCTAGAGATTTCGCTGCC TATCGGAGC

The corresponding amino acid sequence is presented under SEQ ID NO: 2: LDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRS

The nucleotide sequence of the extracellular portion of CD28T is given under SEQ ID NO: 3: CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTG

TCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA

The corresponding amino acid sequence of the extracellular domain of CD28T is shown under SEQ ID NO: 4:

LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP

The nucleotide sequence of the transmembrane domain of CD28 is presented under SEQ ID NO: 5):

TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCG

TGGCTTTTATAATCTTCTGGGTT

The amino acid sequence of the transmembrane domain of CD28 is presented under SEO ID NO:6:

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FWVLVVVGGV LACYSLLVTV AFIIFWV

The nucleotide sequence of the intracellular signaling domain of CD28 is shown under SEQ ID NO: 7:

AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCG

CCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGC CTATCGGAGC

The amino acid sequence of the intracellular signaling domain of CD28 is shown as SEQ ID NO: 8:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

Additional CD28 sequences suitable for use in the present invention include the CD28 nucleotide sequence set forth under SEQ ID NO: 11:

ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGTACCAT CATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCCTCTTCCCCGGGCCATCAAA GCCC

The corresponding amino acid sequence is presented under SEQ ID NO: 12: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

Other suitable extracellular or transmembrane sequences may be derived from CD8. The nucleotide sequence of a suitable extracellular and transmembrane domain of CD8 is provided under SEQ ID NO: 13:

GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGTTCTTGC CGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCTCCTACCATCG CTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCAGGGGGCGCTG TTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACCCCTGGCCG GAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGTAATCACAGGA AC

The corresponding amino acid sequence is presented under SEQ ID NO: 14: AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN

Other suitable intracellular signaling sequences can be obtained from: 41BB. The nucleotide sequence of a suitable intracellular signaling domain of 41-BB is provided under SEQ ID NO: 15:

CGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCG

TTTATGAGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCT GAGGAGGAGGAGGGCGGGTGCGAACTG

The corresponding amino acid sequence is presented under SEQ ID NO: 16: RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Suitable costimulatory domains within the scope of the present invention may be derived from, among other sources, CD28, CD28T, OX40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte functional-associated antigen-1 (LFA-1 (CD1 1a/CD 18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig-alpha (CD79a), DAP-10, Fc-gamma receptor, MHC class I molecule, TNF, TNFr, integrin, lymphocyte activation signaling molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL-2Rgamma, IL-2R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, FTGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4d), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 ligand or fragments or combinations thereof.

Activating domains.

CD3 is a T cell receptor element on naïve T cells and has been shown to be an important intracellular activating element in CARs. In a preferred embodiment, CD3 is CD3-zeta, the nucleotide sequence of which is shown in SEQ ID NO: 9:

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AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCA ACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGC GCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGA GGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAG GCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACT

CAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAG

G

The corresponding amino acid of intracellular CD3-g3ema is represented by SEQ ID NO: 10: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR

Domain orientation.

It should be understood that structurally these domains correspond to positions relative to an immune cell. Thus, these domains can be part of (i) a hinge or extracellular (EC) domain, (ii) a transmembrane (TM) domain and/or (iii) an intracellular (cytoplasmic) domain (IC). The intracellular component often partially comprises a member of the CD3 family, preferably CD3zeta, which is capable of activating a T cell upon binding of an antigen-binding molecule to its target. In one embodiment, the hinge domain typically consists of at least one costimulatory domain as defined herein.

It should also be understood that the hinge region may also contain several or all members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

Illustrative CAR designs in accordance with the present invention are shown in Table 1.

Table 1

Name of the structure scFv Co-stimulatory domain Activation domain 2F3-CD28T-CD28- 41BB 2F3 CD28T, 4-1BB CD3-zeta 2F3-CD28T-CD28 2F3 CD28T CD3-zeta 2F3-CD28T-41BB 2F3 4-1BB CD3-zeta 2F3-C8K-CD28 2F3 CD28T CD3-zeta 2F3-C8K-41BB 2F3 4-1BB CD3-zeta 11C2-CD28TCD28-41BB 11C2 CD28T, 4-1BB CD3-zeta 11C2-CD28T-CD28 11C2 CD28T CD3-zeta 11C2-CD28T-41BB 11C2 4-1BB CD3-zeta

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11C2-C8K-CD28 11C2 CD28T CD3-zeta 11C2-C8K-41BB 11C2 4-1BB CD3-zeta 1A1-CD28T-CD28- 41BB 1A1 CD28T, 4-IBB CD3-zeta 1A1-CD28T-CD28 1A1 CD28T CD3-zeta 1A1-CD28T-41BB 1A1 4-1BB CD3-zeta 1A1-C8K-CD28 1A1 CD28T CD3-zeta 1A1-C8K-41BB 1A1 4-1BB CD3-zeta 7A4-CD28T-CD28- 41BB 7A4 CD28T, 4-IBB CD3-zeta 7A4-CD28T-CD28 7A4 CD28T CD3-zeta 7A4-CD28T-41BB 7A4 4-1BB CD3-zeta 7A4-C8K-CD28 7A4 CD28T CD3-zeta 7A4-C8K-41BB 7A4 4-1BB CD3-zeta 7A5-CD28T-CD28- 41BB 7A5 CD28T, 4-IBB CD3-zeta 7A5-CD28T-CD28 7A5 CD28T CD3-zeta 7A5-CD28T-41BB 7A5 4-1BB CD3-zeta 7A5-C8K-CD28 7A5 CD28T CD3-zeta 7A5-C8K-41BB 7A5 4-1BB CD3-zeta 14C1-CD28TCD28-41BB 14C1 CD28T, 4-IBB CD3-zeta 14C1-CD28T-CD28 14C1 CD28T CD3-zeta 14C1-CD28T-41BB 14C1 4-1BB CD3-zeta 14C1-C8K-CD28 14C1 CD28T CD3-zeta 14C1-C8K-41BB 14C1 4-1BB CD3-zeta

Domains in relation to the cell.

It should be understood that, with respect to a cell bearing a receptor, the engineered T cells of the present invention comprise an antigen-binding molecule (such as an scFv), an extracellular domain (which may include a hinge domain), a transmembrane domain and an intracellular domain. The intracellular domain comprises, at least in part, an activation domain, preferably consisting of a member of the CD3 family, such as CD3-zeta, CD3-tomoh, CD3-gamma or portions thereof. It should further be understood that the antigen-binding molecule (e.g., one or more scFvs) is engineered to be located in the extracellular portion of the molecule/construct such that it is capable of recognizing and binding to its target or targets.

Extracellular domain. The extracellular domain is useful for signaling and for the effective response of lymphocytes to antigen. The extracellular domains used in the present invention may be derived from (i.e., comprise) CD28, CD28T, CD8, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein 1 (PD-1), inducible T-cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3gamma, CD3delta, CD3epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig-alpha (CD79a), DAP-10, Fc-gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling activation molecules lymphocytes (SLAM proteins), NK cell activating receptors, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, K^-beta, K^-gamma, K^-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,

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CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, FTGB2, CD 18, LFA-1 , ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108 ), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof. The extracellular domain may be from either a natural or synthetic source.

As described herein, extracellular domains often include a hinge portion. This is a portion of the extracellular domain sometimes referred to as a spacer region. Various hinge regions can be used in accordance with the present invention, including costimulatory molecules, as discussed above, as well as immunoglobulin (Ig) sequences or other suitable molecules to achieve the desired predetermined distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises CD28T or the CD28 EC domain.

Transmembrane domain. The CAR can be engineered to contain a transmembrane domain that is fused to the extracellular domain of the CAR. Likewise, it can be fused to the intracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution so as to avoid association of such domains with transmembrane domains of the same or other surface membrane proteins so as to minimize interactions with other members of the receptor complex. The transmembrane domain can be from either a natural or synthetic source. If the source is natural, the domain can be from any membrane-associated or transmembrane protein. The transmembrane regions used in the present invention may be derived from (i.e., comprise) CD28, CD28T, CD8, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein 1 (PD-1), inducible T-cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3-gamma, CD3-delta, CD3-epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig-alpha (CD79a), DAP-10, Fc-gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling activation molecules lymphocytes (SLAM proteins), NK cell activating receptors, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, CD8-beta, CD8-beta, CD8-gamma, CD8-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

Optionally, short linkers can form connections between all or some of the extracellular, transmembrane, and intracellular domains of the CAR.

In one embodiment, the transmembrane domain of the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a transmembrane portion of the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence that encodes a transmembrane amino acid sequence contained in SEQ ID NO: 14.

In certain embodiments, the transmembrane domain of a CAR of the present invention is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 5. In one embodiment, the CD28 transmembrane domain comprises a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 6. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6.

Intracellular (cytoplasmic) domain. The intracellular (cytoplasmic) domain of the engineered T cells of the present invention may provide for the activation of at least one of the normal effector functions of an immune cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines.

It will be appreciated that suitable intracellular molecules include (i.e., contain) without limitation CD28, CD28T, CD8, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein 1 (PD-1), inducible T-cell costimulator (ICOS), functional

- 8 048570 lymphocyte-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3-raMMa, CD3-delta, CD3-epsilon. CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig-alpha (CD79a), DAP-10, Fc-gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL-2Rbeta, Sh-2K-gamma, IL-VR-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

In a preferred embodiment, the cytoplasmic domain of the CAR may be engineered to comprise the CD3-zeta signaling domain alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR may comprise a portion of the CD3-zeta chain and a costimulatory signaling region.

The cytoplasmic signal sequences in the cytoplasmic signal portion of the CAR of the present invention may be linked to each other in a random or predetermined order.

In one preferred embodiment, the cytoplasmic domain is engineered to comprise a CD3-zeta signaling domain and a CD28 signaling domain. In another embodiment, the cytoplasmic domain is engineered to comprise a CD3-zeta signaling domain and a 4-1BB signaling domain, wherein the cytoplasmic CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 15 and the amino acid sequence set forth in SEQ ID NO: 16. In another embodiment, the cytoplasmic domain in a CAR of the present invention is engineered to comprise a portion of CD28 and CD3-zeta, wherein the cytoplasmic CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8. The nucleic acid sequence of CD3-zeta is set forth in SEQ ID NO: 9 and the amino acid sequence is set forth in SEQ ID NO: 8.

It should be understood that one preferred orientation of a CAR in accordance with the present invention provides an antigen-binding domain (such as an scFv) in tandem with a costimulatory domain and an activating domain. The costimulatory domain may comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. It should also be understood that multiple costimulatory domains may be used in tandem.

In some embodiments, nucleic acids are provided comprising a promoter operably linked to a first polynucleotide encoding an antigen-binding molecule, at least one costimulatory molecule, and an activating domain.

In some embodiments, the nucleic acid construct is contained in a viral vector. In some embodiments, the viral vector is selected from the group consisting of retroviral vectors, murine leukemia virus vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, and vaccinia virus vectors. In some embodiments, the nucleic acid is contained in a plasmid.

The present invention further relates to isolated polynucleotides encoding chimeric antigen receptors and vectors comprising the polynucleotides. Any vector known in the art may be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (such as pMSVG1), a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV) vector, a lentiviral vector (such as pGAR), or any combination thereof. A map of the pGAR vector is shown in Fig. 6. The sequence of pGAR is as follows:

- 9 048570

CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGTGTGGTGGTTACGCG CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCT TCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAACTTGATTAGGGTG ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGG AGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTA TCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAA AAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTAC AATTTGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGC CTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTG GGTAACGCCAGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGT AATACGACTCACTATAGGGCGACCCGGGGATGGCGCGCCAGTAATCAATTACGGGG TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCTGATGC GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC GGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGGGGTCTCTCTGGTTAGA CCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCA ATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGT AACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGAC TCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGC CAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGATGGGTGCGAGAGCGTCAGT ATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAAATATAAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACG ATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGG GACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAAT ACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGA AGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAA GCCGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTG AATTATATAAATATAAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAG GCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCT GAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGC

- 10 048570

AGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTG GGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCT AGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTG GGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGC AAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAG TTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAAT GATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAA TAGAGTTAGGCAGGGATATTTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAG GGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGA CAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAA AGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACA GACATACAAACTAAAGAATTACAAAAACAAATTACAAAATTCAAAATTTTATCGCG ATCGCGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCA TTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTA GGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCC TGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCA GTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCT GTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGC TCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAAGTCCTTCGAA GTAGATCTTTGTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCTGCCAA GCTTCCGAGCTCTCGAATTAATTCACGGTACCCACCATGGCCTAGGGAGACTAGTCG AATCGATATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAATCCTGGTTGCTGTCTCTT TATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACT TTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAG CTGACGTCCTTTTCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGT CCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCT GCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCC CTTTGGGCCGCCTCCCCGCCTGGTTAATTAAAGTACCTTTAAGACCAATGACTTACA AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCGA ATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAG ACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTC AATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTG GTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGCATG CCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTG AAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATA AGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG

- 11 048570

GGGGAGGTGTGGGAGGTTTTTTGGCGCGCCATCGTCGAGGTTCCCTTTAGTGAGGGT TAATTGCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC GCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCGCAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTG CCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAA GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAC CAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCC CCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCC TCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC ATGTTGTGCAAAAAAGCGGTTAGCTCCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG TTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG AATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA CCCAACTGATCTTCAGCATCTTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAACA GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATT TCCCCGAAAAGTGCCAC (SEQ ID NO: 147)

Suitable additional exemplary vectors include, for example, pBABE-puro, pBABEneo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (empty Puro IRES GFP plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGKGFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

In some embodiments, the engineered immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, or an NK T cell. In some embodiments, the cell is isolated or obtained from peripheral blood. In some embodiments, the cell is isolated or obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is isolated or obtained from bone marrow. In some embodiments, the cell is isolated or obtained from cord blood. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymeric transfection, nanoparticles, or polyplexes.

- 12 048570

In some embodiments, chimeric antigen receptors are expressed in engineered immune cells that comprise nucleic acids of the present application. These chimeric antigen receptors of the present application may, in some embodiments, comprise (i) an antigen-binding molecule (such as an scFv), (ii) a transmembrane region, and (iii) a T-cell activation molecule or region.

Antigen-binding molecules.

Antigen-binding molecules are within the scope of the present invention.

As used herein, the term antigen-binding molecule means any protein that binds a specific target antigen. In this application, the target antigen is the STEAP1 protein or a fragment thereof. Antigen-binding molecules include, but are not limited to, antibodies and binding portions thereof, such as immunologically functional fragments. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of suitable antigen-binding molecules.

In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to STEAP1. In further embodiments, the antigen-binding molecule is an antibody or fragment thereof, including one or more of its complementarity determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv).

The term "immunologically functional fragment (or fragment) of an antigen-binding molecule" is a type of antigen-binding molecule that comprises a portion (regardless of how the portion is produced or synthesized) of an antibody that lacks at least some amino acids present in the full-length chain, but is still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to a target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to the epitope. In some embodiments, the fragments are neutralizing fragments. In some embodiments, the fragments can block or reduce the activity of STEAP1. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chains, and in some embodiments will comprise one heavy chain and/or a light chain or a portion thereof. These fragments may be produced by recombinant DNA techniques or may be obtained, for example, by enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.

Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more CDRs, diabodies (a heavy chain variable domain on the same polypeptide as a light chain variable domain linked by a short peptide linker that is short enough to prevent pairing between the two domains on the same chain), domain antibodies, and single chain antibodies. These fragments may be obtained from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. As will be appreciated by one of skill in the art, the antigen-binding molecule may include non-protein components.

Variants of the antigen-binding molecules are also within the scope of the present invention, such as variable light and/or variable heavy chains, each of which has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% identity to the amino acid sequences of the sequences described herein. In some cases, such molecules comprise at least one heavy chain and one light chain, while in other cases, variant forms comprise two identical light chains and two identical heavy chains (or subportions thereof). One skilled in the art will be able to determine suitable variants of the antigen-binding molecules set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without compromising its activity by targeting regions that are not considered important for activity.

In certain embodiments, the polypeptide structure of the antigen-binding molecules is based on antibodies, including but not limited to monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, fusion antibodies (sometimes referred to herein as antibody conjugates), and fragments thereof, respectively. In some embodiments, the antigen-binding molecule comprises or consists of avimers.

In some embodiments, an antigen-binding molecule that binds to

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STEAP1, is administered alone. In other embodiments, the antigen-binding molecule that binds to STEAP1 is administered as part of a CAR, TCR, or other immune cell. In such immune cells, the antigen-binding molecule that binds to STEAP1 may be under the regulation of the same promoter region or a separate promoter. In some embodiments, the genes encoding the proteinaceous substances and/or the antigen-binding molecule that binds to STEAP1 may be in separate vectors.

The present invention also relates to pharmaceutical compositions comprising an antigen-binding molecule that binds to STEAP1, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or excipient. In some embodiments, the pharmaceutical compositions will include more than one antigen-binding molecule that binds to STEAP1. In certain embodiments, the pharmaceutical compositions will include more than one antigen-binding molecule that binds to STEAP1, wherein the antigen-binding molecules that bind to STEAP1 bind more than one epitope. In some embodiments, the different antigen-binding molecules will not compete with each other for binding to STEAP1.

In other embodiments, the pharmaceutical composition may be selected for parenteral delivery, for inhalation, or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. In certain embodiments, buffers are used to maintain the composition at physiological pH or slightly lower pH, typically a pH in the range of about 5 to about 8. In some embodiments of the present invention, when parenteral administration is contemplated, the therapeutic composition may be in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired antigen-binding molecule that binds to STEAP1, with or without additional therapeutic agents, in a pharmaceutically acceptable carrier medium. In certain embodiments, the parenteral injection carrier medium is sterile distilled water in which the antigen-binding molecule that binds to STEAP1, with or without at least one additional therapeutic agent, is formulated as a sterile isotonic solution that is suitably preserved. In certain embodiments, the preparation may include formulating the desired molecule with polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes that can provide a controlled or sustained release product that can be delivered by depot injection. In certain embodiments, implantable drug delivery products may be used to administer the desired molecule.

In some embodiments, the antigen-binding molecule is used as a diagnostic or validation tool. The antigen-binding molecule can be used to analyze the amount of STEAP1 present in a sample and/or subject. In some embodiments, the diagnostic antigen-binding molecule is not neutralizing. In some embodiments, the antigen-binding molecules disclosed herein are used or provided in an assay kit and/or method for detecting STEAP1 in mammalian tissues or cells for the purpose of screening/diagnosing a disease or disorder associated with changes in STEAP1 levels. The kit can comprise an antigen-binding molecule that binds STEAP1, together with means for displaying the binding of the antigen-binding molecule that binds STEAP1, if present, and optionally the levels of STEAP1 protein.

Antigen-binding molecules should be further understood in light of the following definitions and descriptions.

The Fc region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

The Fab fragment contains one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab' fragment contains one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and a region between the CH1 and CH2 domains such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab') ₂ molecule. The F(ab') ₂ '' fragment contains two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab') 2 fragment consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

The Fv region contains variable regions from both the heavy and light chains, but does not contain constant regions.

Single-chain variable fragment (scFv, also called single-chain antibody) refers to Fv molecules in which the variable regions of the heavy and light chains are linked

- 14 048570 a flexible linker to form a single polypeptide chain that forms the antigen-binding region. See PCT application WO88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference in their entireties.

A bivalent antigen-binding molecule contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. Bivalent antigen-binding molecules may be bispecific. A multispecific antigen-binding molecule is a molecule that targets more than one antigen or epitope. A bispecific, dual-specific, or bifunctional molecule is a hybrid antigen-binding molecule or antibody, respectively, that has two different antigen-binding sites. The two binding sites of a bispecific antigen-binding molecule will bind two different epitopes, which may be located on the same or different target proteins.

An antigen-binding molecule is said to specifically bind its target antigen when the dissociation constant (Kd) is ~1 x 10 ^-7 M. The antigen-binding molecule specifically binds the antigen with high affinity when the Kd is 1-5 x 10 ^-9 M, and with very high affinity when the Kd is 1-5 x 10 ^-10 M. In one embodiment, the antigen-binding molecule has a Kd of 10 ^-9 M. In one embodiment, the dissociation rate is < 1 x 10 ^-5 . In other embodiments, the antigen-binding molecules will bind to human STEAP1 with a Kd of from about 10 ^-7 M to 10 ^-13 M, and in yet another embodiment, the antigen-binding molecules will bind with a Kd of 1.0-5 x 10 ^-10

An antigen-binding molecule is said to be selective when it binds to one target more tightly than to a second target.

The term "antibody" refers to an intact immunoglobulin of any isotype or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, and includes, for example, chimeric, humanized, fully human and bispecific antibodies. An antibody is a type of antigen-binding molecule as defined herein. An intact antibody will typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as antibodies naturally occurring in camelids may contain only heavy chains. Antibodies may be obtained exclusively from a single source or may be chimeric, i.e., different portions of the antibody may be obtained from two different antibodies, as further described below. Antigen-binding molecules, antibodies or binding fragments may be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term antibody includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include, respectively, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, fusion antibodies (sometimes referred to herein as antibody conjugates) and fragments thereof.

The variable regions are typically characterized by the same overall structure of relatively conserved framework regions (FRs) connected by 3 hypervariable regions (i.e., CDRs). The CDRs from the two chains of each pair are typically aligned with the framework regions, which can mediate binding to a specific epitope. From N-terminus to C-terminus, the variable regions of both the light and heavy chains typically contain the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. By convention, the CDRs in the heavy chain are typically referred to as CDR1, CDR2, and CDR3 HC. The CDRs in the light chain are typically referred to as CDR1, CDR2, and CDR3 LC. The assignment of amino acids to each domain typically follows the definitions of Kabat (Seqs of Proteins of Immunological Interest (NIH, Bethesda, MD (1987 and 1991)) or Chothia (J. Mol. Biol., 196:901–917 (1987); Chothia et al., Nature, 342:878–883 (1989)). A variety of assays can be used to define or approximate CDR regions, including not only the Kabat or Chothia definitions but also the AbM definitions.

The term light chain includes a full-length light chain and fragments thereof having a variable region sequence sufficient to provide binding specificity. A full-length light chain includes a variable region domain V _L and a constant region domain C _L . The variable region domain of a light chain is located at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

The term heavy chain includes the full-length heavy chain and fragments thereof having sufficient variable region sequence to provide binding specificity. A full-length heavy chain contains a variable region domain VH and three constant region domains

- 15 048570

CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, and the _CH domains are located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including the IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including the IgA1 and IgA2 subtypes), IgM, and IgE.

The terms variable region or variable domain refer to a portion of the light and/or heavy chains of an antibody, typically comprising approximately 120-130 amino-terminal amino acids in the heavy chain and approximately 100-110 amino-terminal amino acids in the light chain. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

Variability is not uniformly distributed throughout the variable domains of antibodies; it is concentrated in subdomains of each of the variable regions of the heavy and light chains. These subdomains are called hypervariable regions or complementarity determining regions (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called framework regions (FRMs or FRs) and provide a framework for the six CDRs in three-dimensional space to form the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRMs (FR1, FR2, FR3, and FR4), mostly in a β-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRM and, with hypervariable regions from the other chain, contribute to the formation of the antigen-binding site (see Kabat et al., loc. cit.).

The term CDR and its plural refer to the complementarity-determining region, three of which confer binding character to the variable region of the light chain (CDR-L1, CDR-L2, and CDR-L3), and three more confer binding character to the variable region of the heavy chain (CDRH1, CDRH2, and CDR-H3). The CDRs contain most of the residues responsible for specific antibody-antigen interactions and therefore contribute to the functional activity of the antibody molecule: they are the major determinants of antigen specificity.

The precise definition of the boundaries and sizes of CDRs is the subject of various classifications and numbering systems. Accordingly, CDRs may be specified according to Kabat, Chothia, Contact, or any other delineation system that includes the numbering system described herein. Despite the different boundaries, each of these systems has a certain degree of overlap, representing the so-called hypervariable regions in the variable sequences. Consequently, CDR definitions according to these systems may differ in length and delineation relative to the adjacent framework region. See, for example, Kabat (approach based on interspecies sequence variability), Chothia (approach based on crystallographic studies of antigen-antibody complexes) and/or MacCallum (Kabat et al., loc. cit; Chothia et al., J. Mol. Biol, 1987, 196: 901-917 and MacCallum et al., J. Mol. Biol, 1996, 262: 732). Another standard for characterizing the antigen-binding site is the AbM definition used in Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). In cases where two residue identification methods define regions as overlapping but not identical regions, they can be combined to define a hybrid CDR. However, numbering according to the so-called Kabat system is preferred.

Typically, the CDRs form a loop structure that can be classified as a canonical structure. The term canonical structure refers to the basic chain conformation adopted by the antigen-binding loops (CDRs). Comparative structural studies have revealed that five of the six antigen-binding loops have only a limited set of accessible conformations. Each canonical structure can be characterized by the helical angles of the polypeptide backbone. Therefore, the corresponding loops between antibodies can be characterized by a high degree of similarity in three-dimensional structures despite high amino acid variability in most parts of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800). In addition, there is a relationship between the structure adopted by the loop and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop as well as in the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

The term canonical structure may also include aspects related to the linear sequence of the antibody, such as those listed according to Kabat (Kabat et al., loc. cit.). The Kabat numbering scheme (system) is a widely accepted standard for numbering the amino acid residues of the variable domain of an antibody in a sequential manner and is the preferred scheme used in the present invention, as also mentioned elsewhere herein. Additional structural factors may also be used to determine the canonical structure of an antibody. For example, differences that are not fully reflected in the Kabat numbering may be described by the Chothia et al. numbering system and/or revealed by other techniques, such as crystallography and two-dimensional or three-dimensional computer modeling. Accordingly, a given antibody sequence may be placed into a canonical class, which allows, inter alia, the identification of corresponding framework sequences (e.g., based on the requirement to include different canonical structures in a library). The Kabat system of numbering the amino acid sequences of antibodies and the structural features described by Chothia et al., loc. cit, as well as their significance for the interpretation of canonical aspects of antibody structure, are described in the literature. The structural subunits and three-dimensional configurations of the various classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

The light chain CDR3, and particularly the heavy chain CDR3, may constitute the most important determinants of antigen binding within the variable regions of the light and heavy chains. In some antibody constructs, the heavy chain CDR3 appears to constitute the major interface between antigen and antibody. In vitro selection schemes in which only the CDR3 is varied can be used to alter the binding properties of the antibody or to determine which residues contribute to antigen binding. Consequently, the CDR3 is typically the largest source of molecular diversity within the antibody binding site. For example, the length of the HC may be as few as two amino acid residues or more than 26 amino acids.

The term neutralization refers, respectively, to an antigen-binding molecule, scFv or antibody that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be accomplished, for example, by directly blocking the binding site on the ligand or by binding to the ligand and altering the binding ability of the ligand through indirect mechanisms (such as structural or energetic changes in the ligand). In some embodiments, the term can also refer to an antigen-binding molecule that prevents the protein to which it is bound from performing a biological function.

The terms target or antigen refer to a molecule or portion of a molecule capable of binding by an antigen-binding molecule. In some embodiments, a target may have one or more epitopes.

The term "compete," when used in the context of antigen-binding molecules that compete for the same epitope, means competition between antigen-binding molecules as determined by an assay in which the antigen-binding molecule being tested (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of an illustrative antigen-binding molecule to the antigen. Numerous types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (EIA), sandwich competitive assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using 1-125 as a label (Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552) and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). The term epitope includes any determinant capable of binding by an antigen-binding molecule, such as an scFv, antibody or immune cell of the present invention. An epitope is a region of an antigen that is bound by an antigen-binding molecule that targets that antigen and, in the case where the antigen is a protein, includes specific amino acids that directly bind to the antigen-binding molecule.

As used herein, the terms label or labeled refer to the inclusion of a detectable marker, such as by incorporating a radiolabeled amino acid or by attaching to a polypeptide biotin moieties that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In some embodiments, the label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and may be used.

In accordance with the present invention, on-off switches or other types of control switching techniques may be included herein. These techniques may include the use of dimerization domains and optional dimerization activators such

- 17,048,570 domains. These techniques include, for example, those described by Wu et al., Science 2014 350 (6258) using FKBP/Rapalog dimerization systems in certain cells, the contents of which are hereby incorporated by reference in their entireties. Additional dimerization technology is described in, for example, Fegan et al. Chem. Rev. 2010, 110, 3315-3336 and in U.S. Pat. Nos. 5,830,462; 5,834,266; 5,869,337; and 6,165,787, the contents of which are also hereby incorporated by reference in their entireties. Additional dimerization pairs may include cyclosporin A/cyclophilin receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoid/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Additional examples of dimerization technology can be found, for example, in WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, US Patent Nos. 8,486,693, US 2014/0171649 and US 2012/0130076, the contents of which are incorporated herein by reference in their entirety.

Treatment methods.

Using adoptive immunotherapy, naïve T cells can be (i) isolated from the patient, (ii) genetically engineered to express a chimeric antigen receptor (CAR) that binds to at least one tumor antigen, (iii) expanded ex vivo into a larger population of engineered T cells, and (iv) reintroduced into the patient. See, e.g., U.S. Patents 7,741,465 and 6,319,494 to Eshhar et al. (Cancer Immunol, supra); Krause et al. (supra); Finney et al. (supra). After the engineered T cells are reintroduced into the patient, they mediate an immune response against cells expressing the tumor antigen. See, e.g., Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626). This immune response involves the secretion of IL-2 and other cytokines by T cells, clonal expansion of T cells that recognize the tumor antigen, and T cell-mediated specific killing of target-containing cells. See Hombach et al., Journal of Immun. 167: 6123–6131 (2001).

Thus, in some aspects, the present invention provides a method of treating or preventing a condition associated with undesirable and/or elevated levels of STEAP1 in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated antigen-binding molecule, CAR or TCR disclosed herein.

Methods of treating diseases or disorders, including cancer, are provided. In some embodiments, the present invention relates to generating a T cell-mediated immune response in a subject comprising administering to the subject an effective amount of engineered immune cells according to the present application. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the target cell is a tumor cell. In some aspects, the present invention provides a method of treating or preventing a cancer, said method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding molecule described herein. In some aspects, the present invention provides a method of treating or preventing a cancer, said method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor, T cell receptor and/or isolated antigen-binding molecule as described herein.

In some aspects, the present invention provides a pharmaceutical composition comprising at least one antigen-binding molecule as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

The antigen binding molecules, CARs, TCRs, immune cells, etc. of the present invention can be used to treat diseases associated with STEAP1 expression, including but not limited to prostate cancer, and in one preferred embodiment, metastatic castration-resistant prostate cancer.

It should be understood that target doses for CAR'/CAR-T'/TCR' cells can be in the range of 1x10 ⁶ to 2x10 ¹⁰ cells/kg, preferably 2x10 ⁶ cells/kg, more preferably. It should be understood that doses above and below this range may be suitable for certain subjects, and suitable dose levels can be determined by a physician as needed. In addition, multiple doses of cells can be provided in accordance with the present invention.

Also provided are methods for reducing the size of a tumor in a subject, comprising administering to the subject an engineered cell of the present invention, wherein the cell comprises a chimeric antigen receptor, a T cell receptor, or a T cell receptor based on a chimeric antigen receptor, comprising an antigen-binding molecule that binds to an antigen on the tumor. In some embodiments, the subject has a solid tumor or a hematologic malignancy, such as lymphoma or leukemia. In some embodiments, the engineered cell is delivered to the tumor bed. In some embodiments, the cancer is in the bone marrow of the subject.

In some embodiments, the engineered cells are autologous T cells. In some embodiments, the engineered cells are allogeneic T cells. In some embodiments, the engineered cells are heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo.

The methods may further comprise administering one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is a chemotherapeutic lymphodepletion (pretreatment) agent. Useful pretreatment regimens, along with associated useful biomarkers, are described in U.S. Provisional Patent Applications 62/262,143 and 62/167,750, which are incorporated herein by reference in their entireties. They describe, for example, methods for pretreating a patient in need of T-cell therapy comprising administering to the patient certain useful doses of cyclophosphamide (200 mg/ ^m2 /day to 2000 mg/ ^m2 /day) and certain doses of fludarabine (20 mg/ ^m2 /day to 900 mg/ ^m2 /day). A preferred dosing regimen involves treating the patient with approximately 500 mg/ ^m2 /day of cyclophosphamide and approximately 60 mg/ ^m2 /day of fludarabine daily for three days prior to administering a therapeutically effective amount of engineered T cells to the patient.

In other embodiments, the antigen-binding molecule, transduced (or otherwise engineered) cells (such as CAR or TCR), and chemotherapeutic agent are administered in an amount effective to treat the disease or condition in the subject.

In some embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and reduced trimethylolomelamin; Nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; Nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics such as aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-E-norleucine. doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, cynostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal cortex hormone synthesis inhibitors such as aminoglutethimide, mitotane, trilostane; Folate supplementers such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfomitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosin; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Taxoids such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and doxetaxel (Taxotere®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylmycin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin-diftitox); esperamicins; capecitabine and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included in this definition are anti- 19 048570 hormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Combinations of chemotherapeutic agents, including but not limited to CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, are also administered when necessary.

In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered within 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administration of the cell or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.

Various additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab, and CTLA-4 inhibitors such as ipilimumab (Yervoy®).

Additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, casodex (bicalutamide), degarelix, docetaxel, enzalutamide, Erleada® (apalutamide), flutamide, goserelin acetate, Jevtana® (cabazitaxel), leuprolide acetate, Lupron® (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), mitoxantrone hydrochloride, Nilandron® (nilutamide), nilutamide, Provenge® (sipuleucel-T), radium-223 dichloride, sipuleucel-T, taxotere (docetaxel), Viadur (leuprolide acetate), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate), or Zytiga (abiraterone acetate).

In additional embodiments, a composition comprising a CAR-containing immune cell can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, TNF inhibitors, cyclophosphamide, and mycophenolate. Examples of NSAIDs include ibuprofen, naproxen, sodium naproxen, Cox-2 inhibitors, and sialylates. Examples of analgesics include acetaminophen, oxycodone, tramadol, proporxifene hydrochloride. Examples of glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Illustrative biologic response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biologic response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

In some embodiments, the compositions described herein are administered with a cytokine. As used herein, the term cytokine refers to proteins released by one population of cells that act on another cell as intercellular mediators. Examples of cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl variant of human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Müllerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-derived growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta, and -gamma; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-20 048570

12; IL-15, tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of native sequence cytokines.

In some aspects, the present invention provides an antigen-binding molecule that binds to STEAP1 with a Kd of less than 100 pM. In some embodiments, the antigen-binding molecule binds with a _Kd of less than 10 pM. In other embodiments, the antigen-binding molecule binds with a _Kd of less than 5 pM.

Methods of obtaining.

Various known techniques can be used to obtain polynucleotides, polypeptides, vectors, antigen-binding molecules, immune cells, compositions, etc. in accordance with the present invention.

Prior to the in vitro manipulation or genetic modification of immune cells described herein, the cells can be obtained from a subject. In some embodiments, the immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation. The cells can preferably be obtained from the circulating blood of an individual using apheresis. Typically, the apheresis product contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. The cells can be washed with PBS. It should be understood that a washing step can be used, for example, using a semi-automated flow centrifuge, such as a Cobe™ 2991 cell processor, Baxter CytoMate™, etc. After washing, the cells can be resuspended in various biocompatible buffers or another saline solution with or without a buffer. In some embodiments, unwanted components of the apheresis sample can be removed.

In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, such as by PERCOLL™ gradient centrifugation. A particular subset of T cells, such as CD28', CD4', CD8', CD45RA', and CD45RO' T cells, can be further isolated using positive or negative selection methods known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to negatively selected cells. One method for use herein is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

PBMCs can be used directly for genetic modification by immune cells (such as CAR or TCR) using the methods described herein. In some embodiments, after PBMCs are isolated, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets either before or after genetic modification and/or expansion.

In some embodiments, the CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these CD8+ cell types. In some embodiments, expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, the central memory T cells are CD45RO', CD62L, CD8+ T cells. In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and CD127 and positive for granzyme B and perforin. In some embodiments, the CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that share cell surface antigens.

- 21 048570

Immune cells, such as T cells, may be genetically modified after isolation using known methods, or the immune cells may be activated and expanded (or differentiated in the case of progenitor cells) in vitro prior to genetic modification. In another embodiment, immune cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR) and then activated and/or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874; U.S. Patent No. 6,867,041; U.S. Patent No. 6,797,514; and PCT WO2012/079000, the contents of which are incorporated herein by reference in their entireties. Such methods typically involve contacting PBMCs or isolated T cells with a stimulatory agent and a costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, typically attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. The anti-CD3 and anti-CD28 antibodies attached to the same bead serve as surrogate antigen-presenting cells (APCs). One example is the Dynabeads system, a CD3/CD28 activation/stimulation system for physiological activation of human T cells.

In other embodiments, T cells can be activated and stimulated to proliferate using feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent No. 6,040,177; U.S. Patent No. 5,827,642; and WO2012129514, the entire contents of which are incorporated herein by reference.

Certain methods for producing the constructs and engineered immune cells of the present invention are described in PCT Application No. PCT/US15/14520, the contents of which are incorporated herein by reference in their entirety. Additional methods for producing the constructs and cells can be found in U.S. Provisional Patent Application No. 62/244,036, the contents of which are incorporated herein by reference in their entirety.

It should be understood that the PBMCs may further comprise other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of the chimeric receptor as disclosed herein may be introduced into a population of donor human T cells, NK cells or NKT cells. Successfully transduced T cells carrying the expression vector may be sorted using flow cytometry to isolate CD3 positive T cells and then expanded to increase the number of these CAR expressing T cells, in addition to activating the cells with CD3 antibodies and IL-2 or other methods known in the art, as described elsewhere herein. Standard procedures are used to cryopreserve the CAR expressing T cells for storage and/or recovery for use in humans. In one embodiment, transduction, culturing and/or expansion of T cells in vitro is performed in the absence of products of animal origin other than human origin, such as fetal calf serum and fetal bovine serum.

For cloning polynucleotides, a vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify a copy of the polynucleotide contained therein. Cloning vectors may contain sequence components typically including, but not limited to, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. If necessary, these elements may be selected by a person of ordinary skill in the art. For example, the origin of replication may be selected to facilitate autonomous replication of the vector in the host cell.

In some embodiments, the present invention provides isolated host cells comprising the vector provided herein. Host cells comprising the vector may be useful for expressing or cloning a polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, such as Enterobactedaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

The vector can be introduced into the host cell using any suitable methods known in the art, including but not limited to DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microparticle bombardment, receptor-mediated gene delivery, polylysine, histone, chitosan and peptide mediated delivery. Standard methods for transfection and transformation of cells to express the vector of interest

- 22 048 570 are well known in the art. In a further embodiment, a mixture of different expression vectors can be used to genetically modify a donor population of immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a portion of the engineered cells express more than one different CAR.

In one embodiment, the present invention provides a method for storing genetically engineered cells expressing a CAR or TCR targeting a STEAP1 protein. This includes cryopreserving the immune cells so that the cells remain viable upon thawing. A portion of the CAR-expressing immune cells can be cryopreserved by methods known in the art to provide a continuous source of such cells for future treatment of patients suffering from a malignant neoplasm. If necessary, the cryopreserved transformed immune cells can be thawed, grown and expanded to produce more such cells.

As used in this document, the term cryopreservation refers to the preservation of cells by cooling to sub-zero temperatures, such as (usually) 77 Kelvin or -196°C (the boiling point of liquid nitrogen). Cryoprotectants are often used at sub-zero temperatures to prevent cell damage due to freezing at low temperatures or warming to room temperature. Cryopreservatives and optimal cooling rates can protect cells from damage. Cryoprotectants that can be used in accordance with the present invention include, but are not limited to: dimethyl sulfoxide (DMSO) (Lovelock & Bishop, Nature (1959); 183: 1394-1395; Ashwood-Smith, Nature (1961); 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci. (1960); 85: 576), and polyethylene glycol (Sloviter & Ravdin, Nature (1962); 196: 48). A preferred cooling rate is from 1 to 3°C/minute.

The term substantially pure is used to indicate that a high level of a given component is present. Desirably, the component is the predominant component present in the composition. Preferably, it is present at a level of greater than 30%, greater than 50%, greater than 75%, greater than 90%, or even greater than 95%, said level being determined on a dry weight to dry weight basis relative to the entire composition in question. At very high levels (e.g., greater than 90%, greater than 95%, or greater than 99%), the component may be considered to be in pure form. The biologically active substances of the present invention (including polypeptides, nucleic acid molecules, antigen-binding molecules, fragments) may be presented in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. When a composition is substantially free of a given contaminant, the contaminant will be present at a low level (e.g., at a level of less than 10%, less than 5%, or less than 1% based on the dry weight to dry weight ratio specified above).

In some embodiments, the cells are formulated by first harvesting them from the culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a pharmaceutically acceptable carrier) in an amount effective for treatment. A suitable infusion medium may be any isotonic medium composition, typically physiological saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's may also be used. The infusion medium may be supplemented with human serum albumin.

The required number of cells for treatment in the composition is typically at least 2 cells (e.g., at least 1 from the central CD8' memory T cell subset and at least 1 from the CD4+ helper T cell subset) or typically no more than ¹⁰² and no more than 106 cells, no more than 108 inclusive or ¹⁰⁹ cells, and may be more than ¹⁰¹⁰ cells. The number of cells will depend on the desired use for which the composition is intended and the type of cells included therein. The required cell density is typically greater than 106 cells/mL and typically greater than ¹⁰⁷ cells/mL, typically 108 cells/mL or greater. A clinically significant amount of immune cells can be divided into multiple infusions that are cumulatively equal to or greater than 10 ⁵ , 10 6 , 10 ⁷ , 10 8 , 10 9 , 10 ¹⁰ , 10 11 or 10 ¹² cells. In some aspects of the present invention, in particular, since all of the cells infused into the body will be redirected to a specific target antigen (STEAP1), a smaller number of cells in the range of 10 ⁶ /kg (from 10 6 to 10 ¹¹ per patient) can be administered. CAR treatment can be administered multiple times at doses within these ranges. The cells can be autologous, allogeneic or heterologous to the patient undergoing therapy.

The CAR-expressing cell populations of the present invention may 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. The pharmaceutical compositions of the present invention may comprise a CAR or TCR expressing cell population, such as

- 23 048 570 such as T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; excipients (e.g. aluminum hydroxide) and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline solution, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. The parenteral preparation may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

It should be understood that adverse events can be minimized by transducing immune cells (containing one or more CARs or TCRs) with a suicide gene. It may also be desirable to include an inducible on or acceleration switch in the immune cells. Suitable techniques include the use of inducible caspase-9 (US 2011/0286980) or thymidine kinase before, after, or simultaneously with transduction of cells with a CAR construct of the present invention. Additional methods for introducing suicide genes and/or on switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other methods known in the art.

It should be understood that the descriptions in this document are illustrative and explanatory only and are not limiting of the claimed invention. In this application, the use of the singular includes the plural unless otherwise specifically indicated.

The section headings used in this document are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including without limitation patents, patent applications, articles, books, and treatises, are hereby incorporated by reference in their entirety for all purposes. As used in connection with the present invention, unless otherwise specified, the following terms shall have the following meanings.

In this application, the use of or means and/or, unless otherwise specified. In addition, the use of the term including, as well as other forms such as includes and included, is not limiting. In addition, terms such as element or component include both elements and components containing a single unit and elements and components that contain more than one subunit, unless otherwise specifically specified.

The term STEAP1 activity includes any biological effect of STEAP1. In some embodiments, STEAP1 activity includes the ability of STEAP1 to interact with or bind to a substrate or receptor.

The term polynucleotide, nucleotide, or nucleic acid includes both single-stranded and double-stranded polymers of nucleotides. The nucleotides comprising a polynucleotide may be ribonucleotides or deoxyribonucleotides, or a modified form of either type of nucleotide. These modifications include base modifications such as bromuridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphodiselenoate, phosphoroanilothiate, phosphoraniladate, and phosphoramidate linkages.

The term oligonucleotide refers to a polynucleotide containing 200 or fewer nucleotides. Oligonucleotides may be single-stranded or double-stranded, for example, for use in constructing a mutant gene. Oligonucleotides may be sense or antisense oligonucleotides. An oligonucleotide may contain a label, including a radioactive label, a fluorescent label, a hapten, or an antigen label for use in detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

The term regulatory sequence refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such regulatory sequences may depend on the host organism. In particular embodiments, regulatory sequences for prokaryotes may

- 24 048570 include a promoter, a ribosome binding site, and a transcription termination sequence. For example, regulatory sequences for eukaryotes may include promoters containing one or more transcription factor recognition sites, transcription enhancer sequences, and a transcription termination sequence. Regulatory sequences may include leader sequences (signal peptides) and/or fusion partner sequences.

As used in this document, the term functionally linked means that the components to which the term is used are in a relationship that enables them to perform their intended functions under suitable conditions.

The term vector means any molecule or structural unit (e.g., a nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell. The term expression vector or expression construct refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or regulate (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or regulate transcription, translation, and, if introns are present, affect RNA splicing of the coding region operably linked thereto.

The term host cell refers to a cell that has been transformed or is capable of being transformed by a nucleic acid sequence and thereby expresses a gene of interest. The term includes progeny of the parent cell, whether or not the progeny is identical in morphology or genetic structure to the original parent cell, so long as the gene of interest is present.

The term transformation refers to a change in the genetic characteristics of a cell, and a cell has been transformed if it has been modified to contain new DNA or RNA. For example, a cell is transformed when it is genetically modified from its natural state by the introduction of new genetic material through transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombine with the cell's DNA by physical integration into the cell's chromosome, or may be temporarily maintained as an episomal element without replication, or may be independently replicated as a plasmid. A cell is said to be stably transformed if the transforming DNA is replicated upon cell division.

The term transfection refers to the uptake of foreign or exogenous DNA into a cell. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

The term transduction refers to the process by which foreign DNA is introduced into a cell via a viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

The terms polypeptide or protein refer to a macromolecule having the amino acid sequence of a protein, including deletions, additions and/or substitutions of one or more amino acids of the native sequence. The terms polypeptide and protein particularly encompass STEAP1 antigen binding molecules, antibodies or sequences that contain deletions, additions and/or substitutions of one or more amino acids of an antigen-binding protein. The term polypeptide fragment refers to a polypeptide that contains an amino-terminal deletion, a carboxy-terminal deletion and/or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules. Useful fragments include, but are not limited to, one or more CDR regions, heavy and/or light chain variable domains, a portion of other portions of an antibody chain, etc.

The term "isolated" means that the substance (i) is free from at least some other proteins with which it would normally be found, (ii) is substantially free from other proteins from the same source, such as those obtained from the same species, (iii) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (iv) is operably linked (through a covalent or non-covalent interaction) to a polypeptide with which it is not associated in nature, or (v) is not found in nature.

A variant of a polypeptide (e.g., an antigen-binding molecule or an antibody) comprises an amino acid sequence wherein one or more amino acid residues in the amino acid sequence are inserted, deleted, and/or substituted compared to another polypeptide sequence. Variants include fusion proteins.

The term identity refers to the similarity between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by alignment and comparison of the sequences. Percent identity means the percentage of identical amino acid or nucleotide residues in the molecules being compared and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignments (if any) are preferably taken into account using a specific mathematical model or computer program (i.e., algorithm).

To calculate percent identity, the sequences being compared are typically aligned in a manner that yields the greatest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG software package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI, USA). The GAP computer algorithm is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. The sequences are aligned to obtain an optimal match of their corresponding amino acids or nucleotides (the coverage of the match, determined by the algorithm). In certain embodiments, the algorithm also uses a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:1091510919 for the BLOSUM 62 comparison matrix).

As used herein, the twenty standard (e.g., naturally occurring) amino acids and their abbreviated names are consistent with common practice. See Immunology A Synthesis (2nd Edition, Golub and Gren, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, non-natural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids can also be suitable components for the polypeptides of the present invention. Examples of non-conventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamate, epsilon-N, N,N-trimethyllysine. ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation system used herein, the left-hand direction is toward the amino terminus and the right-hand direction is toward the carboxyl terminus in accordance with standard practice and conventions.

Conservative amino acid substitutions may involve non-naturally occurring amino acid residues that are typically introduced through chemical peptide synthesis rather than through synthesis in biological systems. They include peptidomimetics and other reversed or inverted forms of amino acid fragments. Naturally occurring residues can be divided into classes based on common side chain properties:

a) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c) acidic: Asp, Glu;

d) basic: His, Lys, Arg;

e) residues that influence chain orientation: Gly, Pro; and

f) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the replacement of a member of one of these classes with a member of another class. Such residue substitutions may be introduced, for example, into regions of a human antibody that are homologous to non-human antibodies or into non-homologous regions of the molecule.

When making changes to the antigen-binding molecule, costimulatory domain, or activating domain of the engineered T cell, according to certain embodiments, the hydropathic index of amino acids can be taken into account. Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. They are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). See Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydrophobicity index or measure and yet the proteins retain similar biological activity. It is also understood in the art that substitution of such amino acids can be effected on the basis of hydrophilicity, particularly if the biologically functional protein or peptide thus created is intended to be used in immunological embodiments, as is the case with the present invention. Illustrative amino acid substitutions are set forth in Table 2.

- 26 048570

Table 2

Original Residues Illustrative Substitutions Preferred Substitutions

Ala Val, Leu, lie Val Arg Lys, Gin, Asn Lys Asn Gin Gin Asp Glu Glu Cys Sir, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro,Ala Ala His Asn, Gin, Lys, Arg Arg lie Leu, Val, Met, Ala, Phe, norleucine Leu Leu Norleucine, He, Val, Met, Ala, Phe lie Lys Arg, 1,4-diaminobutyric Arg Acid, Gin, Asn Met Leu, Phe, lie Leu Phe Lee, Val, lie, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val lie, Met, Leu, Phe, Leu Ala, norleucine

The term "derivative" refers to a molecule that includes a chemical modification other than the insertion, deletion, or substitution of amino acids (or nucleic acids). In some embodiments, derivatives comprise covalent modifications, including but not limited to chemical linkage to polymers, lipids, or other organic or inorganic moieties. In some embodiments, a chemically modified antigen-binding molecule may have a longer half-life in the bloodstream than an antigen-binding molecule that is not chemically modified. In some embodiments, the derivative antigen-binding molecule is covalently modified to include one or more water-soluble polymeric attachments, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the parent peptide. These types of non-peptide compounds are called peptide mimetics or peptide mimetics. Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber & Freidinger, TINS, p.392 (1985); and Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated herein by reference for any purpose.

The term therapeutically effective amount refers to that amount of the STEAP1 antigen binding molecule that is determined to result in a therapeutic response in a mammal. Such therapeutically effective amounts can be readily determined by one of ordinary skill in the art.

The terms patient and subject are used interchangeably and include human and non-human subjects, as well as subjects with formally diagnosed disorders, subjects without formally recognized disorders, subjects receiving medical care, subjects at risk of developing disorders, etc.

The terms treat and cure include therapeutic treatments, preventive treatments, and routes of administration that reduce the risk of a subject developing a disorder or other risk factor. Treat does not require a complete cure of the disorder and includes embodiments that reduce symptoms or underlying risk factors. The term prevent does not require 100% elimination of the possibility of an event. Rather, it means that

- 27 048570 the likelihood of occurrence of the phenomenon was reduced in the presence of the compound or method.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification methods can be performed according to the manufacturer's specifications, or as commonly performed in the art, or as described herein. The above methods and procedures can generally be performed according to conventional techniques well known in the art and as described in various general and more specific references that are cited and discussed herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

The present invention will be further illustrated by the following sequences.

CD28T, DNA, extracellular, transmembrane, intracellular domains CTTGATAATGAAAAGTC

AAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCC TGGTCCATCCAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTA CTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAAGAAGCCG CCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAA ACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEP ID

NP:1)

CD28T, extracellular, transmembrane, intracellular amino acids:

LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV

AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRS (SEP ID NP:2) CD28T, DNA - extracellular

CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTG TCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA (SEP ID NP:3)

CD28T, amino acids - extracellular

LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP (SEQ GO NP:4)

CD28, DNA, transmembrane domain

TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGT

CACCGTGGCTTTTATAATCTTCTGGGTT (SEP ID NP:5)

CD28, amino acids, transmembrane domain:

FWVLVVVGGV LACYSLLVTV AFIIFWV (SEP ID NP:6)

CD28, DNA, intracellular domain

AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCAC

GCCGCCCTGGCCCCACAAGGAAACACTACCAGCTTACGCACCACCTAGAGATTTC GCTGCCTATCGGAGC (SEP ID NP:7)

CD28, amino acids, intracellular domain

RSKRSRLLHSDYMNMTPRRPGPTRKHYpPYAPPRDFAAYRS (SEP ID NP:8)

CD3-zeta, DNA

AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGA ACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCC AGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAA ATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGG GACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCAC

CTAGG (SEP ID NP:9)

CD3-zeta, amino acids

RVKFSRSADAPAYppGpNpLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPpEGLYNELpKDKMAEAYSEIGMKGERRRGKGHDGLYpGLSTATKDTYDALHMpA LPPR (SEP ID NP: 10)

CD28, DNA

ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGTA

CCATCATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCCTCTTCCCCGGGCCATC AAAGCCC (SEP ID NP: 11)

CD28, amino acids

IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKP (SEP ID NP: 12)

- 28 048570

CD8, DNA, extracellular and transmembrane domains

GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGTT CTTGCCGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCTCCTAC CATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCAGGGGG CGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACCCT GGCCGGAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGTAATCA CAGGAAC (SEQ ID NO: 13)

CD8, amino acids, extracellular and transmembrane domains

AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 14) 4-1BB, DNA, intracellular domain

CGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTACATTTTCAAAC AGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTGCTCCTGCCGC TTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTG (SEQ ID NO: 15)

4-1BB, amino acids, intracellular domain

RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 16)

Clone 2F3, NS, DNA

CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGATGATGAAGCCTGGGGCCTCAG TGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTACCTACTGGATAGAGTGGG TAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGT GGTAATACTGACTTCAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATAC ATCCTCCGACACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCCGT CTATTACTGTACAAGATGGGGGTACTACGGTACTAGGGGGTACTTCAATGTCTGGGG CGCAGGGTCCACGGTCACCGTCTCCTCA (SEQ ID NO: 87) Clone 2F3, HC, amino acids, CDRs underlined

OVQLVQSGAEVKKPGASVKVSCKASGYTFSTYWIEWVRQAPGQRLEWMGEILP GSGNTDFNEKFOGRVTFTADTSSDTAYMELSSLRSEDTAVYYCTRWGYYGTRGYFNV WGQGTLVTVSS (SEQ ID NO: 88) Clone 2F3, NS, amino acids, CDR1:

TYWIE (SEQID NO: 89)

Clone 2F3, HC, amino acids, CDR2:

EILPGSGNTDFNEKFQG (SEQ Ш NO: 90)

Clone 2F3, HC, amino acids, CDR3:

WGYYGTRGYFNV (SEQ ID NO: 91)

Clone 2F3. LC. DNA

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAA GGTCACCATAACGTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTTCCAGCA GAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACCTCCAACCTGGCTTCTGG AGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAG CCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGCGTAGTTTCCC GTACACGTTCGGAGGGGGGACCAAGCTGGAAATTAAA (SEQ ID NO: 92)

Clone 2F3. LC. amino acids (CDRs are underlined)

EIVLTOSPATLLSLSPGERATLSCRASSSVSYMHWFOQKPGOAPRLLIYSTSNLASG

IPARFSGSGSGTDYTLTISSLEPEDFAVYYCOORRSFPYTFGOGTKLEIK (SEQ ID NO: 93) Clone 2F3, LC, CDR1 amino acids:

RASSSVSYMH (SEQ ID NO: 94)

Clone 2F3, LC, CDR2 amino acids:

STSNLAS (SEQ ID NO: 95)

Clone 2F3, LC, CDR3 amino acids:

QQRRSFPYT (SEQ ID NO: 96)

Clone 11C2. NS DNA

- 29 048570

CAGATCCAGTTGGTGCAGTCTGGACCTGAACTGAAGAAGCCTGGAGAGACAG TCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGAACTGGG TGAAGCAGGCTCCAGGAAAGGGTTTACAGTGGATGGGCTGGATGAACACTTATACT GGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACC TCTGCCAGAACTGTCTCTTTGGACATCAACGACCTCAAAAATGAGGACACGGCTACA TATTTCTGTACAAGAGCAGGGGGACAACTCAGGCCCGGGGCTATGGACTACTGGGG

TCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:97)

Clone 11C2, HC, amino acids (CDRs are underlined)

QLVQSGAEVKKPGATVKISCKASGYTFTNYGMNWVQQAPGQGLEWMGWMNT YTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRSEDTAVYFCARAGGQLRPGAMDY WGQGTMVTVSS (SEQ ID NO:98)

Clone 11C2, HC, amino acids, CDR1:

NYGMN (SEQ ID NO:99)

Clone 11C2, HC, amino acids, CDR2:

WMNTYTGEPTYADKFQG (SEQ GO NO: 100)

Clone 11C2, HC, amino acids, CDR3:

AGGQLRPGAMDY (SEQ ID NO: 101)

Clone 11C2. LC. DNA

GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAG GGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTTTAT GAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGTTGCAT CCAATCTAGAATCTGGGATCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGAC TTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTATTGTCAG CAAAGTAATGAGGAACCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 102)

Clone 11C2, LC, amino acids (CDRs are underlined)

DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCOQSNEEPPTFGQGTKLEIK (SEQ ID NO: 103)

Clone 11C2, LC, amino acids, CDR1:

KASQSVDYDGDSFMN (SEQ ID NO: 104)

Clone 11C2, LC, amino acids, CDR2:

VASNLES (SEQ ID NO: 105)

Clone 11C2, LHC, amino acids, CDR3:

QQSNEEPPT (SEQ ID NO: 106)

Clone 1A1. NS, DNA

CAGATACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGTAGCGTCTGGATTCACCTTCAAGAACTATGGCATGCACTGGGTCCG CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGTATGATGGAAGTA ATGAATACTATGGAGACCCCGTGAAGGGCCGATTCACCATCTCCAGGACAACTCC AAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGCCGATGACACGGCTGTGTA TTACTGTGCGAGGTCGGGAATAGCAGTGGCTGGGGCCTTTGACTACTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 107)

Clone 1A1, HC, amino acids (CDRs are underlined)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGLEWMGEINPSSG RTNYNEKFKTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKLGPGPQYYAMDYWGO GTMVTVSS (SEQ ID NO: 108)

Clone 1A1, HC, amino acids, CDR1:

TYWMH (SEQ ID NO: 109)

Clone 1A1, HC, amino acids, CDR2:

EINPSSGRTNYNEKFKT (SEO ID NO: И0)

Clone 1A1, HC, amino acids, CDR3:

LGPGPQYYAMDY (SEQ ID NO: 111)

Clone 1A1, LC, DNA

- 30 048570

GAAATTGTGTTGACGCAGTCTCCAGACACCCTGTCTTTGTCTCCAGGGGAAAAAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTGGCCTGGTACCAG CAGAAACCTGGACAGGCTCCCAGTCTCCTCATCTATGTTGCATCCAGAAGGGCCGCT GGCATCCCTGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGGAGCCTGAAGATTTTGGAATGTTTTACTGTCAACACTATGGTAGGACA CCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGA (SEQ ID NO: 112)

Clone 1 A1, LC, amino acids (CDRs underlined)

DIQMTQSPSSLSASVGDRVTTITCHASONINVWLSWYQQKPGKAPKLLIYKASKLHTGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCOOGQSYPWTFGQGTKLEIK (SEQ ID NO: 113)

Clone 1A1, LC, amino acids, CDR1:

HASONINVWLS (SEQ ID NO: 114)

Clone 1A1, LC, amino acids, CDR2:

KASKLHT (SEQ ID NO: 115)

Clone 1A1, LC, amino acids, CDR3:

OOGQSYPWT (SEQ ID NO: 116)

Clone 7A4, NS, DNA

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATACACTGGGTGCG ACAGGCCCCTGAACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTG GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGGCCAGGGACACGTCC ATCAGCACAGTTTACATGGACCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTA TTACTGTGCGAGAATACCGGTGGTAACTCGGTCTTTGACTACTGGGGCCAGGGAAC

CCTGGTCACCGTCTCCTCA (SEQ ID NO: 117)

Clone 7A4, HC, amino acids (CDRs underlined)

OVQLVQSGAEVKKPGASVKVSCKASGYSFTSYDINWVRQATGQGLEWMGWMNPNNSG NTGYAQKFOGRVTMTRDTSISTAYMELSSLRSEDTAVYYCGRAGYYYYFGMDVWGQG TTVTVSS (SEQ ID NO: 118)

Clone 7A4, HC, amino acids, CDR1:

SYDIN (SEQ ID NO: 119)

Clone 7A4, HC, amino acids, CDR2:

WMNPNSGNTGYAQKFQG (SEQ ID NO: 120)

Clone 7A4, HC, amino acids, CDR3:

AGYYYYFGMDV (SEQ ID NO: 121)

Clone 7A4, LC, DNA

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCC ACCATCAACTGCAAGTCCACCCAGAGTATTTTATACACCTCCAACAATAAGAACTTC TTAGCTTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTCATTTCCTGGGCA TCTATCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGA TTTCGCTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCA ACAATATTTTAGTACTATGTTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACG

A (SEQ ID NO: 122)

Clone 7A4, LC, amino acids (CDRs underlined)

EIVLTQSPGTLSLSPGERATLSCRAGOSVTSSSFAWYQQKPGQAPRLLIYQTSTRATGIPD

RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGGSRSFGQGTKVELKR (SEQ ID NO: 123)

Clone 7A4, LC, amino acids, CDR1:

RAGQSVTSSSFA (SEQ ID NO: 124)

Clone 7A4, LC, amino acids, CDR2:

QTSTRAT (SEQ ID NO: 125)

Clone 7A4, LC, amino acids, CDR3:

OOYGGSRS (SEQ ID NO: 126)

Clone 7A5, NS, DNA

- 31 048570

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTC CCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGTGCATACTACTGGACTTG GATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCCATTACAGTG GGAGCACCTACTCCAACCCGTCCCTCAAGAGTCGAATTACCATATCGTTAGACACGT CTAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACTGCCGCGGACACGGCCGTGT ATTACTGTGCGAGACAAGAGGACTACGGTGGTTTGTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTTTCCTCA (SEQ ID NO: 127)

Clone 7 A5, HC, amino acids (CDRs underlined)

OVOLVQSGAEVKKPGASVKVSCKASGYSFTSYDINWVROATGOGLEWMGWMNPNSG NTGYAOKFOGRVTMTRDTSISTAYMELSSLRSEDTAVYYCGRAGYYYYFGMDVWGOG TTVTVSS (SEQ ID NO: 128)

Clone 7A5, HC, amino acids, CDR1:

SYDIN (SEQ ID NO: 129)

Clone 7A5, HC, amino acids, CDR2:

WMNPNSGNTGYAQKFQG (SEQ ID NO: 130)

Clone 7A5, HC, amino acids, CDR3:

AGYYYYFGMDV (SEQ ID NO: 131)

Clone 7A5, LC, DNA

GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAT CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCACCGACTTAGCCTGGTACCAGCA GATGCCTGGACAGGCTCCCCGGCTCCTCATCTATGATGCTTCCACCAGGGCCACTGG TTTCCCAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCACGCTCACCATCAG CAGCCTGCAGGCTGAAGATTTTGCAGTTTATTACTGTCAACATTATAAAACCTGGCC TCTCACTTTCGGCGGAGGGACTAAGGTGGAGATCAAACGA (SEQ ID NO: 132)

Clone 7A5, LC, amino acids (CDRs underlined)

EIVLTQSPGTLSLSPGERATLSCRAGOSVTSSSLAWYOQKPGQAPRLLIYQTSTRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCOQYGGSRAFGQGTKVELKR (SEQ ID NO: 133) Clone 7A5, LC, amino acids, CDR1:

RAGOSVTSSSLA (SEQ ID NO: 134)

Clone 7A5, LC, amino acids, CDR2:

QTSTRAT (SEQ ID NO: 135)

Clone 7A5, LC, amino acids, CDR3:

OOYGGSRA (SEQ ID NO: 136)

Clone 14C11.NS, DNA

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTC CCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGTGCATACTACTGGACTTG GATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCCATTACAGTG GGAGCACCTACTCCAACCCGTCCCTCAAGAGTCGAATTACCATATCGTTAGACACGT CTAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACTGCCGCGGACACGGCCGTGT ATTACTGTGCGAGACAAGAGGACTACGGTGGTTTGTTTGACTACTGGGGCCAGGGA

ACCCTGGTCACCGTTTCCTCA (SEQ ID NO: 137)

Clone 14C11, HC, amino acids (CDRs are underlined)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSG GTNSAQKFOGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARGWLQTYYFDNWGQGT LVTVSS (SEQ ID NO: 138)

Clone 14C11, HC, amino acids, CDR1:

GYYMH (SEQ ID NO: 139)

Clone 14C1, HC, amino acids, CDR2:

WINPNSGGTNSAQKFQG (SEQ ID _N0 : 140)

Clone 14C1, HC, amino acids, CDR3:

GWLQTYYFDN (SEQ ID NO: 141)

Clone 14C11.LC. DNA

- 32 048570

GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAT CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCACCGACTTAGCCTGGTACCAGCA GATGCCTGGACAGGCTCCCCGGCTCCTCATCTATGATGCTTCCACCAGGGCCACTGG TTTCCCAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCACGCTCACCATCAG CAGCCTGCAGGCTGAAGATTTTGCAGTTTATTACTGTCAACATTATAAAACCTGGCC TCTCACTTTCGGCGGAGGGACTAAGGTGGAGATCAAACGA (SEQ ID NO: 142)

Clone 14C11, LC, amino acids (CDRs underlined)

DIVMTQSPDSLAVSLGERATIYCKSSOTVLTSSNNKNFLAWYQQKLGQPPKLLISWAST RESGVPDRFSGSGSGTDFTLTISSLOAEDVAIYYCQHYYTSPLTFGGGTKVEIKR (SEQ ID NO: 143)

Clone 14C11, LC, amino acids, CDR1:

TVLTSSNNKNFLA (SEQ ID NO: ¹⁴⁴ )

Clone 14C1, LC, amino acids, CDR2:

WASTES (SEQ ID NO: 145)

Clone 14C1, LC, amino acids, CDR3:

QHYYTSPLT (SEQ ID NO: 146)

S1-2F3-CD28T-CD28-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGA GCCAGCGTGAAGGTGAGCTGCAAAGCCAGCGGCTACACCTTCTCCACCTACTGGAT CGAGTGGGTGAGACAGGCCCCGGACAGAGGCTGGAATGGATGGGAGAGATCCTGC CCGGCAGCGGCAACACCGACTTCAACGAGAAGTTCCAGGGCAGAGTGACCTTCACC GCCGATACCAGCAGCGACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGA TACCGCCGTCTACTACTGCACCAGATGGGGCTACTACGGCACCAGGGGCTATTTCAA CGTGTGGGGCCAGGGAACCCTCGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGTG GCGGTGGTTCTGGCGGCGGAGGCTCCGAGATTGTGCTGACCCAGAGCCCTGCTACA CTGAGCCTGAGCCCCGGCGAGAGAGCCACACTGAGCTGCAGAGCCAGCAGCAGCGT GAGCTACATGCACTGGTTCCAGCAAAAGCCCGGCCAGGCCCTAGGCTGCTGATCT ACAGCACATCCAACCTGGCCAGCGGCATCCCTGCCAGATTCAGCGGTTCTGGCTCCG GCACCGACTACACCCTGACCATCTCCAGCCTGGAGCCCGAGGACTTTGCCGTGTATT ACTGCCAGCAGAGGAGGAGCTTCCCCTACACATTCGGCCAGGGCACCAAACTGGAG ATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAA GGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGT GTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTT ATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAAT ATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCT AGAGATTTCGCTGCCTATCGGAGCCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAA GCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGA GGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGG TGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTG TATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAG AGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGT CTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCAT GAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGTTTGTACCAGGGACTCAGC ACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCC AAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCCTTACATGTGGGGACGTGGA

- 33 048570

AGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGC GACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCGGAGGCGCTAAGGAGGCCTGTC CAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGG GCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAG TGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTG TGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGAT GTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTG TGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGT GAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTT GCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGG CAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCT GAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCA GGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACA ACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTT GGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTA A (SEQ ID NO: 17)

S1-2F3-CD28T-CD28-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFST YWIEWVRQAPGQRLEWMGEILPGSGNTDFNEKFQGRVTFTADTSSDTAYMELSSSLRSE DTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPAT LSLSPGERATLSCRASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQRRSFPYTFGQGTKLEIKAAALDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAK EACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTE CVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTV CEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGS DSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVG LVAYIAFKR (SEQ ID NO: 18)

S1-2F3-CD28T-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGA GCCAGCGTGAAGGTGAGCTGCAAAGCCAGCGGCTACACCTTCTCCACCTACTGGAT CGAGTGGGTGAGACAGGCCCCCGGACAGAGGCTGGAATGGATGGGAGAGATCCTG CCCGGCAGCGGCAACACCGACTTCAACGAGAAGTTCCAGGGCAGAGTGACCTTCAC CGCCGATACCAGCAGCGACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGG ATACCGCCGTCTACTACTGCACCAGATGGGGCTACTACGGCACCAGGGGCTATTTCA ACGTGTGGGGCCAGGGAACCCTCGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGAGATTGTGCTGACCCAGAGCCCTGCTAC ACTGAGCCTGAGCCCCGGCGAGAGAGCCACACTGAGCTGCAGAGCCAGCAGCAGCG TGAGCTACATGCACTGGTTCCAGCAAAAGCCCGGCCAGGCCCTAGGCTGCTGATCT ACAGCACATCCAACCTGGCCAGCGGCATCCCTGCCAGATTCAGCGGTTCTGGCTCCG GCACCGACTACACCCTGACCATCTCCAGCCTGGAGCCCGAGGACTTTGCCGTGTATT ACTGCCAGCAGAGGAGGAGCTTCCCCTACACATTCGGCCAGGGCACCAAACTGGAG ATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAA GGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGT

- 34 048570

GTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTT ATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAAT ATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCT AGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCA GCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGA AGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAAC CAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATG GCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCC ACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGG ATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCG CCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGT CTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAAT GTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAG ACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACC GAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGT GTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAAC TACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAG TTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCG ACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAA CGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGG GAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTA CCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGC GTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGA CAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGC CTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO: 19)

S1-2F3-CD28T-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFST YWIEWVRQAPGQRLEWMGEILPGSGNTDFNEKFQGRVTFTADTSSDTAYMELSSSLRSE DTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPAT LSLSPGERATLSCRASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQRRSFPYTFGQGTKLEIKAAALDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLL LLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSD VVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGS GLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEI PGRWrFRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLI PVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:20)

S1-2F3-CD28T-41BB construct, DNA (signal sequence in bold)

- 35 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGA GCCAGCGTGAAGGTGAGCTGCAAAGCCAGCGCTACACCTTCTCCACCTACTGGAT CGAGTGGGTGAGACAGGCCCCCGGACAGGCTGGAATGGATGGGAGAGATCCTG CCCGGCAGCGGCAACACCGACTTCAACGAGAAGTTCCAGGGCAGAGTGACCTTCAC CGCCGATACCAGCAGCGACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGG ATACCGCCGTCTACTACTGCACCAGATGGGGCTACTACGGCACCAGGGGGCTATTTCA ACGTGTGGGGCCAGGGAACCCTCGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGAGATTGTGCTGACCCAGAGCCCTGCTAC ACTGAGCCTGAGCCCCGGCGAGAGAGCCACACTGAGCTGCAGAGCCAGCAGCAGCG TGAGCTACATGCACTGGTTCCAGCAAAAGCCCGGCCAGGCCCTAGGCTGCTGATCT ACAGCACATCCAACCTGGCCAGCGGCATCCCTGCCAGATTCAGCGGTTCTGGCTCCG GCACCGACTACACCCTGACCATCTCCAGCCTGGAGCCCGAGGACTTTGCCGTGTATT ACTGCCAGCAGAGGAGGAGCTTCCCCTACACATTCGGCCAGGGCACCAAACTGGAG ATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAA GGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGT GTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTT ATAATCTTCTGGGTTCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTAC ATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTG CTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGGTGAAGTTTT CCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAG CTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGA CCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATG AGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAG CGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGA AGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGT GGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCC GGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTC TCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCC TCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCAC AACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCA GCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTC AGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATG GATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCC GGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTG TCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCA

CCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCT GAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGATC AGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGA TCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGTCG TGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCG TGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:21)

S1-2F3-CD28T-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 36 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFST YWIEWVRQAPGQRLEWMGEILPGSGNTDFNEKFQGRVTFTADTSSDTAYMELSSSLRSE DTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPAT LSLSPGERATLSCRASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQRRSFPYTFGQGTKLEIKAAALDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMD GPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLD SVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRV CEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWAD AECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRG TTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:22)

S1-2F3-C8K-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGA GCCAGCGTGAAGGTGAGCTGCAAAGCCAGCGGCTACACCTTCTCCACCTACTGGAT CGAGTGGGTGAGACAGGCCCCCGGACAGAGGCTGGAATGGATGGGAGAGATCCTG CCCGGCAGCGGCAACACCGACTTCAACGAGAAGTTCCAGGGCAGAGTGACCTTCAC CGCCGATACCAGCAGCGACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGG ATACCGCCGTCTACTACTGCACCAGATGGGGCTACTACGGCACCAGGGGCTATTTCA ACGTGTGGGGCCAGGGAACCCTCGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGAGATTGTGCTGACCCAGAGCCCTGCTAC ACTGAGCCTGAGCCCCGGCGAGAGAGCCACACTGAGCTGCAGAGCCAGCAGCAGCG TGAGCTACATGCACTGGTTCCAGCAAAAGCCCGGCCAGGCCCTAGGCTGCTGATCT ACAGCACATCCAACCTGGCCAGCGGCATCCCTGCCAGATTCAGCGGTTCTGGCTCCG GCACCGACTACACCCTGACCATCTCCAGCCTGGAGCCCGAGGACTTTGCCGTGTATT ACTGCCAGCAGAGGAGGAGCTTCCCCTACACATTCGGCCAGGGCACCAAACTGGAG ATCAAGGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCCCC

- 37 048570

GCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGAGA CCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGGCCTGGACTT CGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTGCT GTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACAGATCCAAAAGAAGCCGCCT GCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAAC ACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGT TTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAAC GAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACG GGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATA ATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGA GAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTA CGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGA AGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAA TCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGC TTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGG GCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCG CACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACAT TCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCC TTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCT ATGGATATTACCAGACGGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAA GCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGA GTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGT GCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGAC GCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGG ATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACC TGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAG TCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAG CCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:23)

S1-2F3-C8K-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFST YWIEWVRQAPGQRLEWMGEILPGSGNTDFNEKFQGRVTFTADTSSDTAYMELSSSLRSE DTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPAT LSLSPGERATLSCRASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQRRSFPYTFGQGTKLEIKAAAFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDV

EENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEG VAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCA YGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCT VCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVA GVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:24)

S1-2F3-C8K-41BB DNA construct (signal sequence in bold)

- 38 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCCGGA GCCAGCGTGAAGGTGAGCTGCAAAGCCAGCGCTACACCTTCTCCACCTACTGGAT CGAGTGGGTGAGACAGGCCCCCGGACAGGCTGGAATGGATGGGAGAGATCCTG CCCGGCAGCGGCAACACCGACTTCAACGAGAAGTTCCAGGGCAGAGTGACCTTCAC CGCCGATACCAGCAGCGACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGG ATACCGCCGTCTACTACTGCACCAGATGGGGCTACTACGGCACCAGGGGGCTATTTCA ACGTGTGGGGCCAGGGAACCCTCGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGAGATTGTGCTGACCCAGAGCCCTGCTAC ACTGAGCCTGAGCCCCGGCGAGAGAGCCACACTGAGCTGCAGAGCCAGCAGCAGCG TGAGCTACATGCACTGGTTCCAGCAAAAGCCCGGCCAGGCCCTAGGCTGCTGATCT ACAGCACATCCAACCTGGCCAGCGGCATCCCTGCCAGATTCAGCGGTTCTGGCTCCG GCACCGACTACACCCTGACCATCTCCAGCCTGGAGCCCGAGGACTTTGCCGTGTATT ACTGCCAGCAGAGGAGGAGCTTCCCCTACACATTCGGCCAGGGCACCAAACTGGAG ATCAAGGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCCCC GCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGAGA CCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGGCCTGGACTT CGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTGCT GTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACCGCTTTTCCGTCGTTAAGCG GGGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAA CGACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGG TGCGAACTGAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGG CCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTT TGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAA CCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATT CTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTA CCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCT GCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACAT GTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGC TATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGC TAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTG TAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACC ATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCC TTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGA TGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTG AGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGC AAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCA CGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAG AGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACC CGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGA GGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGT CATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGT CTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAA GAGATGACCTAGGTAA (SEQ ID NO:25)

S1-2F3-C8K-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 39 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFST YWIEWVRQAPGQRLEWMGEILPGSGNTDFNEKFQGRVTFTADTSSDTAYMELSSSLRSE DTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPAT LSLSPGERATLSCRASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQRRSFPYTFGQGTKLEIKAAAFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLL TCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKAC NLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDA VCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVD PCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDL IASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:26)

S1-11C2-CD28T-CD28-41BB DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCA CGCCCGGAGATTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGAGCCAC CGTGAAGATCAGCTGCAAGGCCAGCGGCTACACATTCACCAACTACGGCATGAACT GGGTGCAACAGGCCCCCGGCCAGGGACTGGAGTGGATGGGCTGGATGAACACATAC ACCGGCGAGCCCACCTACGCCGACAAGTTCCAGGGCAGGGTGACATTCACACTCGA TACCAGCGCCAGAACCGTGTATATGGAACTGAGCAGCCTGAGGAGCGAGGACACCG CTGTGTACTTCTGCGCAAGGGCTGGAGGCCAGCTGAGACCTGGCGCTATGGACTACT GGGGCCAGGGCACCATGGTGACCGTGAGCTCCGGAGGCGGAGGATCTGGTGGCGGT GGTTCTGGCGGCGGAGGCTCCGACATCGTGCTGACCCAGACACCTCTCTCCCTGTCC GTGACCCCCGGACAGCCTGCCTCCATTTCCTGTAAGGCCTCCCAGAGCGTGGACTAT GACGGCGACAGCTTCATGAACTGGTACCTGCAGAAGCCCGGACAACCCCCCCAGCT GCTGATCTACGTGGCCAGCAACCTGGAGTCCGGCGTGCCTGACAGGTTTTCCGGCTC CGGCAGCGGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAAGCCGAGGACGTGG GCGTGTACTACTGCCAGCAGAGCAACGAGGAGCCCCCTACCTTCGGACAGGGCACC AAGCTGGAGATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCAT TCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCC ATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACC GTGGCTTTTAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGAT TACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTAC GCACCACCTAGAGATTTCGCTGCCTATCGGAGCCGCTTTTCCGTCGTTAAGCGGGGG AGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGAC TCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCG AACTGAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAG AACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGA CAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCC CAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGA AATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAG GGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCA CCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGG GGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATG GATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAG GAGGCCTGTCCAACGGGCCTTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAAC CTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGC CTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGT ACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCA GTGTGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGC CTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAA

- 40 048570

CACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCG ACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGTGT ACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAG CACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCC CTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGG GATCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACT GTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGAT GACCTAGGTAA (SEQ ID NO:27)

S1-11C2-CD28T-CD28-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGATVKISCKASGYTFTNY GMNWVQQAPGQGLEWMGWMNTYTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRS EDTAVYFCARAGGQLRPGAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQTPL SLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLESGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCQQSNEEPPTFGQGTKLEIKAAALDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVS LGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEP CKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQ DKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITR STPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSIL AAVVVGLVAYIAFKR (SEQ ID NO:28)

S1-11C2-CD28T-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGGAGATTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGA GCCACCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACATTCACCAACTACGGCAT GAACTGGGTGCAACAGGCCCCCGGCCAGGGACTGGAGTGGATGGGCTGGATGAACA CATACACCGGCGAGCCCACCTACGCCGACAAGTTCCAGGGCAGGGTGACATTCACA CTCGATACCAGCGGCCAGAACCGTGTATATGGAACTGAGCAGCCTGAGGAGCGAGGA CACCGCTGTGTACTTCTGCGCAAGGGCTGGAGGCCAGCTGAGACCTGGCGCTATGG ACTACTGGGGCCAGGGCACCATGGTGACCGTGAGCTCCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGACATCGTGCTGACCCAGACACCTCTCTCC CTGTCCGTGACCCCCGGACAGCCTGCCTCCATTTCCTGTAAGGCCTCCCAGAGCGTG GACTATGACGGCGACAGCTTCATGAACTGGTACCTGCAGAAGCCCGGACAACCCCC CCAGCTGCTGATCTACGTGGCCAGCAACCTGGAGTCCGGCGTGCCTGACAGGTTTTC CGGCTCCGGCAGCGGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAAGCCGAGG ACGTGGGCGTGTACTACTGCCAGCAGAGCAACGAGGAGCCCCCTACCTTCGGACAG GGCACCAAGCTGGAGATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAAC AATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATC CAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTC GTCACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCAT AGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCA GCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAG ATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCA ACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCT GAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCT GCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGG AGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGG

- 41 048570

ATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGC AGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGG GCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCT GCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTA CACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACC TTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGA TGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTC CATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATA TTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCT CTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCA GACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGT CTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGAGT GCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGAC AGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGC TTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGAC GCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGT CGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:29) S1-11C2-CD28T-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGATVKISCKASGYTFTNY GMNWVQQAPGQGLEWMGWMNTYTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRS EDTAVYFCARAGGQLRPGAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQTPL SLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLESGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCQQSNEEPPTFGQGTKLEIKAAALDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAM DGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCL DSVVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACR VCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWA DAECEEIPGRWrTRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTR GTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:30)

S1-11C2-CD28T-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGGAGATTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGA GCCACCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACATTCACCAACTACGGCAT GAACTGGGTGCAACAGGCCCCCGGCCAGGGACTGGAGTGGATGGGCTGGATGAACA CATACACCGGCGAGCCCACCTACGCCGACAAGTTCCAGGGCAGGGTGACATTCACA

- 42 048570

CTCGATACCAGCGCCAGAACCGTGTATATGGAACTGAGCAGCCTGAGGAGCGAGGA CACCGCTGTGTACTTCTGCGCAAGGGCTGGAGGCCAGCTGAGACCTGGCGCTATGG ACTACTGGGGCCAGGGCACCATGGTGACCGTGAGCTCCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGACATCGTGCTGACCCAGACACCTCTCTCC CTGTCCGTGACCCCCGGACAGCCTGCCTCCATTTCCTGTAAGGCCTCCCAGAGCGTG GACTATGACGGCGACAGCTTCATGAACTGGTACCTGCAGAAGCCCGGACAACCCCC CCAGCTGCTGATCTACGTGGCCAGCAACCTGGAGTCCGGCGTGCCTGACAGGTTTTC CGGCTCCGGCAGCGGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAAGCCGAGG ACGTGGGCGTGTACTACTGCCAGCAGAGCAACGAGGAGCCCCCTACCTTCGGACAG GGCACCAAGCTGGAGATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAAC AATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATC CAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTC GTCACCGTGGCTTTTATAATCTTCTGGGTTCGCTTTTCCGTCGTTAAGCGGGGGAGA AAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAG GAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACT GAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACC AACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAG CGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGG AGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATA GGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGAC TCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTA GGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGAC GTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGG CCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGC CTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGG CGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGG ATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCG AATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGT GCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGC CGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACAC AGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACC CTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACG CGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCAC ACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTC CAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGA TCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGT AGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGA CCTAGGTAA (SEQ ID NO:31)

S1-11C2-CD28T-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 43 048570

MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGATVKISCKASGYTFTNY GMNWVQQAPGQGLEWMGWMNTYTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRS EDTAVYFCARAGGQLRPGAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQTPL SLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLESGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCQQSNEEPPTFGQGTKLEIKAAALDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGAT GRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTV CEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGR CEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRE CTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSS QPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:32)

Design S1-11C2-C8K-CD28, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCA CGCCCGGAGATTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGAGCCAC CGTGAAGATCAGCTGCAAGGCCAGCGGCTACACATTCACCAACTACGGCATGAACT GGGTGCAACAGGCCCCCGGCCAGGGACTGGAGTGGATGGGCTGGATGAACACATAC ACCGGCGAGCCCACCTACGCCGACAAGTTCCAGGGCAGGGTGACATTCACACTCGA TACCAGCGCCAGAACCGTGTATATGGAACTGAGCAGCCTGAGGAGCGAGGACACCG CTGTGTACTTCTGCGCAAGGGCTGGAGGCCAGCTGAGACCTGGCGCTATGGACTACT GGGGCCAGGGCACCATGGTGACCGTGAGCTCCGGAGGCGGAGGATCTGGTGGCGGT GGTTCTGGCGGCGGAGGCTCCGACATCGTGCTGACCCAGACACCTCTCTCCCTGTCC GTGACCCCCGGACAGCCTGCCTCCATTTCCTGTAAGGCCTCCCAGAGCGTGGACTAT GACGGCGACAGCTTCATGAACTGGTACCTGCAGAAGCCCGGACAACCCCCCCAGCT GCTGATCTACGTGGCCAGCAACCTGGAGTCCGGCGTGCCTGACAGGTTTTCCGGCTC CGGCAGCGGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAAGCCGAGGACGTGG GCGTGTACTACTGCCAGCAGAGCAACGAGGAGCCCCCTACCTTCGGACAGGGCACC AAGCTGGAGATCAAGGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACA ACTACCCCCGCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTG TCTCTGAGACCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGG CCTGGACTTCGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGT ACTGCTGCTGTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACAGATCCAAAAG AAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCAC AAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCA GGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAA CTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCG

- 44 048570

CAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAG GGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGG CATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGTTTGTACCAGGGACTC AGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG GCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGT GGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCC CGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCT GTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCG AGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATT CAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAAT GTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCA GATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGG GTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGT ATGTGAGGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTT GCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGC TGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACC TCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAG AGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCT CACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCA TCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTA GGTAA (SEQ ID NO:33)

S1-11C2-C8K-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGATVKISCKASGYTFTNY GMNWVQQAPGQGLEWMGWMNTYTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRS EDTAVYFCARAGGQLRPGAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQTPL SLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLESGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCQQSNEEPPTFGQGTKLEIKAAAFVPVFLPAKPTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRG SLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECC KACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEA DDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAN HVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPE QDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:34)

S1-11C2-C8K-41BB DNA construct (signal sequence in bold)

- 45 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGGAGATTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGA GCCACCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACATTCACCAACTACGGCAT GAACTGGGTGCAACAGGCCCCCGGCCAGGGACTGGAGTGGATGGGCTGGATGAACA CATACACCGGCGAGCCCACCTACGCCGACAAGTTCCAGGGCAGGGTGACATTCACA CTCGATACCAGCGCCAGAACCGTGTATATGGAACTGAGCAGCCTGAGGAGCGAGGA CACCGCTGTGTACTTCTGCGCAAGGGCTGGAGGCCAGCTGAGACCTGGCGCTATGG ACTACTGGGGCCAGGGCACCATGGTGACCGTGAGCTCCGGAGGCGGAGGATCTGGT GGCGGTGGTTCTGGCGGCGGAGGCTCCGACATCGTGCTGACCCAGACACCTCTCTCC CTGTCCGTGACCCCCGGACAGCCTGCCTCCATTTCCTGTAAGGCCTCCCAGAGCGTG GACTATGACGGCGACAGCTTCATGAACTGGTACCTGCAGAAGCCCGGACAACCCCC CCAGCTGCTGATCTACGTGGCCAGCAACCTGGAGTCCGGCGTGCCTGACAGGTTTTC CGGCTCCGGCAGCGGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAAGCCGAGG ACGTGGGCGTGTACTACTGCCAGCAGAGCAACGAGGAGCCCCCTACCTTCGGACAG GGCACCAAGCTGGAGATCAAGGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAA CCCACAACTACCCCCGCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAA CCCCTGTCTCTGAGACCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACT AGAGGCCTGGACTTCGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGC GGGGTACTGCTGCTGTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACCGCTTT TCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATG AGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGA GGAGGAGGGCGGGTGCGAACTGAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAG CGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAA GAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATG GCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCC ACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGG ATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCG CCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGT CTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAAT GTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAG ACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACC GAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGT GTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAAC TACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAG TTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCG ACGAGGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAA CGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGG GAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTA CCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGC GTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGA CAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGC CTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:35)

S1-11C2-C8K-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 46 048570

MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGATVKISCKASGYTFTNY GMNWVQQAPGQGLEWMGWMNTYTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRS EDTAVYFCARAGGQLRPGAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQTPL SLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLESGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCQQSNEEPPTFGQGTKLEIKAAAFVPVFLPAKPTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSG EGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHS GECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAP CVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYS DEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEP EAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:36)

S2-1A1-CD28T-CD28-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCCGGAG CCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTGGATGC ACTGGGTCAGACAGGCTCCCGGACAGGGCCTGGAATGGATGGGCGAAATCAACCCC TCCTCCGGCAGGACCAACTACAACGAGAAGTTCAAGACCAGGTGACCATGACCAG GGACACCAGCACCAGCACCGTGTACATGGAGCTGTCCAGCCTGAGGAGCGAGGACA CCGCCGTGTACTACTGTGCCAAGCTGGGACCCGGCCCCCAGTACTATGCCATGGACT ACTGGGGCCAAGGCACCATGGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGTGGC GGTGGTTCTGGCGGCGGAGGCTCCGACATCCAGATGACCCAGAGCCCCTCCAGCCT GTCCGCTAGCGTGGGCGACAGGGTCACCATTACCTGCCACGCCAGCCAGAACATCA ACGTGTGGCTGAGCTGGTATCAGCAGAAACCCGGCAAGGCTCCCAAGCTGCTGATC TACAAGGCCAGCAAGCTGCACACCGGCGTGCCCAGCAGGTTTAGCGGTTCTGGCTC CGGCACCGACTTCACCCTCACCATCAGCAGCCTGCAGCCCGAAGACTTCGCTACCTA CTACTGCCAGCAGGGACAAAGCTACCCCTGGACCTTCGGCCAGGGAACCAAGCTGG AAATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTG AAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGG GTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTT

- 47 048570

TTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGA ATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCA CCTAGAGATTTCGCTGCCTATCGGAGCCGCTTTTCCGTCGTTAAGCGGGGGAGAAAA AAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAA GAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAG GGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAAC TGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGC AGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGG GTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGC ATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGTTTGTACCAGGGACTCA GCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGG CCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTG GAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCC GCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTG TCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGA GGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTC AGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATG TGTGGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAG ATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGG TGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTAT GTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGC TTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTG GGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTC CTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAG CAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCA CAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATC TTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGG TAA (SEQ ID NO:37)

S2-1A1-CD28T-CD28-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTT YWMHWVRQAPGQGLEWMGEINPSSGRTNYNEKFKTRVTMTRDTSTSTVYMELSSLRS EDTAVYYCAKLGPGPQYYAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSP SSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASKLHTGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGQSYPWTFGQGTKLEIKAAALDNEKSNGTIIHVKGKH LCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSL

GGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQD KQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRS TPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILA AVVVGLVAYIAFKR (SEQ ID NO:38)

S2-1A1-CD28T-CD28 construct, DNA (signal sequence in bold)

- 48 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCCGGAG CCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTGGATGC ACTGGGTCAGACAGGCTCCCGGACAGGGCCTGGAATGGATGGGCGAAATCAACCCC TCCTCCGGCAGGACCAACTACAACGAGAAGTTCAAGACCAGGGTGACCATGACCAG GGACACCAGCACCAGCACCGTGTACATGGAGCTGTCCAGCCTGAGGAGCGAGGACA CCGCCGTGTACTACTGTGCCAAGCTGGGACCCGGCCCCCAGTACTATGCCATGGACT ACTGGGGCCAAGGCACCATGGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGTGGC GGTGGTTCTGGCGGCGGAGGCTCCGACATCCAGATGACCCAGAGCCCCTCCAGCCT GTCCGCTAGCGTGGGCGACAGGGTCACCATTACCTGCCACGCCAGCCAGAACATCA ACGTGTGGCTGAGCTGGTATCAGCAGAAACCCGGCAAGGCTCCCAAGCTGCTGATC TACAAGGCCAGCAAGCTGCACACCGGCGTGCCCAGCAGGTTTAGCGGTTCTGGCTC CGGCACCGACTTCACCCTCACCATCAGCAGCCTGCAGCCCGAAGACTTCGCTACCTA CTACTGCCAGCAGGGACAAAGCTACCCCTGGACCTTCGGCCAGGGAACCAAGCTGG AAATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTG AAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGG GTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTT TTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGA ATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCA CCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCA CCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAG GGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCA AACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAA GATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAA GGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGC TCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCC GGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCC GGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGC GTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGT GAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAAC CAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCC ACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCC TGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGA

AACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTT CAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACA GCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACC GAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCC TGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGA GTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCT GGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAAC CGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGT AGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:39)

S2-1A1-CD28T-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

- 49 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTT YWMHWVRQAPGQGLEWMGEINPSSGRTNYNEKFKTRVTMTRDTSTSTVYMELSSLRS EDTAVYYCAKLGPGPQYYAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSP SSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASKLHTGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGQSYPWTFGQGTKLEIKAAALDNEKSNGTIIHVKGKH LCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDG PRLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDS VTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVC EAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADA ECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGT TDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:40)

S2-1A1-CD28T-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCCGGAG CCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTGGATGC ACTGGGTCAGACAGGCTCCCGGACAGGGCCTGGAATGGATGGGCGAAATCAACCCC TCCTCCGGCAGGACCAACTACAACGAGAAGTTCAAGACCAGGTGACCATGACCAG GGACACCAGCACCAGCACCGTGTACATGGAGCTGTCCAGCCTGAGGAGCGAGGACA CCGCCGTGTACTACTGTGCCAAGCTGGGACCCGGCCCCCAGTACTATGCCATGGACT ACTGGGGCCAAGGCACCATGGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGTGGC GGTGGTTCTGGCGGCGGAGGCTCCGACATCCAGATGACCCAGAGCCCCTCCAGCCT GTCCGCTAGCGTGGGCGACAGGGTCACCATTACCTGCCACGCCAGCCAGAACATCA ACGTGTGGCTGAGCTGGTATCAGCAGAAACCCGGCAAGGCTCCCAAGCTGCTGATC TACAAGGCCAGCAAGCTGCACACCGGCGTGCCCAGCAGGTTTAGCGGTTCTGGCTC CGGCACCGACTTCACCCTCACCATCAGCAGCCTGCAGCCCGAAGACTTCGCTACCTA

CTACTGCCAGCAGGGACAAAGCTACCCCTGGACCTTCGGCCAGGGAACCAAGCTGG AAATCAAGGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTG AAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGG GTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTT TTATAATCTTCTGGGTTCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGT ACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAGGACGGC TGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGGTGAAGTT TTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACG AGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGG GACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAA TGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAG AGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTAC GAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAA GTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAAT CCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCT TCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGG CCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGC ACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATT

- 50 048570

CAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCT TCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTA TGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAG CCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAG TGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTG CACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACG CTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGA TCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCT GATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGT CGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGC CGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:41)

S2-1A1-CD28T-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTT YWMHWVRQAPGQGLEWMGEINPSSGRTNYNEKFKTRVTMTRDTSTSTVYMELSSLRS EDTAVYYCAKLGPGPQYYAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSP SSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASKLHTGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGQSYPWTFGQGTKLEIKAAALDNEKSNGTIIHVKGKH LCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATG RAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCE PCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCE ACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECT RWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQP VVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:42)

S2-1A1-C8K-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCCGGAG CCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTGGATGC ACTGGGTCAGACAGGCTCCCGGACAGGGCCTGGAATGGATGGGCGAAATCAACCCC TCCTCCGGCAGGACCAACTACAACGAGAAGTTCAAGACCAGGTGACCATGACCAG GGACACCAGCACCAGCACCGTGTACATGGAGCTGTCCAGCCTGAGGAGCGAGGACA CCGCCGTGTACTACTGTGCCAAGCTGGGACCCGGCCCCCAGTACTATGCCATGGACT ACTGGGGCCAAGGCACCATGGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGTGGC GGTGGTTCTGGCGGCGGAGGCTCCGACATCCAGATGACCCAGAGCCCCTCCAGCCT GTCCGCTAGCGTGGGCGACAGGGTCACCATTACCTGCCACGCCAGCCAGAACATCA ACGTGTGGCTGAGCTGGTATCAGCAGAAACCCGGCAAGGCTCCCAAGCTGCTGATC TACAAGGCCAGCAAGCTGCACACCGGCGTGCCCAGCAGGTTTAGCGGTTCTGGCTC CGGCACCGACTTCACCCTCACCATCAGCAGCCTGCAGCCCGAAGACTTCGCTACCTA CTACTGCCAGCAGGGACAAAGCTACCCCTGGACCTTCGGCCAGGGAACCAAGCTGG AAATCAAGGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCC CCGCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGA GACCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGGCCTGGAC TTCGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTG CTGTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACAGATCCAAAAGAAGCCGC

- 51 048570

CTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAA CACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAG TTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAAC GAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACG GGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATA ATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGA GAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTA CGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGA AGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAA TCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGC TTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGG GCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCG CACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACAT TCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCC TTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCT ATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAA GCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGA GTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGT GCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGAC GCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGG ATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACC TGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAG TCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAG CCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:43)

S2-1A1-C8K-CD28 construct, amino acids (signal sequence in bold; CDRs underlined) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTT

YWMHWVRQAPGQGLEWMGEINPSSGRTNYNEKFKTRVTMTRDTSTVYMELSSLRS EDTAVYYCAKLGPGPQYYAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSP SSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASKLHTGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGQSYPWTFGQGTKLEIKAAAFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLL TCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKAC NLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDA VCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVD PCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDL IASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:44)

S2-1A1-C8K-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCCGGAG CCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTGGATGC ACTGGGTCAGACAGGCTCCCGGACAGGGCCTGGAATGGATGGGCGAAATCAACCCC TCCTCCGGCAGGACCAACTACAACGAGAAGTTCAAGACCAGGTGACCATGACCAG GGACACCAGCACCAGCACCGTGTACATGGAGCTGTCCAGCCTGAGGAGCGAGGACA CCGCCGTGTACTACTGTGCCAAGCTGGGACCCGGCCCCCAGTACTATGCCATGGACT ACTGGGGCCAAGGCACCATGGTGACCGTGAGCAGCGGAGGCGGAGGATCTGGTGGC GGTGGTTCTGGCGGCGGAGGCTCCGACATCCAGATGACCCAGAGCCCCTCCAGCCT

- 52 048570

GTCCGCTAGCGTGGGCGACAGGGTCACCATTACCTGCCACGCCAGCCAGAACATCA ACGTGTGGCTGAGCTGGTATCAGCAGAAACCCGGCAAGGCTCCCAAGCTGCTGATC TACAAGGCCAGCAAGCTGCACACCGGCGTGCCCAGCAGGTTTAGCGGTTCTGGCTC CGGCACCGACTTCACCCTCACCATCAGCAGCCTGCAGCCCGAAGACTTCGCTACCTA CTACTGCCAGCAGGGACAAAGCTACCCCTGGACCTTCGGCCAGGGAACCAAGCTGG AAATCAAGGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCC CCGCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGA GACCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGGCCTGGAC TTCGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTG CTGTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACCGCTTTTCCGTCGTTAAGC GGGGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAA ACGACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGG GTGCGAACTGAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGG GCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTT TTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAA ACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTAT TCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGT ACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCC CTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCGGGGATCTCTCCTTAC ATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGG GCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGC GCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCC TGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAA CCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAG CCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCTGTGTCGAAGCCGAT GATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTG TGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAA GCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACACAGCGACGAGGCGAAC CACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGA GAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCAC CCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGG AGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACA GTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCT GTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTT AAGAGATGACCTAGGTAA (SEQ ID NO:45)

S2-1A1-C8K-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTT YWMHWVRQAPGQGLEWMGEINPSSGRTNYNEKFKTRVTMTRDTSTSTVYMELSSLRS EDTAVYYCAKLGPGPQYYAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSP SSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASKLHTGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGQSYPWTFGQGTKLEIKAAAFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEG RGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGE CCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCV EADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDE ANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEA PPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:46)

S2-7A4-CD28T-CD28-41BB DNA construct (signal sequence in bold)

- 53 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTTGCTTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGTCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAG GGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGT TGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTAT AATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATAT GACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTA GAGATTTCGCTGCCTATCGGAGCCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAG CTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAG GACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGGT GAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGT ATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGA GGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTC TCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATG AAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGTTTGTACCAGGGACTCAGCA CTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCA AGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAA GAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCG ACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCC AACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGG CGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGT GACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGT GGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATG TGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGT GTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGT GAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTT GCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGG CAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCT GAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCA GGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACA ACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTT GGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTA A (SEQ ID NO:47)

S2-7A4-CD28T-CD28-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 54 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSFAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRSFGQGTKVELKRAAALDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLG GAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCK PCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDK QNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRST PPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAA VVVGLVAYIAFKR (SEQ ID NO:48)

S2-7A4-CD28T-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTTGCTTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGTCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAG GGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGT TGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTAT AATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATAT GACTCCACGCCGCCCTGGCCCAAAGGAAACACTACCAGCCTTAGCACCACCTA GAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAG CGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAA GAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATG GCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCC ACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGG ATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCG CCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGT CTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAAT GTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAG ACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACC GAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGT

- 55 048570

GTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAAC TACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAG TTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCG ACGAGGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAA CGCCAGCTGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGG GAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTA CCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGC GTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGA CAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGC CTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:49)

S2-7A4-CD28T-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSFAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRSFGQGTKVELKRAAALDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPR LLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTF SDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEA GSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTD NLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:50)

S2-7A4-CD28T-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTTGCTTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGTCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAG GGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGT TGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTAT AATCTTCTGGGTTCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTACAT TTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTGCT CCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGGTGAAGTTTTCC AGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCT CAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGAC

- 56 048570

CCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGA GCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAA GGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTG GCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCG GGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCT CCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCT CTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACA ACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAG CGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCA GTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGG ATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCG GCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTC CAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACC GTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGA GTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAG ACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATC GCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTG ACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTG GTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:51)

S2-7A4-CD28T-41BB construct, amino acids (signal sequence in bold; CDRs underlined) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS

YDINWVRQATGQGLEWMGWMNPNNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSFAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRSFGQGTKVELKRAAALDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGR AMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEP CLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTR WADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPV VTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:52)

S2-7A4-C8K-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

- 57 048570

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTTGCTTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGTCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCCCCG CCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGAGAC CTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGGCCTGGACTTC GCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTGCTG TCTCTGGTGATTACCCTCTACTGCAACCACAGAAACAGATCCAAAAGAAGCCGCCTG CTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACA CTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTT TTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACG AGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGG GACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAA TGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAG AGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTAC GAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAA GTGGCAGCGGGGAGGGCCGGGGATTCTCTCCTTACATGTGGGGACGTGGAAGAAAAT CCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCT TCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGG CCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGC ACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATT CAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCT TCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTA TGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAG CCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAG TGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTG CACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACG CTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGA TCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCT GATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGT CGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGC CGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:53)

S2-7A4-C8K-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

- 58 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSFAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRSFGQGTKVELKRAAAFVPVFLPAKPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLT CGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKAC NLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDA VCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVD PCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDL IASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:54)

S2-7A4-C8K-41BB construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTTGCTTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGTCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCCCCG CCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGAGAC CTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGGCCTGGACTTC GCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTGCTG

- 59 048570

TCTCTGGTGATTACCCTCTACTGCAACCACAGAAACCGCTTTTCCGTCGTTAAGCGG GGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAAC GACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGT GCGAACTGAGGGTGAAGTTTTCCAGATCTGCCAGATGCACCAGCGTATCAGCAGGGC CAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTT GGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAAC CCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTC TGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTAC CAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTG CCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATG TGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCT ATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCT AAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGT AACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACC ATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCC TTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGA TGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTG AGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGC AAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCA CGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAG AGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACC CGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGA GGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGT CATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGT CTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAA GAGATGACCTAGGTAA (SEQ ID NO:55)

S2-7A4-C8K-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSFAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRSFGQGTKVELKRAAAFVPVFLPAKPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGR GSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGEC CKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVE ADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEA NHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAP PEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:56)

S2-7A5-CD28T-CD28-41BB DNA construct (signal sequence in bold)

- 60 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTAGCTTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGGCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAG GGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGT TGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTAT AATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATAT GACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTA GAGATTTCGCTGCCTATCGGAGCCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAG CTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAG GACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGGT GAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGT ATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGA GGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTC TCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATG AAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGTTTGTACCAGGGACTCAGCA CTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCA AGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAA GAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCG ACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCC AACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGG CGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGT GACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGT GGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATG TGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGT GTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGT GAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTT GCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGG CAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCT GAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCA GGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACA ACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTT GGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTA A (SEQ ID NO:57)

S2-7A5-CD28T-CD28-41BB construct. amino acids (signal sequence in bold; CDRs underlined)

- 61 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSLAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRAFGQGTKVELKRAAALDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLG GAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCK PCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDK QNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRST PPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAA VVVGLVAYIAFKR (SEQ ID NO:58)

S2-7A5-CD28T-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTAGCTTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT

- 62 048570

CAGACATCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGGCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAG GGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGT TGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTAT AATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATAT GACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTA GAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAG CGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAA GAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATG GCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCC ACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGGCCGGGG ATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCG CCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGT CTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAAT GTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAG ACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACC GAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGT GTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAAC TACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAG TTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCG ACGAGGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAA CGCCAGCTGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGG GAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTA CCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGC GTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGA CAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGC CTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:59)

S2-7A5-CD28T-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSLAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRAFGQGTKVELKRAAALDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPR LLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTF SDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEA GSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTD NLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:60)

S2-7A5-CD28T-41BB construct, DNA (signal sequence in bold)

- 63 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTAGCTTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGGCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAG GGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGT TGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTAT AATCTTCTGGGTTCGCTTTTCCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTACAT TTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTGCT CCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGAGGGTGAAGTTTTCC AGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCT CAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGAC CCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGA GCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAA GGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTG GCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCG GGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCT CCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCT CTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACA ACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAG

CGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCA GTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGG ATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCG GCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTC CAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACC GTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGA GTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAG ACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATC GCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTG ACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTG GTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:61)

S2-7A5-CD28T-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 64 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSLAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRAFGQGTKVELKRAAALDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGR AMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEP CLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTR WADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPV VTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:62)

S2-7A5-C8K-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC

CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC

- 65 048570

AGCTCCTTAGCTTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGGCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCCCCG CCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGAGAC CTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGAGGCCTGGACTTC GCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTGCTG TCTCTGGTGATTACCCTCTACTGCAACCACAGAAACAGATCCAAAAGAAGCCGCCTG CTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACA CTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTT TTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACG AGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGG GACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAA TGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAG AGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTAC GAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAA GTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAAT CCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCT TCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGG CCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGC ACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATT CAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCT TCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTA TGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAG CCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAG TGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTG CACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACG CTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGA TCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCT GATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGT CGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGC CGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:63)

S2-7A5-C8K-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSLAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRAFGQGTKVELKRAAAFVPVFLPAKPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLT CGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKAC NLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDA VCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVD PCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDL IASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:64)

S2-7A5-C8K-41BB DNA construct (signal sequence in bold)

- 66 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACAGCTTCACCAGTTATGATATCA ACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGGTGGATGAACCCG AACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAG GGACACCTCCATAAGCACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGGGAGAGCCGGTTACTACTACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGT GGCTCTGGCGGTGGCGGAAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCGGTCAGAGTGTTACCAGC AGCTCCTTAGCTTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT CAGACATCCACCAGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGATTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTGGCTCACGGGCGTTCGGCCAAGGGACCAAGGTGGAACTCA AACGAGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACCCACAACTACCCCCG CCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACCCCTGTCTCTGAGAC CTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTAGGCCTGGACTTC GCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCGGGGTACTGCTGCTG TCTCTGGTGATTACCCTCTACTGCAACCACAGAAACCGCTTTTCCGTCGTTAAGCGG GGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAAC GACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGT GCGAACTGAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGC CAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTT GGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAAC CCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTC TGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTAC CAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTG CCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATG TGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCT ATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCT AAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGT AACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACC ATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCC TTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGA TGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTG AGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGC AAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCA CGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAG AGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACC CGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGA GGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGT CATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCCGACAATCTGATTCCTGT CTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAA GAGATGACCTAGGTAA (SEQ ID NO:65)

S2-7A5-C8K-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 67 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTS YDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRS EDTAVYYCGRAGYYYYFGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGERATLSCRAGQSVTSSSLAWYQQKPGQAPRLLIYQTSTRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGGSRAFGQGTKVELKRAAAFVPVFLPAKPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGR GSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGEC CKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVE ADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEA NHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAP PEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:66)

S2-14C1-CD28T-CD28-41BB DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCT

- 68 048570

AATAGTGGTGGCACAAACTCTGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAG GGACACGTCCATCAGTACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACA CGGCCGTTTATTACTGTGCGAGAGGATGGCTACAGACGTACTACTTTGACAACTGGG GCCAGGGAACCCTGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGC TCTGGCGGTGGCGGAAGTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTG TCTCTGGGCGAGAGGGCCACCATCTACTGCAAGTCCAGCCAGACTGTTTTGACCAGC TCCAACAATAAGAACTTCTTAGCTTGGTACCAACAGAAACTAGGACAGCCTCCTAA GCTGCTCATTTCCTGGGCCTCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGG CAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGT GGCAATTTATTACTGTCAGCACTATTATACTAGTCCACTCACTTTCGGCGGCGGGAC CAAGGTGGAGATCAAACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAA TCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCA AGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGT CACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAG CGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGC CTTACGCACCACCTAGATTTCGCTGCCTATCGGAGCCGCTTTTCCGTCGTTAAGC GGGGGAGAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAA ACGACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGG GTGCGAACTGAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGG GCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTT TTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAA ACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTAT TCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGT ACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCC CTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGCCGGGGATCTCTCCTTAC ATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGG GCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGC GCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCC TGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAA CCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAG CCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGAT GATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTG TGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAA GCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAAC CACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGA GAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCAC CCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGG AGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACA GTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCT GTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTT

AAGAGATGACCTAGGTAA (SEQ ID NO:67)

S2-14C1-CD28T-CD28-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 69 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPNSGGTNSAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARGWLQTYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDS LAVSLGERATIYCKSSQTVLTSSNNKNFLAWYQQKLGQPPKLLISWASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAIYYCQHYYTSPLTFGGGTKVEIKRAAALDNEKSNGTIIHV KGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLL GVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSA TEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRW ITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYC SILAAVVVGLVAYIAFKR (SEQ ID NO:68)

S2-14C1-CD28T-CD28 construct, DNA (signal sequence in bold) ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCA

CGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC AGTGAAGGTGTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTG GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAATA GTGGTGGCACAAACTCTGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGAC ACGTCCATCAGTACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGC CGTTTATTACTGTGCGAGAGGATGGCTACAGACGTACTACTTTGACAACTGGGGCCA GGGAACCCTGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGCTCTG GCGGTGGCGGAAGTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTC TGGGCGAGAGGGCCACCATCTACTGCAAGTCCAGCCAGACTGTTTTGACCAGCTCCA ACAATAAGAACTTCTTAGCTTGGTACCAACAGAAACTAGGACAGCCTCCTAAGCTG CTCATTTCCTGGGCCTCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGC GGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGC AATTTATTACTGTCAGCACTATTATACTAGTCCACTCACTTTCGGCGGCGGGACCAA GGTGGAGATCAAACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAATCA TTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGC CATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCAC CGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAGAAAGCCGCCTGCTCCATAGCGA TTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTA

- 70 048570

CGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGC AGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGG GACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGAT GGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGA AGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAG GGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTT ATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGG GAGGGCCGGGGATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTAT GGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTT GTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCA CTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGG TGCTAACCAGACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGT CTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTC CGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCA GGACGAAACTACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCC TCGTGTTCAGTTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGA ACCTACAGCGACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGA GGATACCGAACGCCAGCTGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGG AGATCCCTGGGAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACA GCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTAC AGTTGCTGGCGTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGG GCAAACCGACAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGG GCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:69)

S2-14C1-CD28T-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPNSGGTNSAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARGWLQTYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDS LAVSLGERATIYCKSSQTVLTSSNNKNFLAWYQQKLGQPPKLLISWASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAIYYCQHYYTSPLTFGGGTKVEIKRAAALDNEKSNGTIIHV KGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAGATGR AMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEP CLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTR WADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPV VTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:70)

S2-14C1-CD28T-41BB construct, DNA (signal sequence in bold)

- 71 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCT AATAGTGGTGGCACAAACTCTGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAG GGACACGTCCATCAGTACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACA CGGCCGTTTATTACTGTGCGAGAGGATGGCTACAGACGTACTACTTTGACAACTGGG GCCAGGGAACCCTGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGC TCTGGCGGTGGCGGAAGTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTG TCTCTGGGCGAGAGGGCCACCATCTACTGCAAGTCCAGCCAGACTGTTTTGACCAGC TCCAACAATAAGAACTTCTTAGCTTGGTACCAACAGAAACTAGGACAGCCTCCTAA GCTGCTCATTTCCTGGGCCTCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGG CAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGT GGCAATTTATTACTGTCAGCACTATTATACTAGTCCACTCACTTTCGGCGGCGGGAC CAAGGTGGAGATCAAACGAGCCGCTGCCCTTGATAATGAAAAGTCAAACGGAACAA TCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCA AGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGT CACCGTGGCTTTTATAATCTTCTGGGTTCGCTTTTCCGTCGTTAAGCGGGGGAGAAA AAAGCTGCTGTACATTTTCAAACAGCCGTTTATGAGGCCGGTCCAAACGACTCAGGA AGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGAGGAGGAGGGCGGGTGCGAACTGA GGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAA CTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCG CAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAG GGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGG CATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTC AGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG GCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGACGT GGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATGGCC CGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGGCCT GTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTGGCG AGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTGGATT CAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACCGAAT GTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTGTGCA GATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTGCCGG GTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACACAGT ATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGACCCTT GCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTACGCGC TGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCACACC TCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCTCCAG

AGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGGATCCT CACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTGTAGCA TCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATGACCTA GGTAA (SEQIDNO:71)

S2-14C1-CD28T-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 72 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPNSGGTNSAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARGWLQTYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDS LAVSLGERATIYCKSSQTVLTSSNNKNFLAWYQQKLGQPPKLLISWASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAIYYCQHYYTSPLTFGGGTKVEIKRAAALDNEKSNGTHHV KGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRGSLLTCGDVEENPGPMGAG ATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT VCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTG RCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLR ECTRWADAECEEIPGRWrrRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGS SQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:72)

S2-14C1-C8K-CD28 construct, DNA (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCT AATAGTGGTGGCACAAACTCTGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAG GGACACGTCCATCAGTACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACA CGGCCGTTTATTACTGTGCGAGAGGATGGCTACAGACGTACTACTTTGACAACTGGG GCCAGGGAACCCTGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGC TCTGGCGGTGGCGGAAGTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTG TCTCTGGGCGAGAGGGCCACCATCTACTGCAAGTCCAGCCAGACTGTTTTGACCAGC TCCAACAATAAGAACTTCTTAGCTTGGTACCAACAGAAACTAGGACAGCCTCCTAA GCTGCTCATTTCCTGGGCCTCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGG CAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGT GGCAATTTATTACTGTCAGCACTATTATACTAGTCCACTCACTTTCGGCGGCGGGAC CAAGGTGGAGATCAAACGAGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACC CACAACTACCCCCGCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACC CCTGTCTCTGAGACCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTA GAGGCCTGGACTTCGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCG

- 73 048570

GGGTACTGCTGCTGTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACAGATCCA AAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCC CCACAAGGAAACACTACCAGCCTTACGCACCACCTAGATTTCGCTGCCTATCGGA GCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAAC CAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAA GCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAG GAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAAT AGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGA CTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCT AGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGGATCTCTCCTTACATGTGGGGA CGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCGCCACGGGAAGGGCTATGGATG GCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGTCTCTCGGAGGCGCTAAGGAGG CCTGTCCAACGGGCCTCTACACTCACTCCGGTGAATGTTGCAAAGCCTGTAACCTTG GCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAGACAGTCTGTGAACCATGCCTG GATTCAGTGACATTCAGCGATGTTGTCTCAGCCACCGAGCCTTGCAAGCCTTGTACC GAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGTGTCGAAGCCGATGATGCAGTG TGCAGATGTGCCTATGGATATTACCAGGACGAAACTACCGGGCGGTGTGAGGCCTG CCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAGTTGCCAGGATAAGCAAAACA CAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCGACGAGGCGAACCACGTCGAC CCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAACGCCAGCTGAGAGAGTGTAC GCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGGGAGATGGATCACCCGGAGCA CACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTACCCAAGAACCGGAGGCCCCT CCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGCGTGGTGACGACAGTCATGGG ATCCTCACAACCAGTCGTGACGCGGGGCACAACCGACAATCTGATTCCTGTCTACTG TAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGCCTACATCGCCTTTAAGAGATG ACCTAGGTAA (SEQ ID NO:73)

S2-14C1-C8K-CD28 construct, amino acids (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPNSGGTNSAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARGWLQTYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDS LAVSLGERATIYCKSSQTVLTSSNNKNFLAWYQQKLGQPPKLLISWASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAIYYCQHYYTSPLTFGGGTKVEIKRAAAFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV ITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGSGEGRG SLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECC KACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEA DDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAN

HVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPE QDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:74)

S2-14C1-C8K-41BB DNA construct (signal sequence in bold)

- 74 048570

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG CCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCT AATAGTGGTGGCACAAACTCTGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAG GGACACGTCCATCAGTACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACA CGGCCGTTTATTACTGTGCGAGAGGATGGCTACAGACGTACTACTTTGACAACTGGG GCCAGGGAACCCTGGTCACCGTATCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGC TCTGGCGGTGGCGGAAGTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTG TCTCTGGGCGAGAGGGCCACCATCTACTGCAAGTCCAGCCAGACTGTTTTGACCAGC TCCAACAATAAGAACTTCTTAGCTTGGTACCAACAGAAACTAGGACAGCCTCCTAA GCTGCTCATTTCCTGGGCCTCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGG CAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGT GGCAATTTATTACTGTCAGCACTATTATACTAGTCCACTCACTTTCGGCGGCGGGAC CAAGGTGGAGATCAAACGAGCCGCTGCCTTCGTGCCTGTTTTTCTGCCCGCGAAACC CACAACTACCCCCGCCCCTCGGCCCCCAACTCCTGCACCAACTATCGCTTCCCAACC CCTGTCTCTGAGACCTGAGGCATGCCGCCCCGCGGCAGGCGGCGCCGTGCACACTA GAGGCCTGGACTTCGCCTGCGATATTTATATCTGGGCCCCCCTTGCCGGGACATGCG GGGTACTGCTGCTGTCTCTGGTGATTACCCTCTACTGCAACCACAGAAACCGCTTTT CCGTCGTTAAGCGGGGGAGAAAAAAGCTGCTGTACATTTTCAAACAGCCGTTTATG AGGCCGGTCCAAACGACTCAGGAAGAGGACGGCTGCTCCTGCCGCTTTCCTGAGGA GGAGGAGGGCGGGTGCGAACTGAGGGTGAAGTTTTCCAGATCTGCCAGATGCACCAG CGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAA GAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATG GCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCC ACATGCAAGCCCTGCCACCTAGGGCCAAGAGAAGTGGCAGCGGGGAGGGCCGGGG ATCTCTCCTTACATGTGGGGACGTGGAAGAAAATCCGGGGCCTATGGGTGCCGGCG CCACGGGAAGGGCTATGGATGGCCCGCGACTGCTTCTCCTGCTGTTGTTGGGCGTGT CTCTCGGAGGCGCTAAGGAGGCCTGTCCAACGGGCCTCTACACTCACTCCGGTGAAT GTTGCAAAGCCTGTAACCTTGGCGAGGGCGTCGCACAACCTTGTGGTGCTAACCAG ACAGTCTGTGAACCATGCCTGGATTCAGTGACATTCAGCGATGTTGTCTCAGCCACC GAGCCTTGCAAGCCTTGTACCGAATGTGTGGGCCTTCAGTCCATGTCCGCCCCCTGT GTCGAAGCCGATGATGCAGTGTGCAGATGTGCCTATGGATATTACCAGGACGAAAC TACCGGGCGGTGTGAGGCCTGCCGGGTGTGTGAAGCCGGCTCTGGCCTCGTGTTCAG TTGCCAGGATAAGCAAAACACAGTATGTGAGGAGTGTCCAGACGGAACCTACAGCG ACGAGGCGAACCACGTCGACCCTTGCTTGCCGTGCACCGTCTGCGAGGATACCGAA CGCCAGCTGAGAGAGTGTACGCGCTGGGCAGACGCTGAGTGCGAGGAGATCCCTGG GAGATGGATCACCCGGAGCACACCTCCTGAGGGATCAGACAGTACAGCCCCGAGTA CCCAAGAACCGGAGGCCCCTCCAGAGCAGGACCTGATCGCTTCTACAGTTGCTGGC GTGGTGACGACAGTCATGGGATCCTCACAACCAGTCGTGACGCGGGGCACAACCGA CAATCTGATTCCTGTCTACTGTAGCATCTTGGCAGCCGTGGTCGTGGGCCTGGTAGC CTACATCGCCTTTAAGAGATGACCTAGGTAA (SEQ ID NO:75)

S2-14C1-C8K-41BB construct, amino acids (signal sequence in bold; CDRs underlined)

- 75 048570

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPNSGGTNSAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARGWLQTYYFDNWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDS LAVSLGERATIYCKSSQTVLTSSNNKNFLAWYQQKLGQPPKLLISWASTRESGVPDRFS GSGSGTDFTLTISSLQAEDVAIYYCQHYYTSPLTFGGGTKVEIKRAAAFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV ITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAKRSGS GEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTH SGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAP CVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYS DEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEP EAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO:76)

Human STEAP1 NM_012449 (NP_036581). Amino acids

MESRKDrrNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCP SELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM VSrrLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYS LSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTS IPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPI VVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL (SEQ ID NO:77)

CAR signal peptide, DNA

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGC CGCACGCCCG (SEQ ID NO:78)

CAR signal peptide:

MALPVTALLLPLALLLHAARP (SEQ ID NO:79) scFv-linker G4S, DNA

GGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCC (SEQ ID NO:80) scFv linker G4S:

GGGGSGGGGSGGGGS (SEQ ID NO:81) scFv-linker Whitlow, DNA

GGGTCTACATCCGGCTCCGGGAAGCCCGGAAGTGGCGAAGGTAGTACAAAG

GGG (SEQ ID NO:82) scFv-linker Whitlow:

GSTSGSGKPGSGEGSTKG (SEQ ID NO:83)

4-1BB, nucleic acid sequence (intracellular domain)

AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGC

CCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAG

GAAGGTGGGTGCGAGCTG (SEQ ID NO:84)

4-1BB, amino acids (intracellular domain)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:85)

OX40, amino acids

RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:86)

Inclusion via link.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. However, citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussions provided herein, the terms and definitions according to this specification shall control.

Equivalents.

The foregoing description is believed to be sufficient to enable one skilled in the art to practice the present invention. The foregoing description and examples have described in detail certain preferred embodiments of the present invention and have described the best mode contemplated by the inventors of the present invention. However, it should be understood that no matter how detailed the foregoing may appear, the present invention may be practiced in many ways and the present invention should be construed in accordance with the appended claims and any equivalents thereof.

The following examples, including experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the present invention.

Example 1.

PNT-2, LNCaP, PC-3, 22Rvl, C4-2B, and DU145 cell lines were cultured in RPMI1640 (Lonza) + 10% FBS (Corning) + 1X penicillin-streptomycin-L-glutamine (Corning) (R10) and maintained at cell densities of 0.5 to 2.0 x 10 cells/ml. PNT-2 and DU145 serve as negative controls. To examine STEAP1 cell surface expression, cells were incubated with anti-STEAP1 antibody (2F3) or IgG1 isotype control antibody (BD Pharmingen) in staining buffer (BD Pharmingen) for 30 min at 4°C. Cells were then washed and resuspended in propidium iodide staining buffer (BD Pharmingen) prior to data collection. STEAP1 expression on the target cell is shown in Fig. 1.

Example 2.

Plasmids encoding the T7 promoter, CAR construct, and beta-globin stabilizing sequence were linearized by digesting 10 μg of DNA overnight with EcoRI and BamHI (NEB). DNA was then digested for 2 h at 50°C with phenol/chloroform purified proteinase K (Thermo Fisher, 600 U/ml) and precipitated by adding sodium acetate and two volumes of ethanol. The beads were then dried, resuspended in RNase/DNase-free water, and counted using NanoDrop. One μg of linear DNA was then used for in vitro transcription using mMESSAGE mMACHINE T7 Ultra (Thermo Fisher) according to the manufacturer's instructions. RNA was further purified using the MEGAClear kit (Thermo Fisher) according to the manufacturer's instructions and quantified using NanoDrop. mRNA integrity was assessed using agarose gel mobility. PBMCs were isolated from healthy donor leukopaques (Hemacare) using Ficoll-Paque density gradient centrifugation according to the manufacturer's instructions. PBMCs were stimulated with OKT3 (50 ng/ml, Miltenyi Biotec) in R10+IL-2 (300 IU/ml, Proleukin®, Prometheus®, Therapeutics and Diagnostics). Seven days after stimulation, T cells were washed twice in Opti-MEM (Thermo Fisher Scientific) and resuspended to a final concentration of 2.5 x 107 cells/ml in Opti-MEM. Ten μg of mRNA was used for electroporation. Cell electroporation was performed using the Gemini X2 system (Harvard Apparatus BTX) to deliver a single pulse of 400 V for 0.5 ms in 2 mm cuvettes (Harvard Apparatus BTX). Cells were directly transferred to R10+IL-2 medium and allowed to recover for 6 h. To examine CAR expression, T cells were stained with LNGFR or biotinylated protein L (Thermo Scientific) in staining buffer (BD Pharmingen) for 30 min at 4°C. Cells were then washed and stained with anti-LNGFR-PE or PE-streptavidin (BD Pharmingen) in staining buffer for 30 min at 4°C. Cells were then washed and resuspended in staining buffer with propidium iodide (BD Pharmingen) prior to data collection. Expression of CAR to STEAP1 in electroporated T cells is shown in Fig. 2.

Example 3.

To examine the cytolytic activity in electroporated anti-STEAP1 CAR T cells, effector cells were co-cultured with target cells at a 1:1 E:T ratio in R10 medium. Sixteen hours after co-culture, supernatants were analyzed by Luminex (EMD Millipore) and target cell viability was assessed by flow cytometric analysis of propidium iodide (PI) uptake by CD3-negative cells. Cytolytic activity in electroporated CAR T cells is shown in Fig. 3 and cytokine production is shown in Fig. 4.

Example 4.

To assess CAR T cell proliferation in response to STEAP1-expressing target cells, T cells were labeled with CFSE prior to co-culture with target cells at a 1:1 E:T ratio in R10 medium. Five days later, T cell proliferation was assessed by CFSE dilution flow cytometry analysis. Proliferation of anti-STEAP1 CAR T cells is shown in Fig. 5.

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Claims (22)

CLAUSE OF THE INVENTION 1. A chimeric antigen receptor comprising an antigen-binding molecule that specifically binds to STEAP1, a costimulatory domain, and an activating domain that is the CD3-zeta signaling domain, wherein the antigen-binding molecule comprises: a) a heavy chain variable region comprising complementarity determining regions (CDRs) 1, 2 and 3 with the amino acid sequences of SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively, and a light chain variable region comprising CDRs 1, 2 and 3 with the amino acid sequences of SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, respectively; b) a VH region of the clone of SEQ ID NO: 88 and a VL region of SEQ ID NO: 93, wherein the VH and VL region are connected by at least one linker; or c) a VH region having at least 90% identity with the amino acid sequence of SEQ ID NO: 88, and a VL region having at least 90% identity with the amino acid sequence of SEQ ID NO: 93, provided that the VH region comprises CDR1 of the variable heavy chain of SEQ ID NO: 89, CDR2 of the variable heavy chain of SEQ ID NO: 90 and CDR3 of the variable heavy chain of SEQ ID NO: 91, and the VL region comprises CDR1 of the variable light chain of NO: 94, CDR2 of the variable light chain of SEQ ID NO: 95 and CDR3 of the variable light chain of SEQ ID NO: 96, wherein the VH and VL region are connected by at least one linker; and the costimulatory domain is a CD28 costimulatory domain comprising a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues from the sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 4 in combination with SEQ ID NO: 6 or SEQ ID NO: 8, or a CD8 costimulatory domain comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 14 in combination with SEQ ID NO: 8. 2. The chimeric antigen receptor of claim 1, wherein the activating domain comprises CD3. 3. The chimeric antigen receptor according to claim 2, wherein CD3 comprises CD3-zeta. 4. The chimeric antigen receptor of claim 3, wherein CD3-zeta comprises a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues from the sequence of SEQ ID NO: 10. 5. The chimeric antigen receptor of claim 1, wherein the costimulatory domain comprises a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues from the sequence of SEQ ID NO: 2, and the activating domain comprises a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues from the sequence of SEQ ID NO: 10. 6. A polynucleotide encoding a chimeric antigen receptor according to claim 1. 7. An expression vector containing a polynucleotide according to claim 6. 8. The vector according to claim 7, which is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated virus vector, a lentiviral vector, or any combination thereof. 9. An immune cell containing the vector according to item 8. 10. The immune cell of claim 9, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, or an NKT cell. 11. The immune cell of claim 10, wherein the cell is an autologous T cell. 12. The immune cell of claim 10, wherein the cell is an allogeneic T cell. 13. An immune cell according to claim 9, wherein the vector is introduced into a cell isolated from the patient's body or grown from a sample taken from the patient's body. 14. An immune cell according to claim 9, wherein the vector is introduced into a cell isolated from a donor's body or grown from a sample taken from a patient's body. 15. A pharmaceutical composition containing an immune cell according to claim 9. 16. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 22, 24 or 26, wherein the polypeptide specifically binds to STEAP1. 17. An expression vector encoding the polypeptide according to claim 16. 18. An immune cell containing the polypeptide according to claim 16. 19. The immune cell of claim 18, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, or an NKT cell. 20. The T cell according to claim 19, which is an autologous T cell. 21. The T cell according to claim 19, which is an allogeneic T cell. 22. An isolated polynucleotide encoding the polypeptide according to claim 16. -