Chimeric antigen receptors based on lilrb1

Abstract

Provided are chimeric antigen receptors or functional fragments or variants thereof having a hinge, transmembrane region, and / or intracellular domain of LILRB1. Also provided herein are cells comprising the LILRB1-based receptors, and methods of making and using the same.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application 62 / 946,888, filed December 11, 2019, and U.S. Provisional Application 63 / 085,969, filed August 30, 2020, the disclosures of each of which are incorporated herein by reference in their entirety.

[0003] By referencing the sequence list and incorporating

[0004] This application contains a sequence list, which has been submitted in ASCII format via EFS-WEB and is hereby incorporated in its entirety by reference. The ASCII copy created on December 11, 2020, is named A2BI-015-01WO_SeqList.txt and is 228KB in size. Background Technology

[0005] Chimeric antigen receptor (CAR) T-cell therapy and T-cell receptor (TCR) therapy have proven to be effective treatments for a variety of diseases, particularly hematologic malignancies and other cancers. CAR NK cells may also have clinical applications. Conventional CARs deliver stimulatory signals to engineered immune cells, such as T cells or NK cells. In CAR-T cells, this results in killing activity against target cells identified by the antigen-binding domain of the CAR. Inhibitory CARs (iCARs) have been developed as a means of controlling cell activity or restricting the activity of activator CARs to specific cell types. Fedorov et al., Sci. Transl. Med. 5(215):215ra172 (2013). Inhibitory CARs typically have intracellular domains of inhibitory signaling molecules (such as PD-1 or CTLA-4) fused to an antigen-binding domain (e.g., a single-chain variable fragment, scFv) via transmembrane and optionally hinge regions.

[0006] Many alternative iCAR architectures have been described in this art. However, there remains an unmet need for the identification of novel alternative inhibitory receptors and specific inhibitory receptor architectures with superior performance, as well as their associated compositions and methods of use. Summary of the Invention

[0007] In one aspect, this disclosure provides a chimeric antigen receptor or a functional fragment or variant thereof having a hinge, transmembrane region, and / or intracellular domain of LILRB1. The chimeric antigen receptor may comprise a single polypeptide or more than one polypeptide. The receptor may comprise one or more of the following: (a) a LILRB1 hinge domain or a functional fragment or variant thereof; (b) a LILRB1 transmembrane domain or a functional variant thereof; and (c) a LILRB1 intracellular domain or a functional variant thereof, such as the LILRB1 intracellular domain and / or an intracellular domain of at least one immunoreceptor tyrosine inhibitory motif (ITIM) found in the polypeptide sequence of LILRB1. In some embodiments, the receptor comprises at least two ITIMs found in the polypeptide sequence of LILRB1. The ITIMs of LILRB1 are NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). The receptor may contain one, two, three, four, five, six or more of these ITIMs, in any combination, including multiple copies of the same ITIM.

[0008] In some embodiments of the receptor of this disclosure, the intracellular domain comprises two ITIMs, NLYAAV (SEQ ID NO:8) and VTYAEV (SEQ ID NO:9). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO:12. In some embodiments, the intracellular domain comprises two ITIMs, VTYAEV (SEQ ID NO:9) and VTYAQL (SEQ ID NO:10). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO:13. In some embodiments, the intracellular domain comprises two ITIMs, VTYAQL (SEQ ID NO:10) and SIYATL (SEQ ID NO:11). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO:14. In some embodiments, the polypeptide comprises an intracellular domain containing at least three immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), and VTYAQL (SEQ ID NO:10). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:15. In some embodiments, the intracellular domain comprises the ITIMs VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:16. In some embodiments, the intracellular domain comprises ITIM NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 7). In some embodiments, the intracellular domain comprises the sequences of SEQ ID NO: 12-17.

[0009] In some embodiments of the receptor described in this disclosure, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:5. In some embodiments, the LILRB1 transmembrane domain comprises SEQ ID NO:5.

[0010] In some embodiments of the receptor of this disclosure, the polypeptide comprises a LILRB1 hinge domain or a functional fragment or variant thereof. In some embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:93. In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:93. In some embodiments, the LILRB1 hinge domain or a functional segment or variant thereof comprises at least 95% identical sequences to SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84. In some embodiments, the LILRB1 hinge domain comprises identical sequences to SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84.

[0011] In some embodiments of the receptor described in this disclosure, the polypeptide comprises: (a) a LILRB1 hinge domain or a functional fragment or variant thereof, and (b) the LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:20.

[0012] In some embodiments of the receptor described in this disclosure, the polypeptide comprises: (a) a LILRB1 transmembrane domain or a functional variant thereof, and (b) a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:21. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:21.

[0013] In some embodiments of the receptor of this disclosure, the polypeptide comprises: (a) a LILRB1 hinge domain or a functional fragment or variant thereof; (b) a LILRB1 transmembrane domain or a functional variant thereof; and (c) a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immune receptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0014] In some embodiments of the receptor of this disclosure, the polypeptide comprises at least 95% identical sequence to SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, the polypeptide comprises at least 99% identical sequence to SEQ ID NO:20. In some embodiments, the polypeptide comprises at least 99% identical sequence to SEQ ID NO:21. In some embodiments, the polypeptide comprises at least 99% identical sequence to SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, the polypeptide comprises the same sequence as SEQ ID NO:20. In some embodiments, the polypeptide comprises the same sequence as SEQ ID NO:21. In some embodiments, the polypeptide comprises the same sequence as SEQ ID NO:2 or SEQ ID NO:3.

[0015] In some embodiments of the receptor described in this disclosure, the polypeptide comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is an antigen-binding domain other than the LILRB1 extracellular ligand-binding protein. In some embodiments, the polypeptide comprises two or more antigen-binding domains. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the receptor comprises a second polypeptide. In some embodiments, the first polypeptide comprises a first chain of an antibody, and the second polypeptide comprises a second chain of the antibody. In some embodiments, the receptor comprises a Fab fragment of an antibody. In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the heavy chain of an antibody, and (b) the second polypeptide comprises an antigen-binding fragment of the light chain of an antibody. In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the light chain of an antibody, and (b) the second polypeptide comprises an antigen-binding fragment of the heavy chain of an antibody. In some embodiments, the first polypeptide comprises a first chain of a T-cell receptor (TCR), and the second polypeptide comprises a second chain of the TCR. In some embodiments, the receptor comprises an extracellular fragment of a T-cell receptor (TCR). In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the α chain of the TCR, and (b) the second polypeptide comprises an antigen-binding fragment of the β chain of the TCR. In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the β chain of the TCR, and (b) the second polypeptide comprises an antigen-binding fragment of the α chain of the TCR. In some embodiments, the receptor comprises a single-chain TCR. In some embodiments, the scFv comprises a complementarity-determining region (CDR) of any one of SEQ ID NO: 22-33. In some embodiments, the scFv comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 35-46 or 125. In some embodiments, the scFv comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 35, 39, 46 or 125. In some embodiments, the scFv comprises a sequence that is identical to any one of SEQ ID NO: 35-46 or 125. In some embodiments, the scFv comprises a sequence that is identical to any one of SEQ ID NO: 35, 39, 46 or 125. In some embodiments, the heavy chain of the antibody comprises a heavy chain CDR of any one of SEQ ID NO:25-27 or 31-33, and the light chain of the antibody comprises a light chain CDR of any one of SEQ ID NO:22-24 or 28-30.In some embodiments, the heavy chain of the antibody comprises at least 95% identical sequence to the heavy chain portion of any one of SEQ ID NO:35-46 or 125, and the light chain of the antibody comprises at least 95% identical sequence to the light chain portion of any one of SEQ ID NO:35-46 or 125. In some embodiments, the heavy chain of the antibody comprises identical sequence to the heavy chain portion of any one of SEQ ID NO:35-46 or 125, and the light chain of the antibody comprises identical sequence to the light chain portion of any one of SEQ ID NO:35-46 or 125. In some embodiments, the heavy chain of the antibody comprises identical sequence to the heavy chain portion of any one of SEQ ID NO:35, 39, 46 or 125, and the light chain of the antibody comprises identical sequence to the light chain portion of any one of SEQ ID NO:35, 39, 46 or 125.

[0016] In some embodiments of the receptor described in this disclosure, the receptor comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NO:47-71, 77-79, 89-92, 120, or 122. In some embodiments, the receptor comprises an amino acid sequence of SEQ ID NO:47-71, 77-79, 89-92, 120, or 122.

[0017] In some embodiments of the receptor described in this disclosure, the receptor is an inhibitory receptor.

[0018] This disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a receptor or polypeptide of this disclosure.

[0019] This disclosure provides a vector containing a polynucleotide of this disclosure. In some embodiments, the vector further includes a sequence encoding a promoter operatively linked to the polynucleotide.

[0020] This disclosure provides an immune cell comprising a receptor, polynucleotide, polypeptide, or receptor of this disclosure. In some embodiments, immune cell activation is reduced when the cell comes into contact with an antigen or a cell expressing the antigen on its surface. In some embodiments, immune cell activation includes expressing a gene operatively linked to an NFAT promoter. In some embodiments, the immune cell is a T cell. In some embodiments, an activator receptor is further included. In some embodiments, the activator receptor is a chimeric antigen receptor or a T cell receptor.

[0021] This disclosure provides a method for manufacturing immune cells, comprising introducing a polynucleotide or vector of this disclosure into the immune cells. In some embodiments, the immune cells express the receptor. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, immune cell activation is reduced when the cells come into contact with an antigen specific to the chimeric antigen receptor or a cell expressing the antigen on its surface. In some embodiments, immune cell activation includes expressing a gene operatively linked to an NFAT promoter.

[0022] This disclosure provides a method for treating a subject suffering from a disease or disorder, comprising administering to the subject a plurality of immune cells of this disclosure. In some embodiments, the disease or disorder is cancer.

[0023] This disclosure provides a kit comprising the receptor, peptide, polynucleotide, vector, or immune cell of this disclosure.

[0024] This disclosure provides an immune cell comprising a chimeric antigen receptor comprising a polypeptide, wherein the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO:21.

[0025] In some embodiments of the immune cells described in this disclosure, the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO:3. In some embodiments, the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO:2. In some embodiments, the chimeric antigen receptor comprises an antigen-binding domain comprising the sequences CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 according to SEQ ID NO:22-27, respectively. In some embodiments, the chimeric antigen receptor comprises an antigen-binding domain comprising the sequences CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 according to SEQ ID NO:28-33, respectively. In some embodiments, the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO:122. In some embodiments, the polypeptide sequence shares at least 95% or at least 100% identity with any one of SEQ ID NO:35, 39, 46 or 125 in combination with SEQ ID NO:2.

[0026] In some embodiments, the immune cells are T cells. In some embodiments, the T cells comprise chimeric antigen receptors or T cell receptors that specifically bind to targets expressed on tumor cells. In some embodiments, the T cells comprise chimeric antigen receptors or T-cell receptors that specifically bind to a target selected from the following: luteinizing receptor, ανββ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, phosphatidylinositol proteoglycan-3 (GPC3), h5T4, IL-11R, IL-13R-a2, KDR, κ light chain, λ light chain, LeY, LI CAM, MAGE-A1, mesothelin, MHC presenting peptide, MUC1, MUC16, NCAM, NKG2D ligand, Notchl, Notch2 / 3, NY-ESO-1, PRAME, PSCA, PSMA, survivability protein, TAG-72, TEM, TERT, VEGFR2, and ROR1.

[0027] This disclosure provides methods for treating and / or preventing cancer in a subject of need, comprising administering immune cells of this disclosure to the subject. In some embodiments, the methods include treating and / or preventing cancer in a subject of need, comprising administering immune cells of this disclosure to the subject.

[0028] The illustrative CARs provided in this article include, but are not limited to, antibody-based CARs, such as single-chain variable fragment (scFv) CARs, Fab CARs, or others; and T-cell receptor (TCR)-based CARs.

[0029] In other respects, this disclosure provides polynucleotides encoding such receptors; vectors for delivering such polynucleotides; and immune cells having such polynucleotides and receptors.

[0030] In a further aspect, this disclosure provides methods for introducing polynucleotides or vectors encoding such receptors into cells. Advantageously, immune cell activation is reduced when cells come into contact with antigens or cells expressing said antigens on their surface.

[0031] Other aspects and embodiments of the present invention are provided in the following detailed description. Attached Figure Description

[0032] Figure 1 shows an illustrative diagram of the domain arrangement in an embodiment having a ligand-binding domain (LBD), hinge, transmembrane (TM) and intracellular signal transduction domain (ICD).

[0033] Figures 2A-2B illustrate illustrative diagrams of the domain arrangement in embodiments having a ligand-binding domain (LBD), hinge, transmembrane (TM), and intracellular signal transduction domain (ICD). When the ligand-binding domain comprises two peptides, such as the heterodimer LDB from the T-cell receptor, each peptide can be fused to the hinge, TM, and intracellular domain (Figure 2A). Alternatively, only one peptide from the ligand-binding domain can be fused to the hinge, TM, and intracellular domain (Figure 2B).

[0034] Figure 3 illustrates four illustrative embodiments of immune cells having activator-based chimeric antigen receptor (CAR) [101 and 102] or activator-based T cell receptor [103 and 104] and inhibitory scFv-based CAR [101 and 103] or inhibitory TCR-based CAR [102 and 104].

[0035] Figure 4 shows the luminescence (relative luminescence units, RLU) measured based on the NFAT reporter in the presence of 50 μM NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 activated peptide 1 (MP1, μL, μM).

[0036] Figure 5 shows the luminescence (RLU) measured based on NFAT reporter in the presence of various concentrations (μM) of NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0037] Figure 6 shows the luminescence (RLU) measured based on NFAT reporter in the presence of various concentrations (μM) of NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0038] Figure 7 shows the luminescence (RLU) measured based on the NFAT reporter in the presence of 50 μM NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0039] Figure 8 shows the luminescence (RLU) measured based on NFAT reporter in the presence of 50 μM NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0040] Figure 9 shows the luminescence (RLU) measured based on the NFAT reporter in the presence of 5 μM NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0041] Figure 10 shows the luminescence (RLU) measured based on the NFAT reporter in the presence of 50 μM NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0042] Figure 11 shows the luminescence (RLU) measured based on the NFAT reporter in the presence of 50 μM NY-ESO-1 peptide for the indicated construct at different concentrations of MAGE-A3 peptide 1 (MP1, nM).

[0043] Figure 12A is a plot illustrating the effect of the LIR-1 hinge on the ability of the HLA-A*02scFv inhibitory receptor to block KRAS TCR activation in Jurkat cells. H: hinge, T: transmembrane domain, ICD: intracellular domain, s: short. The LIR-1 constructs are described in more detail in Figure 12B. Humanized PA2.1 and humanized BB7.2, with shorter LIR-1 hinges, blocked similarly to the original, longer hinge.

[0044] Figure 12B is a plot and table showing the EC50 shift (+ / - HLA-A*02 target cells) of Jurkat cells expressing the KRAS TCR activator and HLA-A*02scFvLIR-1 inhibitory receptor shown in the bottom table.

[0045] Figure 13A is a plot illustrating the effect of the LIR-1 hinge on the ability of HLA-A*02 inhibitory receptor blockade of KRAS TCR to activate Jurkat cells. H: hinge, TM: transmembrane domain, ICD: intracellular domain, s: short; tr: truncated. The LIR-1 construct is described in more detail in Figure 13B. In the T2-Jurkat assay, mouse PA2.1 with a slightly longer hinge functioned similarly to the original LIR-1 hinge.

[0046] Figure 13B is a plot of the EC50 shifts (+ / -HLA-A*02 target cells) of Jurkat cells expressing the KRAS TCR activator and HLA-A*02scFvLIR-1 inhibitory receptor shown in the bottom table, and a pair of tables, with hinge lengths shown in the left table.

[0047] Figure 14A is a schematic diagram illustrating the T2-Jurkat experiment for evaluating the inhibitor construct.

[0048] Figure 14B is a diagrammatic and tabular representation illustrating the effect of various NY-ESO-1scFv LBD inhibitor modules (PD-1, CTLA-4, LIR-1) on the EC50 of the MAGE-A3 CAR activator (MP1-LBD 1-CAR), measured by MAGE peptide titration of cells loaded with a fixed (50 μM) concentration of the NY-ESO-1 inhibitor peptide. In each of Figures 14B-14F, NFAT-luciferase signaling in Jurkat cells transfected with the activator CAR alone or in combination with each inhibitor receptor was measured after 6 hours of co-culturing with T2 cells loaded with both the activator and inhibitor peptides. Baseline (Jurkat only) varied with the different individual activator constructs and may be particularly high for CAR; in most cases, inhibitor receptor expression suppressed baseline in the absence of its ligand. Activator peptide concentrations ranged from 0 μM to 10 μM. -6 μM to 10 2 μM, and the emission measurement range is 0 RLU to 80000 RLU.

[0049] Figure 14C is a plot, table, and diagram illustrating the effect of LIR-1 receptors with various scFv LBDs (ESO, MP1 LBD 1, MP1 LBD 2, HPV E6 LBD 1, HPVE6 LBD 2, HPV E7) on the EC50 of the MAGE-A3 CAR activator (MP1-CAR) when loaded with a fixed (50 μM) concentration of the corresponding blocking peptide (as shown in Figure 14B). RLU = relative optical units; error bars indicate ±SD (n = 2). Activator peptide concentrations range from 0 μM to 10 μM. -3 μM to 10 2 μM, and the emission measurement range is 0 RLU to 100000 RLU.

[0050] Figure 14D is a diagram, table, and illustration showing the effect of LIR-1 receptors with NY-ESO-1scFv LBD on the EC50 of different MAGE-A3 CAR activators (MP1-LBD1-CAR or MP2-CAR) when loaded with 50 μM NY-ESO-1 blocking peptide. The activator peptide concentration ranges from 0 μM to 10 μM. -4 μM to 10 2 μM, and the emission measurement range is 0 RLU to 80000 RLU.

[0051] Figure 14E is a diagram, table, and illustration showing the effect of LIR-1 receptors with NY-ESO-1scFv LBD on the EC50 of different TCR activators (MP1-TCR, MP2-TCR, HPV E6-TCR) when loaded with 50 μM NY-ESO-1 blocking peptide. The activator peptide concentration ranges from 0 μM to 10 μM. -4 μM to 10 2 μM, and the normalized luminescence measurement range is 0 RLU to 150 RLU.

[0052] Figure 14F is a plot, table, and diagram illustrating the effect of LIR-1 receptors with NY-ESO-1TCR LBD on the EC50 of MAGE-A3 CAR and TCR activators (MP1-LBD1-CAR, MP1-TCR) when loaded with 50 μM NY-ESO-1 blocking peptide. RLU = relative optical units; error bars indicate ±SD (n = 2). Activator peptide concentrations range from 0 μM to 10 μM. -7 μM to 10 1 μM, and the normalized luminescence measurement range is 0 RLU to 150 RLU.

[0053] Figure 15 is a plot showing the effect of blocking peptide loading (50 μM each of NY-ESO-1, MAGE-A3, HPV E6, and HPV E7) on activated MAGE-A3 CAR. MP2-CAR [0 μM], EC50 = 44 nM; MP2-CAR [50 μM HPVp2], EC50 = 495 nM. RLU = relative optical units; error bars indicate ±SD (n = 2).

[0054] Figure 16A is a series of plots illustrating the NFAT-luciferase signaling of Jurkat cells transfected with either the activator MAGE-A3 CAR alone or in combination with various amounts of NY-ESO-1scFv LBD inhibitors after 6 h of co-culture with T2 cells loaded with activator and inhibitor peptides (the DNA ratio of activator receptor component to inhibitor receptor component is shown on the left as A:B, i.e., activator receptor:inhibitor receptor). T2 cells were loaded with titrations of the activator MAGE-A3 peptide and fixed amounts of the inhibitor NY-ESO-1 peptide. The concentrations of the activator and / or inhibitor peptides ranged from 0 μM, followed by 10... -6 μM to 10 2 μM, and the emission measurement range is 0 RLU to 200,000 RLU.

[0055] Figure 16B is a series of plots illustrating NFAT-luciferase signaling in Jurkat cells transfected with the activator MAGE-A3 CAR alone or in combination with various amounts of NY-ESO-1scFv LBD inhibitor. T2 cells were loaded with a titration of the inhibitor NY-ESO-1 peptide and a fixed concentration of the activator MAGE-A3 peptide above the Emax concentration (approximately 0.1 mM). The concentrations of the activator and / or inhibitor peptide ranged from 10... -5 μM to 10 2 μM, and the emission measurement range is 0 RLU to 200,000 RLU.

[0056] Figure 16C is a series of plots and two tables showing NFAT-luciferase signaling in Jurkat cells transfected with the activator MAGE-A3 CAR alone or in combination with various amounts of the NY-ESO-1scFv LBD inhibitor. The x-values ​​from Figure 16B, representing the concentration of the inhibitor NY-ESO-1 peptide, are normalized relative to a constant concentration of the activator MAGE peptide used for each curve and plotted on the x-axis. For each curve, the ratio of the inhibitor peptide to the activator peptide required for 50% blockade (IC50) is indicated. For all DNA ratios, the required B:A peptide ratio is less than 1, indicating that for this pair of activator CAR and inhibitor, similar (or fewer) inhibitor pMHC antigens are needed on the target cells to block the activator pMHC antigen. The activator and / or inhibitor peptide concentrations range from 10... -10 μM to 10 4 μM, and the emission measurement range is 0 RLU to 200,000 RLU.

[0057] Figure 16D is a table and plot showing that it is possible to block CD19-CAR activator with pMHC inhibitors at pMHC antigen densities similar to those required to activate pMHC CAR. Jurkat cells transfected with CD19 CAR alone or in combination with various amounts of NY-ESO-1 inhibitors (DNA ratios shown) for 6 h after co-culturing with T2 cells expressing endogenous levels of CD19 antigen loaded with inhibitor peptides were used to assess NFAT-luciferase signaling. IC50 was estimated from the inhibition curves to range from 0.1–1.0 mM, corresponding to approximately 1,500–3,500 pMHC / cell. RLU = relative optical units; error bars indicate ±SD (n = 2).

[0058] Figure 17A is a plot showing the effect of NY-ESO-1-LIR-1 inhibitor on the EC50 of MAGE-A3 CAR (MP1-CAR) activation when loaded with various concentrations of the NY-ESO-1 inhibitor peptide. The EC50 shift increases with increasing inhibitor peptide (NY-ESO-1). A shift is typically observed in the presence of the negative control HPV peptide (binding HLA-A*02 but not the NY-ESO-1 inhibitor scFv) and is thought to be caused by competition of the control peptide for binding sites on the T2HLA-A*02 molecule, reducing the number of activator targets. Activator concentrations range from 0 μM to 10 μM. -4 μM to 10 2 μM, and the emission measurement range is 0 RLU to 140000 RLU.

[0059] Figure 17B is a plot showing the effect of modified LIR-1 receptors without ICD or with mutant ICD and NY-ESO-1scFv LBD on the EC50 of MAGE-A3 CAR activator (MP2-CAR) when loaded with 10 μM NY-ESO-1 blocking peptide. The activator concentration range is 0 μM, followed by 10 μM. -4 μM to 10 2 μM, and the emission measurement range is 0 RLU to 140000 RLU.

[0060] Figures 17C-17E are a series of plots illustrating the effects of various NY-ESO-1scFv LBD inhibitor receptors (CTLA-4 (Figure 17C), PD-1 (Figure 17D), and LIR-1 (Figure 17E)) on the EC50 of the MAGE-A3 CAR activator (MP1-LBD 1-CAR) with or without stimulation of the inhibitor. Jurkat cells transfected with the activator CAR alone or in combination with each inhibitor were tested for NFAT-luciferase signaling after 6 h of co-culture with T2 cells loaded with the peptide. T2 cells were loaded with a titration of the activating MAGE peptide, and tests were performed with and without an additional constant amount (50 μM) of the NY-ESO-1 inhibitor peptide. RLU = relative optical units; error bars indicate ±SD (n = 2). Activator concentrations ranged from 0 μM to 10 μM. -6 μM to 10 2 μM, and the emission measurement range is 0 RLU to 100000 RLU.

[0061] Figure 18A is a diagram and a pair of plots showing that co-culturing Jurkat cells transfected with HPV E7-CAR or HPV E7-CAR&A2-LIR-1 with beads displaying various ratios of activator (HPV E7) and blocker (NY-ESO-1) antigens indicates cis-blocking rather than trans-blocking.

[0062] Figure 18B is a plot showing the HLA-A*02-LIR-1 inhibitor receptor blocking CD19-CAR activator at various activator-to-blocker ratios. The ratios range from 0 to 10, and the luminescence emission (RLU) ranges from 0 to 70,000.

[0063] Figure 18C is a plot showing the surface expression of the titrated HLA-A*02(A2)LIR-1 blocker receptor.

[0064] Figure 18D is a plot showing that the scFv targeting HLA-A*02 can also act as an activator when fused with the activator CAR. T2 cells expressing endogenous HLA-A*02 act as the target. RLU = relative optical units; error bars indicate ±SD (n=2).

[0065] Figure 19A is a plotted graph showing the effect of the LIR-1 receptor with NY-ESO-1scFvLBD on the EC50 of different TCR activators (MP1-TCR, MP2-TCR, HPVE6-TCR) when loaded with the NY-ESO-1 blocking peptide. For Figure 14E, the Emax of each group was normalized relative to the curve showing the response of the activator only. The activator concentration range was 0 μM, followed by 10 μM. -4 μM to 10 2 μM, and the emission measurement range is 0 RLU to 140000 RLU.

[0066] Figure 19B is a plot showing the effect of the LIR-1 blocking receptor with NY-ESO-1TCR LBD on the EC50 of MAGE-A3CAR and TCR activators (MP1-LBD 1-CAR, MP1-TCR). The Emax of each group was normalized relative to the curve showing the response of the activator only. RLU = relative optical units; error bars indicate ±SD (n = 2). Activator concentrations ranged from 0 μM, then 10 μM. -7 μM to 10 1 μM, and the emission measurement range is 0 RLU to 140000 RLU.

[0067] Figure 20A is a plot showing an approximately 25-fold EC50 shift in primary T cells (donor 1) transduced with HPV E7-TCR activator and ESO-LIR-1 inhibitor in a primary T cell killing assay (HPV E7 TCR, EC50 = 0.044 nM; HPV E7 TCR + ESO-LIR-1, EC50 = 1.1 nM). The assay was performed using MCF7 target cells loaded with the peptide at a 3:1 E:T ratio. Luciferase measurements represent live target cells at 48 hours.

[0068] Figure 20B is a plot showing the blocking of NY-ESO-1 CAR activator in Jurkat cells using T2 target cells loaded with NY-ESO-1 peptide and HLA-A*02-LIR-1 at various activator:blocker DNA ratios. RLU = relative optical units; error bars indicate ±SD (n = 2).

[0069] Figure 21A is a series of images and plots illustrating the differentiation of “tumor” cells from “normal” cells using primary T cells (donor 1) transduced with CD19 CAR activator and HLA-A*02 inhibitor in an in vitro cytotoxicity assay, and demonstrating selective killing of “tumor” cells at a 3:1E:T ratio in a mixed target cell assay. The images shown were captured at 72 hours. Untransduced T cells, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1 are shown. Measurements were performed between 0 and 150 hours, and normalized fluorescent protein intensities (GFP or RFP) ranged from 0 to 10.

[0070] Figure 21B is a series of plots illustrating the similar selective killing of tumor cells by primary T cells (donor 1) at a 3:1 E:T ratio across various tumor cell to “normal” cell ratios in an Incucyte imaging assay. RLU values ​​were normalized relative to a mixture of target cells grown in the absence of primary T cells. Untransduced T cells, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1 are shown. Measurements were performed between 0 and 150 hours, and normalized fluorescent protein intensities (GFP or RFP) ranged from 0 to 3 × 10⁻⁶ in the top row from left to right. 7 0 to 2×10 7 0 to 1.6 × 10 7 0 to 7×10 6 0 to 2×10 6 Bottom row, from left to right: 0 to 7 × 10 5 0 to 2×10 5 0 to 4×10 5 0 to 6×10 5 0 to 7×105 .

[0071] Figure 21C is a series of plots illustrating the similar selective killing of tumor cells by primary T cells (donor 1) at a 3:1 E:T ratio under various tumor cell to "normal" cell ratios during quantitative target cell lysis and IFNγ secretion. Untransduced T cells, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1 are shown.

[0072] Figure 22A is a pair of plots showing that Jurkat cells transfected with MSLN LBD1-CAR or MSLN LBD1-CAR & A2-LIR-1, co-cultured with K562 cells expressing MSLN or MSLN & HLA-A*02, showed that only the A2-LIR-1 blocker blocked activation induced by high-density antigens in the presence of HLA-A*02.

[0073] Figure 22B is a pair of plots showing that, in the presence of HLA-A*02, the killing of endogenous MSLN+HeLa cells by MLSN LBD1-CAR T cells was blocked by the A2-LIR-1 inhibitor.

[0074] Figure 22C is a pair of plots illustrating the killing of endogenous MSLN+ HeLa cells by MLSN LBD2-CAR T cells. The T cell killing effect of the A2-LIR-1 blocker is partially controlled by the activator LBD.

[0075] Figure 23 is a plot showing that the LIR-1 blocker receptor has almost no effect on the cytotoxic efficacy of the activator in the absence of the blocker antigen. Primary T cells (donor 1) transduced with both the HPV E7-TCR activator and the ESO-LIR-1 blocker showed similar cytotoxic efficacy in the absence of the NY-ESO-1 blocker antigen to those transduced with only the HPV E7-TCR activator. Luciferase measurements represent live target cells. RLU = relative optical units; error bars indicate ±SD (n = 2).

[0076] Figure 24A is a pair of plots showing that A2-LIR-1 blocks Jurkat activation in A2+ Raji cells but not in WT Raji cells. The histogram shows that Raji WT “tumor” cells and Raji A2+ “normal” cells have the same CD19 surface expression, while HLA-A*02 is expressed only in Raji A2 “normal” cells.

[0077] Figure 24B is a plot showing that A2-LIR-1 blocks Jurkat activation in A2+Raji cells without blocking WTRaji cells. Jurkat cells transfected with CD19 or CD19+A2-LIR-1 were co-cultured with WT(A2-)Raji cells or A2+Raji cells at various cell ratios. RLU = relative optical units; error bars indicate ±SD (n=2).

[0078] Figures 25A-25B are each a series of images illustrating the reversibility of blockade by the LIR-1 inhibitory receptor. Primary T cells (donor 2) transduced with a CD19 CAR activator and an HLA-A*02 blocker showed reversible blockade (Figure 25A) and activation (Figure 25B) in an in vitro cytotoxicity assay at a 3:1E:T ratio after three rounds of antigen exposure (AB-A-AB and A-AB-A). The cytotoxicity assay of primary T cells was reproduced using three HLA-A*02 negative donors. The images shown were captured at 72 hours.

[0079] Figures 25C-25D are each a pair of plots illustrating the quantification of target cell lysis (Figure 25C) and IFNγ (Figure 25D) in response to repeated exposure to multiple rounds of normal and target cells, 3:1E:T (T cells from donor 2). The conditions shown are untransduced T cells, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1. Error bars indicate ± SEM (n=2). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, determined using two-way ANOVA followed by Tukey's multiple comparison test. In this experiment, the IFNγ response attenuated over time, while cytotoxicity remained robust.

[0080] Figures 26A-26B are each a pair of plots illustrating the killing of cytotoxic T cells and the secretion of IFNγ when co-cultured with a single donor (donor 3). T cells transduced with the cytotoxic CD19 CAR activator and HLA-A*02 blocker showed reversible blockade in cytotoxicity assays and IFNγ at 9:1E:T after multiple rounds of antigen exposure. We note that the survival and activity of T cells from this donor decreased over time in culture. The conditions shown are untransduced T cells, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1. Cytotoxicity (Figure 26A) and IFNγ (Figure 26B) results correspond to Figures 25C-25D. Error bars indicate ±SEM (n=2). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, determined using two-way ANOVA followed by Tukey multiple comparison test.

[0081] Figure 27A is a plot showing that primary T cells transduced with CD19 CAR activator and HLA-A*02 blocker showed approximately 20-fold expansion upon CD3 / 28 stimulation within 10 days.

[0082] Figure 27B is a diagram illustrating an experiment demonstrating that CAR-T cells expressing the LIR-1 inhibitor receptor selectively kill tumors in a xenograft model. "Tumor cells" (A2-negative Raji cells) or "normal cells" (A2-positive Raji cells) were subcutaneously administered to HLA-A*02NSG mice, and the Raji xenografts were approximately 70 mm in size. 3 At that time, primary T cells (human, HLA-A*02 negative donor 4) were injected into the tail vein.

[0083] Figures 27C-27E are each a pair of plots showing caliper readings (Figure 27C), peripheral blood human T cell counts obtained by flow cytometry (Figure 27D), and survival rate (Figure 27E). Error bars indicate SEM (n=7). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, determined using two-way ANOVA followed by Tukey's multiple comparison test.

[0084] Figure 28A is a series of plots illustrating flow cytometry analysis of enriched and expanded primary T cells prepared for tail vein injection in mice. Tumor volume was measured from 10 days (-10) before T cell injection to 40 days (40) after T cell injection. Tumor volume ranged from 0 to 2500 mm. 3 .

[0085] Figure 28B is a series of plots showing tumor measurements obtained by calipers for individual mice in each group.

[0086] Figure 28C is a series of plots illustrating the correlation between huCD3+ T cells in mouse blood and tumor growth. The graph compares tumor volume at 10 and 17 days after huCD3+ T cell injection with that of other T cells.

[0087] Figure 28D is a pair of plots showing the counts of huCD4+ T cells and huCD8+ T cells in peripheral blood obtained by flow cytometry. Hollow rings represent mice transplanted with “normal” cells; solid rings represent mice transplanted with tumor cells. Samples with fewer than 100 cells were excluded from the analysis. Error bars indicate ± SEM (n = 7 in all groups, except for the CD19+ / A2-Raji group treated with CD19-CAR+A2-LIR-1 T cells, where n = 6).

[0088] Figure 29A is a series of images illustrating the histological analysis of T-cell infiltration in the tumor. Representative images of tumor samples collected, sectioned, and stained against huCD3 at the end of the study are shown.

[0089] Figure 29B is a plot showing the quantitative T cell infiltration using ImageJ. T cell infiltration was significantly higher in CD19+ / A2- tumors for T cells with either CD19-CAR or CD19-CAR+A2-LIR-1 compared to untransduced cells. However, in CD19+ / A2+ tumors, there was no significant difference in CD19-CAR+A2-LIR-1 T cells compared to untransduced cells. There was also a significant decrease in CD19-CAR+A2-LIR-1 T cell infiltration between CD19+ / A2- and CD19+ / A2+ tumors. Qualitatively, CD19-CAR+A2-LIR-1 T cells were less prevalent in CD19+ / A2+ tumors compared to CD19-CAR-only T cells; however, this difference was not statistically significant. Similarly, saline samples were quantified to show background staining levels. The datasets were analyzed using standard one-way ANOVA, while individual pairs between “tumor” and “normal” were analyzed using unpaired t-tests. ns = not significant, *p<0.05, **p<0.01. Detailed Implementation

[0090] This disclosure describes receptors having one or more domains from a member of the leukocyte immunoglobulin-like receptor subfamily B (LILRB1, sometimes referred to as LIR1 or LIR-1). Many receptors, engineered cells, and their uses are considered herein. The inventors have discovered that chimeric receptors comprising an antigen-binding domain and one or more LILRB1 domains (including the LILRB1 intracellular domain) can inhibit immune cell signaling even in the presence of activating chimeric antigen receptors (CARs) or T-cell receptors (TCRs).

[0091] As used herein, the term "chimeric antigen receptor" or "CAR" can refer to, for example, an artificial T-cell receptor, a chimeric T-cell receptor, or a chimeric immune receptor, and includes engineered receptors that have been artificially and specifically transplanted onto specific immune effector cells such as helper T cells (CD4+), cytotoxic T cells (CD8+), or NK cells. CARs can be used to confer specificity to T cells with monoclonal antibodies, thereby allowing the generation of large numbers of specific T cells, for example, for adoptive cell therapy. In specific embodiments, the CAR directs the cells to be specific to tumor-associated antigens. In some embodiments, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing an antigen-binding region. In some embodiments, the CAR comprises a fusion of a single-chain variable fragment (scFv) or scFab derived from a monoclonal antibody, fused to the transmembrane domain and one or more intracellular signaling domains. The fusion may also include a hinge. Either heavy-light (HL) scFv or light-heavy (LH) scFv can be used. The specificity of the CAR design can be derived from a receptor ligand (e.g., a peptide). Depending on the type of intracellular domain, a CAR can be an activating or inhibitory receptor. In some embodiments, such as when the CAR is an activating receptor, the CAR contains domains for additional co-stimulatory signaling, such as CD3, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, co-expressed molecules may include co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally eliminate T cells upon prodrug administration, homing receptors, cytokines, and cytokine receptors. As used herein, the characteristics attributed to a chimeric antigen receptor can be understood to refer to the receptor itself or the host cell containing said receptor.

[0092] As used herein, a “TCR” (sometimes also referred to as a “TCR complex” or “TCR / CD3 complex”) is a protein complex comprising one or more of the following: a TCRα chain, a TCRβ chain, and invariant CD3 chains (ζ, γ, δ, and ε) (sometimes referred to as subunits). The TCRα and TCRβ chains may be disulfide-linked to act as heterodimers to bind to the peptide-MHC complex. Once the TCRα / β heterodimer binds to the peptide-MHC, it induces a conformational change in the associated invariant CD3 subunits in the TCR complex, leading to their phosphorylation and association with downstream proteins, thereby transducing the primary stimulus signal. In an exemplary TCR complex, the TCRα and TCRβ peptides form a heterodimer, CD3ε and CD3δ form a heterodimer, CD3ε and CD3γ form a heterodimer, and the two CD3ζ forms a homodimer.

[0093] The term "stimulus" refers to a primary response induced by the binding of a stimulating domain or stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating a signal transduction event (such as, but not limited to, signal transduction via the TCR / CD3 complex). Stimuli can mediate altered expression of certain molecules and / or reorganization of cytoskeleton structures.

[0094] The term "stimulatory molecule" or "stimulatory domain" refers to a molecule or portion thereof that, when naturally expressed by T cells, provides one or more primary cytoplasmic signaling sequences that stimulately regulate the activation of the TCR complex at least one aspect of the T cell signaling pathway. The TCRα and / or TCRβ chains of the wild-type TCR complex do not contain stimulatory domains and require association with CD3 subunits such as CD3ζ to initiate signaling. In one aspect, the primary stimulatory signal is initiated, for example, by the binding of the TCR / CD3 complex to the major histocompatibility complex (MHC) bound to the peptide, and this leads to a T cell response, including but not limited to proliferation, activation, differentiation, etc. One or more stimulatory domains as described herein may be fused to the intracellular portion of any one or more subunits of the TCR complex, including TCRα, TCRβ, CD3δ, CD3γ, and CD3ε.

[0095] As used herein, a "domain capable of providing a stimulatory signal" means any domain that can provide a stimulatory signal, directly or indirectly, that enhances or increases the effectiveness of signal transduction mediated by the TCR complex to enhance at least one aspect of T cell signaling. A domain capable of providing a stimulatory signal can provide such a signal directly; for example, a domain capable of providing a stimulatory signal is a primary stimulatory domain or a co-stimulatory domain. Alternatively or additionally, a domain capable of providing a stimulatory signal can function indirectly. For example, the domain may be a scaffold that recruits stimulatory proteins to the TCR, or it may provide enzymatic activity, such as kinase activity, that functions to provide a stimulatory signal through downstream targets.

[0096] As used herein, a "domain capable of providing an inhibitory signal" means any domain that can provide an inhibitory signal, directly or indirectly, which inhibits or reduces the effectiveness of signal transduction mediated by the TCR complex. A domain capable of providing an inhibitory signal can completely or partially reduce or block at least one aspect of T cell signaling or function. A domain capable of providing an inhibitory signal can provide such a signal directly; for example, a domain capable of providing an inhibitory signal provides a primary inhibitory signal. Alternatively or additionally, a domain capable of providing a stimulatory signal can act indirectly. For example, the domain can recruit additional inhibitory proteins to the TCR or can provide enzymatic activity that acts through downstream targets to provide an inhibitory signal.

[0097] Scope: Throughout this disclosure, various aspects of the invention may be presented in the form of scope. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, it should be considered that the description of scope specifically discloses all possible sub-scopes and individual numerical values ​​within said scope. For example, a description of a scope (such as from 1 to 6) should be considered as having specifically disclosed sub-scopes (such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc.) and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95%-99% identity includes something having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96%-99%, 96%-98%, 96%-97%, 97%-99%, 97%-98%, and 98%-99% identity. This applies regardless of the width of the range.

[0098] Generally, "sequence identity" or "sequence homology" refers to the exact correspondence between the respective nucleotides or amino acids of two polynucleotide or polypeptide sequences. Typically, techniques used to determine sequence identity involve determining the nucleotide sequence of the polynucleotide and / or the amino acid sequence it encodes, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percentage of identity." The percentage of identity between two sequences (nucleic acid or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. The percentage of identity can also be determined, for example, by comparing sequence information using an advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health. The BLAST procedure is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). In short, the BLAST procedure defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The procedure can be used to determine the percentage of identity across the full length of the compared proteins. Default parameters are provided to optimize the search for short query sequences, for example, in the blastp procedure. The desired degree of sequence identity ranges from approximately 80% to 100% and integer values ​​in between. Typically, the percentage of identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0099] As used herein, a "subsequence" refers to a continuous amino acid or nucleotide of a certain length that forms part of the sequence described herein. When compared with the full-length sequence, a subsequence may be identical to a part of the full-length sequence, or may be less than 100% identical to a part of the full-length sequence it is compared with (e.g., 50% or 90% identical to the full-length sequence, etc.).

[0100] The term "exogenous" is used in this article to refer to any molecule originating outside the organism, including nucleic acids, proteins or peptides, small molecule compounds, etc. Conversely, the term "endogenous" refers to any molecule originating inside the organism (i.e., naturally produced by the organism).

[0101] When a polynucleotide is positioned to have a functional relationship with another polynucleotide, the polynucleotide is operatively linked to the other polynucleotide. For example, if a promoter or enhancer affects the transcription of a sequence, it is operatively linked to the coding sequence. When polynucleotides encoding a peptide and another peptide are operatively linked, the peptide is "operatively linked" to the other peptide, preferably they are in the same open reading frame.

[0102] A promoter is a DNA sequence required to turn a gene on or off. Promoters are located immediately upstream of and / or overlap with the transcription start site, and are typically between one hundred and several hundred base pairs in length.

[0103] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated herein by reference. In case of conflict, this application shall prevail, including any definitions herein. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or suggestion of any kind that they constitute valid prior art or form part of common general knowledge in any country of the world.

[0104] In this specification, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, to include fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated. When immediately preceding a number or value, the term “about” means that the number or value is within ±10%.

[0105] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were specifically and individually indicated for inclusion by reference.

[0106] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1)

[0107] This disclosure describes receptors having one or more domains from leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1 or LIR1). Many receptors, engineered cells, and their uses are considered herein.

[0108] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) (also known as leukocyte immunoglobulin-like receptor B1, along with ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2LIR-1, MIR-7, and PIR-B) is a member of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to subfamily B LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, one transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces negative signals of stimuli that suppress the immune response. LILRB1 is thought to regulate inflammatory responses and cytotoxicity and play a role in limiting autoreactivity. Multiple transcript variants encoding different isotypes of LILRB1 exist, all of which are considered within the scope of this disclosure.

[0109] In some embodiments of the receptor having one or more domains of LILRB1, one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence or subsequence of SEQ ID NO:1. In some embodiments, one or more domains of LILRB1 comprise an amino acid sequence identical to the sequence or subsequence of SEQ ID NO:1. In some embodiments, one or more domains of LILRB1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence or subsequence of SEQ ID NO:1. In some embodiments, one or more domains of LILRB1 consist of an amino acid sequence identical to the sequence or subsequence of SEQ ID NO:1.

[0110] In some embodiments of a receptor having one or more domains of LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence or subsequence of SEQ ID NO:34.

[0111] In some embodiments of a receptor having one or more domains of LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence identical to the sequence or subsequence of SEQ ID NO:34.

[0112] receptor

[0113] In various embodiments, a chimeric antigen receptor comprising a polypeptide is provided, wherein the polypeptide comprises one or more of the following: a LILRB1 hinge domain or a functional fragment or variant thereof; a LILRB1 transmembrane domain or a functional variant thereof; and a LILRB1 intracellular domain or an intracellular domain comprising at least one or at least two immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0114] Intracellular domains

[0115] This disclosure provides a chimeric antigen receptor comprising a polypeptide. In some embodiments, the polypeptide comprises an intracellular domain. In some embodiments, the intracellular domain is the LILRB1 intracellular domain or a functional variant thereof.

[0116] As used herein, "intracellular domain" refers to the cytoplasmic or intracellular domain of a protein, such as a receptor, that interacts with the cell interior and performs cytoplasmic functions. As used herein, "cytoplasmic function" refers to the function of a protein or protein complex performed in the cytoplasm of the cell. For example, intracellular signal transduction cascades are cytoplasmic functions.

[0117] As used herein, “immunoreceptor tyrosine inhibitory motif” or “ITIM” refers to a conserved amino acid sequence with a common sequence such as S / I / V / LxYxxI / V / L (SEQ ID NO:124), which is present in the cytoplasmic tails of many inhibitory receptors of the immune system. Following interaction between an inhibitory receptor possessing an ITIM and its ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as phosphotyrosine phosphatases SHP-1 and SHP-2, or inositol phosphatases known as SHIPs.

[0118] In some embodiments, the polypeptide comprises an intracellular domain containing at least one immunoreceptor tyrosine inhibitory motif (ITIM), at least two ITIMs, at least three ITIMs, at least four ITIMs, at least five ITIMs, or at least six ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0119] In some embodiments, the polypeptide comprises an intracellular domain containing at least one ITIM selected from the group consisting of: NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0120] In a further specific embodiment, the polypeptide comprises an intracellular domain containing at least two immune receptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0121] In some embodiments, the intracellular domain comprises two ITIM NLYAAV (SEQ ID NO:8) and VTYAEV (SEQ ID NO:9). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO:12. In some embodiments, the intracellular domain comprises or is substantially composed of a sequence identical to SEQ ID NO:12.

[0122] In some embodiments, the intracellular domain comprises two ITIM VTYAEV (SEQ ID NO: 9) and VTYAQL (SEQ ID NO: 10). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the intracellular domain comprises or is substantially composed of a sequence identical to or substantially composed of SEQ ID NO: 13.

[0123] In some embodiments, the intracellular domain comprises two ITIM VTYAQL (SEQ ID NO:10) and SIYATL (SEQ ID NO:11). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO:14. In some embodiments, the intracellular domain comprises or is substantially composed of the same sequence as SEQ ID NO:14.

[0124] In some embodiments, the intracellular domain comprises ITIM NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), and VTYAQL (SEQ ID NO:10). In some embodiments, the intracellular domain comprises at least 95% of the sequence identical to SEQ ID NO:15. In some embodiments, the intracellular domain comprises or is substantially composed of the same sequence as SEQ ID NO:15.

[0125] In some embodiments, the intracellular domain comprises ITIM VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). In some embodiments, the intracellular domain comprises at least 95% of the sequence identical to SEQ ID NO:16. In some embodiments, the intracellular domain comprises or is substantially composed of the same sequence as SEQ ID NO:16.

[0126] In some embodiments, the intracellular domain comprises ITIM NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). In some embodiments, the intracellular domain comprises at least 95% of the sequence identical to SEQ ID NO:17. In some embodiments, the intracellular domain comprises the same sequence as SEQ ID NO:17 or is substantially composed of it.

[0127] In some embodiments, the intracellular domain comprises at least 95% of the sequence identical to that of the LILRB1 intracellular domain (SEQ ID NO:7). In some embodiments, the intracellular domain comprises or is substantially composed of the same sequence as the LILRB1 intracellular domain (SEQ ID NO:7).

[0128] The LILRB1 intracellular domain or functional variant thereof disclosed herein may have at least one, at least two, at least four, at least five, at least six, at least seven, or at least eight ITIMs. In some embodiments, the LILRB1 intracellular domain or functional variant thereof has two, three, four, five, or six ITIMs.

[0129] In a particular embodiment, the polypeptide comprises an intracellular domain containing two, three, four, five, or six immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0130] In a particular embodiment, the polypeptide comprises an intracellular domain containing at least three immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0131] In a particular embodiment, the polypeptide comprises an intracellular domain containing three immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0132] In a particular embodiment, the polypeptide comprises an intracellular domain containing four immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0133] In a particular embodiment, the polypeptide comprises an intracellular domain containing five immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0134] In a particular embodiment, the polypeptide comprises an intracellular domain containing six immunoreceptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0135] In a particular embodiment, the polypeptide comprises an intracellular domain containing at least seven immune receptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0136] In some embodiments, the intracellular domain comprises a TCRα intracellular domain. In some embodiments, the intracellular domain comprises both a TCRα intracellular domain and a LILRB1 intracellular domain, as described herein. In some embodiments, the TCRα intracellular domain comprises Ser-Ser. In some embodiments, the TCRα intracellular domain is encoded by a TCCAGC sequence.

[0137] In some embodiments, the intracellular domain comprises a TCRβ intracellular domain. In some embodiments, the intracellular domain comprises a TCRβ intracellular domain and a LILRB1 intracellular domain, as described herein. In some embodiments, the TCRβ intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or identical to MAMVKRKDSR (SEQ ID NO: 94). In some embodiments, the TCRβ intracellular domain comprises or is substantially composed of MAMVKRKDSR (SEQ ID NO: 94). In some embodiments, the TCRβ intracellular domain is encoded by the following sequence: ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 95).

[0138] Transmembrane domain

[0139] This disclosure provides a chimeric antigen receptor comprising a polypeptide. In some embodiments, the polypeptide comprises a transmembrane domain. In some embodiments, the transmembrane domain is the LILRB1 transmembrane domain or a functional variant thereof.

[0140] As used in this article, a "transmembrane domain" refers to a protein domain that spans the cell membrane. Transmembrane domains are typically composed primarily of nonpolar amino acids and can cross the lipid bilayer one or more times. Transmembrane domains usually contain α-helices, whose conformation maximizes internal hydrogen bonding.

[0141] Transmembrane domains isolated from or derived from any source are envisioned within the scope of fusion proteins described in this disclosure.

[0142] In a particular embodiment, the polypeptide contains a LILRB1 transmembrane domain or a functional variant thereof.

[0143] In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:5. In some embodiments, the LILRB1 transmembrane domain comprises a sequence identical to SEQ ID NO:5. In some embodiments, the LILRB1 transmembrane domain is substantially composed of a sequence identical to SEQ ID NO:5.

[0144] In some embodiments of the chimeric antigen receptor described in this disclosure, the transmembrane domain is not the LILRB1 transmembrane domain. In some embodiments, the transmembrane domain is a domain associated with one of the other domains of the fusion protein, or is isolated from or derived from a protein identical to one of the other domains of the fusion protein.

[0145] The transmembrane domain can be derived from natural or recombinant sources. In the case of a natural source, the domain can originate from any membrane-binding or transmembrane protein. Exemplary transmembrane domains may include at least one or more of the following transmembrane regions: for example, the α, β, or ζ chain of a TCR, CD3δ, CD3ε, or CD3γ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0146] In some embodiments, the transmembrane domain comprises a TCRα transmembrane domain. In some embodiments, the TCRα transmembrane domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the same amino acid sequence as VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 96). In some embodiments, the TCRα transmembrane domain comprises or is substantially composed of VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 96). In some embodiments, the TCRα transmembrane domain is encoded by the following sequence: GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 97).

[0147] In some embodiments, the transmembrane domain comprises a TCRβ transmembrane domain. In some embodiments, the TCRβ transmembrane domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence TILYEILLGKATLYAVLVSALVL (SEQ ID NO:98). In some embodiments, the TCRβ transmembrane domain comprises or is substantially composed of TILYEILLGKATLYAVLVSALVL (SEQ ID NO:98). In some embodiments, the TCRβ transmembrane domain is encoded by the following sequence: ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO:99).

[0148] In some embodiments, the TCRα and / or TCRβ transmembrane domains contain one or more mutations that weaken or eliminate the interaction between the TCR and the TCRCD3 subunit. In some embodiments, the TCRα transmembrane domain contains the R253L mutation. In some embodiments, the TCRβ transmembrane domain contains the K288L mutation.

[0149] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% identity, or the same amino acid sequence as the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:100). In some embodiments, the CD28 transmembrane domain comprises or is substantially composed of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:100). In some embodiments, the CD28 transmembrane domain encodes a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 99% identity, or the same nucleotide sequence as the following:

[0150] TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 101).

[0151] In some embodiments, the transmembrane domain may be attached to an extracellular chimeric antigen receptor via a hinge (e.g., a hinge derived from a human protein), such as an antigen-binding domain or a ligand-binding domain. For example, in some embodiments, the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0152] Hinge structural domain

[0153] This disclosure provides a chimeric antigen receptor comprising a polypeptide. In some embodiments, the polypeptide comprises a hinge domain. In some embodiments, the hinge domain is a LILRB1 hinge domain or a functional variant thereof.

[0154] The LILRB1 protein has four immunoglobulin (Ig)-like domains, designated D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises the LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the same sequence as SEQ ID NO:18. In some embodiments, the LILRB1 D3D4 domain comprises or is substantially composed of SEQ ID NO:18.

[0155] In some embodiments, the polypeptide comprises a LILRB1 hinge domain or a functional fragment or variant thereof. In some embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:4, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises at least 95% of the same sequence as SEQ ID NO:4, SEQ ID NO:18, or SEQ ID NO:19.

[0156] In some embodiments, the LILRB1 hinge structure domain contains the same sequence as SEQ ID NO:4, SEQ ID NO:18, or SEQ ID NO:19.

[0157] In some embodiments, the LILRB1 hinge structure domain is substantially composed of the same sequence as SEQ ID NO:4, SEQ ID NO:18, or SEQ ID NO:19.

[0158] In some embodiments of the chimeric antigen receptor of this disclosure, the polypeptide comprises a hinge that is not isolated from or derived from LILRB1.

[0159] In some embodiments, the hinge is isolated from or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% identity, or the same amino acid sequence as TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:102). In some embodiments, the CD8α hinge comprises TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:102). In some embodiments, the CD8α hinge is substantially composed of TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:102). In some embodiments, the CD8α hinge is encoded by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 99% identity, or identical to the following sequence: accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO:103).

[0160] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or the same amino acid sequence as the sequence CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:104). In some embodiments, the CD28 hinge comprises or is substantially composed of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:104). In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 99% identity, or identical to the following sequence: tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO:105).

[0161] Combination of LILRB1 domains

[0162] In some embodiments, the chimeric antigen receptor of this disclosure comprises a polypeptide containing more than one LILRB1 domain or a functional equivalent thereof. For example, in some embodiments, the polypeptide comprises a LILRB1 transmembrane domain and an intracellular domain, or a LILRB1 hinge domain, a transmembrane domain, and an intracellular domain.

[0163] In certain embodiments, the polypeptide comprises a LILRB1 hinge domain or a functional fragment or variant thereof, and a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to SEQ ID NO:20. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:20. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO:20.

[0164] In another embodiment, the polypeptide comprises: a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11). In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two ITIMs, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0165] In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain and an intracellular domain. In some embodiments, the polypeptide comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to SEQ ID NO:21. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:21. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO:21.

[0166] In a preferred embodiment, the polypeptide comprises: a LILRB1 hinge domain or a functional fragment or variant thereof; a LILRB1 transmembrane domain or a functional variant thereof; and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immune receptor tyrosine inhibitory motifs (ITIMs), wherein each ITIM is independently selected from LYAAV (SEQ ID NO:8), VTYAE (SEQ ID NO:9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO:11).

[0167] In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to, or at least 99% identical to, SEQ ID NO:2 or SEQ ID NO:3, or identical to, SEQ ID NO:2 or SEQ ID NO:3.

[0168] In some embodiments, the polypeptide comprises a sequence that is at least 99% identical to, or at least 99% identical to, or identical to, SEQ ID NO:20.

[0169] In some embodiments, the polypeptide comprises a sequence that is at least 99% identical to, or at least 99% identical to, or identical to SEQ ID NO:21.

[0170] extracellular domain

[0171] This disclosure provides chimeric antigen receptors comprising peptides. In some embodiments, the peptide includes a ligand-binding domain, such as an antigen-binding domain. Suitable antigen-binding domains include, but are not limited to, antigen-binding domains derived from antibodies, antibody fragments, scFvs, antigen-binding domains derived from T-cell receptors, etc. All forms of antigen-binding domains known in the art are contemplated within the scope of this disclosure.

[0172] As used herein, “extracellular domain” refers to the extracellular portion of a protein. For example, the TCRα chain and TCRβ chain each contain an extracellular domain, which includes a constant region and a variable region involved in peptide-MHC recognition. An “extracellular domain” may also include a fusion domain, such as a fusion domain between an additional domain capable of binding to and targeting a specific antigen and an endogenous extracellular domain of a TCR subunit.

[0173] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that binds specifically to an antigen. Antibodies can be complete immunoglobulins or fragments thereof from polyclonal or monoclonal sources, and can be derived from natural or recombinant sources.

[0174] The term "antibody fragment" or "antibody-binding domain" refers to at least a portion of an antibody or a recombinant variant thereof, said at least a portion containing an antigen-binding domain (i.e., the antigen-determining variable region of the complete antibody) sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single-domain antibodies (abbreviated as "sdAb") (VL or VH), camel VHH domains, and multispecific antibodies formed from antibody fragments.

[0175] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously linked via a short flexible polypeptide linker and can be represented as a single polypeptide chain, and wherein the scFv retains the specificity of the complete antibody from which it is derived.

[0176] The “heavy chain variable region” or “VH” of an antibody (or, in the case of a single-domain antibody, such as a nanobody, “VHH”) refers to a segment of the heavy chain containing three CDRs inserted between flanking extensions called framework regions. These framework regions are typically more conserved than the CDRs and form a scaffold that supports the CDRs.

[0177] Unless otherwise specified, as used herein, scFv may have VL and VH variable regions in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0178] The term "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in the antibody molecule in their naturally occurring conformation. The carpa (“κ”) light chain and the lambda (“λ”) light chain refer to two main isotypes of antibody light chains.

[0179] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as antibodies expressed by phage or yeast expression systems. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding an antibody and said DNA molecule expressing an antibody protein, or by synthesizing an amino acid sequence specifying said antibody, wherein said DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequencing technologies available and well-known in the art.

[0180] In some embodiments, such as those in which the receptor comprises a first polypeptide and a second polypeptide, the antigen-binding domain is isolated from or derived from the extracellular domain of a T-cell receptor (TCR) or an antibody.

[0181] In a preferred embodiment, the polypeptide comprises an antigen-binding domain, for example, an antigen-binding domain other than that of the LILRB1 antigen-binding protein. An illustrative embodiment of a receptor having a single antigen-binding domain is depicted in Figure 1. Chimeric antigen receptors contemplated in this disclosure have two, three, four, or more antigen-binding domains. The antigen-binding domains may be provided on the same or different chains of the chimeric antigen receptor. In an embodiment, the chimeric antigen receptor is DARIC, as described, for example, in Leung et al. JCI Insight. 2019 June 6; 4(11):e124430; WO2015017214A1; and WO 2017156484A1.

[0182] In some embodiments, the receptor is an inhibitory chimeric antigen receptor (iCAR). Various methods and compositions applicable to the embodiments disclosed herein include those provided in US 2018 / 0044399A1; WO 2018148454A1; and WO2017087723A1, each of which is incorporated herein for all purposes.

[0183] In some implementations, the antigen-binding domain comprises a single-stranded variable fragment (scFv).

[0184] In some embodiments, the receptor comprises a second polypeptide. This disclosure provides a receptor having two polypeptides, each polypeptide having a portion of a ligand-binding domain (e.g., a homolog of heterodimeric LDB, such as a TCRα / β or Fab-based LBD) and each polypeptide having an intracellular domain, as depicted in Figure 2A. This disclosure further provides a receptor having two polypeptides, each polypeptide having a portion of a ligand-binding domain (e.g., a homolog of heterodimeric LDB, such as a TCRα / β or Fab-based LBD), and a portion of the ligand-binding domain is fused to a hinge or transmembrane domain, while another portion of the ligand-binding domain does not have an intracellular domain, as depicted in Figure 2B. Further variations include receptors where each polypeptide has a hinge domain, and where each polypeptide has both a hinge and a transmembrane domain. In some embodiments, the hinge domain is absent. In other embodiments, the hinge domain is a proximal extracellular region (MPER), such as the LILRB1 D3D4 domain. In any of the embodiments disclosed herein, the domains may be merged adjacent to each other, with joints between them.

[0185] In some embodiments, the first polypeptide comprises a first chain of the antibody, and the second polypeptide comprises a second chain of the antibody.

[0186] In some embodiments, the receptor comprises a Fab fragment of the antibody. In one embodiment, the antibody's heavy chain contains an antigen-binding fragment, and the second polypeptide comprises an antigen-binding fragment of the antibody's light chain. In another embodiment, the first polypeptide comprises an antigen-binding fragment of the antibody's light chain, and the second polypeptide comprises an antigen-binding fragment of the antibody's heavy chain.

[0187] In some embodiments, the first polypeptide comprises a first chain of a T-cell receptor (TCR), and the second polypeptide comprises a second chain of the TCR. In some embodiments, the receptor comprises an extracellular fragment of the T-cell receptor (TCR). In some embodiments, the first polypeptide comprises an antigen-binding fragment of the α-chain of the TCR, and the second polypeptide comprises an antigen-binding fragment of the β-chain of the TCR. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the β-chain of the TCR, and the second polypeptide comprises an antigen-binding fragment of the α-chain of the TCR.

[0188] In some implementations, the receptor comprises a single-chain TCR, such as, but not limited to, those disclosed in WO 2017091905A1.

[0189] Explanatory antigen-binding domain

[0190] Various single variable domains known in the art or disclosed herein are suitable for implementation. Such scFvs include (e.g., but not limited to) the following mouse and humanized scFv antibodies (complementarity-determining regions underlined) that bind HLA-A*02 in a peptide-independent manner:

[0191] C-001765

[0192] MMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPRT SGGGTKLEIKGGGGSGGGGSGGGGSGGQVQLQQSGPELVKPGASVRISCK ASGYTFTSYHIH WVKQRPGQGLEWIG WIYPGNVNTEYNEKFKGK ATLTADKSSSTAYMHLSSLTSEDSAVYFCAR EEITYAMDY WGQGTSVTVSSYG(SEQ ID NO:35); or

[0193] DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPRT SGGGTKLEIKGGGGSGGGGSGGGGSGGQVQLQQSGPELVKPGASVRISCK ASGYTFTSYHIH WVKQRPGQGLEWIG WIYPGNVNTEYNEKFKGK ATLTADKSSSTAYMHLSSLTSEDSAVYFCAR EEITYAMDYWGQGTSVTVSS (SEQ ID NO:125, the corresponding polynucleotide sequence is provided as SEQ ID NO:127)

[0194] C-002159

[0195] QLVQSGAEVKKPGSSVKVSCK ASGYTFTSYHIH WVRQAPGQGLEWMG WIYPGNVNTEYNEKFKGK ATITADKSTSTAYMELSSLRSEDTAVYYCAR EEITYAMDY WGQGTTVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGERATLSC ​ WYQQKPGQAPRLLIY ​ FSGSGSGTDFTLTISRLEPEDFAVYYC ​ FGGGTKVEIK (SEQ ID NO:36)

[0196] C-002160

[0197] QLVQSGAEVKKPGSSVKVSCK ​ WVRQAPGQGLEWMG ​ ATITADKSTSTAYMELSSLRSEDTAVYYCAR ​ WGQGTTVTVSSGGGGSGGGGSGGGGSGGDIVMTQTPLSLPVTPGEPASISC ​ WYLQKPGQSPQLLIY ​ FSGSGSGTDFTLKISRVEAEDVGVYYC ​ FGGGTKVEIK (SEQ ID NO:37)

[0198] C-002161

[0199] QLVESGGGLVKPGGSLRLSCA ​ WVRQAPGKGLEWVG ​ FTISRDDSKNTLYLQMNSLKTEDTAVYYCAR ​ WGQGTTVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITC ​ WYQQKPGKAPKLLIY ​ FSGSGSGTDFTLTISSLQPEDFATYYC ​FGGGTKVEIK(SEQ ID NO:38)

[0200] C-002162

[0201] QLVQSGAEVKKPGSSVKVSCK ​ WVRQAPGQGLEWIG ​ ATITADESTNTAYMELSSLRSEDTAVYYCAR ​ WGQGTLVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSTLSASVGDRVTITC ​ WYQQKPGKAPKLLIY ​ FSGSGSGTEFTLTISSLQPDDFATYYC ​ FGQGTKVEVK(SEQ ID NO:39)

[0202] C-002163

[0203] QLVQSGAEVKKPGSSVKVSCK ​ WVRQAPGQGLEWIG ​ ATLTADKSTNTAYMELSSLRSEDTAVYFCAR ​ WGQGTLVTVSSGGGGSGGGGSGGGGSGGDVQMTQSPSTLSASVGDRVTITC ​ WYQQKPGKAPKLLIY ​ FSGSGSGTEFTLTISSLQPDDFATYYC ​ FGQGTKVEVK(SEQ ID NO:40)

[0204] C-002164

[0205] QVQLQQSGPELVKPGASVKMSCKA ​ WVKQRPGQGLEWIG ​ KTTLTADKSSSTAYMLLSSLTSEDSAIYFCAR ​ WGQGTSVTVSSGGGGSGGGGSGGGGSGGDVLMTQTPLSLPVSLGDQVSISC ​ WYLQKPGQSPKLLIY ​ FSGSGSGTDFTLKISRVEAEDLGVYYC ​ FGGGTKLEIK(SEQ ID NO:41)

[0206] C-002165

[0207] QLQLQESGPGLVKPSETLSLTCTV ​ IQWIRQPPGKGLEWIG ​ RATISVDTSKNQFSLNLDSVSAADTAIYYCAR ​ WGKGSTVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITC ​ WYQQKPGKAPKLLIY ​ FSGSGSGTDFTFTISSLQPEDIATYYC ​ FGPGTKVDIK(SEQ ID NO:42)

[0208] C-002166

[0209] EVQLVQSGAELKKPGSSVKVSCKA ​ WVKQAPGQGLEWIG ​ KATLTVDKSTNTAYMELSSLRSEDTAVYYCAR ​ WGQGTLVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSTLSASVGDRVTITC ​ WYQQKPGKAPKLLIY ​ FSGSGSGTDFTLTISSLQPDDFATYYC ​ FGQGTKVEVK(SEQ ID NO:43)

[0210] C-002167

[0211] QVQLVQSGAEVKKPGSSVKVSCKA ​ WVRQAPGQGLEWMG ​ RVTITADKSTSTAYMELSSLRSEDTAVYYCAR ​ WGQGTTVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGERATLSC ​ WYQQKPGQAPRLLIY ​ FSGSGSGTDFTLTISRLEPEDFAVYYC ​ FGGGTKVEIK(SEQ ID NO:44)

[0212] C-002168

[0213] QVTLKQSGAEVKKPGSSVKVSCTA ​ WVRQAPGQGLEWLG ​ KVTITADKSMDTSFMELTSLTSEDTAVYYCAR ​ WGQGTLVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGERATLSC ​ WYQQKPGQAPRLLIS ​ FSGSGSGTDFTLTISRLEPEDFAVYYC ​ FGGGTKVEIK(SEQ ID NO:45)

[0214] C-002169

[0215] QVQLVQSGAEVKKPGASVKVSCKA ​ WVRQAPGQRLEWMG ​ KVTITRDTSASTAYMELSSLRSEDTAVYYCAR ​ WGQGTLVTVSSGGGGSGGGGSGGGGSGGDIVMTQTPLSLPVTPGEPASISC ​ WYLQKPGQSPQLLIY ​ FSGSGSGTDFTLKISRVEAEDVGVYYC ​ FGGGTKVEIK(SEQ ID NO:46)

[0216]

[0217] In some embodiments, the scFv contains a complementarity-determining region (CDR) of any one of SEQ ID NO:22-33. In some embodiments, the scFv contains a sequence that is at least 95% identical to any one of SEQ ID NO:22-33. In some embodiments, the scFv contains a sequence that is identical to any one of SEQ ID NO:22-33. In some embodiments, the heavy chain of the antibody contains a heavy chain CDR of any one of SEQ ID NO:25-27 or 31-33, and the light chain of the antibody contains a light chain CDR of any one of SEQ ID NO:22-24 or 28-30. In some embodiments, the heavy chain of the antibody contains a sequence that is at least 95% identical to the heavy chain portion of any one of SEQ ID NO:35-46 or 125, and wherein the light chain of the antibody contains a sequence that is at least 95% identical to the light chain portion of any one of SEQ ID NO:35-46 or 125. In some embodiments, the heavy chain contains all SEQ ID NO:25-27, and the light chain contains all SEQ ID NO:22-24. In some embodiments, the heavy chain contains all SEQ ID NO:31-33, and the light chain contains all SEQ ID NO:28-30.

[0218] In some embodiments, the heavy chain of the antibody comprises the same sequence as the heavy chain portion of any one of SEQ ID NO:35-46 or 125, and the light chain of the antibody comprises the same sequence as the light chain portion of any one of SEQ ID NO:35-46 or 125.

[0219] In some embodiments, the ScFv comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or identical to any one of SEQ ID NO:35-46 or 125. B- and T-lymphocyte attenuation factor (BTLA) domains

[0220] In some embodiments, the polypeptide comprises a B- and T-lymphocyte attenuation factor (BTLA) hinge domain, a transmembrane domain, an intracellular domain, or a functional variant, derivative, or combination thereof.

[0221] In some embodiments, the polypeptide comprises a BTLA intracellular domain. In some embodiments, the BTLA intracellular domain comprises the following sequence

[0222] RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 87). In some embodiments, the BTLA intracellular domain comprises SEQ ID NO: 87 or a sequence having at least 95% identity with it. In some embodiments, the BTLA intracellular domain is substantially composed of SEQ ID NO: 87.

[0223] In some embodiments, the BTLA transmembrane domain and intracellular domain comprise a sequence that is at least 95% identical to the following sequence: LLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 88). In some embodiments, the BTLA transmembrane domain and intracellular domain comprise or are substantially composed of the sequence SEQ ID NO: 88.

[0224] signal peptide

[0225] In some embodiments, the polypeptide comprises a signal peptide. For example, the polypeptide comprises a VK1 signal peptide. In some embodiments, the signal peptide is an N-terminal signal peptide. In some embodiments, the signal peptide comprises a sequence that is at least 95% identical to the sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signal peptide comprises the sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signal peptide is encoded by a sequence that is at least 95% identical to or identical to the following sequence: ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT (SEQ ID NO: 129).

[0226] antigen

[0227] Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides) can be used as an antigen against the LILRB1-based receptor described herein. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion thereof encoding a protein that elicits an immune response therefore encodes what is referred to herein as an "antigen." Furthermore, those skilled in the art will understand that an antigen does not need to be encoded solely by the full-length nucleotide sequence of a gene. Moreover, those skilled in the art will understand that an antigen does not need to be encoded by a "gene" at all. It will be apparent that antigens can be synthesized, or can be derived from biological samples, or can be macromolecules other than peptides. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0228] In some embodiments, the antigen-binding domain specifically binds to a target selected from etiolate receptors, ανββ integrins, TNF receptor superfamily member 17 (BCMA), CD276 molecule (B7-H3), natural killer cytotoxic receptor 3 ligand 1 (B7-H6), carbonic anhydrase 9 (CAIX), CD19 molecule (CD19), transmembrane 4-domain A1 (CD20), CD22 molecule (CD22), TNF receptor superfamily member 8 (CD30), CD33 molecule (CD33), CD37 molecule (CD37), CD44 molecule (CD44), CD44v6, CD44v7 / 8, CD70 molecule (CD70), interleukin-3 receptor subunit α (CD123), multiligand proteoglycan 1 (CD138), L1 cell adhesion molecule (CD171), CEA cell adhesion molecule (CEA), delta-like canonical Notch ligand 4 (DLL4), epithelial cell adhesion molecule (EGP- 2) Epithelial cell adhesion molecule (EGP-40), chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor receptor (EGFR), EGFR family (including ErbB2 (HER2)), EGFRvIII, epithelial cell adhesion molecule (EPCAM), EPH receptor A2 (EphA2), EpCAM, fibroblast activator protein α (FAP), folate receptor α (FBP), fetal acetylcholine receptor, coiled receptor 7 (Fzd7), diganglioside GD2 (GD2), ganglioside GD3 (GD3), phosphatidylinositol proteoglycan-3 (GPC3), trophoblast glycoprotein (h5T4), interleukin-11 receptor subunit α (IL-11R), interleukin-13 receptor subunit α2 (IL13R-a2), kinase insertion domain receptor (KDR), κ light chain, λ light chain, LeY, L1 cell adhesion molecule (L1 CAM), MAGE-A1, mesothelin, MHC presenting peptide, cell surface-associated mucin 1 (MUC1), cell surface-associated mucin 16 (MUC16), neural cell adhesion molecule 1 (NCAM), cytotoxic cell lectin-like receptor K1 (NKG2D) ligand, Notch1, Notch2 / 3, NY-ESO-1, PRAME nuclear receptor transcription factor (PRAME), prostate stem cell antigen (PSCA), folate hydrolase 1 (PSMA), survivin, TAG-72, TEM, telomerase reverse transcriptase (TERT), kinase insertion domain receptor (VEGFR2), and receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0229] In some embodiments, the antigen-binding domain specifically binds to a target selected from CD33, CD38, human leukocyte antigen (HLA), organ-specific antigen, blood-brain barrier-specific antigen, epithelial-mesenchymal transition (EMT) antigen, E-cadherin, cytokeratin, opioid-binding protein / cell adhesion molecule (OPCML), HYLA2, colorectal cancer deletion type (DCC), scaffold / stromal attachment zone binding protein 1 (SMAR1), cell surface carbohydrates, and mucin-type O-glycans.

[0230] In some implementations, the extracellular domain of the LILRB1-based receptor described herein includes an antigen-binding domain that is specific to antigens lost in the subject's cells through loss of heterozygosity.

[0231] As used in this article, “loss of heterozygosity (LOH)” refers to a genetic change that occurs frequently in cancer, in which one of the two alleles is missing, leaving a single monoallelic (hemiszygous) locus.

[0232] In some embodiments, the LILRB1-based receptor includes an antigen-binding domain specific to minor histocompatibility antigens (MiHAs). MiHAs are peptides derived from proteins containing nonsynonymous differences between alleles and represented by common HLA alleles. These nonsynonymous differences can be caused by SNPs, deletions, frameshift mutations, or insertions in the coding sequence of the gene encoding the MiHA. Exemplary MiHAs may have a length of approximately 9-12 amino acids and can bind to both MHC class I and MHC class II proteins. Binding of the TCR to an MHC complex representing the MiHA can activate T cells. The genetic and immunological characteristics of MiHAs are known to those skilled in the art, and specific MiHAs are described in PCT / US2020 / 045228, the contents of which are incorporated herein by reference.

[0233] In some implementations, the LILRB1-based receptor includes an antigen-binding domain that is specific to antigens lost in the subject's cancer cells via Y chromosome loss.

[0234] In some embodiments, the LILRB1-based receptor comprises an antigen-binding domain specific to HLA class I alleles. Major histocompatibility complex (MHC) class I is a protein complex that presents antigens to cells of the immune system, thereby triggering an immune response. The human leukocyte antigens (HLA) corresponding to MHC class I are HLA-A, HLA-B, and HLA-C. HLA-E is considered in the art to be a non-classical MHC class I molecule. In some embodiments, the LILR1-based receptor antigen comprises HLA class I alleles. In some embodiments, HLA class I alleles are lost in target cells such as cancer cells via loss of heterozygosity (LOH).

[0235] HLA-A is a group of human leukocyte antigens (HLA) encoded by the HLA-A locus, representing the major histocompatibility complex (MHC). HLA-A is one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer comprising a heavy α chain and a smaller β chain. The α chain is encoded by variants of HLA-A, while the β chain (β2-microglobulin) is invariant. Several thousand HLA-A variants exist, all of which fall within the scope of this disclosure.

[0236] In some embodiments, the LILRB1-based receptor includes an antigen-binding domain specific to the HLA-B allele. The HLA-B gene has many possible alterations (alleles). Hundreds of forms (alleles) of the HLA-B gene are known, each assigned a specific number (such as HLA-B27).

[0237] In some embodiments, the LILRB1-based receptor includes an antigen-binding domain specific to the HLA-C allele. HLA-C belongs to HLA class I heavy chain paralogs. These class I molecules are heterodimers composed of heavy and light chains (β-2 microglobulins).

[0238] In some implementations, the HLA class I alleles have widespread or ubiquitous RNA expression.

[0239] In some implementations, the HLA class I allele has a known or typically high minor allele frequency.

[0240] In some implementations, the HLA class I allele does not require a peptide-MHC antigen, for example, when the HLA class I allele is recognized by a pan-HLA ligand-binding domain.

[0241] In some embodiments, the LILRB1-based receptor includes an antigen-binding domain specific to the HLA-A allele. In some embodiments, the HLA-A allele comprises HLA-A*O2. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*O2 are suitable for embodiments and are described herein.

[0242] In some embodiments, the antigen-binding domain specifically binds to the HLA-A*02 antigen. In some embodiments, the antigen-binding domain specifically binds to the HLA-A*02 antigen in a peptide-independent manner.

[0243] Polynucleotides and carriers

[0244] In other respects, this disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a receptor of this disclosure. In some embodiments, the polynucleotide encodes one or more of the following: a LILRB1 hinge domain, a LILRB1 transmembrane domain, and a LILRB1 intracellular domain, or a functional derivative or fragment thereof.

[0245] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to any one of SEQ ID NO: 1-7 or 12-21. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to any one of SEQ ID NO: 47-71, 77-79, 89-92, 120, or 122. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to the heavy chain or light chain portion of any one of SEQ ID NO: 35-46 or 125. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to the heavy chain or light chain portion of any one of SEQ ID NO: 35, 39, 46, or 125. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is identical to the heavy chain or light chain portion of any one of SEQ ID NO: 35, 39, 46, or 125. In another aspect, this disclosure provides a vector comprising a polynucleotide encoding a receptor of this disclosure.

[0246] In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:121 or 123. In some embodiments, the polynucleotide comprises SEQ ID NO:121 or 123.

[0247] In some embodiments, the polynucleotide comprises sequences of the LILRB1 hinge, transmembrane, and intracellular domains. In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:126. In some embodiments, the polynucleotide comprises the sequence SEQ ID NO:126.

[0248] Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their dissemination in progeny cells. Lentiviral vectors have additional advantages over vectors derived from oncoretroviruses (such as murine leukemia virus) because they can transduce non-proliferating cells (such as hepatocytes). They also have the added advantage of low immunogenicity.

[0249] Expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operatively linking the nucleic acid encoding the receptor or a portion thereof to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0250] The polynucleotide encoding the receptor can be cloned into various types of vectors. For example, the polynucleotide can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0251] Furthermore, the expression vector can be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0252] Various virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject, either in vivo or in vitro. Various retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Various adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0253] In some embodiments, the vector contains a promoter. The vector may also contain additional regulatory elements. These additional regulatory elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these are located in a region 30–110 base pairs (bp) upstream of the start site, but it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, so that promoter function is preserved when the elements are flipped or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act synergistically or independently to activate transcription.

[0254] An example of a suitable promoter is the Immediate Early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is Elongation Growth Factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus Immediate Early promoter, Rous sarcoma virus promoter, and human gene promoters (such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch capable of turning on the expression of the polynucleotide sequence operatively linked to it when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0255] To assess receptor expression, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells attempting to be transfected or infected via a viral vector. In other respects, the selectable marker may be carried on a separate piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene may be side-linked with appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0256] Methods for introducing genes into cells and expressing them in the cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be transferred into host cells by physical, chemical, or biological means.

[0257] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0258] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0259] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane capsule).

[0260] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as DNA blotting and RNA blotting, RT-PCR and PCR; and "biochemical" assays, such as detecting the presence or absence of a specific peptide, for example by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of this invention.

[0261] engineered cells

[0262] In another aspect, this disclosure provides immune cells containing nucleic acid sequences or vectors encoding the receptor of this disclosure and / or expressing the receptor of this disclosure.

[0263] In embodiments, immune cell activation is reduced when cells come into contact with an antigen of the LILRB1-based receptor of this disclosure or with cells expressing said antigen on their surface. In embodiments, immune cell activation includes the expression of a gene operatively linked to an NFAT promoter. Immune cell activation and / or inhibition of activation can be measured by a variety of other methods known in the art. In some embodiments, the immune cells contain additional exogenous receptors, such as activator receptors, such as chimeric antigen receptors (CARs) or TCRs.

[0264] In the implementation scheme, the immune cells are T cells.

[0265] As used herein, the term "immune cell" refers to cells involved in the innate or adaptive (acquired) immune system. Exemplary innate immune cells include phagocytes, such as neutrophils, monocytes, and macrophages; natural killer (NK) cells; polymorphonuclear leukocytes, such as neutrophils, eosinophils, and basophils; and monocytes, such as monocytes, macrophages, and mast cells. Immune cells that play a role in acquired immunity include lymphocytes, such as T cells and B cells.

[0266] As used herein, "T cell" refers to a type of lymphocyte derived from bone marrow precursors that develop in the thymus. Several different types of T cells develop upon migration to the thymus, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, and stem memory T cells. Those skilled in the art can distinguish between different types of T cells based on the expression of markers. Methods for differentiating T cell types are readily apparent to those skilled in the art.

[0267] Methods for manufacturing engineered cells

[0268] In another aspect, this disclosure provides a method comprising introducing a polynucleotide of this disclosure into a cell using a vector of this disclosure. In one embodiment, the resulting cells express a LILRB1-based receptor encoded by the polynucleotide. In another embodiment, the cells are immune cells. In yet another embodiment, the immune cells are T cells.

[0269] Methods for transforming immune cell populations (such as T cells) using the vectors described in this disclosure will be readily apparent to those skilled in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi) according to the manufacturer's instructions. T cells can be cultured at a density of 1 x 10^6 cells / mL in X-Vivo 15 medium supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1:1 cell-to-bead ratio) and 300 units / mL IL-2 (Miltenyi). After 2 days, T cells can be transduced with viral vectors such as lentiviral vectors using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. Cells can then be cultured for an additional 5 days in IL-2 or other cytokines (such as a combination of IL-7 / 15 / 21) before enrichment. Methods for isolating and culturing other immune cell populations, such as B cells or other T cell populations, will be readily apparent to those skilled in the art. While this approach outlines a potential pathway, it should be noted that these methods are rapidly evolving. For example, excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to produce a cell population with >99% CD3+ high transduction.

[0270] Methods for activating and culturing populations of T cells containing receptors, polynucleotides, or vectors contained in this disclosure will be apparent to those skilled in the art.

[0271] Whether before or after genetic modification, T cells can typically be activated and expanded using methods described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; 10040846; and U.S. Patent Application No. 2006 / 0121005.

[0272] In some embodiments, the T cells of this disclosure are expanded and activated in vitro. Typically, the T cells of this disclosure are expanded in vitro by contact with a surface coated with an agent that stimulates CD3 / TCR complex-related signaling and a ligand that stimulates co-stimulatory molecules on the T cell surface. Specifically, the T cell population can be stimulated, as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of helper molecules on the T cell surface, a ligand binding to said helper molecule is used. For example, the T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Anti-CD3 and anti-CD28 antibodies can be used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besan (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0273] In some embodiments, primary and co-stimulatory signals for T cells can be provided in different ways. For example, the agent providing each signal can be in solution or conjugated to a surface. When conjugated to a surface, the agent can be conjugated to the same surface (i.e., in a “cis” configuration) or separate surfaces (i.e., in a “trans” configuration). Alternatively, one agent can be conjugated to a surface while the other is in solution. In some embodiments, the agent providing the co-stimulatory signal is bound to the cell surface, and the agent providing the primary activation signal is in solution or conjugated to a surface. In some embodiments, both agents can be in solution. In another embodiment, the agent can be in a soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind to the agent. In this regard, regarding artificial antigen-presenting cells (aAPCs) contemplated in this invention for activating and amplifying T cells, see, for example, U.S. Patent Application Publications 20040101519 and 20060034810.

[0274] In some embodiments, the two agents are immobilized on beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). For example, the agent providing the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or its antigen-binding fragment; and both agents are co-immobilized on the same bead at equal molecular weights. In one embodiment, each antibody is used to bind to the beads for CD4+ T cell expansion and T cell growth in a 1:1 ratio. In some embodiments, the CD3:CD28 antibody ratio bound to the beads ranges from 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibody binds to the particles compared to the anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than 1. In some embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1.

[0275] T cells or other target cells can be stimulated using particle-to-cell ratios of 1:500 to 500:1 and all integer values ​​therebetween. As will be readily apparent to those skilled in the art, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads can bind only a few cells, while larger beads can bind many cells. In some embodiments, a cell-to-particle ratio ranging from 1:100 to 100:1 and all integer values ​​therebetween is used, and in other embodiments, the ratio, including 1:9 to 9:1 and all integer values ​​therebetween, can also be used to stimulate T cells. In some embodiments, a 1:1 cell-to-bead ratio is used. Those skilled in the art will understand that many other ratios can be applied to this invention. In particular, the ratio will vary depending on the particle size and the size and type of the cells.

[0276] In another embodiment, cells such as T cells are combined with drug-coated beads, followed by separation of the beads and cells, and then cell culture. In an alternative embodiment, the drug-coated beads and cells are not separated before culture, but are cultured together. In another embodiment, the beads and cells are first concentrated by applying a force such as magnetism, resulting in increased binding of cell surface markers, thereby inducing cell stimulation.

[0277] For example, cell surface proteins can be linked by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached to contact T cells. In one embodiment, cells (e.g., CD4+ T cells) and beads (e.g., DYNABEADS CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer solution. Similarly, those skilled in the art will readily appreciate that any cell concentration can be used. In some embodiments, it may be desirable to significantly reduce the volume in which the particles are mixed with the cells (i.e., increase the cell concentration) to ensure maximum cell-particle contact. For example, in one embodiment, a concentration of approximately 2 billion cells / ml is used. In another embodiment, a concentration greater than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In another embodiment, a concentration of 125 million or 150 million cells / ml can be used. In some embodiments, a concentration of 1x10⁻⁶ cells / ml is used. 6 Cells cultured at a density of cells / mL.

[0278] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, beads and T cells are cultured together for 2–3 days. Suitable conditions for T cell culture include appropriate culture media (e.g., Limit Essentials Medium or RPMI 1640 or X-vivo 15 (Lonza)) that may contain factors required for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to a skilled craftsman for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma protein powder, and reducing agents (such as N-acetylcysteine ​​and 2-mercaptoethanol). The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or one or more cytokines in sufficient quantities for T cell growth and expansion. In some embodiments, the culture medium comprises X-VIVO-15 medium supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep), and 300 units / ml IL-2 (Miltenyi).

[0279] Maintain T cells under conditions that support their growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0280] In some embodiments, the T cells of the recipient comprising this disclosure are autologous. The T cell source is obtained from the subject prior to amplification and genetic modification. Immune cells such as T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T cell lines available in the art can be used. In some embodiments of the invention, any number of techniques known to those skilled in the art (such as Ficoll) can be used. TM T cells were obtained from blood units collected from the subjects (isolation).

[0281] In some embodiments, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected via apheresis may be washed to remove plasma fractions and placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution is calcium-deficient and may be magnesium-deficient, or may lack many (if not all) divalent cations. As will be readily appreciated by those skilled in the art, the washing step can be performed by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, unwanted components of the ablation sample can be removed, and the cells can be directly resuspended in the culture medium.

[0282] In some implementations, this is achieved by lysing red blood cells and depleting monocytes, for example, via PERCOLL. TM Immune cells, such as T cells, are isolated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugation. Specific subsets of immune cells, such as T cells, B cells, or CD4+ T cells, can be further isolated using positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubating with anti-CD4 conjugated beads for a period sufficient to allow for positive selection of the desired T cells.

[0283] Enriching immune cell populations, such as T cell populations, through negative selection can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. One approach is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells through negative selection, a cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0284] To separate a desired population of immune cells by positive or negative selection, the concentrations of cells and surfaces (e.g., particles, such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of the beads mixed with the cells (i.e., increase the cell concentration) to ensure maximum contact between the cells and the beads.

[0285] In some implementations, cells can be incubated on a rotator at varying speeds for varying durations at 2°C-10°C or at room temperature.

[0286] PBMCs used for stimulation of T cells or from which immune cells such as T cells are isolated can also be frozen after the washing step. Not wanting to be limited by theory, freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes from the cell population and, to some extent, monocytes. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be available in this case, one approach involves using PBS containing 20% ​​DMSO and 8% human serum albumin; or a medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO; or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, then freezing the cells to -80°C at a rate of 1° / min and storing them in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled freezing at -20°C or immediately in liquid nitrogen.

[0287] Measurement of signal transduction

[0288] In some embodiments, immune cell activation is reduced when immune cells come into contact with an antigen corresponding to a LILRB1-based receptor of this disclosure or with cells expressing said antigen on their surface. In some embodiments, immune cell activation includes the expression of a gene operatively linked to an NFAT promoter. Nuclear factors (NFATs) of activated T cells are a family of transcription factors that have shown importance in immune responses. The NFAT transcription factor family consists of five members: NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFATs play a role in regulating inflammation.

[0289] As used herein, the NFAT promoter is a promoter that is regulated (i.e., activated or inhibited) when NFAT is expressed in the cell. NFAT target promoters are described in Badran, BM et al. (2002) J. Biological Chemistry Vol. 277: 47136-47148 and contain NFAT-shared sequences such as GGAAA.

[0290] Methods for assessing the effect of receptor activation on gene expression are known in the art and include the use of reporter genes whose expression can be quantified. Reporter genes are used to identify potentially transfected or transduced cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue and encodes a polypeptide whose expression manifests as some readily detectable property (e.g., enzyme activity). Reporter gene expression is measured at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al. 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared or commercially available using known techniques. Typically, constructs exhibiting the highest level of reporter gene expression with a minimum 5' flanking region are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of drugs to regulate promoter-driven transcription. In an exemplary embodiment, an NFAT promoter operatively linked to a reporter gene is used to evaluate the expression of receptors for NFAT signaling in this disclosure.

[0291] ​

[0292] This disclosure provides pharmaceutical compositions comprising immune cells containing a LILRB1-based receptor of this disclosure, a pharmaceutically acceptable diluent, a carrier, or an excipient.

[0293] Such compositions may contain buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; and preservatives.

[0294] Methods of treating diseases

[0295] This document provides a method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of immune cells containing a LILRB1-based receptor as described herein. In some embodiments, the immune cells further comprise an activator receptor, such as an activator CAR or TCR.

[0296] Additional methods of treating subjects and combinations of activator receptors with inhibitory receptors are described in PCT / US2020 / 045228, the contents of which are incorporated herein by reference in their entirety.

[0297] In some embodiments, the subject in need has cancer. In some embodiments, the method of treating the subject includes administering to the subject a plurality of immune cells comprising the LILRB1 receptor of this disclosure. In some embodiments, the plurality of immune cells further comprises an activator receptor, such as a CAR or TCR. In some embodiments, the CAR or TCR comprises an antigen-binding domain specific to a tumor antigen. The activator receptor specific to a cancer antigen may comprise an antigen-binding domain isolated from or derived from any antibody or antigen-binding domain known in the art, including but not limited to urelumab, utomilumab, oleclumab, naptumomab, ascrinvacumab, tacatuzumab, nesvacumab, and vanucizumab. umab), belimumab, tabalumab, tibulizumab, belantamab, igovozumab, oregovomab, sofituzumab, mogamulizumab, talacotuzumab, tavolimab, vonlerolizumab, ipilimumab, dutto Duvortuxizumab, Clonatuximab, Denintuzumab, Inebilizumab, Loncastuximab, Tapitumomab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Ofamumab, Rituximab imab), tosimomumab, veltuzumab, samalizumab, bectumomab, epazolizumab, inotuzumab, moxetumomab, pinatuzumab, gomiliximab, lumiliximab, camidanlumab, basiliximab, enomumab, daklizumab,Varlilumab, Enoblituzumab, Omburtamab, Bentuximab, Iratumumab, Gelatuzumab, Lintuzumab, Vadastuximab, Lilotomab, Otlertuzumab, Tetulomab, Daremumab, Isatuximab, Bivatuzumab, Abituzumab, Intuximab (Intetumumab), Lovotuzumab, Itolizumab, Cusatuzumab, Vorsetuzumab, Milatuzumab, Polotuzumab, Iladatuzumab, Galiximab, Altumomab, Asimomab, Labetuzumab, Cibisatamab, Zolbetuximab, Latozumab Monoclonal antibodies (lacnotuzumab), cabiralizumab, emactuzumab, gimsilumab, lenzilumab, otilimab, mavrilimumab, trimethoprim, ulocuplumab, tepoditamab, rovalpituzumab, demcizumab, drozitumab, parsatuzumab The following are listed: cetuximab, depatuxizumab, futuximab, imgatuzumab, laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab, zalutumumab, modotuximab, amivantamab, tomuzotuximab, and losatuxizumab.Adacumumab, citatuzumab, ezetoxumab, oportuzumab, solitomab, tucotuzumab, caputuzumab, ifabotuzumab, duligotuzumab, elgemtumab, lumretuzumab, patrituzumab, seribantumab, zenocutuzumab Aprutumab, bemarituzumab, vantictumab, dinutuximab, ecromoxicillin, mitumomab, codrituzumab, glembatumumab, zatuximab, ertumaxomab, margetuximab, timigutuzumab, gancota (mab), pertuzumab, trastuzumab, ficlatuzumab, rilotumumab, telisotuzumab, emibetuzumab, cetuzumab, dalotuzumab, figitumumab, ganitumab, robatumumab, teprotumumab, flotetuzumab, bermekimab, cergutuzumab Volociximab, etaracizumab, relatlimab, carlumab, amatuximab, clivatuzumab, gatipotuzumab, pemtumomab, cantuzumab, pankomab, racotumomab, brontictuzumab, tarextumabm, vesencumabCamrelizumab, cetrelimab, nivolumab, pembrolizumab, pildizumab, cemiplimab, spartalizumab, atelizumab, averuzumab, durvalumab, cetuzumab, tenatumomab, fresolimumab, brolucizumab, bevacizumab, ranibizumab, varisacumab, faricimab, icrucumab, alacizumab, and ramucirumab.

[0298] In some embodiments, the LILRB1-based receptor of this disclosure includes an antigen-binding domain specific to an antigen lost in cancer cells through loss of heterozygosity. In some embodiments, the antigen is a minor histocompatibility antigen (MiHA). In some embodiments, the antigen is an HLA class I allele. In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA class I allele comprises HLA-E. In some embodiments, the HLA class I allele is the HLA-A*02 allele. In some embodiments, the antigen is not expressed in target cells due to Y chromosome loss. In some embodiments, the receptor-specific antigen for LILRB1 is the HLA-A*02 antigen.

[0299] In some implementations, the subject in need has cancer. Cancer is a disease in which abnormal cells divide uncontrollably and spread to nearby tissues. In some implementations, the cancer includes liquid tumors or solid tumors. Exemplary liquid tumors include leukemia and lymphoma. Other cancers that are liquid tumors can be, for example, those that occur in the blood, bone marrow, and lymph nodes, and can include, for example, leukemia, myeloid leukemia, lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, melanoma, and multiple myeloma. Leukemia includes, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and hairy cell leukemia. Exemplary solid tumors include sarcomas and carcinomas. Cancer can actually occur in organs within the body, including the blood, bone marrow, lungs, breast, colon, bone, central nervous system, pancreas, prostate, and ovaries. Other cancers that are solid tumors include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell carcinoma of the skin, kidney cancer, head and neck cancer, laryngeal cancer, squamous cell carcinoma forming on the moist mucous membrane lining of the nose, mouth, and larynx, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the condition treated by the methods described herein is a metastasis of the following cells: melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, stomach cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, kidney cancer cells, head and neck cancer cells, laryngeal cancer cells, squamous cell carcinoma cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells.

[0300] Any cancer in which multiple cancer cells express a first activator ligand but not a second inhibitor ligand is contemplated within the scope of this disclosure. For example, CEA-positive cancers that can be treated using the methods described herein include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung adenocarcinoma, head and neck cancer, diffuse large B-cell carcinoma, or acute myeloid leukemia.

[0301] Cancer treatment can lead to a reduction in tumor size. This reduction in tumor size can also be referred to as "tumor regression." Preferably, after treatment, the tumor size decreases by 5% or more relative to its pre-treatment size; more preferably, by 10% or more; even more preferably, by 20% or more; more preferably, by 30% or more; even more preferably, by 40% or more; and even more preferably, by 50% or more; and most preferably, by a reduction of greater than 75% or more. The size of the tumor can be measured by any reproducible means. The size of the tumor can be measured as its diameter.

[0302] Cancer treatment can lead to a reduction in tumor volume. Preferably, after treatment, the tumor volume is reduced by 5% or more relative to its pre-treatment size; more preferably, the tumor volume is reduced by 10% or more; even more preferably, by 20% or more; more preferably, by 30% or more; even more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by more than 75% or more. Tumor volume can be measured by any reproducible means.

[0303] Cancer treatment results in a reduction in the number of tumors. Preferably, after treatment, the number of tumors is reduced by 5% or more compared to the number before treatment; more preferably, the number of tumors is reduced by 10% or more; even more preferably, by 20% or more; more preferably, by 30% or more; even more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by more than 75%. The number of tumors can be measured by any reproducible means of measurement. The number of tumors can be measured by counting tumors that are visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0304] Cancer treatment can lead to a reduction in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or more compared to the number before treatment; more preferably, the number of metastatic lesions is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by more than 75%. The number of metastatic lesions can be measured by any reproducible means of measurement. The number of metastatic lesions can be measured by counting metastatic lesions visible to the naked eye or visible at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0305] Cancer treatment can lead to an increase in the mean survival time of the treated subject population compared to a population that only receives the carrier. Preferably, the increase in mean survival time is greater than 30 days; more preferably, greater than 60 days; even more preferably, greater than 90 days; and most preferably, greater than 120 days. The increase in mean survival time of the population can be measured by any reproducible means. The increase in mean survival time of the population can be measured, for example, by calculating the length of the mean survival time of the population after treatment with the active compound has begun. The increase in mean survival time of the population can also be measured, for example, by calculating the length of the mean survival time of the population after the completion of the first round of treatment with the active compound.

[0306] Compared to an untreated group of subjects, cancer treatment can lead to an increase in the mean survival time of the treated group. Preferably, the increase in mean survival time is greater than 30 days; more preferably, greater than 60 days; even more preferably, greater than 90 days; and most preferably, greater than 120 days. The increase in mean survival time of the population can be measured by any reproducible means. The increase in mean survival time of the population can be measured, for example, by calculating the length of mean survival time of the population after treatment with the active compound has begun. The increase in mean survival time of the population can also be measured, for example, by calculating the length of mean survival time of the population after the completion of the first round of treatment with the active compound.

[0307] Cancer treatment can lead to an increase in the mean survival time of the treated subject population compared to a population receiving monotherapy with a compound not of the present invention or a pharmaceutically acceptable salt, prodrug, metabolite, analogue, or derivative thereof. Preferably, the increase in mean survival time is greater than 30 days; more preferably, greater than 60 days; even more preferably, greater than 90 days; and most preferably, greater than 120 days. The increase in mean survival time of the population can be measured by any reproducible means. The increase in mean survival time of the population can be measured, for example, by calculating the mean survival time of the population after treatment with the active compound has begun. The increase in mean survival time of the population can also be measured, for example, by calculating the mean survival time of the population after the completion of the first round of treatment with the active compound.

[0308] Cancer treatment can lead to a lower mortality rate in the treated subject population compared to a population receiving only the carrier. Cancer treatment can lead to a lower mortality rate in the treated subject population compared to an untreated population. Cancer treatment can lead to a lower mortality rate in the treated subject population compared to a population receiving monotherapy with a drug that uses a pharmaceutically acceptable salt, prodrug, metabolite, analogue, or derivative of a compound not of the present invention or its 1a derivative. Preferably, the mortality rate reduction is greater than 2%; more preferably, greater than 5%; even more preferably, greater than 10%; and most preferably, greater than 25%. The reduction in mortality rate in the treated subject population can be measured by any reproducible means. The reduction in population mortality rate can be measured, for example, by calculating the average number of disease-related deaths per unit time in the population after treatment with the active compound has begun. The reduction in population mortality rate can also be measured, for example, by calculating the average number of disease-related deaths per unit time in the population after the completion of the first round of treatment with the active compound.

[0309] Cancer treatment can lead to a reduction in tumor growth rate. Preferably, after treatment, the tumor growth rate is reduced by at least 5% relative to the pre-treatment value; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The tumor growth rate can be measured by any reproducible means. The tumor growth rate can be measured based on the change in tumor diameter per unit time.

[0310] Cancer treatment can lead to a reduction in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, less than 10%; even more preferably, less than 20%; more preferably, less than 30%; even more preferably, less than 40%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth can be measured by any reproducible means. Tumor regrowth can be measured, for example, by measuring the increase in tumor diameter after previous tumor shrinkage following treatment. A reduction in tumor regrowth is indicated by the absence of tumor recurrence after treatment cessation.

[0311] Treatment or prevention of cell proliferation disorders can lead to a reduction in the cell proliferation rate. Preferably, after treatment, the cell proliferation rate is reduced by at least 5%; more preferably, at least 10%; more preferably, at least 20%; more preferably, at least 30%; more preferably, at least 40%; more preferably, at least 50%; even more preferably, at least 50%; and most preferably, at least 75%. The cell proliferation rate can be measured by any reproducible measurement method. The cell proliferation rate can be measured, for example, by measuring the number of dividing cells per unit time in a tissue sample.

[0312] Treatment or prevention of cell proliferation disorders can lead to a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, at least 10%; more preferably, at least 20%; more preferably, at least 30%; more preferably, at least 40%; more preferably, at least 50%; even more preferably, at least 50%; and most preferably, at least 75%. The proportion of proliferating cells can be measured by any reproducible measurement method. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells in a tissue sample relative to the number of undivided cells. The proportion of proliferating cells can be equivalent to the mitotic index.

[0313] Treatment or prevention of cell proliferation disorders can lead to a reduction in the size of the region or area of ​​cell proliferation. Preferably, after treatment, the size of the region or area of ​​cell proliferation is reduced by at least 5% relative to its pre-treatment size; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The size of the region or area of ​​cell proliferation can be measured by any reproducible measurement method. The size of the region or area of ​​cell proliferation can be measured as the diameter or width of the region or area of ​​cell proliferation.

[0314] Treatment or prevention of cell proliferation disorders can result in a reduction in the number or proportion of cells with abnormal appearance or morphology. Preferably, after treatment, the number of cells with abnormal morphology is reduced by at least 5% relative to their pre-treatment size; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The abnormal cell appearance or morphology can be measured by any reproducible measurement method. Abnormal cell morphology can be measured by microscopic examination, for example, using an inverted tissue culture microscope. Abnormal cell morphology can take the form of nuclear pleomorphism.

[0315] reagent kits and products

[0316] This disclosure provides kits and articles comprising polynucleotides and vectors encoding the receptors described herein. In some embodiments, the kits include articles such as vials, syringes, and instructions for use.

[0317] In some embodiments, the kit comprises a polynucleotide or vector containing a sequence encoding one or more chimeric antigen receptors of this disclosure. For example, the polynucleotide or vector contains one or more sequences of the LILRB1 domain as described herein.

[0318] In some embodiments, the kit comprises a plurality of immune cells, which contain chimeric antigen receptors as described herein. In some embodiments, the plurality of immune cells comprises a plurality of T cells.

[0319] Polypeptide sequences of elements of an illustrative chimeric antigen receptor

[0320]

[0321]

[0322] Explanatory chimeric antigen receptor polypeptide sequence

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] This specification describes numerous exemplary configurations, methods, parameters, etc. However, it should be understood that such description is not intended to be a limitation on the scope of this disclosure, but is intended to be provided as a description of exemplary embodiments.

[0331] Example

[0332] Example 1: Comparison of LILRB1-based inhibitory scFv-CAR with PD-1, KIR3DL2, and KIR3DL3

[0333] A reactive inhibitory construct of NY-ESO-1 was generated by fusing the NY-ESO-1 ligand-binding scFv domain (C-266) with the receptor domain, wherein the receptor domain includes a hinge, transmembrane region, and / or intracellular domain of the leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1); cytotoxic cell immunoglobulin-like receptor 3DL2 (KIR3DL2); cytotoxic cell immunoglobulin-like receptor 3DL3 (KIR3DL3); and / or the hinge, transmembrane region, and / or intracellular domain of the B- and T-lymphocyte attenuating factor BTLA. Gene fragments were assembled using Golden Gate cloning and inserted downstream of the eF1α promoter contained in the lentiviral expression plasmid (pLenti1).

[0334] As reporter cells, Jurkat cells encoding the NFAT luciferase reporter were maintained in RPMI medium supplemented with 10% FBS, 1% Pen / Strep, and 0.4 mg / mL G418 / genimycin. T2 cells (ATCC CLR-1992) were maintained in IMDM medium + 20% FBS and 1% Pen / Strep. For each construct to be evaluated, Jurkat cells were transfected using the following settings, according to the manufacturer's protocol: 3 pulses, 1500 V, 10 msec, via a 100 μL format Neon electroporation system (Thermo Fisher).

[0335] Co-transfection was performed using 3 μg of activated CAR construct (C-563) or TCR construct (CT-139) and 3 μg of inactivated CAR construct or empty vector (pLenti0) per million cells, and the cells were recovered in RPMI medium supplemented with 20% heat-inactivated FBS and 0.1% Pen / Strep.

[0336] The peptides (MAGE-A3(FLWGPRALV)(SEQ ID NO:106) and modified NY-ESO-1(SLLMWITQV)(SEQ ID NO:107)) were synthesized via Genscript. The activating peptide MAGE-A3 was serially diluted 5-fold starting at 50 μM. The inactivating peptide NY-ESO-1 was diluted to 50 μM, 5 μM, 0.5 μM, or 0.05 μM, and these constant amounts were added to the serially diluted MAGE-A3 buffer and subsequently loaded onto 10,000 T2 cells in 15 μL of RPMI supplemented with 1% BSA and 0.1% Pen / Strep. Jurkat cells were incubated in 384-well, low-friction, white, flat-bottomed polystyrene (TC) microplates. The next day, 10,000 Jurkat cells were resuspended in 15 μL of RPMI supplemented with 10% heat-inactivated FBS and 0.1% Pen / Strep, added to the peptide-loaded T2 cells, and co-cultured for 6 hours. Jurkat luminescence was evaluated using a one-step luciferase assay system (BPS Bioscience). Assays were performed in duplicate.

[0337] Comparison of LILRB1 with PD-1, KIR3DL2, and KIR3DL3

[0338] Figure 4 illustrates the testing of the constructs provided in Table 1. The data show that scFv-LILRB1 inhibits CAR activation in a trans-regulatory manner. Because constructs with the LILRB1 domain exhibited inhibition of signal transduction at higher concentrations of the MAGE-A3 activator peptide, the data suggest that CARs with hinges, transmembrane domains, and intracellular domains derived from LILRB1 are superior to CARs derived from the same domains from PD-1, KIR3DL2, or KIR3DL3.

[0339] Table 1

[0340]

[0341] Inhibition induced by LILRB1-based CARs requires antigen recognition via its ligand-binding domain.

[0342] Figure 5 illustrates the tests performed on the selected constructs from Table 1. The data show that LILRB1 inhibits signal transduction in a dose-dependent manner. The response to the activating peptide MAGE-A3 shifts downwards with increasing concentrations of the inhibitory peptide NY-ESO-1. This indicates that the inhibitory effect of LILRB1-based CARs depends on antigen binding, and that the ligand-binding domain is specific to the antigen.

[0343] LILRB1 CAR inhibits signal transduction via T cell receptors (TCRs).

[0344] Figure 6 illustrates the testing of selected constructs from Table 1, specifically the TCR specific to the activating peptide MAGE-A3, rather than the CAR used in previous experiments. The LILRB1-based inhibitory CAR inhibits TCR-mediated signaling in a dose-dependent manner.

[0345] LIRLB1 CAR inhibition is maintained when two of the four natural ITIMs are present.

[0346] Figure 7 illustrates the testing performed on the intracellular domain of LILRB1 with an inactivating mutation in the ITIM motif of LILRB1. The ITIM indicated by the asterisks in Table 2 was inactivated using a tyrosine mutation to phenylalanine (Y→F).

[0347] Table 2

[0348]

[0349] Inactivation of all four ITIMs resulted in a nonfunctional repressive CAR (C2182). Inactivation of only two of the four ITIMs preserved the inhibitory function of the CAR at the tested concentrations (C1760 and C1762). Inactivation of all four ITIMs (C1759) indicates that ITIMs are essential for repressive function. When all four ITIMs were mutated, the molecule lost its repressive function. When only two of the four ITIMs were mutated, repressive activity was preserved.

[0350] Figure 8 illustrates the tests performed on different combinations of LILRB1 ITIMs corresponding to the intracellular domains of native LILRB1. As shown in Table 3, CARs with a total of four or six copies of the third and fourth ITIMs of LILRB1 achieved inhibitory activity comparable to that of the native LILRB1 intracellular domains. Inhibitory activity was also observed when only one copy of each of the third and fourth ITIMs was used (C2179), or when two copies of each ITIM were used (C2180), or when multiple copies were used (C2302 or C2180).

[0351] Table 3

[0352]

[0353] Example 2: LILRB1 hinge and transmembrane domain enhance the inhibitory activity of BTLA-based inhibitory CARs.

[0354] BTLA-based CARs and LILRB1 / BTLA-based CARs suppress signal transduction via NFAT.

[0355] B- and T-lymphocyte attenuating factor (BTLA), also known as CD272 (differentiation cluster 272), interacts with the B7 homolog B7H4. Unlike CTLA-4 and PD-1, BTLA is also a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14). Inhibitory signaling of BTLA occurs in response to the binding of B7H4 or TNFRSF14.

[0356] The full-length BTLA protein was cloned into a construct (C2220) containing an extracellular scFv domain, resulting in constructs (C2219) replacing the extracellular domain of BTLA with the extracellular domain of LILRB1, and constructs (C2218) replacing the extracellular and transmembrane domains of BTLA with the extracellular and transmembrane domains of LILRB1. These constructs were then tested, as shown in Table 4 and Figure 9. The LILRB1-BTLA fusion exhibited inhibitory signaling comparable to that of a LILRB1-based CAR.

[0357] Table 4

[0358]

[0359] Example 3: Comparison of LILRB1 hinge and transmembrane with CD8 or CD28

[0360] LILRB1-based CARs with LILRB1 hinges and transmembrane regions outperform CD8 hinges and CD28 transmembrane regions.

[0361] The LILRB1-based CARs are compared with CARs that have the LILRB1 intracellular domain but also have the CD8 hinge and CD28 transmembrane region, as shown in Table 5 and Figure 10.

[0362] Table 5

[0363]

[0364] The results produced by the LILRB1 hinge and transmembrane region were surprisingly superior to those of the construct with the CD8 hinge and CD28 transmembrane region. Emin was reduced. Overall dynamic range was increased. Inhibition efficacy was increased.

[0365] Table 6: Summary of the results shown in Examples 1-3.

[0366] ​ ​ ​ <![CDATA[E min [RLU]]> <![CDATA[EC 50 [nM]]]> <![CDATA[E max [RLU]]> - C563 50 30,000 10 100,000 - CT139 50 0 20 100,000 C1761 C563 50 1,000 >10,000 NA C1761 CT139 50 400 >1,000 NA C1759 C563 50 20,000 200 100,000 C1760 C563 50 5,000 60 100,000 C1762 C563 50 5,000 600 90,000 C2184 C563 50 1,000 >1,000 NA C2183 C563 50 4,000 >1,000 NA C2182 C563 50 30,000 30 90,000 C2302 C563 50 0 >10,000 NA C2181 C563 50 0 >10,000 NA C2180 C563 50 0 >10,000 NA C2179 C563 50 0 >10,000 NA C2218 C563 50 1,000 >1,000 NA C2219 C563 50 2,000 >1,000 NA C2220 C563 50 3,000 >1,000 NA C2153 C563 50 20,000 2,000 60,000 C2107 CT139 5 0 100 100,000 C2106 CT139 5 0 40 100,000

[0367] Example 4: TCR-based inhibitory chimeric antigen receptor

[0368] Construct design and cloning

[0369] A high-affinity anti-HLA-A*02:01 / NY-ESO-1 1G4α95:LY T-cell receptor (TCR) variant was used to generate a NY-ESO-1-reactive inhibitory construct. Charged residues (R253 and K258) in the TCRα TM and charged residues (K288) in the TCRβ TM were mutated to leucine. Then, LILRB1 ITIM (residues 484-650) were attached to the mutated TCRα or TCRβ. Internally, an anti-HLA-A*02:01 / MAGE-A3 single-stranded variable fragment (scFv) was generated. The anti-HLA-A2*02:01 / MAGE-A3 chimeric antigen receptor (CAR) used in this study contained an anti-HLA-A*02:01 / MAGE-A3 scFv, a CD8 hinge, a CD28 TM, and intracellular domains (ICDs) of CD28, 41BB, and CD3ζ. All fragments (including the 5' and 3' BsmBI sites) were amplified using Q5 polymerase (New England Biolabs) and digested with DpnI (Thermo Scientific) at 37°C for 60 min. The resulting PCR fragments were purified using Nucleospin gel and a PCR purification kit (Macherey-Nagel). Plasmid assembly was performed in a reaction containing BsmBI (Thermo Scientific), T4 DNA ligase (Thermo Scientific), 10 mM ATP, and 1x FastDigest buffer (Thermo Scientific).

[0370] Jurkat NFAT activation assay

[0371] Jurkat T lymphocytes containing the firefly luciferase gene under the control of the activator T cell nuclear factor (NFAT) transcription factor (BPS Bioscience) were co-transfected with plasmids encoding TCR and / or scFv-fusion constructs using the Neon transfection system (Thermo Fisher). Electroporated cells were incubated in RPMI medium supplemented with 20% fetal bovine serum (FBS) (HIA-FBS) heat-inactivated at 56°C for 60 min and 0.1% penicillin-streptomycin (P / S) (Gibco). Different amounts of the modified NY-ESO-1 peptide alone (SLLMWITQV) (SEQ ID NO:107), the modified MAGE-A3 peptide alone (FLWGPRALV) (SEQ ID NO:106), or the modified NY-ESO-1 peptide alone in RPMI supplemented with 1% BSA and 0.1% P / S (Gibco) and different amounts of MAGE-A3 peptide were loaded into TAP-deficient T2 lymphoblasts (ATCC RL-1992). All peptides used in the synthesis assays were assessed for purity >95% by mass spectrometry (Genscript). Jurkat was loaded at 0.8 x 10⁻⁶ at 18 h post-transfection. 6 Cells / mL were resuspended in RPMI supplemented with 10% HIA-FBS and 1% P / S. In 384-well plates, 12,000 Jurkat cells were co-cultured with 12,000 peptide-loaded T2 cells / well at 37°C and 5% CO2 for 6 hours. NFAT-mediated luciferase production was measured by adding 15 μL of a one-step luciferase assay kit (BPS Bioscience). Luminescence was detected using a microplate reader (Tecan) after 20 minutes.

[0372] TCR-based inhibitory CAR using LILRB1 intracellular domain

[0373] As shown in Table 7 and Figure 11, co-expression of TCR-based CARs with only the extracellular domain of TCR or with the transmembrane region of TCR (with mutations to polar residues) produces functional repressive CARs.

[0374] Table 7

[0375]

[0376] TCR-based CARs were trans-inhibited from anti-HLA-A*02:01MAGE-A3 CAR. Jurkat-NFAT luciferase reporter cells were transfected as follows: (1) with anti-HLA-A*02:01 / MAGE-A3 CAR (C563) alone, (2) with MAGE-A3 CAR (C563) and TCRα (R253L / K258L)-LILRB1 fusion and TCRβ (K288L)-LILRB1 ICD fusion TCR inhibitory fusion construct (C2156+C2157), or (3) with MAGE-A3 CAR and TCRαECD / TCRβECD-LIRT1(TM+ICD) fusion (C2057+C2058) TCR inhibitory fusion construct. The effects of two inhibitory variants on NFAT activation were measured by co-culturing transfected Jurkat cells with T2 cells loaded with a combination of 50 μM NY-ESO-1 peptide and different amounts of MAGE-A3 peptide. The data are summarized in Table 8.

[0377] Table 8: Trans effects of TCR inhibitory fusion constructs on MAGE-A3 CAR

[0378]

[0379]

[0380] The truncated TCRα or TCRβ extracellular domain (ECD) fusion with LILRB1™-ICD inhibits CAR activation. When TCR transmembrane mutations eliminate TCR-CD3 subunit interactions, the TCRα or TCRβ extracellular domain (ECD) fusion with TCR™-LILRB1 ICD also acts as an inhibitory CAR. Experiments show that the TCRα and TCRβ chains can be used to generate inhibitory chimeric antigen receptors by interfering with the recruitment of stimulatory factors by the CD3 subunits.

[0381] Example 5: Method used in Examples 6-14

[0382] Cell culture

[0383] Jurkat cells encoding the NFAT luciferase reporter were obtained from BPS Bioscience. All other cell lines used in this study were obtained from ATCC. In culture, Jurkat cells were maintained in RPMI medium supplemented with 10% FBS, 1% Pen / Strep, and 0.4 mg / mL G418 / genimycin. T2, MCF7, Raji, K562, and HeLa cells were maintained according to ATCC guidelines. “Normal” Raji cells were prepared by transducing Raji cells with HLA-A*02 lentivirus (custom lentivirus, Alstem) at an MOI of 5. HLA-A*02 positive Raji cells were sorted using a FACSMelody cell sorter (BD).

[0384] plasmid construction

[0385] NY-ESO-1-reactive inhibitory constructs were generated by fusing the NY-ESO-1scFv LBD to the receptor domain, the receptor domain comprising the hinge, transmembrane region, and / or intracellular domain of leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1 (LIR-1)); programmed cell death protein 1 (PDCD1 (PD-1)); or cytotoxic T-lymphocyte protein 4 (CTLA4 (CTLA-4)). All activating CAR constructs contained scFvs fused to the CD8α hinge, CD28™, and CD28, 4-1BB, and CD3ζICD. The CD19-activating CAR scFv was derived from FMC63 mouse hybridomas. The MSLN-activating CAR scFv was derived from human M5 (LBD1) and humanized SS1 (LBD2) as described above. Gene fragments were combined using Golden Gate cloning and inserted downstream of the human EF1α promoter contained in the lentiviral expression plasmid.

[0386] Jurkat cell transfection

[0387] Jurkat cells were transiently transfected using a 100µL Neon electroporation system (Thermo Fisher Scientific) following the manufacturer's protocol: 3 pulses, 1500V, 10msec. Co-transfection was performed with 1–3µg of activator CAR or TCR construct and 1–3µg of scFv or TCRα / TCRβLIR-1 inhibitor construct or empty vector / 1e6 cells, and the cells were recovered in RPMI medium supplemented with 20% heat-inactivated FBS and 0.1% Pen / Strep. To confirm inhibitor surface expression, Jurkat cells were stained with 10µg / mL streptavidin-PE-HLA-A*02-pMHC tetramer at 4°C in PBS containing 1% BSA for 60 min 18–24 hours post-transfection and characterized by flow cytometry (BD FACScantoII).

[0388] Jurkat-NFAT-luciferase activation study

[0389] Peptides MAGE-A3 (MP1; FLWGPRALV; SEQ ID NO: 106), MAGE-A3 (MP2; MPKVAELVHFL; SEQ ID NO: 108), HPV E6 (TIHDIILECV; SEQ ID NO: 109), HPV E7 (YMLDLQPET; SEQ ID NO: 110), and the modified NY-ESO-1ESO (ESO; SLLMWITQV; SEQ ID NO: 107) were synthesized by Genscript. The activating peptides were serially diluted starting at 50 μM. The blocking peptide NY-ESO-1 was diluted to 50 μM (unless otherwise specified), added to the serially diluted activating peptide buffer, and subsequently loaded onto 1e4 T2 cells in 15 μL of RPMI supplemented with 1% BSA and 0.1% Pen / Strep. Jurkat cells were incubated in 384-well, low-friction, white, flat-bottomed polystyrene (TC) microplates. The next day, 1e⁴ Jurkat cells were resuspended in 15 μL of RPMI supplemented with 10% heat-inactivated FBS and 0.1% Pen / Strep, added to peptide-loaded T2 cells, and co-cultured for 6 hours. Jurkat luminescence was evaluated using a one-step luciferase assay system (BPSBioscience). For assays involving high-density targets, Jurkat cells were similarly transfected and co-cultured with tumor cells expressing the target antigen at various Jurkat cell:tumor cell ratios. Assays were repeated in duplicate.

[0390] Primary T cell transduction, expansion and enrichment

[0391] Leukopak purchased from Collection protocols and donor informed consent were approved by the Institutional Review Board (IRB) and strictly monitored. HIPAA compliance and approved protocols were also followed. Frozen PBMCs were thawed in a 37°C water bath and cultured at 1e6 cells / mL in LymphoONE (Takara) containing 1% human serum, and activated with TransAct (Miltenyi) T cells supplemented with IL-15 (10 ng / mL) and IL-21 (10 ng / mL) at a 1:100 ratio. After 24 hours, lentiviruses were added to the PBMCs at MOI=5. For each lentivirus, activator and blocker receptors were co-transduced simultaneously at MOI=5. The PBMCs were cultured for an additional 2–3 days to allow the cells to expand under TransAct stimulation. After expansion, primary T cells transduced with activator and blocker were enriched by positive selection against blocker-positive T cells using anti-PE microbeads (Miltenyi) according to the manufacturer's instructions. In short, primary T cells were incubated with 10 μg / mL streptavidin-PE-HLA-A*02-pMHC tetramer in MACS buffer (0.5% BSA + 2 mM EDTA in PBS) at 4°C for 60 minutes. The cells were washed three times in MACS buffer and passed through an LS column (Miltenyi) to separate blocker-positive cells (a mixture of blocker-only cells and activator + blocker cells) from untransduced cells and activator-only cells.

[0392] Primary T cell in vitro cytotoxicity study

[0393] For cytotoxicity studies using pMHC targets, enriched primary T cells were incubated with 2e3 Biosettia-expressing Biosettia MCF7 cells loaded with titrated target peptides as described above at an effector:target ratio of 3:1 for 48 hours. Live luciferase-expressing MCF7 cells were quantified using the Promega Biosettia reporter assay system. For cytotoxicity studies using non-pMHC targets, enriched primary T cells were incubated with 2e3 WT Raji cells (“tumor” cells) or HLA-A*02-transduced Raji cells (“normal” cells) at an effector:target ratio of 3:1 for up to 6 days. Using the IncuCyte live-cell imaging system, WT “tumor” Raji cells stably expressing GFP and Biosettia luciferase, or HLA-A*02-transduced “normal” Raji cells stably expressing RFP and Biosettia luciferase, were imaged alongside unlabeled primary T cells. The fluorescence intensity of live Raji cells over time was quantified using the IncuCyte imaging software. For reversibility studies, similarly, enriched primary T cells were co-cultured with “normal” or “tumor” Raji cells for 3 days and imaged. After 3 days, T cells were isolated from the remaining Raji cells using CD19 negative selection and reseeded with fresh “normal” or “tumor” Raji cells as described. Live Raji cells expressing luciferase were quantified in individual wells at 72 hours using the Promega dual luciferase reporter assay system. For studies assessing IFNγ secretion, the BD Human IFNγ flex kit was used in accordance with the manufacturer’s exact instructions to test for IFNγ in the supernatant collected after 48 hours of co-culture.

[0394] Mouse xenotransplantation research

[0395] Frozen PBMCs were thawed in a 37°C water bath and incubated overnight in serum-free TexMACS medium (Miltenyi) before activation. PBMCs were activated using T-cell TransAct (Miltenyi) and TexMACS medium supplemented with IL-15 (20 ng / mL) and IL-21 (20 ng / mL) at a concentration of 1.5e6 cells / mL. After 24 hours, lentivirus was added to the PBMCs at an MOI of 5. PBMCs were cultured for an additional 8–9 days to allow for cell expansion under TransAct stimulation. Following expansion, T cells were enriched on A2-LIR-1 cells for an additional 2–5 days using anti-PE microbeads (Miltenyi) targeting streptavidin-PE-HLA-A*02-pMHC before in vivo injection. The expression of CD19 scFv activator and HLA-A*02LIR-1 inhibitor in enriched T cells was verified by sequential staining with CD19-Fc (1:100; R&D Systems) and goat anti-human IgG-FITC (1:200; Invitrogen) for activators and with streptavidin-APC-HLA-A*02-pMHC (10 μg / mL) for inhibitors using flow cytometry (BD FACScanto II).

[0396] In vivo experiments were conducted by Explora BioLabs under a protocol approved by the Institutional Animal Care and Use Committee (IACUC). Five- to six-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(HLA-A / H2-D / B2M)1Dvs / SzJ(NSG-HLA-A2 / HHD) mice were purchased from Jackson Labs. Animals were acclimatized to their living environment for at least 3 days prior to the start of the study. 100 μL of 2e6 WT Raji cells or HLA-A*02-transduced Raji cells were injected subcutaneously into the right flank of each animal. Tumors reached an average size of 70 mm. 3When V = L x W x W / 2, animals were randomly divided into 5 groups (n = 7) and administered 2e6 or 1e7 T cells via tail vein. Tumor measurements were performed 3 times weekly after T cell injection, and blood was collected for flow cytometry analysis at 10 and 17 days. One animal from the WT Raji group, which received 1e7 CD19-CAR+A2-LIR-1 T cells, was excluded from the study due to a failed tail vein injection; the absence of human T cells in the blood was subsequently confirmed by flow cytometry. At each time point, human T cells in the blood were quantified by flow cytometry (BD FACScantoII) after RBC lysis. Cells were stained with anti-mouse CD45-FITC (clone 30-F11), anti-human CD3-PE (clone SK7), anti-human CD4-APC (clone OKT4), and anti-human CD8-PerCP-Cy5.5 (clone RPA-T8). All antibodies were obtained from Biolenging and used at a 1:100 dilution. DAPI (Invitrogen) was used to exclude dead cells from the analysis. For histological analysis, tumor samples were fixed, sectioned, and stained for huCD3 (clone EP449E). Image quantification was performed using ImageJ software.

[0397] Statistical analysis

[0398] Statistical analyses were performed using GraphPad Prism software. Unless otherwise specified, all peptide and cell titration studies are presented as mean ± standard deviation (SD), while in vitro and in vivo studies using primary T cells are presented as mean ± standard error of mean (SEM). Peptide and cell titration curves were fitted using four-parameter nonlinear regression analysis. EC50 values ​​were calculated directly from the curves. Unless otherwise specified, all other data groups were analyzed using ordinary two-way ANOVA followed by Tukey's multiple comparison test.

[0399] Example 6: Determining the effect of the LIR-1 hinge on blocking activity

[0400] The effect of different LIR-1 hinges on the ability of the HLA-A*02scFv LIR-1 inhibitory receptor to block killing by Jurkat cells expressing KRAS TCR activators was determined using the Jurkat NFat luciferase assay described above. The effects of humanized PA2.1 scFv LIR-1 receptors and humanized BB7.2 scFv LIR-1 with shorter LIR-1 hinges were determined in Jurkat cells as previously described, and the results are shown in Figures 12A-12B. Jurkat cells were transfected with KRAS TCR activator receptors and / or HLA-A*02scFv LIR-1 inhibitory receptors (humanized PA2.1 or humanized BB7.2) with various LIR-1-derived hinges and co-cultured with T2 target cells that were HLA:A11 positive or HA:A11 and HLA:A02 positive. Inhibitory receptors with shorter and longer hinges exhibit similar behavior (Figs. 12A-12B). In T2-Jurkat assays, inhibitory receptors with mouse PA2.1 scFv and slightly longer hinges were also found to be functionally similar to those with shorter LIR-1 hinges (Figs. 13A-13B). Hinge sequences are shown in black in Figs. 12B and 13B, with gray SS in Fig. 13B representing the linker between the antigen-binding domain and the hinge, and gray VIGIL representing the origin of the LIR-1 transmembrane domain. The hinge, transmembrane domain, and intracellular domain of the inhibitory receptor are all derived from LIR-1. Figs. 12A-12B and 13A-13B demonstrate that the LIR-1 hinge length can vary without adversely affecting the LIR-1 inhibitory receptor. Shorter hinges can provide an advantage when the nucleic acid sequence encoding the LIR-1 inhibitory receptor is packaged in a lentiviral vector for delivery.

[0401] Table 9. Sequence of Constructs

[0402]

[0403]

[0404]

[0405]

[0406] Example 7: Comparison of LIR-1, CTLA-4 and PD-1 inhibitory receptors

[0407] A NY-ESO-1-reactive inhibitory construct was generated by fusing the NY-ESO-1scFv LBD to the receptor's domain, the domain of which includes the hinge, transmembrane region, and / or intracellular domain of leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1 (LIR-1)); programmed cell death protein 1 (PDCD1 (PD-1)); or cytotoxic T-lymphocyte protein 4 (CTLA4 (CTLA-4)). The MAGE-A3-activated CAR construct contains a scFv fused to the CD8α hinge, CD28™, and the intracellular domains (ICDs) of CD28, 4-1BB, and CD3ζ. Gene fragments were assembled using a Golden Gate clone and inserted downstream of the human EF1α promoter contained in a lentiviral expression plasmid.

[0408] Initially, peptide-MHC (pMHC) targets for both activator and blocker receptors were used because pMHC allows for convenient quantification of systematic pharmacology (Figure 14A). Specifically, HLA-A*02-NY-ESO-1 binding was used. (SLLMWITQC / V) The single-chain variable fragment (scFv) serves as the inhibitor receptor ligand binding domain (LBD) and will target HLA-A*02-MAGE-A3. (FLWGPRALV) The second scFv of pMHC (Gallo, undisclosed) is part of a third-generation CAR activator receptor. Activator sensitivity was read out in Jurkat effector cells expressing luciferase after activation with NFAT, and the EC50 value reported the half-maximal activator peptide concentration required for the response. Both the PD-1 and CTLA-4 intracellular domains (ICDs) mediate EC50 shifts of less than approximately 10x in activation in Jurkat cells, measured by titrating peptides loaded as stimulants onto T2 cells (Fig. 14B).

[0409] Multiple potential inhibitor (blocker) receptor constructs were screened, and the constructed LIR-1 blocker was found to have stronger blocking properties than PD-1 and CTLA-4. This blocker receptor comprises the intracellular domain, transmembrane (TM) domain, and hinge domain of the LIR-1 (LILRB1) receptor, one of several LIR family molecules encoded by the human genome. The LIR-1 blocker fused to NY-ESO-1LBD (hereinafter referred to as LIR-1) mediates an EC50 shift of >5,000x (Fig. 14B, Fig. 17A-17D). Control titration of the unrelated HLA-A*02 binding peptide provides an estimate of the shift caused by competition for available HLA molecules with the peptide loaded on T2 cells, typically contributing less than about 10x to the total shift (Fig. 15). The EC50 shift values ​​reported here are typically compared to the EC50 of constructs containing only the activator. Furthermore, for a given pair of activator / blocker receptors, the midpoint of the titration for inhibition is approximately constant and depends on the ratio of activating to blocking peptides, presumably directly related to the target-antigen ratio (Figures 16A-16D). In most of these experiments, the target concentration estimates studied ranged from approximately 1,000 to 10,000 copies / cell.

[0410] Example 8: LIR-1 inhibitory receptor with multiple scFv ligand-binding domains

[0411] The activity of the LIR-1 inhibitory receptor was tested using multiple antigen-binding domains specific to other pMHC targets. For four different pMHC targets, a total of six different scFvs grafted onto LIR-1 mediated significant EC50 shifts ranging from 10x to 1,000x (Fig. 14C). The LIR-1 receptor is also robust regarding its interaction with activator receptors; its blocking behavior is applicable to multiple targets and scFvs (Fig. 14D). Blockade is ligand-dependent (Fig. 17A), although many LIR-1 constructs produce lower basal / tonic signaling when paired with specific activator receptors. EC50 shift is dependent on the presence of a fusion ICD, as LIR-1 constructs completely lacking an ICD or containing mutations in key elements of the ICD have no effect (Fig. 17B). However, ligand-independent blocking activity has little effect on EC50 activation in the absence of a ligand (Figs. 16A–16D). The LIR-1 inhibitory receptor is a modular, adaptive, ligand-gated system that functions across multiple target and antigen-binding domains.

[0412] Example 9: LIR-1 inhibitory domains fused to TCRα and TCRβ

[0413] The LIR-1 inhibitory receptor was tested when fused with the TCRα and TCRβ subunits or when combined with the TCR activator receptor. The TCRs target three different pMHC targets, two from MAGE-A3 and one from HPV (see Methods, above). In each case, LIR-1 caused a significant shift in the activated EC50, estimated to be >1,000x (Fig. 14E; Fig. 19A). Furthermore, the NY-ESO-1TCR LBD also produced a significant EC50 shift upon fusion with LIR-1 (Fig. 14F; Fig. 19B). In fact, MAGE-A3... (FLWGPRALV) CAR or TCR with NY-ESO-1 (SLLMWITQV) All combinations of scFv or TCR exhibit large shifts. Therefore, the LIR-1 inhibitory receptor demonstrates regulation encompassing both CAR and TCR.

[0414] Example 10: LIR-1 inhibitory receptors respond to target antigens present in cis configuration relative to activator receptor target antigens.

[0415] The ability of LIR-1 blocker receptors to inhibit activation induced by activator receptors was determined when activator and inhibitor targets were present in cis configuration. In the first assay, a simplified stimulus consisting of target-loaded beads approximately the size of a cell (d ≈ 2.8 μm) was used. Jurkat cells expressing both activator and blocker receptors were activated only by beads containing the A (activator) target, and not by beads with dual A / B (activator / blocker) targets (Fig. 18A). Interestingly, effector cells were activated by a mixture of A+ and B+ beads, even when A+ beads comprised only 20% of the total. This indicates that cells expressing both activator and blocker receptors are: (i) blocked by the blocker receptor when the target is present in cis configuration on the same surface; and (ii) activated by individual A+ beads in an excess of B+ beads.

[0416] Example 11: LIR-1 inhibitory receptor and cell surface antigen

[0417] The ability of LIR-1 inhibitor receptor blockade to respond to activation by non-pMHC targets, representing surface antigens that can extend to 100,000 epitopes per cell, was determined. scFvs binding to the B-cell marker CD19, the solid tumor antigen mesothelin (MSLN), or HLA-A*02 were tested in a peptide-independent manner. In these cases, the target antigen concentration was uncontrolled, as was the case with exogenous peptides and pMHC. Conversely, different DNA concentrations were used in transient transfection assays to alter the ratio of activator to blocker expression. Although assay sensitivity prevented the study of the full range of EC50 shifts, shifts in Emax exceeding 10x were observed. These experiments demonstrate that the properties of the LIR-1 receptor in dual-receptor systems are generally the same for high-density targets (Fig. 18B; Figs. 16–16D; Fig. 21A), and that the blocker receptor blocks A receptor activation to the extent reflected by its relative surface level on effector cells (Fig. 18C). The LIR-1 receptor also functions in a modular manner in this high-antigen-density environment. When scFv targeting HLA-A*02 fuses to activator receptors or blocker receptors, it acts as an activator (Fig. 18D) or a blocker, respectively. The LIR-1 receptor is flexible enough to adapt to both low and high target densities, and in principle, allows for optimization of pMHC targets as well as non-pMHC surface antigens.

[0418] Example 12: LIR-1 inhibitory receptor in primary T cells

[0419] The ability of the LIR-1 receptor to block primary T cell activation was determined. First, pMHC targets were used. After enriching transduced T cells via physical selection, engineered T cells expressing luciferase were used as live cell reads to measure the expression of both the activator and blocker receptors. Using HPV TCR as an activator, NY-ESO-1scFv fused to LIR-1 shifted the cell count in peptide-loaded MCF7 tumor cells by approximately 25x relative to the peptide concentration curve (Fig. 20A; Fig. 23). Therefore, the behavior of the LIR-1 receptor demonstrated in the Jurkat cell activation assay extends to primary T cell function, including cytotoxicity.

[0420] To establish proof of concept, the HLA-A*02LIR-1 construct was shown to act as an inhibitor in Jurkat cells and T2 target cells in the presence of a pMHC-dependent activator (Fig. 20B). For the activator, CD19 scFv was used as the CAR. This activator / inhibitor pair was shown to function together as robustly as other pairs previously tested in Jurkat cells (Fig. 18B). T model tumor cells differentially expressing the LIR-1 receptor ligand, and CD19-positive, HLA-A*02-negative Raji cells were used. For modeling corresponding normal cells, the same cell line stably expressing the HLA-A*02 gene was used (Fig. 24A). If the target cells expressed only CD19, the cell line activated Jurkat cells; however, if they expressed both CD19 and HLA-A*02, they did not activate Jurkat cells; i.e., the HLA-A*02 inhibitor receptor blocks activation induced by the CD19-CAR in a ligand-dependent manner (Fig. 24B).

[0421] The CD19 / HLA-A*02 receptor pair also plays a role in primary T cells (Fig. 21). Engineered T cells killed Raji cells expressing CD19 in the absence of HLA-A*02 expression. Raji cells expressing both CD19 and HLA-A*02 were blocked from cytotoxicity. Importantly, primary T cells carrying the aforementioned receptor pair distinguished CD19+ “tumor” cells from CD19+ / HLA-A*02+ “normal” cells in mixed cultures. These findings reflect results from the aforementioned bead experiments, but in more complex cellular environments where the cytotoxicity of effector cells is concentrated on the “tumor” target, even when surrounded by “normal” cells. Given that no intentional optimization for maximum selectivity was performed on either receptor, the degree of selectivity is impressive. This behavior was confirmed using the secondary antigen MSLN (Fig. 22B).

[0422] Example 13: Reversibility of inhibition of LIR-1 inhibitory receptors

[0423] The ability of the LIR-1 inhibitory receptor to function reversibly—that is, a cycle from a blocking state to an activated state and back to a blocking state—was tested. Effector T cells expressing both the LIR-1 receptor and the activator receptor were tested to observe whether they could function reversibly and repeatedly. Effector cells were co-cultured with Raji cells to simulate the encounter between tumor (CD19+) or normal (CD19+ / HLA-A*02+) cells. After each round of exposure to target cells, Raji cells were removed from the culture and a new population of target cells was introduced. At the end of each round, cytotoxicity and interferon-gamma (IFNγ) levels were measured. In both block-kill-block and kill-block-kill arrangements, T cells functioned as needed by this type of cell therapy (Figs. 25A-25D). They reversibly cycled from a block state to cytotoxicity and back, depending on the target cells they were exposed to. This result indicates that T cells expressing both activator receptors and LIR-1 inhibitory receptors are not fixed in one state (blocked or activated) but can switch back and forth as they integrate signals from normal and tumor cells. Furthermore, these experiments were replicated in primary T cells from multiple donors (Figs. 21A-21D; 25A-25D; 26A-26B), demonstrating robust function of the LIR-1 inhibitory receptor despite the complexity, heterogeneity, and donor-to-donor variability of these primary T cells.

[0424] Example 14: In a mouse model, human T cells expressing activator receptor and LIR-1 blocker receptor selectively target cancer cells.

[0425] The ability of engineered CD19 / HLA-A*02 activator / blocker pairs in primary T cells to allow for the in vitro expansion of T cells to a large number using standard CD3 / CD28 stimulation was determined (Fig. 27A; Fig. 28A). Therefore, T cells expressing both the activator receptor and LIR-1 receptor c can be expanded to a number sufficient for animal experiments and ultimately for patient use.

[0426] In vitro testing of CD19 / HLA-A*02 activator / blocker combinations demonstrated selective killing of CD19+ tumor cells without affecting CD19+ / HLA-A*02+ cells in a murine xenograft carcinoma model (Figure 27B). Two identical Raji cell lines (one CD19+ and one CD19+ / HLA-A*02+) were injected into the flanks of immunocompromised (NGS-HLA-A2.1) mice. T cells engineered with the test constructs were injected at two doses: 2e6 T cells (not shown) or 1e7 T cells. Tumor growth over time and persistence of implanted T cells were analyzed. Only CD19+ tumor cells were killed in the mice, and tumor control was tracked by the number of transferred T cells, promoting host mouse survival (Figures 27C - 27E; Figures 28B - 28C). CD19+ / HLA-A*02+ cells designed to model normal cells were unaffected. The human CD4+ / CD8+ cell ratio in the blood of mice carrying engineered cells was tracked with its control counterpart; i.e., in "tumor" transplanted mice, CD4+ cells > CD8+ cells, as in CD19 CAR positive control mice; and, in "normal" transplanted mice, CD4+ cells < CD8+ cells, similar to mice with untransduced T cells (Figure 28D). Additionally, huCD3+ cells in the tumor (Figures 29A - 29B) were inversely correlated with tumor volume, and engineered T cells expressing both receptors behaved similarly to untransduced T cells in "normal" grafts (low infiltration) and similar to CD19 CAR in "tumor" grafts (high infiltration). Finally, the mice appeared normal in typical clinical observations (Table 10). Taken together, these results indicate that in a simplified scenario designed to mimic different normal cell types and tumor cell types that would be encountered in patients, the LIR-1 inhibitory receptor can function in vivo to inhibit activator receptors.

[0427] Table 10. Clinical observations of mice during in vivo experiments

[0428]

[0429]

[0430] Tumor cells and "normal" cells were injected on study day 0, and treatment was initiated on study day 10. Clinical observations (CO) for ill health, stress, and pain were made three times per week. Per IACUC guidelines, mice were euthanized if the tumor reached >2000 mm 3 ,. Codes: N = normal; 19A = abnormal tumor [N = necrosis, O = open], EUT = lethal. Severity codes: 0 = absent, 1 = moderate, 2 = severe. Sequence Listing <110> A2 Biotherapy Company <120> LILRB1-based chimeric antigen receptor <130> A2BI-015 / 04WO 331656-2036 <150> US 62 / 946,888 <151> 2019-12-11 <150> US 63 / 085,969 <151> 2020-08-30 <160> 129 <170> PatentIn version 3.5 <210> 1 <211> 670 <212> PRT <213> Homo sapiens <400> 1 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540 Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val 545 550 555 560 Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565 570 575 Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590 Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605 Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620 Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val 625 630 635 640 ​​​​​​​​​​​​​​​​​​​​​​​​​​Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45 Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val 50 55 60 Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu 65 70 75 80 Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr 85 90 95 Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly 100 105 110 Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala 115 120 125 Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro 130 135 140 Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro 145 150 155 160 Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu 165 170 175 Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys 180 185 190 Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala 195 200 205 Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr 210 215 220 Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser 225 230 235 240 Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 245 250 <210> 3 <211> 233 <212> PRT <213> Homo sapiens <400> 3 Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly Pro 1 5 10 15 Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Ser[[ID=三十一]] 20 25 30 Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly Ile 35 40 45<00​​​​​​​​Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr 85 90 95 Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu 100 105 110 Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val 115 120 125 Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr 130 135 140 Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro 145 150 155 160 Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala 165 170 175 Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro 180 185 190 Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu 195 200 205 Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro 210 215 220 Ser Ile Tyr Ala Thr Leu Ala Ile His 225 230 <210> 4 <211> 63 <212> PRT <213> Homo sapiens <400> 4 Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp 1 5 10 15 Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro 20 25 30 Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45 Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 50 55 60 <210> 5 <211> 23 <212> PRT <213> Homo sapiens <400> 5 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Phe Leu Ile Leu 20 <210> 6 <400> 6 000 <210> 7 <211> 167 <212> PRT <213> Homo sapiens <400> 7 Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala 1 5 10 15 Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg 20 25 30 Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn 35 40 45 Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu Met 50 55 60 Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala 65 70 75 80 Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser 85 90 95 Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu 100 105 110 Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp 115 120 125 Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr 130 135 140 Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile 145 150 155 160 Tyr Ala Thr Leu Ala Ile His 165 <210> 8 <211> 6 <212> PRT <213> Homo sapiens <400> 8 Asn Leu Tyr Ala Ala Val 1 5 <210> 9 <211> 6 <212> PRT <213> Homo sapiens <400> 9 Val Thr Tyr Ala Glu Val 1 5 <210> 10 <211> 6 <212> PRT <213> Homo sapiens <400> 10 Val Thr Tyr Ala Gln Leu 1 5 <210> 11 <211> 6 <212> PRT <213> Homo sapiens <400> 11[[ID=四十八]] Ser Ile Tyr Ala Thr Leu 1 5 <210> 12 <211> 35 <212> PRT <213> Homo sapiens <400> 12 Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu 1 5 10 15 Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 20 25 30<00014??>Ala Glu Val 35 <210> 13 <211> 58 <212> PRT <213> Homo sapiens <400> 13 Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala 1 5 10 15 Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg 20 25 30 Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu 35 40 45 Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu 50 55 <210> 14 <211> 36 <212> PRT <213> Homo sapiens <400> 14 Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr 1 5 10 15 Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile 20 25 30 Tyr Ala Thr Leu 35 <210> 15 <211> 87 <212> PRT <213> Homo sapiens <400> 15 Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu 1 5 10 15 Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 20 25 30 Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro 35 40 45 Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu 50 55 60 Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln 65 70 75 80 Asp Val Thr Tyr Ala Gln Leu 85 <210> 16 <211> 88 <212> PRT <213> Homo sapiens <400> 16 Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala 1 5 10 15 Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg 20 25 30 Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu 35 4​Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg 50 55 60 Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala 65 70 75 80 Val Pro Ser Ile Tyr Ala Thr Leu 85 <210> 17 <211> 117 <212> PRT <213> Homo sapiens <400> 17 Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu 1 5 10 15 Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 20 25 30 Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro 35 40 45 Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu 50 55 60 Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln 65 70 75 80 Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala 85 90 95 Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser 100 105 110 Ile Tyr Ala Thr Leu 115 <210> 18 <211> 20 <212> PRT <213> Homo sapiens <400> 18 Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp 1 5 10 15 Pro Leu Glu Leu 20 <210> 19 <211> 43 <212> PRT <213> Homo sapiens <400> 19 Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly Pro 1 5 10 15 Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Ser 20 25 30 Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 35 40 <210> 20​​​​​​​​​​​1 5 10 15 Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro 20 25 30 Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45 Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val 50 55 60 Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu 65 70 75 80 Leu Leu Phe Leu Ile Leu 85 <210> 21 <211> 190 <212> PRT <213> Homo sapiens <400> 21 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 20 25 30 Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 35 40 45 Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 50 55 60 Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln 65 70 75 80 Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 85 90 95 Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg 100 105 110 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 115 120 125 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 130 135 140 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 145 150 155 160 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 165 170 175 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 180 185 190 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR <400> 22 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> twenty three <211> 12 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR <400> twenty three Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg 1 5 10 <210> twenty four <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR <400> twenty four Phe Gln Gly Ser His Val Pro Arg Thr 1 5 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR <400> 25 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Ile His 1 5 10 <210> 26 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR <400> 26 Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly Lys <210> 27 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR <400> 27 Glu Glu Ile Thr Tyr Ala Met Asp Tyr 1 5 <210> 28 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR <400> 28 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Asp 1 5 10 15 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR <400> 29 Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg 1 5 10 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR <400> 30 Met Gln Gly Ser His Val Pro Arg Thr 1 5 <210> 31 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR <400> 31 Ser Gly Tyr Thr Phe Thr Ser Tyr His Met His 1 5 10 <210> 32 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR <400> 32 Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 33 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR <400> 33 Glu Gly Thr Tyr Tyr Ala Met Asp Tyr 1 5 <210> 34 <211> 3229 <212> DNA <213> Homo sapiens <400> 34 aaatgagttt taaaaaggct tgtccaggaa gcacatatgg gagctggtca ctctgcattt 60 tgggccctcc tggaggtgtt tagaccttcc gagagagaaa ctgagacaca tgagagggaa 120 gaaatgactc agtggtgaga ccctgtggag tcccacccac aaccagcaca ctgtgaccca 180 ctgcacaaac ctctagccca cagctcactt cctcctttaa gaagagaaga gaaaagagga 240 gaggagagga ggaacagaaa agaaaagaaa agaaaaagtg ggaaacaaat aatctaagaa 300 tgaggagaaa gcaagaagag tgaccccctt gtgggcactc cattggtttt atggcgcctc 360 tactttctgg agtttgtgta aaacaaaaat attatggtct ttgtgcacat ttacatcaag 420 ctcagcctgg gcggcacagc cagatgcgag atgcgtctct gctgatctga gtctgcctgc 480 agcatggacc tgggtcttcc ctgaagcatc tccagggctg gagggacgac tgccatgcac 540 cgagggctca tccatccaca gagcagggca gtgggaggag acgccatgac ccccatcctc 600 acggtcctga tctgtctcgg gctgagtctg ggcccccgga cccacgtgca ggcagggcac 660 ctccccaagc ccaccctctg ggctgaacca ggctctgtga tcacccaggg gagtcctgtg 720 accctcaggt gtcagggggg ccaggagacc caggagtacc gtctatatag agaaaagaaa 780 acagcaccct ggattacacg gatcccacag gagcttgtga agaagggcca gttccccatc 840 ccatccatca cctgggaaca cacagggcgg tatcgctgtt actatggtag cgacactgca 900 ggccgctcag agagcagtga ccccctggag ctggtggtga caggagccta catcaaaccc 960 accctctcag cccagcccag ccccgtggtg aactcaggag ggaatgtaac cctccagtgt 1020 gactcacagg tggcatttga tggcttcatt ctgtgtaagg aaggagaaga tgaacaccca 1080 caatgcctga actcccagcc ccatgcccgt gggtcgtccc gcgccatctt ctccgtgggc 1140 cccgtgagcc cgagtcgcag gtggtggtac aggtgctatg cttatgactc gaactctccc 1200 tatgagtggt ctctacccag tgatctcctg gagctcctgg tcctaggtgt ttctaagaag 1260 ccatcactct cagtgcagcc aggtcctatc gtggcccctg aggagaccct gactctgcag 1320 tgtggctctg atgctggcta caacagattt gttctgtata aggacgggga acgtgacttc 1380 cttcagctcg ctggcgcaca gccccaggct gggctctccc aggccaactt caccctgggc 1440 cctgtgagcc gctcctacgg gggccagtac agatgctacg gtgcacacaa cctctcctcc 1500 gagtggtcgg cccccagcga ccccctggac atcctgatcg caggacagtt ctatgacaga 1560 gtctccctct cggtgcagcc gggccccacg gtggcctcag gagagaacgt gaccctgctg 1620 tgtcagtcac agggatggat gcaaactttc cttctgacca aggagggggc agctgatgac 1680 ccatggcgtc taagatcaac gtaccaatct caaaaatacc aggctgaatt ccccatgggt 1740 cctgtgacct cagcccatgc ggggacctac aggtgctacg gctcacagag ctccaaaccc 1800 tacctgctga ctcaccccag tgaccccctg gagctcgtgg tctcaggacc gtctgggggc 1860 cccagctccc cgacaacagg ccccacctcc acatctggcc ctgaggacca gcccctcacc 1920 cccaccgggt cggatcccca gagtggtctg ggaaggcacc tgggggttgt gatcggcatc 1980 ttggtggccg tcatcctact gctcctcctc ctcctcctcc tcttcctcat cctccgacat 2040 cgacgtcagg gcaaacactg gacatcgacc cagagaaagg ctgatttcca acatcctgca 2100 ggggctgtgg ggccagagcc cacagacaga ggcctgcagt ggaggtccag cccagctgcc 2160 gatgcccagg aagaaaacct ctatgctgcc gtgaagcaca cacagcctga ggatggggtg 2220 gagatggaca ctcggagccc acacgatgaa gacccccagg cagtgacgta tgccgaggtg 2280 aaacactcca gacctaggag agaaatggcc tctcctcctt ccccactgtc tggggaattc ctggacacaa aggacagaca ggcggaagag gacaggcaga tggacactga ggctgctgca tctgaagccc cccaggatgt gacctacgcc cagctgcaca gcttgaccct cagacgggag gcaactgagc ctcctccatc ccaggaggg ccctctccag ctgtgcccag catctacgcc actctggcca tccactagcc cagggggga cgcagacccc acactccatg gagtctgga tgcatgggag ctgcccccc agtggacacc attggacccc acccagcctg gatctacccc 2640 aggagactct gggaactttt aggggtcact caattctgca gtataata ctaatgtctc 2760. 2760. 2760. 2760. 2760. 2760. 2760. 2760. 2760 agtcagaaag tgcattaaac tgaatcacaa tgtaaatatt acacatcaag cgatgaaact ggaaaactac aagccacgaa tgaatgaatt aggaaaaa aaaagtagga aatgaatgat cttggctttc ctataagaaa tttagggcag ggcacggtgg ctcacgcctg taattccagc actttgggag gccgaggcgg gcagatcacg agttcaggag atcgagacca tcttggccaa catggtgaaa ccctgtctct cctaaaaata caaaaattag ctggatgtgg tggcagtgcc 3060 tgtaatccca gctatttggg aggctgaggc aggagaatcg cttgaaccag ggagtcagag 3120 gtttcagtga gccaagatcg caccactgct ctccagcctg gcgacagagg gagactccat 3180 ctcaaattaa aaaaaaaaaa aaaaaagaaa gaaaaaaaaa aaaaaaaaa 3229 <210> 35 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> C-001765 scFv Antibody Construct <400> 35 Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln 1 5 10 15 Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly 20 25 30 Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys 35 40 45 Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg 65 70 75 80 Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His 85 90 95 Val Pro Arg Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gln 115 120 125 Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 130 135 140 Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His 145 150 155 160 Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 165 170 175 Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys 180 185 190 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 195 200 205 His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 210 215 220 Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230 235 240 Val Thr Val Ser Ser Tyr Gly 245 <210> 36 <211> 245 <212> PRT <213> Artificial sequence <220> <223> C-002159 scFv antibody construct <400> 36 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Ile 20 25 30 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys Gly 50 55 60<00017​​​​​​​​​​​​​​115 120 125 Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln 145 150 155 160 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg 180 185 190 Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr 210 215 220 Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys Val Glu Ile Lys 245 <210> 37 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> C-002160 scFv Antibody Construct <400> 37 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Ile 20 25 30 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys Gly 50 55 60 Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 65 70 75 80 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu 130 135 140 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 145 150 155 160 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln 165 170 175 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 180 185 190 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 210 215 220 Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys Val Glu Ile Lys 245 <210> 38 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> C-002161 scFv Antibody Construct <400> 38 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Ile 20 25 30 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp 35 40 45 Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln 145 150 155 160 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg 180 185 190 Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220 Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys Val Glu Ile Lys 245 <210> 39 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> C-002162 scFv Antibody Construct <400> 39 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Ile 20 25 30 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp 35 40 45 Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys Gly 50 55 60 Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr Met Glu 65 70 75 80 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu 130 135 140 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln 145 150 155 160 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg 180 185 190 Phe Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu 195 200 205 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr 210 215 220 Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gln Gly Thr 225 230 235 240 Lys Val Glu Val Lys 245 <210> 40 <211> 245 <212> PRT <213> Artificial sequence <220> <223> C-002163 scFv antibody construct <400> 40 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Met 20 25 30 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr 35 40 45 Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys Gly 50 55 60 Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr Met Glu 65 70 75 80 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg 85 90 95 Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Asp Val Gln Met Thr Gln Ser Pro Ser Thr Leu 130 135 140 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ser Ser Gln 145 150 155 160 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Met Glu Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg 180 185 190<N / A>Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu 195 200 205 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr 210 215 220 Tyr Cys His Gln Gly Ser His Val Pro Arg Thr Phe Gly Gln Gly Thr 225 230 235 240 Lys Val Glu Val Lys ​​​​​​​​​​​​​​​​​​​​Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 His Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Asp Val Leu Met Thr Gln Thr Pro Leu 130 135 140 Ser Leu Pro Val Ser Leu Gly Asp Gln Val Ser Ile Ser Cys Arg Ser 145 150 155 160 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175 Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190 Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 210 215 220 Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys 245 <210> 42 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> C-002165 scFv Antibody Construct <400> 42 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 His Ile Gln Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Asn Leu Asp Ser Val Ser Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Lys Gly Ser 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser 145 150 155 160 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190 Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 210 215 220 Thr Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Pro 225 230 235 240 Gly Thr Lys Val Asp Ile Lys 245 <210> 43 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> C-002166 scFv Antibody Construct <400> 43 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 His Ile Gln Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr[[ID=…]] 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser 145 150 155 160 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190 Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Val Glu Val Lys 245 <210> 44 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> C-002167 scFv antibody construct <400> 44 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly 130 135 140 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ser 145 150 155 160 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser 180 185 190 Asn Arg Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 210 215 220 Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu Ile Lys 245 <210> 45 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> C-002168 scFv Antibody Construct [[ID= forty]]<400> 45 Gln Val Thr Leu Lys Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Thr Ser Tyr It should be noted that in the above translation, "forty" in [[ID= forty]] is a misrepresentation in the original text, which should be . This is just to show the translation result according to the content you provided.20 25 30 His Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Met Asp Thr Ser Phe 65 70 75 80 Met Glu Leu Thr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly 130 135 140 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ser 145 150 155 160 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Ala Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Ser Lys Val Ser 180 185 190 Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 210 215 220 Val Tyr Tyr Cys Gln Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu Ile Lys 245 <210> 46 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> C-002169 scFv Antibody Construct <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 His Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Asp Ile Val Met Thr Gln Thr Pro Leu 130 135 140 Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser 145 150 155 160 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Asp Trp Tyr 165 170 175 Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser 180 185 190 Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly 210 215 220 Val Tyr Tyr Cys Met Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu Ile Lys 245 <210> 47 <211> 511 <212> PRT <213> Artificial sequence <220> <223> C563 chimeric antigen receptor construct <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Pro Phe Gly Asp Trp Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Ser Tyr Ser Phe Val Leu Thr Phe Gly Gly Gly Thr Lys 225 230 235 240 Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 245 250 255 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 260 265 270 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 275 280 285 Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 290 295 300 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 305 310 315 320 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 325 330 335 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 340 345 350 Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 355 360 365 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 370 375 380 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 385 390 395 400 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln 405 410 415 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 420 425 430 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 435 440 445 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 450 455 460 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 465 470 475 480 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 485 490 495 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 510 <210> 48 <211> 403 <212> PRT <213> Artificial sequence <220> <223> C1759 chimeric antigen receptor construct <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly[[ID=​​​​​​​​​Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Phe Gly Ser Phe Arg Ala Leu Pro Cys Val Trp Ser Asn Ser Ser 245 250 255 Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser Ser Ser Trp Pro 260 265 270 Ser Pro Thr Glu Pro Ser Ser Lys Ser Gly Ile Cys Arg His Leu His 275 280 285 Val Leu Ile Gly Thr Ser Val Val Ile Phe Leu Phe Ile Leu Leu Leu 290 295 300 Phe Phe Leu Leu Tyr Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val 305 310 315 320 Met Asp Gln Glu Pro Ala Gly Asp Arg Thr Val Asn Arg Gln Asp Ser 325 330 335 Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu Asp His Cys 340 345 350 Val Phe Ile Gln Arg Lys Ile Ser Arg Pro Ser Gln Arg Pro Lys Thr 355 360 365 Pro Leu Thr Asp Thr Ser Val Tyr Thr Glu Leu Pro Asn Ala Glu Pro 370 375 380 Arg Ser Lys Val Val Ser Cys Pro Arg Ala Pro Gln Ser Gly Leu Glu 385 390 395 400 Gly Val Phe <210> 49 <211> 358 <212> PRT <213> Artificial sequence <220> <223> C1760 chimeric antigen receptor construct <400> 49 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Phe Gly Ser Phe Arg Ala Leu Pro His Ala Trp Ser Asp Pro Ser 245 250 255 Asp Pro Leu Pro Val Ser Val Thr Gly Asn Ser Arg Asn Leu His Val 260 265 270 Leu Ile Gly Thr Ser Val Val Ile Ile Pro Phe Ala Ile Leu Leu Phe 275 280 285 Phe Leu Leu His Arg Trp Cys Ala Asn Lys Lys Asn Ala Val Val Met 290 295 300 Asp Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Arg Glu Asp Ser Asp 305 310 315 320 Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu Asn His Cys Val 325 330 335 Phe Thr Gln Arg Lys Ile Thr Arg Pro Ser Gln Arg Pro Lys Thr Pro 340 345 350 Pro Thr Asp Thr Ser Val 355 <210> 50 <211> 494 <212> PRT <213> Artificial sequence <220> <223> C1761 chimeric antigen receptor construct <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 325 330 335 Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 340 345 350 Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 355 360 365 Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln 370 375 380 Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 385 390 395 400 Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg 405 410 415 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 420 425 430 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 435 440 445 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 450 455 460 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 465 470 475 480 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 485 490 <210> 51 <211> 385 <212> PRT <213> Artificial Sequence <220> <223> C1762 Chimeric Antigen Receptor Construct <400> 51 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser 245 250 255 Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly 260 265 270 Gly Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile 275 280 285 Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln 290 295 300 Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr 305 310 315 320 Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val 325 330 335 Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser 340 345 350 Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro 355 360 365 Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro 370 375 380 Leu 385 <210> 52 <211> 439 <212> PRT <213> Artificial sequence <220> <223> C2057 chimeric antigen receptor construct <400> 52 Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp 1 5 10 15 Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30 Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45 Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60 Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg 65 70 75 80 Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95 Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg 100 105 110 Pro Leu Tyr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser 115 120 125 Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Val Ile Gly Ile Leu Val 245 250 255 Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe Leu Ile Leu 260 265 270 Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala 275 280 285 Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg 290 295 300 Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn 305 310 315 320 Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu Met 325 330 335 Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala 340 345 350 Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser 355 360 365 Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu 370 375 380 Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp 385 390 395 400 Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr 405 410 415 Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile 420 425 430 Tyr Ala Thr Leu Ala Ile His 435 <210> 53 <211> 466 <212> PRT <213> Artificial sequence <220> <223> C2058 chimeric antigen receptor construct <400> 53 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu 275 280 285 Leu Leu Leu Leu Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln 290 295 300 Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro 305 310 315 320 Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg 325 330 335 Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val 340 345 350 Lys His Thr Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro 355 360 365 His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser 370 375 380 Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu 385 390 395 400 Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp 405 410 415 Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln 420 425 430 Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser 435 440 445 Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala 450 455 460 Ile His 465 <210> 54 <211> 421 <212> PRT <213> Artificial sequence <220> <223> C2070 chimeric antigen receptor construct <400> 54 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Gly Gly Gly Gly Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg 325 330 335 Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe 340 345 350 Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro 355 360 365 Glu Pro Pro Val Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile 370 375 380 Val Phe Pro Ser Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser 385 390 395 400 Ala Asp Gly Pro Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His 405 410 415 Cys Ser Trp Pro Leu 420 <210> 55 <211> 416 <212> PRT <213> Artificial sequence <220> <223> C2071 chimeric antigen receptor construct <400> 55 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Gly Gly Gly Gly Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln 325 330 335 Glu Pro Ala Gly Asp Arg Thr Val Asn Arg Gln Asp Ser Asp Glu Gln 340 345 350 Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu Asp His Cys Val Phe Ile 355 360 365 Gln Arg Lys Ile Ser Arg Pro Ser Gln Arg Pro Lys Thr Pro Leu Thr 370 375 380 Asp Thr Ser Val Tyr Thr Glu Leu Pro Asn Ala Glu Pro Arg Ser Lys 385 390 395 400 Val Val Ser Cys Pro Arg Ala Pro Gln Ser Gly Leu Glu Gly Val Phe 405 410 415 <210> 56 <211> 493 <212> PRT <213> Artificial sequence <220> <223> C2072 chimeric antigen receptor construct <400> 56 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Gly Gly Gly Gly Ser Arg His Arg Arg Gln Gly Lys His Trp Thr 325 330 335 Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly 340 345 350 Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala 355 360 365 Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro 370 375 380 Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro 385 390 395 400 Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu 405 410 415 Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys 420 425 430 Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala 435 440 445 Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr 450 455 460 Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser 465 470 475 480 Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 485 490 <210> 57 <211> 704 <212> PRT <213> Artificial sequence <220> <223> C2106 chimeric antigen receptor construct <400> 57 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 245 250 255 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 260 265 270 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 275 280 285 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 290 295 300 Val Ile Thr Leu Tyr Cys Asn His Asp Arg Glu Lys Lys Pro Arg Gln 305 310 315 320 His Ser Gly Asp His Glu Asn Leu Met Asn Val Pro Ser Asp Lys Glu 325 330 335 Met Phe Ser Arg Ser Val Thr Ser Leu Ala Thr Asp Ala Pro Ala Ser 340 345 350 Ser Glu Gln Asn Gly Ala Leu Thr Asn Gly Asp Ile Leu Ser Glu Asp 355 360 365 Ser Thr Leu Thr Cys Met Gln His Tyr Glu Glu Val Gln Thr Ser Ala 370 375 380 Ser Asp Leu Leu Asp Ser Gln Asp Ser Thr Gly Lys Pro Lys Cys His 385 390 395 400 Gln Ser Arg Glu Leu Pro Arg Ile Pro Pro Glu Ser Ala Val Asp Thr 405 410 415 Met Leu Thr Ala Arg Ser Val Asp Gly Asp Gln Gly Leu Gly Met Glu 420 425 430 Gly Pro Tyr Glu Val Leu Lys Asp Ser Ser Ser Gln Glu Asn Met Val 435 440 445 Glu Asp Cys Leu Tyr Glu Thr Val Lys Glu Ile Lys Glu Val Ala Ala 450 455 460 Ala Ala His Leu Glu Lys Gly His Ser Gly Lys Ala Lys Ser Thr Ser 465 470 475 480 Ala Ser Lys Glu Leu Pro Gly Pro Gln Thr Glu Gly Lys Ala Glu Phe 485 490 495 Ala Glu Tyr Ala Ser Val Asp Arg Asn Lys Lys Cys Arg Gln Ser Val 500 505 510 Asn Val Glu Ser Ile Leu Gly Asn Ser Cys Asp Pro Glu Glu Glu Ala 515 520 525 Pro Pro Pro Val Pro Val Lys Leu Leu Asp Glu Asn Glu Asn Leu Gln 530 535 540 Glu Lys Glu Gly Gly Glu Ala Glu Glu Ser Ala Thr Asp Thr Thr Ser 545 550 555 560 Glu Thr Asn Lys Arg Phe Ser Ser Leu Ser Tyr Lys Ser Arg Glu Glu 565 570 575 Asp Pro Thr Leu Thr Glu Glu Glu Ile Ser Ala Met Tyr Ser Ser Val 580 585 590 Asn Lys Pro Gly Gln Leu Val Asn Lys Ser Gly Gln Ser Leu Thr Val 595 600 605 Pro Glu Ser Thr Tyr Thr Ser Ile Gln Gly Asp Pro Gln Arg Ser Pro 610 615 620 Ser Ser Cys Asn Asp Leu Tyr Ala Thr Val Lys Asp Phe Glu Lys Thr 625 630 635 640 Pro Asn Ser Thr Leu Pro Pro Ala Gly Arg Pro Ser Glu Glu Pro Glu 645 650 655 Pro Asp Tyr Glu Ala Ile Gln Thr Leu Asn Arg Glu Glu Glu Lys Ala 660 665 670 Thr Leu Gly Thr Asn Gly His His Gly Leu Val Pro Lys Glu Asn Asp 675 680 685 Tyr Glu Ser Ile Ser Asp Leu Gln Gln Gly Arg Asp Ile Thr Arg Leu 690 695 700 <210> 58 <211> 702 <212> PRT <213> Artificial sequence <220>0002926<223> C2107 chimeric antigen receptor construct <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 245 250 255 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 260 265 270 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Trp 275 280 285 Gly Ser Leu Ala Ala Val Ala Ile Phe Phe Val Ile Thr Phe Leu Ile 290 295 300 Phe Leu Cys Ser Ser Cys Asp Arg Glu Lys Lys Pro Arg Gln His Ser 305 310 315 320 Gly Asp His Glu Asn Leu Met Asn Val Pro Ser Asp Lys Glu Met Phe 325 330 335 Ser Arg Ser Val Thr Ser Leu Ala Thr Asp Ala Pro Ala Ser Ser Glu 340 345 350 Gln Asn Gly Ala Leu Thr Asn Gly Asp Ile Leu Ser Glu Asp Ser Thr 355 360 365 Leu Thr Cys Met Gln His Tyr Glu Glu Val Gln Thr Ser Ala Ser Asp 370 375 380 Leu Leu Asp Ser Gln Asp Ser Thr Gly Lys Pro Lys Cys His Gln Ser 385 390 395 400 Arg Glu Leu Pro Arg Ile Pro Pro Glu Ser Ala Val Asp Thr Met Leu 405 410 415 Thr Ala Arg Ser Val Asp Gly Asp Gln Gly Leu Gly Met Glu Gly Pro 420 425 430 Tyr Glu Val Leu Lys Asp Ser Ser Ser Gln Glu Asn Met Val Glu Asp 435 440 445 Cys Leu Tyr Glu Thr Val Lys Glu Ile Lys Glu Val Ala Ala Ala Ala 450 455 460 His Leu Glu Lys Gly His Ser Gly Lys Ala Lys Ser Thr Ser Ala Ser 465 470 475 480 Lys Glu Leu Pro Gly Pro Gln Thr Glu Gly Lys Ala Glu Phe Ala Glu 485 490 495 Tyr Ala Ser Val Asp Arg Asn Lys Lys Cys Arg Gln Ser Val Asn Val 500 505 510 Glu Ser Ile Leu Gly Asn Ser Cys Asp Pro Glu Glu Glu Ala Pro Pro 515 520 525 Pro Val Pro Val Lys Leu Leu Asp Glu Asn Glu Asn Leu Gln Glu Lys 530 535 540 Glu Gly Gly Glu Ala Glu Glu Ser Ala Thr Asp Thr Thr Ser Glu Thr 545 550 555 560 Asn Lys Arg Phe Ser Ser Leu Ser Tyr Lys Ser Arg Glu Glu Asp Pro 565 570 575 Thr Leu Thr Glu Glu Glu Ile Ser Ala Met Tyr Ser Ser Val Asn Lys 580 585 590 Pro Gly Gln Leu Val Asn Lys Ser Gly Gln Ser Leu Thr Val Pro Glu 595 600 605 Ser Thr Tyr Thr Ser Ile Gln Gly Asp Pro Gln Arg Ser Pro Ser Ser 610 615 620 Cys Asn Asp Leu Tyr Ala Thr Val Lys Asp Phe Glu Lys Thr Pro Asn 625 630 635 640 Ser Thr Leu Pro Pro Ala Gly Arg Pro Ser Glu Glu Pro Glu Pro Asp 645 650 655 Tyr Glu Ala Ile Gln Thr Leu Asn Arg Glu Glu Glu Lys Ala Thr Leu 660 665 670 Gly Thr Asn Gly His His Gly Leu Val Pro Lys Glu Asn Asp Tyr Glu 675 680 685 Ser Ile Ser Asp Leu Gln Gln Gly Arg Asp Ile Thr Arg Leu 690 695 700 <210> 59 <211> 481 <212> PRT <213> Artificial sequence <220> <223> C2153 chimeric antigen receptor construct <400> 59 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 245 250 255 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 260 265 270 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp 275 280 285 Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 290 295 300 Thr Val Ala Phe Ile Ile Phe Trp Val Leu Arg His Arg Arg Gln Gly 305 310 315 320 Lys His Trp Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala 325 330 335 Gly Ala Val Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser 340 345 350 Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys 355 360 365 His Thr Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His 370 375 380 Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg 385 390 395 400 Pro Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe 405 410 415 Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr 420 425 430 Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu 435 440 445 His Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln 450 455 460 Glu Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile 465 470 475 480 His <210> 60 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> C2156 Chimeric Antigen Receptor Construct <400> 60 Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp 1 5 10 15 Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30 Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45 Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60 Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg 65 70 75 80 Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95 Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg 100 105 110 Pro Leu Tyr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser 115 120 125 Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Leu Ile Leu 245 250 255 Leu Leu Leu Val Ala Gly Phe Asn Leu Leu Met Thr Leu Leu Leu Trp 260 265 270 Ser Ser Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser 275 280 285 Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro 290 295 300 Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp 305 310 315 320 Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu 325 330 335 Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln 340 345 350 Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met 355 360 365 Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp 370 375 380 Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser 385 390 395 400 Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu 405 410 415 Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro 420 425 430 Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 435 440 <210> 61 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> C2157 Chimeric Antigen Receptor Construct <400> 61 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 [[ID=]33]Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Leu Ile Leu Leu Gly Leu Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Leu Ile Leu 290 295 300 Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala 305 310 315 320 Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg 325 330 335 Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn 340 345 350 Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu Met 355 360 365 Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala 370 375 380 Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser 385 390 395 400 Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu 405 410 415 Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp 420 425 430 Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr 435 440 445 Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile 450 455 460 Tyr Ala Thr Leu Ala Ile His 465 470 <210> 62 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> C2158 Chimeric Antigen Receptor Construct <400> 62 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Pro Phe Gly Asp Trp Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Ser Tyr Ser Phe Val Leu Thr Phe Gly Gly Gly Thr Lys 225 230 235 240 Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 245 250 255 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 260 265 270 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 275 280 285 Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 290 295 300 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Leu Arg His Arg 305 310 315 320 Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln 325 330 335 His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln 340 345 350 Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala 355 360 365 Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg 370 375 380 Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala Glu Val Lys 385 390 395 400 His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser 405 410 415 Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln 420 425 430 Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr 435 440 445 Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro 450 455 460 Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr 465 470 475 480 Leu Ala Ile His <210> 63 <211> 414 <212> PRT <213> Artificial sequence <220> <223> C2179 chimeric antigen receptor construct <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Arg Pro Arg Arg 325 330 335 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 340 345 350 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 355 360 365 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 370 375 380 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 385 390 395 400 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 405 410 <210> 64 <211> 576 <212> PRT <213> Artificial sequence <220> <223> C2180 chimeric antigen receptor construct <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 325 330 335 Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 340 345 350 Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 355 360 365 Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln 370 375 380 Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 385 390 395 400 Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg 405 410 415 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 420 425 430 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 435 440 445 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 450 455 460 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 465 470 475 480 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His Arg Pro 485 490 495 Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu 500 505 510 Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu 515 520 525 Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His 530 535 540 Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu 545 550 555 560 Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 565 570 575 <210> 65 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> C2181 Chimeric Antigen Receptor Construct <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Arg Pro Arg Arg 325 330 335 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 340 345 350 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 355 360 365 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 370 375 380 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 385 390 395 400 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His Arg Pro 405 410 415 Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu 420 425 430 Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu 435 440 445 Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His 450 455 460 Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu 465 470 475 480 Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 485 490 495 <210> 66 <211> 494 <212> PRT <213> Artificial sequence <220> <223> C2182 chimeric antigen receptor construct <400> 66 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 325 330 335 Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 340 345 350 Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 355 360 365 Ala Asp Ala Gln Glu Glu Asn Leu Phe Ala Ala Val Lys His Thr Gln 370 375 380 Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 385 390 395 400 Pro Gln Ala Val Thr Phe Ala Glu Val Lys His Ser Arg Pro Arg Arg 405 410 415 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 420 425 430 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 435 440 445 Ala Ser Glu Ala Pro Gln Asp Val Thr Phe Ala Gln Leu His Ser Leu 450 455 460 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 465 470 475 480 Ser Pro Ala Val Pro Ser Ile Phe Ala Thr Leu Ala Ile His 485 490 <210> 67 <211> 494 <212> PRT <213> Artificial sequence <220> <223> C2183 chimeric antigen receptor construct <400> 67 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 325 330 335 Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 340 345 350 Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 355 360 365 Ala Asp Ala Gln Glu Glu Asn Leu Phe Ala Ala Val Lys His Thr Gln 370 375 380 Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 385 390 395 400 Pro Gln Ala Val Thr Phe Ala Glu Val Lys His Ser Arg Pro Arg Arg 405 410 415 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 420 425 430 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 435 440 445 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 450 455 460 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 465 470 475 480 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 485 490 <210> 68 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> C2184 Chimeric Antigen Receptor Construct <400> 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 325 330 335 Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 340 345 350 Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 355 360 365 Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln 370 375 380 Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 385 390 395 400 Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg 405 410 415 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 420 425 430 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 435 440 445 Ala Ser Glu Ala Pro Gln Asp Val Thr Phe Ala Gln Leu His Ser Leu 450 455 460 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 465 470 475 480 Ser Pro Ala Val Pro Ser Ile Phe Ala Thr Leu Ala Ile His 485 490 <210> 69 <211> 438 <212> PRT <213> Artificial sequence <220> <223> C2218 chimeric antigen receptor construct <400> 69 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg Arg His Gln Gly Lys Gln Asn Glu 325 330 335 Leu Ser Asp Thr Ala Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu 340 345 350 Lys Ser Glu Gln Thr Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu 355 360 365 Leu Ser Glu Thr Gly Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg 370 375 380 Met Gln Glu Gly Ser Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn 385 390 395 400 Lys Pro Gly Ile Val Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro 405 410 415 Asn Ser Arg Leu Ala Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala 420 425 430 Ser Ile Cys Val Arg Ser 435 <210> 70 <211> 437 <212> PRT <213> Artificial sequence <220> <223> C2219 chimeric antigen receptor construct <400> 70 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Leu Leu Pro Leu Gly Gly Leu Pro Leu Leu Ile Thr Thr Cys Phe 305 310 315 320 Cys Leu Phe Cys Cys Leu Arg Arg His Gln Gly Lys Gln Asn Glu Leu 325 330 335 Ser Asp Thr Ala Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys 340 345 350 Ser Glu Gln Thr Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu 355 360 365 Ser Glu Thr Gly Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met 370 375 380 Gln Glu Gly Ser Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys 385 390 395 400 Pro Gly Ile Val Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro Asn 405 410 415 Ser Arg Leu Ala Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser 420 425 430 Ile Cys Val Arg Ser 435 <210> 71 <211> 397 <212> PRT <213> Artificial sequence <220> <223> C2220 chimeric antigen receptor construct <400> 71 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Thr Asp Val Lys Ser Ala Ser Glu Arg Pro Ser Lys Asp Glu Met 245 250 255 Ala Ser Arg Pro Trp Leu Leu Tyr Arg Leu Leu Pro Leu Gly Gly Leu 260 265 270 Pro Leu Leu Ile Thr Thr Cys Phe Cys Leu Phe Cys Cys Leu Arg Arg 275 280 285 His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg Glu Ile 290 295 300 Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr Glu Ala Ser Thr 305 310 315 320 Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly Ile Tyr Asp Asn 325 330 335 Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser Glu Val Tyr Ser 340 345 350 Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val Tyr Ala Ser Leu 355 360 365 Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu Ala Arg Asn Val Lys 370 375 380 Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg Ser 385 390 395 <210> 72 <400> 72 000 <210> 73 <400> 73 000 <210> 74 <400> 74 000 <210> 75 <400> 75 000 <210> 76 <400> 76 000 <210> 77 <211> 578 <212> PRT <213> Artificial Sequence <220> <223> C2302 Chimeric Antigen Receptor Construct <400> 77 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220 Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser 245 250 255 Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser 260 265 270 Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu 275 280 285 Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 290 295 300 Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu 305 310 315 320 Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Arg Pro Arg Arg 325 330 335 Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 340 345 350 Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 355 360 365 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 370 375 380 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 385 390 395 400 Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His Arg Pro 405 410 415 Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu 420 425 430 Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu 435 440 445 Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His 450 455 460 Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu 465 470 475 480 Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 485 490 495 Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu 500 505 510 Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp 515 520 525 Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln 530 535 540 Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser 545 550 555 560 Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala 565 570 575 Ile His <210> 78 <211> 600 <212> PRT <213> Synthetic Sequence <220> <223> CT138 Chimeric Antigen Receptor Construct <400> 78 Met Gly Pro Val Thr Cys Ser Val Leu Val Leu Leu Leu Met Leu Arg 1 5 10 15 Arg Ser Asn Gly Asp Gly Asp Ser Val Thr Gln Thr Glu Gly Leu Val 20 25 30 Thr Leu Thr Glu Gly Leu Pro Val Met Leu Asn Cys Thr Tyr Gln Thr 35 40 45 Ile Tyr Ser Asn Pro Phe Leu Phe Trp Tyr Val Gln His Leu Asn Glu 50 55 60 Ser Pro Arg Leu Leu Leu Lys Ser Phe Thr Asp Asn Lys Arg Thr Glu 65 70 75 80 His Gln Gly Phe His Ala Thr Leu His Lys Ser Ser Ser Ser Phe His 85 90 95 Leu Gln Lys Ser Ser Ala Gln Leu Ser Asp Ser Ala Leu Tyr Tyr Cys 100 105 110 Ala Phe Asp Thr Asn Thr Tyr Lys Val Ile Phe Gly Lys Gly Thr His 115 120 125 Leu His Val Leu Pro Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln 130 135 140 Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser 180 185 190 Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp 195 200 205 Ile Phe Lys Glu Thr Asn Thr Thr Tyr Pro Ser Ser Asp Val Pro Cys 210 215 220 Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn 225 230 235 240 Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val 245 250 255 Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser Arg Ala 260 265 270 Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala 275 280 285 Gly Asp Val Glu Glu Asn Pro Gly Pro Met Arg Val Arg Leu Ile Ser 290 295 300 Ala Val Val Leu Cys Ser Leu Gly Thr Gly Leu Val Asp Met Lys Val 305 310 315 320 Thr Gln Met Pro Arg Tyr Leu Ile Lys Arg Met Gly Glu Asn Val Leu 325 330 335 Leu Glu Cys Gly Gln Asp Met Ser His Glu Thr Met Tyr Trp Tyr Arg 340 345 350 Gln Asp Pro Gly Leu Gly Leu Gln Leu Ile Tyr Ile Ser Tyr Asp Val 355 360 365 Asp Ser Asn Ser Glu Gly Asp Ile Pro Lys Gly Tyr Arg Val Ser Arg 370 375 380 Lys Lys Arg Glu His Phe Ser Leu Ile Leu Asp Ser Ala Lys Thr Asn 385 390 395 400 Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Ser Thr Asn Thr Glu Val 405 410 415 Phe Phe Gly Lys Gly Thr Arg Leu Thr Val Val Glu Asp Leu Arg Asn 420 425 430 Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile 435 440 445 Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe 450 455 460 Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His 465 470 475 480 Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser 485 490 495 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn 500 505 510 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu 515 520 525 Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile 530 535 540 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser 545 550 555 560 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 565 570 575 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met 580 585 590 Ala Met Val Lys Arg Lys Asn Ser 595 600 <210> 79 <211> 605 <212> PRT <213> Artificial sequence <220> <223> CT139 chimeric antigen receptor construct <400> 79 Met Val Leu Val Thr Ile Leu Leu Leu Ser Ala Phe Phe Ser Leu Arg 1 5 10 15 Gly Asn Ser Ala Gln Ser Val Asp Gln Pro Asp Ala His Val Thr Leu 20 25 30 Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Ser Tyr Ser Tyr Ser Ala 35 40 45 Ala Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln Ser Leu Gln 50 55 60 Phe Leu Leu Lys Tyr Ile Thr Gly Asp Thr Val Val Lys Gly Thr Lys 65 70 75 80 Gly Phe Glu Ala Glu Phe Arg Lys Ser Asn Ser Ser Phe Asn Leu Lys 85 90 95 Lys Ser Pro Ala His Trp Ser Asp Ser Ala Lys Tyr Phe Cys Ala Leu 100 105 110 Glu Gly Pro Asp Thr Gly Asn Tyr Lys Tyr Val Phe Gly Ala Gly Thr 115 120 125 Arg Leu Lys Val Ile Ala His Ile Gln Asn Pro Glu Pro Ala Val Tyr 130 135 140 Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr 145 150 155 160 Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr 165 170 175 Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys 180 185 190 Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln 195 200 205 Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro 210 215 220 Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu 225 230 235 240 Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys 245 250 255 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser Arg 260 265 270 Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln 275 280 285 Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Ile Gln Thr Leu 290 295 300 Cys Cys Val Ile Phe Tyr Val Leu Ile Ala Asn His Thr Asp Ala Gly 305 310 315 320 Val Thr Gln Thr Pro Arg His Glu Val Ala Glu Lys Gly Gln Thr Ile 325 330 335 Ile Leu Lys Cys Glu Pro Val Ser Gly His Asn Asp Leu Phe Trp Tyr 340 345 350 Arg Gln Thr Lys Ile Gln Gly Leu Glu Leu Leu Ser Tyr Phe Arg Ser 355 360 365 Lys Ser Leu Met Glu Asp Gly Gly Ala Phe Lys Asp Arg Phe Lys Ala 370 375 380 Glu Met Leu Asn Ser Ser Phe Ser Thr Leu Lys Ile Gln Pro Thr Glu 385 390 395 400 Pro Arg Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Phe Gly Thr Ala 405 410 415 Ser Ala Glu Thr Leu Tyr Phe Gly Ser Gly Thr Arg Leu Thr Val Leu 420 425 430 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 435 440 445 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 450 455 460 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 465 470 475 480 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys 485 490 495 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 500 505 510 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 515 520 525 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 530 535 540 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 545 550 555 560 Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu 565 570 575 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 580 585 590 Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser 595 600 605 <210> 80 <211> 64 <212> PRT <213> Homo sapiens <400> 80 Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp 1 5 10 15 Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro 20 25 30 Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45 ...

Claims

1. A chimeric antigen receptor comprising a polypeptide, wherein the polypeptide comprises: a) an antigen binding domain comprising a scFv comprising a CDR-L1, a CDR-L2, a CDR-L3, a CDR-H1, a CDR-H2, and a CDR-H3, wherein: i) the CDR-L1 consists of SEQ ID NO: 22, the CDR-L2 consists of SEQ ID NO: 23, the CDR-L3 consists of SEQ ID NO: 24, the CDR-H1 consists of SEQ ID NO: 25, the CDR-H2 consists of SEQ ID NO: 26, and the CDR-H3 consists of SEQ ID NO: 27, ii) the CDR-L1 consists of SEQ ID NO: 28, the CDR-L2 consists of SEQ ID NO: 29, the CDR-L3 consists of SEQ ID NO: 30, the CDR-H1 consists of SEQ ID NO: 31, the CDR-H2 consists of SEQ ID NO: 32, and the CDR-H3 consists of SEQ ID NO: 33, or iii) the CDR-L1 consists of SEQ ID NO: 28, the CDR-L2 consists of KVSNRFSGVPAR, the CDR-L3 consists of SEQ ID NO: 30, the CDR-H1 consists of SEQ ID NO: 31, the CDR-H2 consists of SEQ ID NO: 32, and the CDR-H3 consists of SEQ ID NO: 33; b) a LILRB1 hinge domain comprising SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 93; c) a LILRB1 transmembrane domain comprising SEQ ID NO: 5; and d) a LILRB1 endodomain comprising at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs) or an endodomain comprising at least two ITIMs, wherein each ITIM is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:

11.

2. The receptor of claim 1, wherein the polypeptide comprises the LILRB1 endodomain.

3. The receptor of claim 1, wherein the polypeptide comprises an endodomain comprising at least two ITIMs, and the at least two ITIMs comprise SEQ ID NO: 8 and SEQ ID NO:

9. ​ ​ ​ ​ ​ ​ ​ 4. The receptor of claim 3, wherein the endodomain comprises the same sequence as SEQ ID NO:

12.

5. The receptor of claim 1, wherein the polypeptide comprises an endodomain comprising at least two ITIMs, and the at least two ITIMs comprise SEQ ID NO: 9 and SEQ ID NO:

10.

6. The receptor of claim 5, wherein the endodomain comprises the same sequence as SEQ ID NO:

13.

7. The receptor of claim 1, wherein the polypeptide comprises an endodomain comprising at least two ITIMs, and the at least two ITIMs comprise SEQ ID NO: 10 and SEQ ID NO:

11.

8. The receptor of claim 7, wherein the endodomain comprises the same sequence as SEQ ID NO:

14.

9. The receptor of claim 1, wherein the polypeptide comprises an endodomain comprising at least two ITIMs, and the at least two ITIMs comprise at least three ITIMs.

10. The receptor of claim 9, wherein the at least three ITIMs comprise SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

10.

11. The receptor of claim 10, wherein the endodomain comprises the same sequence as SEQ ID NO:

15.

12. The receptor of claim 9, wherein the at least three ITIMs comprise SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:

11.

13. The receptor of claim 12, wherein the endodomain comprises the same sequence as SEQ ID NO:

16.

14. The receptor of claim 1, wherein the polypeptide comprises an endodomain comprising at least two ITIMs, and the at least two ITIMs comprise SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:

11.

15. The receptor of claim 14, wherein the endodomain comprises the same sequence as SEQ ID NO:

17.

16. The receptor of claim 14, wherein the endodomain comprises the same sequence as LILRB1 endodomain SEQ ID NO:

7.

17. The receptor of claim 1, wherein the polypeptide comprises the same sequence as SEQ ID NO:

20.

18. The receptor of claim 1, wherein the polypeptide comprises the same sequence as SEQ ID NO:

21.

19. The receptor of claim 1, wherein the polypeptide comprises the same sequence as SEQ ID NO: 2 or SEQ ID NO:

3.

20. The receptor of claim 1, wherein the scFv comprises a sequence identical to any one of SEQ ID NOs: 35, 37, 46, or 125.

21. The receptor of claim 1, wherein the scFv comprises a heavy chain and a light chain, and wherein the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 35-46 or 125, and wherein the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 35, 37, 39, 46, or 125.

22. The receptor of claim 1, wherein the receptor comprises the amino acid sequence of SEQ ID NOs: 89-92, 120, or 122.

23. A polynucleotide comprising a nucleic acid sequence encoding the receptor of any one of claims 1-22.

24. A vector comprising the polynucleotide of claim 23.

25. The vector of claim 24, further comprising a sequence encoding a promoter operably linked to the polynucleotide.

26. An immune cell comprising the receptor of any one of claims 1-22, the polynucleotide of claim 23, or the vector of claim 24 or 25.

27. The immune cell of claim 26, wherein activation of the immune cell is reduced when the cell is contacted with an antigen or a cell expressing the antigen on its surface.

28. The immune cell of claim 27, wherein the activation of the immune cell comprises expression of a gene operably linked to an NFAT promoter.

29. The immune cell of any one of claims 26-28, wherein the immune cell is a T cell.

30. The immune cell of any one of claims 26-28, further comprising an activator receptor.

31. The immune cell of claim 30, wherein the activator receptor is a chimeric antigen receptor or a T cell receptor.

32. A method of manufacturing an engineered immune cell, comprising introducing the polynucleotide of claim 23 or the vector of claim 24 or 25 into an immune cell.

33. The method of claim 32, wherein the engineered immune cell expresses the receptor.

34. The method of claim 32, wherein the immune cell is a T cell.

35. The method of any one of claims 32-34, wherein activation of the engineered immune cell is reduced when the engineered immune cell is contacted with an antigen specific for the chimeric antigen receptor or a cell expressing the antigen on its surface.

36. The method of claim 35, wherein the activation of the engineered immune cell comprises expression of a gene operably linked to an NFAT promoter.

37. A kit comprising the receptor of any one of claims 1-22, the polynucleotide of claim 23, the vector of claim 24 or 25, or the immune cell of any one of claims 26-31.

Images