Chimeric antigen receptor for human cytomegalovirus

Abstract

Disclosed herein are CMV-specific CARs. In some embodiments, the invention relates to a method of treating, reducing, or inhibiting a cytomegalovirus infection in a subject, comprising administering to the subject (a) an expression vector encoding a CMV-specific CAR as described herein, or (b) one or more cells transduced with the expression vector.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 914,408, filed October 11, 2019, which is incorporated herein by reference in its entirety.

[0002]

[0003] Reference to sequence listing submitted via EFS-WEB

[0004] The contents of the ASCII text file of the Sequence Listing, 85.9kb in size and filename "20201012_034044_209WO1_ST25", created on October 7, 2020, was submitted electronically via EFS-Web herewith, and is hereby incorporated by reference in its entirety.

[0003]

[0005] Acknowledgments for government support

[0006] This invention was made with federal support under Grant No. AI103960 awarded by the National Institutes of Health. The federal government has certain rights in this invention. [Background technology]

[0004]

[0007] Background of the Invention

[0008] 1. Field of the Invention

[0009] Compositions and methods for treating cytomegalovirus infection.

[0005]

[0010] 2. Description of Related Technology

[0011] Human cytomegalovirus (CMV) infection is widespread, affecting approximately 60% of adults in the United States and nearly 100% in other parts of the world. In most healthy individuals, the infection is lifelong but immunologically suppressed and asymptomatic. In some perinatally infected individuals and in those who are severely immunosuppressed (due to iatrogenic / therapeutic immunosuppression for conditions such as AIDS or bone marrow or organ transplantation), disseminated infection can occur and can be associated with significant morbidity and mortality due to end-organ damage.

[0006]

[0012] The primary immune mechanism controlling CMV infection in healthy hosts is cell-mediated immunity, particularly CD8+ T lymphocytes (C8TL). Autologous immunotherapy using expanded CMV-specific C8TL has provided proof-of-concept that C8TL can treat CMV in immunocompromised hosts, but this approach is not generally applicable due to various technical limitations. However, CMV-targeted chimeric antigen receptor (CAR) gene therapy may be readily applicable to the generation of CMV-specific C8TL in patients, similar to the increasing use of CAR T cell gene therapy for various cancers. Summary of the Invention [Problem to be solved by the invention]

[0007]

[0013] To date, there is only one CMV-specific CAR reported in the literature, and to our knowledge, it has not progressed into clinical trials. [Means for solving the problem]

[0008]

[0014] Summary of the Invention

[0015] "CMV-specific CAR": In some embodiments, the present invention provides a CMV-specific CAR having the following SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26: G-X1-X2-X3-X4-X5-X6-X7-X8-X9 formula H1 (SEQ ID NO:21) wherein X1 is F or Y, X2 is S or T, X3 is L or F, X4 is S or T, X5 is D, I, N, S, or T, X6 is F, Y, or S, X7 is G, Y, or W, X8 is present or absent, and if present, X8 is L or I, and X9 is present or absent, and if present, X9 is G; Formula H2 (SEQ ID NO: 22) wherein X10 is D, N, S, or W, X11 is D, N, P, T, or W, X12 is D, G, N, Y, or S, X13 is D, G, or T, X14 is D, G, N, or S, X15 is E, K, S, or Y, and X16 is present or absent, and if present, X16 is P or T;X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31 X23 is A, F, G, L, P, or Y; X24 is A, D, I, Q, S, V, or Y; X25 is present or absent, and if present, X25 is E, F, N, S, or Y; X26 is present or absent, and if present, X26 is A, G, , M, L, or P; X27 is present or absent, and if present, X27 is D, L, or Y; X28 is present or absent, and if present, X28 is A, F, L, or Y; X29 is present or absent, and if present, X29 is D, F, G, or M; X30 is present or absent, and if present, X30 is C, D, or Y; and X31 is present or absent, and if present, X31 is S or Y; X32-X33-X34-X35-X36-X37-X38-X39-X40-X41-X42 X is present or absent, and if present, X is D, G, or N; X is present or absent, and if present, X is G, N, or Y; X is present or absent, and if present, X is K, N, or S; X is present or absent, and if present, X is F, Y, or T; and X is present or absent, and if present, X is Y; Formula L2 (SEQ ID NO: 25) wherein X43 is D, L, R, T, or Y and X44 is A, T, or V;X45-X46-X47-X48-X49-X50-P-X51-T A cytomegalovirus-specific chimeric antigen receptor (CMV-specific CAR) comprising a single-chain antibody sequence or fragment thereof having the formula L3 (SEQ ID NO: 26), wherein X45 is S, Q, or W, X46 is H, N, or Q, X47 is D, G, S, or Y, X48 is H, N, R, S, T, or Y, X49 is E, H, K, R, S, or T, X50 is D, F, L, S, V, or W, and X51 is L, P, W, or Y. In some embodiments, a) Formula H1 (SEQ ID NO:21) is GFSLSTYGIG (SEQ ID NO:27), GFSLTTSGLG (SEQ ID NO:28), GFTFSDYY (SEQ ID NO:29), GYTFTIYG (SEQ ID NO:30), GYTFTNFG (SEQ ID NO:31), GYTFTSYG (SEQ ID NO:32), GYTFTSYW (SEQ ID NO:33), GYTFTIYW (SEQ ID NO:34), or GYTFTSYW (SEQ ID NO:35); b) Formula H2 (SEQ ID NO:22) is IDPSDSET (SEQ ID NO:36), IDPSDSET (SEQ ID NO:37), IDPSDSET (SEQ ID NO:38), INTYTGEP (SEQ ID NO:39), ISDDGNYT (SEQ ID NO:40), ISNGGGST (SEQ ID NO:41), IWWDDDK (SEQ ID NO:42), or IWWNDNK (SEQ ID NO:43); c) Formula H3 (SEQ ID NO:23) is ARE HYYGINPLLGC (SEQ ID NO:44), ARGWLLPVFAY (SEQ ID NO:45), ARKGYYGSSGYFDY (SEQ ID NO:46), ARRGDGLYSMDY (SEQ ID NO:47), ARTGYFDV (SEQ ID NO:48), SNGYSSFAY (SEQ ID NO:49), VRPKRDFQYLYAMDY (SEQ ID NO:50), VRSLYDYDEGYYFDS (SEQ ID NO:51), or ASSGTGAY (SEQ ID NO:52); d) formula L1 (SEQ ID NO:24) is ESIDSYGNSF (SEQ ID NO:53), KSVSTSGYSY (SEQ ID NO:54), QGISNY (SEQ ID NO:55), QSIGNN (SEQ ID NO:56), QSISDY (SEQ ID NO:57), QSLVHSNGNTY (SEQ ID NO:58), QSVSND (SEQ ID NO:59), QSISNN (SEQ ID NO:60), or QSLLDSDGKTY (SEQ ID NO:61);e) formula L2 (SEQ ID NO:25) is DTS (SEQ ID NO:62), LAS (SEQ ID NO:63), RAS (SEQ ID NO:64), TVS (SEQ ID NO:65), YAS (SEQ ID NO:66), YTS (SEQ ID NO:67), or LVS (SEQ ID NO:68); and / or f) formula L3 (SEQ ID NO:26) is QHSRELPWT (SEQ ID NO:69), QNGHTFPPT (SEQ ID NO:70), QQDYSSPWT (SEQ ID NO:71), QQSNEDPLT (SEQ ID NO:72), QQSNRWPWT (SEQ ID NO:73), QQYSKLPYT (SEQ ID NO:74), SQSTHVPYT (SEQ ID NO:75), QQSNSWPLT (SEQ ID NO:76), or WQGTHFPYT (SEQ ID NO:77). In some embodiments, formula H2 (SEQ ID NO:22) is INTYTGEP (SEQ ID NO:39) and formula L2 (SEQ ID NO:25) is YAS (SEQ ID NO:66). In some embodiments, the single chain antibody or fragment thereof comprises SEQ ID NO:30 with 0, 1, 2, or 3 amino acid substitutions, additions, or deletions, SEQ ID NO:39 with 0, 1, 2, or 3 amino acid substitutions, additions, or deletions, SEQ ID NO:46 with 0, 1, 2, or 3 amino acid substitutions, additions, or deletions, SEQ ID NO:59 with 0, 1, 2, or 3 amino acid substitutions, additions, or deletions, SEQ ID NO:66 with 0, 1, or 2 amino acid substitutions, additions, or deletions, and SEQ ID NO:71 with 0, 1, 2, or 3 amino acid substitutions, additions, or deletions. In some embodiments, the single chain antibody or fragment thereof comprises SEQ ID NO:30, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:59, SEQ ID NO:66, and SEQ ID NO:71. In some embodiments, the single chain antibody or fragment thereof comprises or consists of a VH chain and a VL chain selected from those shown in Table 1. In some embodiments, the single chain antibody or fragment thereof comprises or consists of SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the CMV-specific CAR comprises or consists of SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, or SEQ ID NO: 91, preferably SEQ ID NO: 89;

[0009]

[0016] In some embodiments, the present invention relates to nucleic acid molecules encoding a CMV-specific CAR as described herein, such as those described in the section entitled "CMV-Specific CARs" above.

[0010]

[0017] In some embodiments, the present invention relates to an expression vector comprising a nucleic acid molecule encoding a CMV-specific CAR as described herein, such as that described in the section entitled "CMV-Specific CAR" above.

[0011]

[0018] In some embodiments, the present invention relates to a host cell or its progeny cells comprising one or more expression vectors as described herein. In some embodiments, the host cell or progeny cells are CD8+ T lymphocytes, hematopoietic stem cells, or hematopoietic progenitor cells. In some embodiments, the host cell or progeny cells express one or more chimeric antigen receptors encoded by the one or more expression vectors.

[0012]

[0019] In some embodiments, the invention relates to a method of treating, reducing, or inhibiting a cytomegalovirus infection in a subject, comprising administering to the subject (a) an expression vector encoding a CMV-specific CAR as described herein, such as those described above in the section entitled "CMV-Specific CARs," or (b) one or more cells transduced with the expression vector.

[0013]

[0020] In some embodiments, the invention relates to a method of treating, reducing, or inhibiting a cytomegalovirus infection in a subject, comprising administering to the subject (a) an expression vector encoding a CMV-specific CAR as described herein, such as those described above in the section entitled "CMV-Specific CARs," or (b) one or more cells transduced with the expression vector.

[0014]

[0021] In some embodiments, the invention relates to a method of treating, reducing, or inhibiting a cytomegalovirus infection in a subject, the method comprising transplanting into the subject one or more cells expressing one or more CMV-specific CARs as described herein, such as those described in the section above entitled "CMV-Specific CARs."

[0015]

[0022] In some embodiments, the present invention relates to a method of treating, reducing, or inhibiting a cytomegalovirus infection in a subject, the method comprising transplanting into the subject one or more host cells as described herein or their progeny.

[0016]

[0023] In some embodiments, the invention relates to a method of killing a cell infected with cytomegalovirus, comprising contacting the infected cell with (a) one or more cells that express one or more CMV-specific CARs as described herein, such as those described in the section above entitled "CMV-specific CARs," or (b) one or more cells that comprise an expression vector encoding one or more CMV-specific CARs.

[0017]

[0024] In some embodiments, the invention relates to a method of reducing cytomegalovirus replication in a cell or a subject, comprising contacting a cell with, or administering to a subject, (a) one or more cells that express one or more CMV-specific CARs as described herein, such as those described above in the section entitled "CMV-specific CARs," or (b) one or more cells that comprise an expression vector encoding one or more CMV-specific CARs.

[0018]

[0025] In some embodiments, the invention relates to (a) the use of one or more CMV-specific CARs as described herein, such as those described above in the section entitled "CMV-Specific CARs," (b) the use of one or more nucleic acid molecules encoding a CMV-specific CAR as described herein, such as those described above in the section entitled "CMV-Specific CARs," (c) the use of one or more expression vectors comprising a nucleic acid molecule encoding a CMV-specific CAR as described herein, such as those described above in the section entitled "CMV-Specific CARs," and / or (d) the use of one or more host cells or progeny thereof comprising one or more expression vectors as described herein.

[0019]

[0026] In some embodiments, the invention relates to (a) the use of one or more CMV-specific CARs as described herein, such as those described in the section entitled "CMV-Specific CARs" above, in the manufacture of a medicament for the treatment of a cytomegalovirus infection; (b) the use of one or more nucleic acid molecules encoding a CMV-specific CAR as described herein, such as those described in the section entitled "CMV-Specific CARs" above, in the manufacture of a medicament for the treatment of a cytomegalovirus infection; (c) the use of one or more expression vectors comprising a nucleic acid molecule encoding a CMV-specific CAR as described herein, such as those described in the section entitled "CMV-Specific CARs" above, in the manufacture of a medicament for the treatment of a cytomegalovirus infection; and / or (d) the use of one or more host cells, or progeny thereof, comprising one or more expression vectors as described herein, in the manufacture of a medicament for the treatment of a cytomegalovirus infection.

[0020]

[0027] In some embodiments, the invention relates to (a) the use of one or more CMV-specific CARs as described herein, such as those described in the section above entitled "CMV-Specific CARs," for the treatment of cytomegalovirus infection; (b) the use of one or more nucleic acid molecules encoding a CMV-specific CAR as described herein, such as those described above in the section above entitled "CMV-Specific CARs," for the treatment of cytomegalovirus infection; (c) the use of one or more expression vectors comprising a nucleic acid molecule encoding a CMV-specific CAR as described herein, such as those described above in the section above entitled "CMV-Specific CARs," for the treatment of cytomegalovirus infection; and / or (d) the use of one or more host cells, or progeny thereof, comprising one or more expression vectors as described herein, for the treatment of cytomegalovirus infection.

[0021]

[0028] In some embodiments, the present invention relates to (a) one or more CMV-specific CARs as described herein, such as those described in the section entitled "CMV-Specific CARs" above; (b) one or more nucleic acid molecules encoding a CMV-specific CAR as described herein, such as those described in the section entitled "CMV-Specific CARs" above; (c) one or more expression vectors comprising a nucleic acid molecule encoding a CMV-specific CAR as described herein, such as those described in the section entitled "CMV-Specific CARs" above; and / or (d) one or more host cells or progeny thereof comprising one or more expression vectors as described herein, for the treatment of cytomegalovirus infection.

[0022]

[0029] In some embodiments, the subject is a human. In some embodiments, the subject has an immunodeficiency disorder. In some embodiments, the cytomegalovirus is a human cytomegalovirus.

[0023]

[0030] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated into and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0024]

[0031] DESCRIPTION OF THE DRAWINGS

[0032] The invention will be further understood with reference to the following drawings: [Brief explanation of the drawings]

[0025] [Figure 1]

[0033] NAb recognition of CMV-infected EpCs. EpCs were infected with CMV TB40 / E (MOI: 5). Cells were fixed and immunostained with 1B2 (PC-specific) or 62-11 (gH-specific) NAbs at 48 hours (middle curve) and 72 hours (right curve) postinfection (PI). Uninfected EpCs served as controls (left curve). [Figure 2]

[0034] Schematic of a typical CAR: A single-chain antibody serves as the binding domain (gray, top part), which is tethered to the CD8 transmembrane domain (black) by an immunoglobulin hinge-CH2-CH3 region spacer (white), which is linked to the 4-1BB and CD3ζ co-signaling and signaling domains (gray, bottom part). [Figure 3]

[0035] Schematic of an exemplary CAR construct. [Figure 4]

[0036] CAR expression in transduced primary C8TL by Western blot analysis. Primary C8TL were transduced with lentiviral vectors delivering the indicated CAR genes. Lysates were then evaluated by Western blot staining for CD3ζ. The filled arrow indicates the approximate size of the CD3ζ-containing CAR molecule, and the open arrow indicates the approximate size of native (unmodified) CD3ζ. Note that the 21F6 and 54E11 CARs are identical duplicates. [Figure 5A-1]

[0037] CAR expression in transduced primary C8TL by flow cytometry. Primary C8TL were transduced with a lentiviral vector delivering the gene for the indicated CAR. Cells were then stained for cell surface human antibody expression and analyzed by flow cytometry. [Figure 5A-2]

[0037] CAR expression in transduced primary C8TL by flow cytometry. Primary C8TL were transduced with a lentiviral vector delivering the gene for the indicated CAR. The cells were then stained for cell surface human antibody expression and analyzed by flow cytometry. [Figure 5B]

[0038] CAR expression in transduced primary C8TL by flow cytometry. Primary C8TL were transduced with a lentiviral vector delivering the gene for the indicated CAR. Cells were then stained for cell surface human antibody expression and analyzed by flow cytometry. Not shown: 62-11. [Figure 6]

[0039] Recognition of CMV-infected target cells by CAR-transduced C8TL. Primary C8TL transduced with the 21E9 CAR were exposed to CMV-infected or CMV-uninfected target cells and assessed by flow cytometry for intracellular IFN-γ / TNF-α and cell surface CD107a staining. [Figure 7]

[0040] Suppression / elimination of CMV by CAR-transduced primary C8TL. Cells acutely infected with CMV (using a GFP-expressing strain of CMV) were cultured with either no cells, non-transduced primary C8TL, or CAR-expressing C8TL and added 4 days post-infection. Imaging was performed 3 days later. The colors of the original figure have been inverted to improve black and white reproduction. [Figure 8]

[0041] Figures 8-11: Intracellular cytokine and cell surface CD107a expression by CAR-transduced primary C8TL upon exposure to CMV-infected cells.

[0042] Representative dot plots are shown for the production of both intracellular IFN-γ and TNF-α by untransduced (top row) or CAR-transduced (bottom row) C8TL exposed to uninfected (left column) or acutely infected with CMV TR (right column) ARPE-19 cells. [Figure 9]

[0043] Representative histograms of cell surface expression of CD107a on untransduced (top) or CAR-transduced (bottom) C8TL exposed to uninfected (left histogram) or CMV TR-infected (right histogram) ARPE-19 cells are shown. [Figure 10]

[0044] The net percentage of untransduced or CAR-transduced C8TL producing both intracellular IFN-γ and TNF-α in response to CMV-infected target cells (after subtracting the response to uninfected target cells) is plotted. [Figure 11]

[0045] The net percentage of untransduced or CAR-transduced C8TL expressing cell surface CD107a after exposure to ARPE-19 cells acutely infected with CMV is plotted. Similar results were observed with ARPE-19 cells infected with CMV TB40 / E (not shown). These results are representative of three experiments with three different C8TL donors; the other four CARs demonstrated minimal activity in the other two experiments (not shown). [Figure 12]

[0046] Proliferation of CAR-transduced primary C8TL upon exposure to CMV-infected cells. Primary C8TL transduced with the indicated CARs were labeled with CellTrace Violet dye and cocultured with uninfected (open gray histograms) or CMV TB40 / E-infected (filled black histograms) ARPE-19 cells for 6 days, and analyzed by flow cytometry for dye expression after 7 days. These results are representative of two independent experiments with two different C8TL donors, each performed in biological duplicate. [Figure 13]

[0047] Killing of CMV-infected target cells by CAR-transduced primary C8TL. Specific lysis of CMV TB40 / E-infected cells was subtracted from background specific lysis of uninfected cells (less than 6%, except for 2-80, which had background levels of 20% and 32% at effector-to-target ratios of 20:1 and 40:1, respectively). In three independent experiments with three different C8TL donors, only 21E9 consistently exhibited targeted killing of CMV-infected cells. [Figure 14]

[0048] Suppression of CMV replication in cell culture by CAR-transduced primary C8TL. ARPE-19 cells were acutely infected with CMV TR (expressing GFP) for 4 days and then cocultured with either no cells, non-transduced primary C8TL, or CAR-transduced C8TL (at a ratio of 10 C8TL per target cell) and subsequently imaged 8 hours later. Similar results were observed with CMV strain TB40 / E (not shown). These results are representative of three independent experiments with three different C8TL donors. The colors in the original figures have been inverted to improve black-and-white reproducibility. DETAILED DESCRIPTION OF THE INVENTION

[0026]

[0049] Detailed Description of the Invention

[0050] CMV entry into fibroblasts (FB) and epithelial / endothelial cells (EpC / EnC) occurs via distinct entry pathways that are blocked by neutralizing antibodies (NAbs) of varying potency and cell-type specificity. CMV infection of FBs depends on the major essential envelope glycoprotein complexes (gC) gM / gN, gB, and gH / gL / gO. In contrast to entry into FBs, CMV infection of EpC / EnCs requires an additional complex formed by gH / gL, UL128, UL130, and UL131A (PC). NAbs targeting the major gC block both CMV entry pathways; however, NAbs that primarily recognize conformational epitopes formed by two or more of the UL128 / UL130 / UL131A (UL128 / 130 / 131A) subunits of the PC cannot prevent entry into the FB but do have the ability to interfere with EpC / EnC infection, which is dramatically greater than that of NAbs targeting the major gC. Both gH- and PC-specific antibodies recognize gC expressed on the surface of CMV-infected cells (Fig. 1).

[0027]

[0051] As disclosed herein, engineered cells expressing a chimeric antigen receptor (CAR) specific for cytomegalovirus (CMV) exhibit activity against CMV-infected cells and suppress CMV replication. Specifically, primary CD8+ T lymphocytes (primary C8TL) were transduced with a CAR construct encoding a CAR specific for CMV (CMV-specific CAR). As used herein, "C8TL" includes naive CD8+ T lymphocytes and cytotoxic T cells.

[0028]

[0052] An exemplary CMV-specific CAR comprises a single-chain antibody (SCA) or fragment thereof specific for a CMV antigen, a CH2-CH3 hinge region (e.g., derived from a human IgG4 antibody), a transmembrane domain (e.g., a CD8 transmembrane domain), and a CD3ζ intracellular domain, with or without a costimulatory domain (e.g., derived from 4-1BB or CD28). See Figure 2.

[0029]

[0053] SCA sequences were derived from gH- and PC-specific NAbs disclosed in U.S. Patent Application Publication No. 20180230200; Lehmann et al. (2019) J Virol 93(17): e00931-19; Chiuppesi, et al. (2015) J Virol 89(23): 11884-98; and Wussow, et al. (2014) PLoS Pathog 10(11): e1004524, which are incorporated by reference in their entireties.

[0030]

[0054] The amino acid sequences of the VH and VL chains of the antibodies used in the construction of the SCA sequence of an exemplary CMV-specific CAR (CDR sequences are underlined) are provided in Table 1:

[0031] [Table 1]

[0032] [Table 2]

[0033]

[0055] CAR constructs are produced that encode CMV-specific CARs with SCA sequences.As used herein, " CAR constructs " refers to the expression vectors that are designed to be capable of expressing given CAR constructs, such as CMV-specific CARs, when provided to cells.CAR constructs are inserted into lentiviral vectors to deliver their CAR gene sequences to cells.Figure 3 shows a schematic representation of exemplary CAR constructs.

[0034]

[0056] When purified primary C8TL cells were transduced with the vector containing the CAR construct, expression of the CMV-specific CAR was detected by both Western blot staining for CD3ζ components (Figure 4) and flow cytometry, where cell surface expression of the CMV-specific CAR was assessed by staining for the SCA sequence (Figures 5A and 5B), because T cells do not normally express cell surface antibodies. These results demonstrate successful expression of the CMV-specific CAR in primary C8TL cells.

[0035]

[0057] Functional testing of primary C8TL concentrates transduced with a CMV-specific CAR confirmed their ability to recognize CMV-infected cells and mediate antiviral activity. When C8TL transduced with a CMV-specific CAR were exposed to CMV-infected cells, the C8TL released effector cytokines and expressed CMV-specific cytolytic activity markers (Figure 6), thus indicating that the CMV-specific CAR functionally guided their recognition of CMV-infected cells. When added to cells infected with a GFP-expressing strain of CMV, the transduced primary C8TL significantly reduced the concentration of infected cells (Figure 7), further confirming the specific recognition and elicitation of antiviral activity by the CMV-specific CAR.

[0036]

[0058] Thus, in some embodiments, the present invention provides the following SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26: G-X1-X2-X3-X4-X5-X6-X7-X8-X9 Formula H1 (SEQ ID NO: 21) wherein X1 is F or Y, X2 is S or T, X3 is L or F, X4 is S or T, X5 is D, I, N, S, or T, X6 is F, Y, or S, X7 is G, Y, or W, X8 is present or absent, and if present, X8 is L or I, and X9 is present or absent, and if present, X9 is G. I-X10-X11-X12-X13-X14-X15-X16 Formula H2 (SEQ ID NO: 22) wherein X10 is D, N, S, or W, X11 is D, N, P, T, or W, X12 is D, G, N, Y, or S, X13 is D, G, or T, X14 is D, G, N, or S, X15 is E, K, S, or Y, and X16 is present or absent, and if present, X16 is P or T. X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31 Formula H3 (SEQ ID NO: 23) [Wherein X17 is A, S, or V, X18 is R, N, or S, X19 is E, G, K, P, R, or S, X20 is G, H, K, L, Y, or W, X21 is D, L, R, S, or Y, X22 is D, F, G, L, S, or Y, X23 is A, D, F, G, L, P, or Y, X24 is A, D, I, Q, S, V, or Y, X25 is present or absent, and if present, X25 is E, F, N, S, or Y, and X26 is present or absent. X26, if present, is A, G, M, L, or P; X27 is present or absent, and if present, X27 is D, L, or Y; X28 is present or absent, and if present, X28 is A, F, L, or Y; X29 is present or absent, and if present, X29 is D, F, G, or M; X30 is present or absent, and if present, X30 is C, D, or Y; and X31 is present or absent, and if present, X31 is S or Y. X32-X33-X34-X35-X36-X37-X38-X39-X40-X41-X42 Formula L1 (SEQ ID NO: 24) wherein X32 is E, K, or Q, X33 is G or S, X34 is I, L, or V, X35 is D, G, S, or V, X36 is D, H, N, S, or T, X37 is D, N, S, or Y, X38 is present or absent, and if present, X38 is D, G, or N, X39 is present or absent, and if present, X39 is G, N, or Y, X40 is present or absent, and if present, X40 is K, N, or S, X41 is present or absent, and if present, X41 is F, Y, or T, and X42 is present or absent, and if present, X42 is Y. X43-X44-S Formula L2 (SEQ ID NO: 25) wherein X43 is D, L, R, T, or Y, and X44 is A, T, or V. X45-X46-X47-X48-X49-X50-P-X51-T Formula L3 (SEQ ID NO: 26) X45 is S, Q, or W; X46 is H, N, or Q; X47 is D, G, S, or Y; X48 is H, N, R, S, T, or Y; X49 is E, H, K, R, S, or T; X50 is D, F, L, S, V, or W; and X51 is L, P, W, or Y.

[0037]

[0059] In some embodiments, SEQ ID NO: 21 is GFSLSTYGIG (SEQ ID NO: 27), GFSLTTSGLG (SEQ ID NO: 28), GFTFSDYY (SEQ ID NO: 29), GYTFTIYG (SEQ ID NO: 30), GYTFTNFG (SEQ ID NO: 31), GYTFTSYG (SEQ ID NO: 32), GYTFTSYW (SEQ ID NO: 33), GYTFTIYW (SEQ ID NO: 34), or GYTFTSYW (SEQ ID NO: 35), preferably SEQ ID NO: 30.

[0038]

[0060] In some embodiments, SEQ ID NO:22 is IDPSDSET (SEQ ID NO:36), IDPSDSET (SEQ ID NO:37), IDPSDSET (SEQ ID NO:38), INTYTGEP (SEQ ID NO:39), ISDDGNYT (SEQ ID NO:40), ISNGGGST (SEQ ID NO:41), IWWDDDK (SEQ ID NO:42), or IWWNDNK (SEQ ID NO:43), preferably SEQ ID NO:39.

[0039]

[0061] In some embodiments, SEQ ID NO: 23 is AREHYYGINPLLGC (SEQ ID NO: 44), ARGWLLPVFAY (SEQ ID NO: 45), ARKGYYGSSGYFDY (SEQ ID NO: 46), ARRGDGLYSMDY (SEQ ID NO: 47), ARTGYFDV (SEQ ID NO: 48), SNGYSSFAY (SEQ ID NO: 49), VRPKRDFQYLYAMDY (SEQ ID NO: 50), VRSLYDYDEGYYFDS (SEQ ID NO: 51), or ASSGTGAY (SEQ ID NO: 52), preferably SEQ ID NO: 46.

[0040]

[0062] In some embodiments, SEQ ID NO:24 is ESIDSYGNSF (SEQ ID NO:53), KSVSTSGYSY (SEQ ID NO:54), QGISNY (SEQ ID NO:55), QSIGNN (SEQ ID NO:56), QSISDY (SEQ ID NO:57), QSLVHSNGNTY (SEQ ID NO:58), QSVSND (SEQ ID NO:59), QSISNN (SEQ ID NO:60), or QSLLDSDGKTY (SEQ ID NO:61), preferably SEQ ID NO:59.

[0041]

[0063] In some embodiments, SEQ ID NO: 25 is DTS (SEQ ID NO: 62), LAS (SEQ ID NO: 63), RAS (SEQ ID NO: 64), TVS (SEQ ID NO: 65), YAS (SEQ ID NO: 66), YTS (SEQ ID NO: 67), or LVS (SEQ ID NO: 68), preferably SEQ ID NO: 66.

[0042]

[0064] In some embodiments, SEQ ID NO:26 is QHSRELPWT (SEQ ID NO:69), QNGHTFPPT (SEQ ID NO:70), QQDYSSPWT (SEQ ID NO:71), QQSNEDPLT (SEQ ID NO:72), QQSNRWPWT (SEQ ID NO:73), QQYSKLPYT (SEQ ID NO:74), SQSTHVPYT (SEQ ID NO:75), QQSNSWPLT (SEQ ID NO:76), or WQGTHFPYT (SEQ ID NO:77), preferably SEQ ID NO:67.

[0043]

[0065] In some embodiments, the VH sequence of the SCA comprises SEQ ID NOs: 30, 39, and 46.

[0044]

[0066] In some embodiments, the VL sequence of SCA comprises SEQ ID NOs: 59, 66, and 71.

[0045]

[0067] In some embodiments, the SCA has a VH sequence comprising SEQ ID NOs: 30, 39, and 46, and a VL sequence comprising SEQ ID NOs: 59, 66, and 71.

[0046]

[0068] In some embodiments, the VH sequence of the SCA is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, preferably SEQ ID NO:11.

[0047]

[0069] In some embodiments, the VL sequence of SCA is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, preferably SEQ ID NO:12.

[0048]

[0070] In some embodiments, the SCA has a VH sequence comprising SEQ ID NO:11 and a VL sequence comprising SEQ ID NO:12.

[0049]

[0071] In some embodiments, the leader sequence has 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity to MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 78).

[0050]

[0072] In some embodiments, the flexible linker has 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity to SGGGGSGGGGSGGGGS (SEQ ID NO: 79).

[0051]

[0073] In some embodiments, the IgG4 hinge-CH2-CH3 region spacer is [ka] and 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity with

[0052]

[0074] In some embodiments, the CD8 transmembrane region has 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity to DIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 81).

[0053]

[0075] In some embodiments, the 4-1BB co-signaling region has 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity to KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 82).

[0054]

[0076] In some embodiments, the CD3 zeta signaling region is [ka] and 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity with

[0055]

[0077] The sequence of the CMV-specific CAR exemplified herein is: 1B2 CAR: [ka] 2-80 CAR: [ka] 12E2 CAR: [ka] 13B5 CAR: [ka] 18F10 CAR: [ka] 21E9 CAR: [ka] 21F6_54E11(21F6)CAR: [ka] 62-11 CAR: [ka]

[0056]

[0078] Thus, in some embodiments, the CMV-specific CAR comprises SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, or SEQ ID NO: 91, preferably the CMV-specific CAR comprises SEQ ID NO: 89.

[0057]

[0079] In some embodiments, C8TL transduced with a CMV-specific CAR may be administered to a subject. In some embodiments, stem cells, such as hematopoietic stem and progenitor cells (HSPCs), may be transduced with a CAR construct encoding a CMV-specific CAR and transplanted into a subject undergoing treatment. As used herein, "HSPC-based CAR cells" refer to cells and their progeny engineered to express a CAR by transducing HSPCs with a CAR construct. As used herein, "HSPCs" refer to hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs).

[0058]

[0080] As used herein, a given percentage of "sequence identity" refers to the percentage of nucleotides or amino acid residues that are identical between sequences when compared over a given comparison window and optimally aligned for maximum correspondence, as determined by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm described in Altschul et al., (1990) J Mol Biol 215:403-410. Software for performing BLAST (e.g., BLASTP and BLASTN) analyses is publicly available at the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The comparison window can span a given portion, e.g., a functional domain, or any given number of contiguous nucleotides or amino acid residues of one or both sequences. Alternatively, the comparison window can span the entire length of the sequences being compared. For the purposes of this specification, if no given comparison window (e.g., over 80% of the given sequence) is provided, the sequence identity described is over 100% of the given sequence. In addition, for the percentage of sequence identity of the proteins provided herein, the percentage is determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and default parameters available at blast.ncbi.nlm.nih.gov / Blast.cgi. See also Altschul, et al., (1997) Nucleic Acids Res 25:3389-3402; and Altschul, et al., (2005) FEBS J 272:5101-5109.

[0059]

[0081] Optimal sequence alignment for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.

[0060]

[0082] As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably to refer to two or more amino acids linked together. A group or series of amino acid abbreviations is used to represent a peptide. Unless specifically indicated, peptides are designated with the N-terminus at the left and sequences are written from N-terminus to C-terminus.

[0061]

[0083] As used herein, "antibody" refers to naturally occurring and synthetic immunoglobulin molecules and immunologically active portions thereof (i.e., molecules that contain an antigen-binding site that specifically binds a molecule to which the antibody is directed). Thus, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments, as well as variants (including derivatives) of antibodies, antibody multimers, and antibody fragments. Examples of molecules described herein by the term "antibody" include single-chain Fv (scFv), Fab fragments, Fab' fragments, F(ab'), disulfide-linked Fv (sdFv), Fv, and fragments comprising or consisting of either a VL domain or a VH domain.

[0062]

[0084] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is obtained from a rabbit-based hybridoma. As used herein, a compound (e.g., a receptor or antibody) "specifically binds" to a given target (e.g., a ligand) if it reacts or associates with that given target more frequently, more rapidly, for a longer duration, and / or with a higher binding affinity than when it reacts or associates with another given target and / or compared to indiscriminate binding and / or background binding that causes nonspecific binding. As used herein, "nonspecific binding" and "background binding" refer to interactions that are not dependent on the presence of a specific structure.

[0063]

[0085] As used herein, "binding affinity" refers to the tendency of a compound to associate with (or dissociate from) a given target, and can be expressed in terms of its dissociation constant, Kd. In some embodiments, an antibody has a binding affinity of 10 or more for its given target. -5 Below, 10 -6 Below 10, preferably -7 Less than or equal to 10, more preferably -8 Below, even more preferably 10 -9 and most preferably 10 -10The binding affinity can be determined using methods known in the art, such as equilibrium dialysis, equilibrium binding, gel filtration, immunoassays, surface plasmon resonance, and spectroscopy, using experimental conditions that are illustrative of the conditions under which a compound and a given target may come into contact and / or interact. The dissociation constant can be used to determine the binding affinity of a compound for a given target relative to another specified target. Alternatively, the binding affinity of a compound for a given target relative to another specified target can be determined using methods known in the art, such as immunoassays, in vivo or in vitro assays for functional activity, etc. Thus, in some embodiments, the binding affinity of an antibody for a given target is at least 1-fold higher, preferably at least 5-fold higher, more preferably at least 10-fold higher, and most preferably at least 100-fold higher than its binding affinity for another specified target.

[0064]

[0086] The following examples are intended to illustrate, but not limit, the present invention. [Example]

[0065]

[0087] Example

[0088] Eight novel CMV-specific CARs were constructed using anti-CMV neutralizing antibody sequences targeting the pentameric complex (PC). These CMV-specific CARs were transduced into primary C8TL cells using lentiviral vectors. Activity against CMV-infected target cells was assessed by cytokine (interferon-γ and tumor necrosis factor-α) release, upregulation of surface CD107a, and inhibition of infected cell proliferation, cytolysis, and viral replication. While some CARs demonstrated variable functional activity across these assays, one CMV-specific CAR based on the antibody 21E9 consistently outperformed all measures. These results support the development of CMV-specific CARs for therapeutic use against CMV and potentially other applications where CMV-driven immunotherapy is beneficial.

[0066]

[0089] Materials and Methods

[0090] Anti-CMV antibody sequence

[0091] The complete variable region sequence of a neutralizing antibody against CMV was used to create a single-chain antibody gene synthesized as a codon-optimized gene (Table 1) encoding the heavy and light chain variable regions separated by a linker, plus an upstream leader sequence derived from granulocyte-macrophage colony-stimulating factor.

[0067]

[0092] cell line

[0093] The cell lines 293T and ARPE-19 were maintained in Dulbecco's modified essential medium supplemented with L-glutamine (2 mM), penicillin (100 U / mL), streptomycin (100 U / mL), and heat-inactivated fetal bovine serum (10%) using methods known in the art. Primary C8TL from healthy donors were generated from total peripheral blood mononuclear cells (PBMCs); briefly, PBMCs were purified using a MACS column isolation kit with positive selection according to the manufacturer's protocol (Miltenyi, San Diego, CA), and stimulated with anti-CD3 antibodies for 5 days in the presence of irradiated feeder PBMCs and 50 U / mL recombinant human interleukin-2 (NIH AIDS Reagent Repository) to obtain CD3 T cells with a purity of >95%. + / CD8 + Cells were obtained. Experiments were repeated using PBMCs from three healthy donors provided by the UCLA AIDS Institute Virology Core Facility as Institutional Review Board-exempt materials without any demographic information.

[0068]

[0094] Construction of chimeric antigen receptor genes and lentiviral vectors

[0095] First, we modified the lentiviral vector pCCLcMNDU3c-X2, kindly provided by DB Kohn, to contain the fixed region of a previously reported human CAR (excluding the leader and single-chain antibody sequences), consisting of an IgG4-based spacer (plus a silent mutation creating a unique ApaI restriction site), a CD8 transmembrane domain, a co-signaling domain derived from 4-1BB (CD137), and a signaling domain derived from the human CD3 complex ζ chain (CD247). Additionally, a P2A ribosomal skip sequence plus a furin cleavage site, followed by the thermostable antigen mouse CD24 reporter (HSA), was inserted immediately downstream of the CAR sequence. This modified vector was digested with HpaI and ApaI restriction enzymes, and the PCR-amplified leader-single-chain antibody sequences were inserted using the In-Fusion Cloning Kit (Takara, Mountain View, CA). The sequence of the final vector was then confirmed. Lentiviral stocks were generated by cotransfecting these constructs into HEK-293T cells with the vesicular stomatitis virus envelope glycoprotein G protein and an HIV-based packaging vector, and stored in aliquots at −80°C until use.

[0069]

[0096] CAR transduction of primary C8TL

[0097] Cells were transduced with CAR-delivering lentiviral vectors. Briefly, polystyrene 24-well tissue culture plates were precoated with RetroNectin (Takara Bio, Mountain View, CA). The lentiviral vector was added at a concentration of approximately 50 ng of p24 antigen in a 100 μL volume and centrifuged at 2000 g for 2 hours. Then, 10 cells were transduced. 6Cells were added and maintained in RPMI 1640 (R10-50) supplemented with L-glutamine (2 mM), penicillin (100 U / mL), streptomycin (100 U / mL), heat-inactivated fetal bovine serum (10%), HEPES buffer (10 mM), and 50 U / mL recombinant human interleukin-2. These cells were maintained and periodically restimulated with goat anti-human F(ab)2 antibody (catalog no. 109-006-006, Jackson ImmunoResearch Laboratories) and enriched to at least 70% purity before use in functional assays.

[0070]

[0098] Western blot for CAR expression

[0099] Transduced C8TL were evaluated for CAR expression by Western blot. Briefly, 2 million transduced cells were lysed and subjected to protein electrophoresis, followed by transfer to a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The membrane was probed using a mouse anti-human CD247 monoclonal antibody (No. 551033; BD Pharmingen, San Jose, CA) and a SuperSignal West Pico detection kit (Pierce, Rockford, IL), which revealed bands for both endogenous CD247 and higher molecular weight CD247-containing CAR proteins.

[0071]

[0100] Flow cytometry analysis of cell surface CAR expression

[0101] Detection of cell surface CAR expression on transduced cells was performed using methods known in the art. Briefly, cells were stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-human F(ab)2 antibody (catalog number 109-006-003, Jackson ImmunoResearch Laboratories, West Grove, PA) or an isotype control antibody, fixed, and analyzed by flow cytometry (MACSQuantVYB, Miltenyi, Sunnyvale, CA) to determine cell surface CAR expression (FlowJo, Ashland, OR).

[0072]

[0102] Preparation of CMV strains and virus stocks

[0103] GFP-tagged human cytomegalovirus (CMV) strains TB40 / E and TR, expressing a GFP reporter under the SV40 promoter, were derived from BAC DNA provided by T.E. Shenk and E.A. Murphy. CMV stocks were generated after virus propagation in ARPE-19 cells (American Type Tissue Culture Collection) by standard ultracentrifugation procedures, and titers on ARPE-19 cells were determined by immunostaining using the IE1-specific monoclonal antibody p63-27, provided by William Britt, and the Vectastain ABC kit (Vector Laboratories) according to the manufacturer's instructions.

[0073]

[0104] Flow cytometry analysis of intracellular cytokine and cell surface CD107a expression by CAR-transduced primary C8TL

[0105] ARPE-19 cells were plated in a 24-well plate at 2 × 10 5 Cells were seeded at 1000 x g / well and infected with CMV at a multiplicity of infection (MOI) of 3 24 hours later, while mock-infected cells served as a negative control. Five days later, when cells were uniformly infected as reflected by GFP expression, 10 cells transduced with CAR were transduced. 6Primary C8TL cells were added and further incubated for 1 hour; stimulation with a leukocyte activation cocktail (BD Biosciences, San Jose, CA) served as a positive control for activation. Brefeldin A and monensin (0.5 μl Golgi Plug and 0.5 μl Golgi Stop, BD Biosciences, San Jose, CA) and allophycocyanin (APC)-conjugated anti-CD107a antibody (catalog no. 328620, Biolegend, San Diego, CA) were then added. After 5 hours of co-incubation, CAR-transduced C8TL cells were removed from each well for analysis. Surface staining was then performed with anti-human antibodies, including fluorescein isothiocyanate (FITC)-conjugated anti-IgG F(ab)2 (catalog no. 109-096-003, Jackson ImmunoResearch Laboratories) and phycoerythrin (PE)-conjugated anti-CD8 (catalog no. 300908, Biolegend, San Diego, CA), followed by fixation and permeabilization (Cytofix / Cytoperm, BD Biosciences, San Jose, CA). Subsequently, intracellular cytokine staining was performed with anti-human antibodies, including Alexa Fluor-647-conjugated anti-interferon-γ (catalog no. 506507, Biolegend, San Diego, CA) and Alexa Fluor-700-conjugated anti-tumor necrosis factor-α (catalog no. 502928, Biolegend, San Diego, CA). Cells were analyzed by FACSCelesta (BD Biosciences, San Jose, CA) using FlowJo software (BD Biosciences, San Jose, CA).

[0074]

[0106] Flow cytometry analysis of cell proliferation

[0107] Using ARPE-19 cells as stimulator cells, CMV-mediated proliferation of CAR-transduced C8TL was tested using methods known in the art with the following modifications: 105 ARPE-19 cells were seeded and the next day infected with HCMV TB40 / E or TR at an MOI of 1.5 and cultured for 3 days to achieve >90% infection as determined by GFP fluorescence (or mock-infected as a negative control). CAR-transduced C8TL cells were labeled with CellTrace Violet according to the manufacturer's instructions (Thermo Fisher Scientific, Grand Island, NY), and 5 × 10 CAR-transduced C8TL cells were cultured. 5 3×10 cells 6 irradiated feeder PBMCs were added to each well and cultured in R10-50 for 7 days with medium changes every 3 days. Anti-CD3 antibody stimulation served as a positive control. 5 x 10 cells were collected from each well for analysis. 6 Cells were harvested, co-stained for CD8 and human F(ab), fixed with 1% paraformaldehyde, and analyzed by flow cytometry for CD8 + Population gating and analysis were performed (MACSQuant VYB, Miltenyi Biotech, Sunnyvale, CA and FlowJo software, BD Biosciences, San Jose, CA).

[0075]

[0108] Chromium release assay of cytolytic activity against CMV-infected cells by CAR-transduced primary C8TL

[0109] Killing of CMV-infected target cells by CAR-transduced primary C8TL was performed using methods described in the art with the following modifications: 51 ARPE-19 cells were cultured at 10 cells per T25 tissue culture flask and tested in the Cr release assay. 6 The following day, cells were infected with CMV TB40 / E or TR at a multiplicity of infection of 1.5 (or mock-infected as a negative control), and then cultured for 4 days to achieve greater than 90% infection as determined by GFP expression. Cells were detached by incubation in 5 mM EDTA in phosphate-buffered saline at 37°C for 15 min and then cultured for 10 min at 37°C for use in chromium release assays. 51 Cr labeled.

[0076]

[0110] Evaluation of suppression of CMV replication by CAR-transduced primary C8TL

[0111] Inhibition of CMV replication was assessed by inoculating ARPE-19 cells at 2 × 10 in 96-well plates. 4 Cells were seeded at 1000 x 1000 cells / well and the next day infected with CMV TB40 / E or TR at an MOI of 1. Four days later, each well received 2 x 10 CAR-transduced (or mock-transduced) cells. 5 Primary C8TL cells were added and co-incubated for 8 hours before imaging using an Axio Observer Z1 inverted fluorescence microscope (Carl Zeiss, Pleasanton, CA) equipped with a linear motorized stage.

[0077]

[0112] result

[0113] Genetic construction of a chimeric antigen receptor based on a neutralizing antibody against CMV

[0114] We utilized the sequences of previously identified neutralizing monoclonal antibodies targeting different CMV PC proteins as targeting regions in our chimeric antigen receptor (CAR) designs (Table 1). These included antibodies targeting epitopes in the gH subunit (21E9, 2-80, 18F10, 62-11), conformational epitopes in the UL128 / 130 / 131A subunit (1B2, 12E2, 21F6), and a linear epitope in UL128 (13B5), all of which have been previously confirmed to bind to CMV-infected cells (not shown). The genes for single-chain versions of these antibodies were incorporated into a scaffold previously described by the inventors for an HIV-1-specific CAR, consisting of a single-chain antibody targeting region, an IgG4 constant region-based spacer, a CD8 transmembrane domain, and cytoplasmic signaling domains derived from 4-1BB and CD3ζ (Figure 2). These novel CAR genes were inserted into lentiviral vectors for transduction of primary C8TL.

[0078]

[0115] Anti-CMV antibody-based CAR is expressed in transduced primary C8TL

[0116] Primary CD8 from healthy donors + Cells were transduced and tested for expression of the novel CARs. Western blot analysis of CD3ζ expression demonstrated detection of CAR expression as a larger CD3ζ-expressed protein in addition to the native CD3ζ protein (Figure 4). Cell surface expression of the CARs was further confirmed by staining for immunoglobulin domains (not present on native T cells) on the surface of the transduced cells and detection by flow cytometry (Figure 5B). Both detection modes revealed that all eight novel CARs were expressed in primary C8TL.

[0079]

[0117] CAR-transduced primary C8TL are specifically triggered to release cytokines and upregulate cell surface CD107a by CMV-infected target cells

[0118] To test whether CARs recognize their target proteins on the surface of CMV-infected cells, CAR-transduced primary C8TL cells were exposed to ARPE-19 cells acutely infected with CMV TR and evaluated for specific production of IFN-γ and TNF-α. Non-transduced C8TL cells demonstrated minimal cytokine production in response to ARPE-19 cells, regardless of CMV infection. In contrast, at least two CARs, 21E9 and 2-80 (both targeting gH), showed specific cytokine release in response to CMV-infected cells but not to non-CMV-infected cells (Figures 8 and 10). Concurrent determination of cell surface expression of the degranulation marker CD107a correlated with cytokine release (Figures 9 and 11), again demonstrating that CARs 21E9 and 2-80, and to a lesser extent 12E2 and 21F6 (targeting the UL128 / 130 / 131A subunit), mediated CMV-specific cell induction. Subsequent studies focused on these four CARs. Similar results were seen in target ARPE-19 cells infected with CMV TB40 / E-infected cells (not shown).

[0080]

[0119] CAR-transduced primary C8TL proliferate in response to CMV-infected target cells

[0120] The functionality of CAR-transduced primary C8TL was further evaluated by examining the proliferation of these cells in response to CMV-infected cells (Figure 12). The 21E9 CAR mediated moderate proliferation when exposed to CMV-infected cells, but did not exhibit proliferation in response to uninfected cells. The other three CARs (2-80, 12E2, 21F6) did not mediate appreciable proliferation. Thus, at least one CAR resulted in CMV-specific proliferation of transduced C8TL.

[0081]

[0121] CAR-transduced primary C8TL mediate moderate cytolysis of CMV-infected target cells

[0122] CMV-infected cells are intrinsically resistant to C8TL cytolysis. We investigated the ability of CAR-redirected primary C8TL to mediate CMV-specific cytolysis. 51 The CARs were tested by chromium release assay (Figure 13). The only CAR that consistently mediated specific killing of CMV-infected target cells across multiple experiments was 21E9, which mediated moderate levels of cell lysis at high effector:target ratios. Other CARs produced lower, inconsistent levels of killing across multiple experiments.

[0082]

[0123] CAR-transduced primary C8TL suppress CMV replication

[0124] The antiviral activity of CAR-transduced C8TL was determined by co-culture with ARPE-19 cells acutely infected with CMV using a GFP-expressing CMV TR (Figure 14). All four CARs tested exhibited quantifiable antiviral activity, accompanied by a decrease in the concentration of fluorescent CMV-infected cells, even after incubation for as short as 8 hours. Across multiple experiments, CAR 21E9 mediated the most consistent and potent antiviral activity. Similar results were observed in ARPE-19 cells infected with CMV TB40 / E (not shown).

[0083]

[0125] Consideration

[0126] To date, only one CMV-specific CAR has been reported in detail. This CAR targets CMV glycoprotein B (gB), and C8TL transfected with RNA encoding the CAR were shown to be CMV-specific by responding to infected cells with the release of IFN-γ and TNF-α and a modest upregulation of surface CD107a. Cells transfected with prior art CARs were shown to kill target cells expressing recombinant gB, but subsequently ceased to kill CMV-infected cells, likely due to a viral escape mechanism for cell lysis.

[0084]

[0127] Here, we designed and screened eight candidate CMV-specific CARs that target other viral proteins on infected cells. These antibodies were previously generated in mice using a modified vaccinia Ankara virus (MVA) vector expressing the PC sequence from TB40 / E and were shown to bind to PC with high affinity. All of the exemplified CMV-specific CARs exhibited functional activity. However, at least one of the CMV-specific CARs (based on the gH-specific antibody 21E9) had consistent activity across multiple functional assays against cells infected with both CMV TB40 / E and TR strains (which belong to two different gH genotypes).

[0085]

[0128] All of these CMV-specific CARs exhibited sufficient expression levels on transduced cells, but their functionality varied. The 21E9-based CAR consistently demonstrated excellent activity in all functional tests, while the 2-80-based CAR showed activity in most assays, though this appeared to result in nonspecific background activity. Other CMV-specific CARs targeting other PC subunits demonstrated minimal overall activity. It is unclear whether this indicates that gH is a superior target for CARs compared with other PC proteins. While the 21E9 and 2-80 antibodies have approximately 10-fold lower binding affinity than the other antibodies, affinity itself is likely not the primary determinant of CAR activity, as two other gH-specific antibodies with similar affinity, 62-11 and 18F10, produced CARs with lower activity. Target protein expression may be a factor; gH appears to be more abundant on CMV-infected cells than UL128, UL130, and UL131A. Another possible factor is that the gH-specific antibodies utilized here recognize gH in multiple contexts, including monomeric gH, gH bound to gL, and gH associated with PC, perhaps allowing for broader target binding and recognition. Finally, it is noteworthy that 21E9 and 2-80 target a different site on gH than other gH-specific antibodies, suggesting that this epitope region may be more accessible to antibodies in the context of a CMV-specific CAR.

[0086]

[0129] The data herein demonstrate modest but reproducible CAR-mediated C8TL killing of CMV-infected cells at levels similar to those observed by Rauser et al., who demonstrated cytolysis of infected cells by endogenous CMV-specific C8TL. Overall, the role of infected cell killing may not be critical for the in vivo efficacy of CMV-specific CARs, as adoptive transfer of natural CMV-specific C8TL clearly demonstrates their antiviral effect, suggesting that targeted non-cytolytic mechanisms may be sufficient.

[0087]

[0130] Beyond their direct utility in anti-CMV therapy, CMV-specific CARs may have other applications by exploiting the pathogenesis of chronic CMV infection. In normal immunocompetent hosts, the virus establishes a chronic, lifelong infection that is mostly latent, but with frequent, low-level, asymptomatic reactivations, which stimulate relatively high levels of persistently circulating functional anti-CMV T cells. In this respect, CMV acts similarly to an endogenous vaccine, boosting and maintaining cellular immunity against itself. A strategy under consideration for cancer immunotherapy is to exploit this process by transducing CMV-specific T cells isolated from peripheral blood with an anti-CD19 CAR, thus linking anti-tumor responses to anti-CMV responses by creating bispecific T cells that recognize both CMV and CD19. A functional anti-CMV CAR could potentially achieve the same goal by coexpressing the anti-CMV CAR with a T cell receptor or a CAR targeting another virus or tumor, without the need to isolate CMV-specific T cells.

[0088]

[0131] Overall, the data herein demonstrate that the CMV-specific CARs as disclosed herein, and in particular the 21E9 CAR, are strong candidates for testing as immunotherapeutic interventions or prevention for disseminated CMV infection and / or for combination with T cell receptor or CAR gene immunotherapy for other diseases, given that the CMV-specific CARs exhibit CMV-targeted functions in terms of eliciting cytokine release, proliferation, killing infected cells, and suppressing viral replication.

[0089]

[0132] References

[0133] The following references are incorporated herein by reference in their entirety, except that in the event that the scope and meaning of a term conflicts with a definition expressly set forth herein, the definition expressly set forth herein shall control: Staras SA,Dollard SC,Radford KW,Flanders WD,Pass RF,Cannon MJ.Seroprevalence of cytomegalovirus infection in the United States,1988-1994.Clin Infect Dis 2006;43:1143-51. Majeed A,Latif A,Kapoor V,et al.Resistant Cytomegalovirus Infection in Solid-organ Transplantation:Single-center Experience,Literature Review of Risk Factors,and Proposed Preventive Strategies.Transplantation proceedings 2018;50:3756-62. ElHelou G,Razonable RR.Letermovir for the prevention of cytomegalovirus infection and disease in transplant recipients:an evidence-based review.Infection and drug resistance 2019;12:1481-91. Riddell SR,Walter BA,Gilbert MJ,Greenberg PD.Selective reconstitution of CD8+ cytotoxic T lymphocyte responses in immunodeficient bone marrow transplant recipients by the adoptive transfer of T cell clones.Bone marrow transplantation 1994;14 Suppl 4:S78-84. Riddell SR,Watanabe KS,Goodrich JM,Li CR,Agha ME,Greenberg PD.Restoration of viral immunity in immunodeficientHumans by the adoptive transfer of T cell clones.Science 1992;257:238-41. Walter EA,Greenberg PD,Gilbert MJ,et al.Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.N Engl J Med 1995;333:1038-44. Proff J,Walterskirchen C,Brey C,et al.Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in anHLA-Recognition Independent Manner.Frontiers in microbiology 2016;7:844. Full F,Lehner M,Thonn V,et al.T cells engineered with a cytomegalovirus-specific chimeric immunoreceptor.J Virol 2010;84:4083-8. Hahn G,Revello MG,Patrone M,et al.Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes.J Virol 2004;78:10023-33. Wang D,Shenk T.Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism.Proc Natl Acad Sci U S A 2005;102:18153-8. Chiuppesi F,Wussow F,Johnson E,et al.Vaccine-Derived Neutralizing Antibodies to theHuman Cytomegalovirus gH / gL Pentamer Potently Block Primary Cytotrophoblast Infection.J Virol 2015;89:11884-98. Diamond DJ,Chiuppesi F,Wussow F.MVA-gh / gL-PC vaccine derived antibodies neutralizingHuman cytomegalovirus infectivity and methods thereof.United States:City ofHope National Medical Center,2018.Patent #10,487,139. Bennett MS,Joseph A,NgHL,GoldsteinH,Yang OO.Fine-tuning of T-cell receptor avidity to increaseHIV epitope variant recognition by cytotoxic T lymphocytes.Aids 2010;24:2619-28. Ali A,Kitchen SG,Chen IS,NgHL,Zack JA,Yang OO.HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies.J Virol 2016;90:6999-7006. Logan AC,Nightingale SJ,Haas DL,Cho GJ,Pepper KA,Kohn DB.Factors influencing the titer and infectivity of lentiviral vectors.Hum Gene Ther 2004;15:976-88. Yang S,Cohen CJ,Peng PD,et al.Development of optimal bicistronic lentiviral vectors facilitatesHigh-level TCR gene expression and robust tumor cell recognition.Gene Ther 2008;15:1411-23. Ali A,Jamieson BD,Yang OO.Half-genomeHuman immunodeficiency virus type 1 constructs for rapid production of reporter viruses.J Virol Methods 2003;110:137-42. Murphy E,Yu D,Grimwood J,et al.Coding potential of laboratory and clinical strains ofHuman cytomegalovirus.Proc Natl Acad Sci U S A 2003;100:14976-81. O’Connor CM,Murphy EA.A myeloid progenitor cell line capable of supportingHuman cytomegalovirus latency and reactivation,resulting in infectious progeny.J Virol 2012;86:9854-65. Britt WJ.Human cytomegalovirus:propagation,quantification,and storage.Current protocols in microbiology 2010;Chapter 14:Unit 14E 3. Wussow F,Chiuppesi F,Martinez J,et al.Human cytomegalovirus vaccine based on the envelope gH / gL pentamer complex.PLoS Pathog 2014;10:e1004524. Andreoni M,Faircloth M,Vugler L,Britt WJ.A rapid microneutralization assay for the measurement of neutralizing antibody reactive withHuman cytomegalovirus.J Virol Methods 1989;23:157-67. Yang OO,Kalams SA,Rosenzweig M,et al.Efficient lysis ofHuman immunodeficiency virus type 1-infected cells by cytotoxic T lymphocytes.J Virol 1996;70:5799-806. Bennett MS,NgHL,Dagarag M,Ali A,Yang OO.Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells.J Virol 2007;81:4973-80. Stripecke R,Gerasch L,Theobald S,et al.CAR T Cells Targeted with aHigh Affinity Scfv Against theHCMV Glycoprotein Gb As Adoptive T Cell Therapy afterHematopoietic Stem Cell Transplantation.Blood 2016;128:5721. Shedlock DJ,Talbott KT,Wu SJ,et al.Vaccination with synthetic constructs expressing cytomegalovirus immunogens isHighly T cell immunogenic in mice.Human vaccines & immunotherapeutics 2012;8:1668-81. Buscher N,Paulus C,Nevels M,Tenzer S,Plachter B.The proteome ofHuman cytomegalovirus virions and dense bodies is conserved across different strains.Medical microbiology and immunology 2015;204:285-93. Barrios Y,Knor S,Lantto J,Mach M,Ohlin M.Clonal repertoire diversification of a neutralizing cytomegalovirus glycoprotein B-specific antibody results in variants with diverse anti-viral properties.Mol Immunol 2007;44:680-90. Lantto J,Fletcher JM,Ohlin M.Binding characteristics determine the neutralizing potential of antibody fragments specific for antigenic domain 2 on glycoprotein B ofHuman cytomegalovirus.Virology 2003;305:201-9. Lantto J,Lindroth Y,Ohlin M.Non-germ-line encoded residues are critical for effective antibody recognition of a poorly immunogenic neutralization epitope on glycoprotein B ofHuman cytomegalovirus.Eur J Immunol 2002;32:1659-69. Tempest PR,White P,Buttle M,Carr FJ,Harris WJ.Identification of framework residues required to restore antigen binding during reshaping of a monoclonal antibody against the glycoprotein gB ofHuman cytomegalovirus.International journal of biological macromolecules 1995;17:37-42. Ohlin M,OwmanH,Rioux JD,Newkirk MM,Borrebaeck CA.Restricted variable region gene usage and possible rheumatoid factor relationship amongHuman monoclonal antibodies specific for the AD-1 epitope on cytomegalovirus glycoprotein B.Mol Immunol 1994;31:983-91. Newkirk MM,GramH,Heinrich GF,Ostberg L,Capra JD,Wasserman RL.Complete protein sequences of the variable regions of the clonedHeavy and light chains of aHuman anti-cytomegalovirus antibody reveal a striking similarity toHuman monoclonal rheumatoid factors of the Wa idiotypic family.J Clin Invest 1988;81:1511-8. Gilbert MJ,Riddell SR,Plachter B,Greenberg PD.Cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product.Nature 1996;383:720-2. Rauser G,EinseleH,Sinzger C,et al.Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants.Blood 2004;103:3565-72. Sylwester AW, Mitchell BL, Edgar JB, et al.Broadly targetedHuman cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.J Exp Med 2005;202:673-85. Wang X,Wong CW,Urak R,et al.CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV-Specific T Cells.Clinical cancer research:an official journal of the American Association for Cancer Research 2015;21:2993-3002.

[0090]

[0134] All technical and scientific terms used in this application have meanings commonly used in the art unless otherwise specified.

[0091]

[0135] Unless specifically indicated, peptides are designated with the N-terminus at the left and sequences are written N-terminus to C-terminus. Similarly, unless specifically indicated, nucleic acid sequences are designated with the 5'-terminus at the left and sequences are written 5' to 3'.

[0092]

[0136] As used herein, the terms "subject," "patient," and "individual" are used interchangeably to refer to humans and non-human animals. The terms "non-human animal" and "animal" refer to all non-human vertebrates, e.g., non-human mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subject and test animals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0093]

[0137] As used herein, the term "diagnosing" refers to the physical and active step of providing, i.e., communicating verbally or in writing (e.g., on paper or electronic media), diagnostic information to another party, e.g., a patient. Similarly, "making a prognosis" refers to the physical and active step of providing, i.e., communicating verbally or in writing (e.g., on paper or electronic media) prognostic information to another party, e.g., a patient.

[0094]

[0138] The use of the singular may include the plural unless specifically stated otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise.

[0095]

[0139] As used herein, "and / or" means "and" or "or." For example, "A and / or B" means "A, B, or both A and B," and "A, B, C, and / or D" means "A, B, C, D, or a combination thereof," where "A, B, C, D, or a combination thereof" refers to any subset of A, B, C, and D, such as a single-member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C, etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D, etc.), or all four members (e.g., A, B, C, and D).

[0096]

[0140] As used herein, the phrase "one or more of," e.g., "one or more of A, B, and / or C," means "one or more of A," "one or more of B," "one or more of C," "one or more of A and one or more of B," "one or more of B and one or more of C," "one or more of A and one or more of C," and "one or more of A, one or more of B, and one or more of C."

[0097]

[0141] The phrase "comprising or consisting of A" is used as a means to avoid excessive page count and translation costs, and in some embodiments means that the given item in question comprises or consists of A. For example, the sentence "In some embodiments, the composition comprises or consists of A" should be interpreted as if it were written as two separate sentences: "In some embodiments, the composition comprises A. In some embodiments, the composition consists of A."

[0098]

[0142] Similarly, a sentence listing a series of alternatives should be interpreted as if the series were provided with each given alternative provided in a separate sentence. For example, the sentence "In some embodiments, the composition comprises A, B, or C" should be interpreted as if it were written as three separate sentences: "In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C." As another example, the sentence "In some embodiments, the composition comprises at least A, B, or C" should be interpreted as if it were written as three separate sentences: "In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C."

[0099]

[0143] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference to the same extent as if each were individually incorporated as such.

[0100]

[0144] Having thus described exemplary embodiments of the present invention, those skilled in the art should note that what is within the scope of this disclosure is merely exemplary, and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but rather is limited only by the scope of the following claims.

Claims

1. A cytomegalovirus-specific chimeric antigen receptor (CMV-specific CAR) having the following CDR sequences: VH CDR1 of SEQ ID NO: 30, VH CDR2 of SEQ ID NO: 39, VH CDR3 of SEQ ID NO: 46, VL CDR1 of SEQ ID NO: 59, VL CDR2 of SEQ ID NO: 66, and A CMV-specific CAR comprising a single chain antibody (scFv) sequence having a VL CDR3 of SEQ ID NO:

71.

2. The CMV-specific CAR of claim 1, comprising or consisting of SEQ ID NO:

89.

3. 10. Use of (a) an expression vector encoding the CMV-specific CAR of claim 1, or (b) one or more cells transduced with the expression vector, in the manufacture of a therapeutic medicament for treating, reducing, or inhibiting a cytomegalovirus infection in a subject.

4. 10. A pharmaceutical composition for treating, reducing, or inhibiting a cytomegalovirus infection in a subject, comprising: (a) an expression vector encoding a CMV-specific CAR according to claim 1 or 2; or (b) one or more cells transduced with the expression vector.

5. 10. A pharmaceutical composition for treating, reducing, or inhibiting a cytomegalovirus infection in a subject, the pharmaceutical composition comprising one or more cells expressing one or more CMV-specific CARs of claim 1 or 2, the cells being transplanted into the subject.

6. An expression vector comprising a nucleic acid sequence encoding the CMV-specific CAR according to claim 1 or 2.

7. A host cell comprising one or more expression vectors according to claim 6.

8. The host cell of claim 7 , wherein the host cell is a CD8+ T lymphocyte, a hematopoietic stem cell, or a hematopoietic progenitor cell.

9. A cell expressing the CMV-specific CAR according to claim 1 or 2.

10. 9. The cell of claim 7 or 8, which expresses one or more chimeric antigen receptors encoded by the one or more expression vectors.

11. 11. A pharmaceutical composition for treating, reducing, or inhibiting a cytomegalovirus infection in a subject, said pharmaceutical composition comprising one or more cells according to any one of claims 7 to 10, said cells being transplanted into said subject.

12. 12. The pharmaceutical composition of claim 11, wherein the subject is a human and / or the cytomegalovirus is a human cytomegalovirus.

13. The pharmaceutical composition of claim 11 or 12, wherein the subject has an immunodeficiency disorder.

14. 10. A pharmaceutical composition for killing cells infected with cytomegalovirus, the pharmaceutical composition comprising: (a) one or more cells expressing one or more CMV-specific CARs according to claim 1 or 2; or (b) one or more cells comprising an expression vector encoding the one or more CMV-specific CARs, the pharmaceutical composition being contacted with the infected cells.

15. A pharmaceutical composition for reducing cytomegalovirus replication in a cell or a subject, the pharmaceutical composition comprising: (a) one or more cells expressing one or more CMV-specific CARs according to claim 1 or 2; or (b) one or more cells comprising an expression vector encoding the one or more CMV-specific CARs; 1) if the cytomegalovirus is in the cell, the cell is contacted with the one or more cells or the expression vector; or 2) A pharmaceutical composition, wherein the one or more cells or the expression vector are administered to the subject if the cytomegalovirus is in the subject.

Images