CMV-HIV specific chimeric antigen receptor T cells

Abstract

The present disclosure relates, inter alia, to compositions comprising T cells expressing both a chimeric antigen receptor (CAR) targeting HIV and a T cell receptor targeting cytomegalovirus (CMV), and methods of making and using the same.

Description

[Technical field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Application No. 63 / 330,726, filed April 13, 2022. The entire contents of the above are incorporated herein by reference. Technical Field The present disclosure relates to T cells expressing both a chimeric antigen receptor (CAR) that targets HIV and a T cell receptor that targets cytomegalovirus (CMV). [Background technology]

[0002] Combination antiretroviral therapy (ART) achieves undetectable plasma viremia (Non-Patent Document 1), but cannot cure HIV infection because latent viral reservoirs containing replicative HIV-1 persist (Non-Patent Document 2). Alternative cellular immune strategies to adoptive immunotherapy have been proposed using the expansion of either endogenous HIV-specific T cells or autologous T cells or natural killers (NK) redirected to HIV-infected cells (Non-Patent Documents 3-5). The first generation of HIV-CAR T cells, in which the extracellular domain of the CD4 receptor on the surface of T cells was engineered, was developed almost 25 years ago (Non-Patent Documents 6, 7). The CD4-based CAR T cells were tested in three clinical trials in HIV-seropositive individuals (Non-Patent Documents 4, 8-10), but this strategy was discontinued due to lack of clinical efficacy. The lack or minimal antiviral effect may be explained by certain factors, such as limited CAR activity in the absence of intracellular costimulatory signaling domains, poor exposure to HIV antigens as CAR recipients continue ART, resulting in low proliferation and minimal persistence of CAR T cells, or high susceptibility of CD4-CAR-expressing T cells to HIV infection. Notably, the first generation CD4-CAR T cells were detected at low levels in the recipients after 10 years, suggesting that long-term persistence is possible (Non-Patent Document 11). Since then, optimizing CARs by adding the costimulatory domains CD28 or 4-1BB has improved efficacy and persistence with promising therapeutic outcomes for symptoms of B-cell malignancies (Non-Patent Documents 12-15). Furthermore, a series of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein gp120 have been identified in non-progressors of HIV infection and used to develop bNAb-derived CAR T cells that efficiently kill gp120-expressing cells in vitro (Non-Patent Documents 16, 17). To limit the emergence of resistance to HIV, the bNAb-based CAR T cells should be effective against almost all strains of HIV. In particular, et al. (2013) isolated a bNAb named N6 that potently neutralizes 98% of HIV-1 isolates, including 16 of 20 that have evolved to circumvent common mechanisms of resistance. As currently being tested in the CD4-based CAR T cell clinical trial [NCT03617198], CAR T cell numbers must be high to achieve therapeutic benefit. Another approach to enhance CAR T cell persistence involves the inclusion of viral antigens as stimulators of CAR T cells (Non-Patent Documents 19-23). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Guenthard, HF, Saag, MS, Benson, CA, del Rio, C., Eron, JJ, Gallant, JE, Hoy, JF, Mugavero, MJ, Sax, PE, Thompson, MA, Gandhi, RT, et al. (2016). Antiretroviral Drugs for Treatment and Prevention of HIV Infection in Adults: 2016 Recommendations of the International Antiviral Society-USA Panel. Jama 316, 191-210. 10.1001 / jama.2016.8900. [Non-Patent Document 2] Sadowski, I., and Hashemi, FB (2019). Strategies to eradicate HIV from infected patients: elimination of latent provirus reservoirs. Cellular and molecular life sciences : CMLS 76, 3583-3600. 10.1007 / s00018-019-03156-8. [Non-Patent Document 3] Hege, K.M., Cooke, K.S., Finer, M.H., Zsebo, K.M., and Roberts, M.R. (1996). Systemic T cell-independent tumor immunity after transplantation of universal receptor-modified bone marrow into SCID mice. The Journal of experimental medicine 184, 2261-2269. 10.1084 / jem.184.6.2261. Non-Patent Document 4 Wagner, T.A. (2018). Quarter Century of Anti-HIV CAR T Cells. Current HIV / AIDS reports 15, 147-154. 10.1007 / s11904-018-0388-x. Non-Patent Document 5 Patel, S., Hanajiri, R., Grant, M., Saunders, D., Van Pelt, S., Keller, M., Hanley, P.J., Simon, G., Nixon, D.F., Hardy, D., Jones, R.B., et al. (2020). HIV-Specific T Cells Can Be Generated against Non-escaped T Cell Epitopes with a GMP-Compliant Manufacturing Platform. Molecular therapy. Methods & clinical development 16, 11-20. 10.1016 / j.omtm.2019.10.001. Non-Patent Document 6 Romeo, C., and Seed, B. (1991). Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 64, 1037-1046.

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[0004] The present disclosure is based, at least in part, on the discovery that T cells expressing a chimeric antigen receptor (CAR) specific for HIV and a T cell receptor specific for cytomegalovirus (CMV) ("CMV-HIV T cells"), in some cases, can be used in conjunction with a CMV vaccine (e.g., one or more CMV antigens or a nucleic acid encoding one or more CMV antigens) to treat a subject infected with HIV. A major problem still faced by HIV patients today is maintaining the activation and persistence of CAR T cells in vivo. HIV patients taking antiretroviral drugs face this problem, and to date, there are no data showing an effective method to expand and maintain CAR T cells in such patients, as cell function is impaired. Furthermore, it is difficult to isolate and expand a population of T cells specific for CMV for many reasons. For example, this specific T cell population is usually very low in a subject, so it can be very difficult to produce sufficient CMV CAR T cells to administer to a subject. The compositions and methods disclosed herein enhance the proliferation and persistence of CAR T cells, including CMV-HIV T cells.

[0005] Disclosed herein, inter alia, is a nucleic acid molecule encoding a chimeric antigen receptor that includes: an scFv that binds to HIV Env; a spacer domain; a transmembrane domain; a costimulatory domain; and a CD3 zeta signaling domain. In various embodiments, the scFv comprises a VL domain comprising a light chain CDR1 comprising QTSQGVGSDLH (SEQ ID NO: 1), a light chain CDR2 comprising HTSSVED (SEQ ID NO: 2), a light chain CDR3 comprising QVLQF (SEQ ID NO: 3), and a VH domain comprising a heavy chain CDR1 comprising AHILF (SEQ ID NO: 4), a heavy chain CDR2 comprising WIKPQYGAVNFGGGFRD (SEQ ID NO: 5), and a heavy chain CDR3 comprising DRSYGDSSWALDA (SEQ ID NO: 6); the scFV comprises (a) a light chain variable domain that is at least 90%, 95% or 98% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7); and (b) a light chain variable domain that is at least 90%, 95% or 98% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7). a heavy chain variable domain that is at least 90%, 95%, or 98% identical to GRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 8); wherein the scFV comprises a light chain variable domain that comprises YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7), and a heavy chain variable domain that comprises RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVS (SEQ ID NO: 73);and a VL domain that is 95% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7) and includes the following CDR sequences: QTSQGVGSDLH (SEQ ID NO: 1) (VL-CDR1), HTSSVED (SEQ ID NO: 2) (VL-CDR2), and QVLQF (SEQ ID NO: 3) (VL-CDR3); and KKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 8) and comprises a VH domain containing the following CDR sequences: AHILF (SEQ ID NO: 4) (VH-CDR1), WIKPQYGAVNFGGGFRD (SEQ ID NO: 5) (VH-CDR2), and DRSYGDSSWALDA (SEQ ID NO: 6) (VH-CDR3);

[0006] Disclosed herein is a nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an scFv comprising or consisting of the following: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSAGGGSGGGSGGGSGGGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 9); a spacer comprising a sequence selected from the group consisting of SEQ ID NOs: 24 to 34; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15 to 23; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 36 to 40, and a CD3ζ signaling domain comprising SEQ ID NO: 35.

[0007] In any of the nucleic acid molecules: the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-34, or an amino acid sequence having 0, 1, 2, 3, 4, or 5 single amino acid substitutions thereto; the transmembrane domain is selected from the group consisting of: CD4 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane domain, and CD3 zeta transmembrane domain; the costimulatory domain is selected from the group consisting of: CD28 costimulatory domain, 41-BB costimulatory domain, OX40 costimulatory domain, and 2B4 costimulatory domain; the chimeric receptor comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 single amino acid substitutions thereto (SEQ ID NO: 10);In some embodiments, the amino acid substitutions are conservative, and in some embodiments, there are no amino acid substitutions in the CDRs, and the nucleic acid molecule further comprises an interdomain linker of 1-5 amino acids between one or more of the scFV and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the costimulatory domain, and the costimulatory domain and the CD3ζ signaling domain, wherein the interdomain linker may consist of 1-5 glycines (SEQ ID NO: 75); in some embodiments, the CAR further comprises an interdomain linker consisting of the sequence GGG located between the costimulatory domain and the CD3ζ signaling domain;

[0008] In one embodiment, the CAR comprises an scFv comprising or consisting of SEQ ID NO:9 or a variant thereof in which one, two, three, four or five amino acids have been substituted, but said substitutions are not in the CDRs; a spacer comprising a sequence selected from the group consisting of SEQ ID NOs:24-34 or a variant thereof in which one, two, three, four or five amino acids have been substituted; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs:15-23 or a variant thereof in which one, two, three, four or five amino acids have been substituted; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs:36-40 or a variant thereof in which one, two, three, four or five amino acids have been substituted; and a CD3ζ signaling domain comprising SEQ ID NO:35 or a variant thereof in which one, two, three, four or five amino acids have been substituted. In one embodiment, the amino acid substitutions are conservative.

[0009] Disclosed herein is an immune cell carrying any nucleic acid molecule. Disclosed herein is an immune cell expressing any CAR. In various embodiments, the immune cells are T cells that express a CMV-specific T cell receptor (CMV-specific T cells). Disclosed herein are populations of cells that include CMV-specific T cells carrying any nucleic acid molecule. Disclosed herein are populations of CMV-specific T cells that express any HIV CAR. In various embodiments, at least 20%, 30%, 40%, or 50% of the CMV-specific T cells are CD8+ T cells.

[0010] Also provided herein is a method for preparing a population of T cells specific for CMV that expresses an HIV chimeric antigen receptor (CAR), comprising: pos Disclosed is a method comprising the steps of isolating a population comprising PBMCs from a blood sample obtained from a subject, contacting the cell population with a CMV antigen to stimulate CMV-specific T cells, isolating a subpopulation of IFNγ-secreting T cells from the cell population (e.g., a subpopulation of IFNγ-secreting T cells (e.g., CMV-specific cells) from the cell population, and transfecting cells in the subpopulation of IFNγ-secreting T cells with a vector comprising a nucleic acid molecule according to any one of claims 1 to 14.

[0011] In an embodiment, a method for preparing T cells specific for cytomegalovirus (CMV) and expressing a chimeric antigen receptor (CAR) is disclosed herein. In an embodiment, the method includes: (a) providing a cell population comprising T cells (e.g., PBMCs) from a cytomegalovirus CMV seropositive human donor; (b) exposing the cell population (e.g., PBMCs) to at least one CMV antigen; (c) treating the exposed cells to produce a cell population enriched for CMV-specific stimulator cells; and (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR to prepare T cells specific for CMV and expressing a CAR. In various cases, treating the exposed cells (e.g., using selection) to generate a population of cells enriched for stimulator cells specific for CMV includes treating the stimulated cells to generate a population of cells enriched for cells expressing an activation marker (e.g., IFN-γ or IL-13), the PBMCs are cultured for less than 5 days (less than 4, 3, 2, 1 day) prior to exposure to a CMV antigen, the cells are exposed to a CMV antigen for less than 3 days (less than 48 hours, 36 hours, 24 hours), the CMV antigen is a pp65 protein or an antigenic portion thereof, and the CMV antigen is selected from two or more different antigenic CMV antigens. comprising a pp65 peptide, transducing the enriched population of cells does not include CD3 stimulation, transducing the enriched population of cells does not include CD28 stimulation, transducing the enriched population of cells does not include CD3 or CD28 stimulation, the enriched population of cells is at least 40% (e.g., 50%, 60%, 70%) IFN-γ positive, at least 20% (e.g., 25%, 30%, 35%) CD8 positive, and at least 20% (e.g., 25%, 30%, 35%) CD4 positive; the enriched population of cells is cultured for less than 10 days (less than 9, 8, 7, 5, 3, 2 days) prior to transducing the enriched population of cells with a vector encoding a CAR. In some cases, the T cells are derived from a CMV positive donor and exposed to a CMV antigen, such as CMV pp65 or a mixture of CMV protein peptides (e.g., a 10-20 amino acid peptide that is a fragment of pp65) in the presence of IL-2 to generate a population of stimulator cells. In some cases, the population of stimulator cells is treated to prepare a population of cells expressing IFN-γ.

[0012] In various embodiments, a subpopulation of CMV T cells (e.g., IFNγ-secreting T cells expressing a T cell receptor specific for CMV) are cultured in the presence of exogenous IL-2 and / or exogenous IL-15 before, after, or before and after transduction; IL-2 is added to 50 U / mL and IL-15 is added to 1 ng / mL; the subpopulation of IFNγ-secreting T cells are cultured in the presence of at least one antiretroviral drug before, after, or before and after transduction; the at least one antiretroviral drug is selected from the group consisting of 1) an HIV protease inhibitor (e.g., tipranavir, atazanavir, indinavir, darunavir, or fosamprenavir); and 2) an HIV fusion inhibitor, HIV entry inhibitor, HIV attachment inhibitor, HIV post-attachment inhibitor (e.g., maraviroc, Ibalizumab, or cyclosporine). buiyk, fostemsavir) and the cells are not cultured in the presence of a reverse transcriptase inhibitor; the blood sample is from an HIV-infected subject (e.g., a subject who has previously been administered one or more antiretroviral drugs). Preferably, they are cultured in the presence of darunavir and enfuvirtide and in the absence of reverse transcriptase inhibitors and / or antiretroviral drugs that interfere with lentivirus replication or the replication of a viral vector carrying a sequence encoding an HIV-targeting chimeric antigen receptor.

[0013] Also disclosed is a method of treating a subject infected with HIV, comprising administering (a) a population of CMV-specific T cells expressing a chimeric antigen receptor, the population comprising: an scFv that binds to HIV Env; a spacer domain; a transmembrane domain; a costimulatory domain; and a CD3ζ signaling domain; and, optionally, (b) at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen. In an embodiment, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered at the same time that the CMV-HIV CAR T cells are administered. In an embodiment, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered prior to administration of the CMV-HIV CAR T cells. In an embodiment, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered after administration of the CMV-HIV CAR T cells. In an embodiment, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered before and after administration of the CMV-HIV CAR T cells. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered in a single dose or in multiple doses, hi some embodiments, the CMV-HIV CAR T cells are administered in a single dose or in multiple doses.

[0014] A useful dose of CMV HIV T cells is approximately 5 × 10 6 , 10×10 6 , 15×10 6 , 20×10 6 , 25×10 6 , 30×10 6 , 35×10 6 , 40×10 6 , 45×10 6 , 50×10 6 , 55×10 6 , 60×10 6 , 65×10 6 , 70×10 6 , 75×10 6 , 80×10 6 , 85×106 , 90×10 6 , 95×10 6 , and 100×10 6 In some embodiments, a single dose of CMV-HIV CAR T cells is administered to the patient. In some embodiments, a second dose of CMV-HIV CAR T cells is administered to the patient. Suitable modes of administration include injection, infusion, drip infusion, or ingestion. Injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracerebroventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion. In some embodiments, the subject is administered the population of CMV HIV T cells in a single intravenous (IV) infusion.

[0015] In some embodiments, the subject is administered at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen prior to administration of the CMV-HIV CAR T cells. In some embodiments, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to administration of the CMV-HIV CAR T cells. In some embodiments, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 28, 36, 48, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, and / or 360 hours prior to administration of the CMV-HIV CAR T cells. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered about 1, 2, 3, or 4 weeks prior to administration of the CMV-HIV CAR T cells. In some embodiments, the subject is administered at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen after administration of the CMV-HIV CAR T cells. In some embodiments, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration of the CMV-HIV CAR T cells. In some embodiments, the at least one CMV antigen or the nucleic acid molecule encoding at least one CMV antigen is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 28, 36, 48, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, and / or 360 hours after administration of the CMV-HIV CAR T cells. In an embodiment, the at least one CMV antigen or nucleic acid molecule encoding at least one CMV antigen is administered about 1, 2, 3, or 4 weeks after administration of the CMV-HIV CAR T cells. In an embodiment, this can be further followed by administration of at least one CMV antigen or nucleic acid molecule encoding at least one CMV antigen prior to administration of the CMV-HIV CAR T cells, thus in an embodiment, the subject is administered at least one CMV antigen or nucleic acid molecule encoding at least one CMV antigen prior to administration of the CMV-HIV CAR T cells, and is administered at least one CMV antigen or nucleic acid molecule encoding at least one CMV antigen after administration of the CMV-HIV CAR T cells. Useful CMV vaccines include one or more CMV antigens or one or more nucleic acids encoding one or more CMV antigens. The CMV antigens can be CMV proteins, fragments of CMV proteins, modified CMV proteins, fragments of modified CMV proteins, mutant CMV proteins or fragments thereof, fused CMV proteins or fragments thereof. In an embodiment, a useful CMV vaccine includes one or more nucleic acids encoding one or more CMV antigens. Examples of CMV antigens include pp65, IE1 exon 4 (IE1 / e4), IE2 exon 5 (IE2 / e5), fusions thereof, and antigenic fragments thereof, and variants thereof having 1, 2, 3, 4, or 5 amino acid modifications. In an embodiment, the 1, 2, 3, 4, or 5 amino acid modifications include 1-2 amino acid substitutions or 1-5 amino acid substitutions. In an embodiment, the amino acid substitutions are conservative. In an embodiment, the CMV antigen includes a sequence selected from SEQ ID NOs: 57-64, and variants thereof having 1, 2, 3, 4, or 5 amino acid modifications. In some embodiments, the 1, 2, 3, 4, or 5 amino acid modifications comprise 1-2 amino acid substitutions or 1-5 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative. In some embodiments, the CMV antigen may comprise a fragment of any of SEQ ID NOs: 57-64. The fragment may comprise or consist of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 consecutive amino acids of any of SEQ ID NOs: 57-64. In various embodiments, the CMV antigen comprises a CMV protein or fragment thereof (e.g., CMVpp65 peptide); the nucleic acid molecule comprises a viral vector encoding a fusion protein comprising (a) a CMVpp65 peptide or protein, and (b) exon 4 (e4) of CMV protein 1E1 and exon 5 (e5) of CMV protein 1E2. Non-limiting examples of nucleic acids encoding CMV antigens include DNA, RNA, mRNA, vectors, viral vectors, lentiviral vectors, MVA vectors, bacterial artificial chromosomes (BAC), vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors, and others known in the art. Useful nucleic acids may encode one or more CMV antigens. In some embodiments, the nucleotide sequence of the CMV antigen is optimized. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered in a single dose or multiple doses before or after administering the CMV-specific T cells. In some embodiments, an effective amount of at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered to the subject. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered in an amount sufficient to elicit an immune response in the subject.

[0016] In some embodiments, the subject is also treated with antiretroviral therapy (ART). In some embodiments, the ART regimen is reduced or stopped after administration of the CMV CAR T cells. In some embodiments, the ART regimen is temporarily stopped for 4 days prior to leukapheresis to collect peripheral blood mononuclear cells (PBMCs). In some embodiments, the subject resumes the ART regimen immediately after leukapheresis. After leukapheresis, the apheresis product may be incubated overnight with one or more CMV antigens and / or CMV peptides. In an embodiment, CMV-specific T cells are enriched based on interferon gamma (IFNγ) positivity. In an embodiment, the cells are then transduced with a self-inactivating lentiviral vector encoding a CAR disclosed herein (e.g., vHIVR(N6)(EQ)BBζ-T2A-EGFRt_epHIV7; SEQ ID NO: 44). In an embodiment, the population of CMV / HIV-CAR T cells is expanded in vitro for about 2 weeks in the presence of IL-2, IL-15, and an ART cocktail inhibitor. In an embodiment, the expanded population of CMV / HIV-CAR T cells is cryopreserved.

[0017] Amino acid modification refers to amino acid substitutions, insertions, and / or deletions in a protein or peptide sequence. An "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. Substitutions may be made in a non-conservative manner (i.e., by changing a codon from an amino acid belonging to a group of amino acids of a particular size or characteristic to an amino acid belonging to another group) or in a conservative manner (i.e., by changing a codon from an amino acid belonging to a group of amino acids of a particular size or characteristic to an amino acid belonging to the same group) to alter the amino acids in the resulting protein. Such conservative changes generally do not appreciably alter the structure and function of the resulting protein. The following are examples of various groups of amino acids: 1) Amino acids with non-polar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; 2) Amino acids with uncharged polar R groups: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (pH 6.0). Another group may be amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine. Examples are provided so that the disclosed invention can be more fully understood. The materials and methods used in the following examples are described in detail in the examples. The examples disclosed herein are provided to illustrate the methods and compositions provided herein and should not be construed as limiting the scope thereof in any way. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety for any and all purposes. Other features and advantages of the disclosed compositions and methods will become apparent from the following detailed description and drawings, and from the claims. Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Brief description of the drawings]

[0018] [Figure 1]Graph showing functional characterization of N6-CAR T cells. (A) Schematic of N6-CAR construct. The construct contains the GM-CSF receptor-α chain signal sequence (GMCSFRss) under the control of the EF1α promoter, CD3ζ and T2A-linked truncated human EGFR (EGFRt) followed by a single chain variable fragment (scFv) of anti-gp120bNAb N6 linked to the CD4 transmembrane (tm) and 4-1BB costimulatory domains via an IgG4 (EQ) spacer. (B) Primary T cells were activated with CD3 / CD28 microbeads and transduced with lentiviral vector encoding N6-CAR. After approximately 15 days of in vitro expansion, CAR expression and T cell subsets were analyzed by flow cytometry using antibodies against EGFR, CD3, CD4 and CD8. Representative FACS plots of four HIVneg donor-derived CAR T cell products are shown. (C) N6-CAR T cells or mock T cells from HIVneg donors were co-cultured with eGFP-expressing 8E5-gp120 cells at various total T cell:target (E:T) ratios for 96 hours and then immunostained for CD3 and eGFP. Cytotoxicity is calculated as follows: 100%-(% of residual tumor cells in CAR T cell group / % of residual tumor cells in mock T or negative targets). n=3 donors. (D) N6-CAR T cell products were labeled with CellTrace™ Violet dye (CTV) and stimulated with 8,000 rad irradiated 8E5-gp120 at a 1:1 E:T ratio for 8 days prior to CTV analysis (blue line). 8,000 rad irradiated lymphoblast cells expressing the CD3 agonist OKT3 (LCL-OKT3) were used as a positive control (red line) and medium as a negative control (black line). CTV dilutions from EGFR+ gated cells (lower panel) and EGFR gated cells (upper panel) are shown. Representative data of five HIVneg donor-derived CAR T cell lines are shown. [Diagram 2]Development of the 8E5-gp120 cell line. Parental 8E5 cells were derived from HIV-infected lymphoblastoid cells and lack a single integrated copy of the HIV genome reverse transcriptase (RT). (A) Flow cytometry analysis using anti-gp120 monoclonal antibody staining showed that approximately 30% of parental cells expressed gp120. (B) Cells were transduced with lentiviral vectors encoding eGFP and firefly luciferase (ffLuc), after which eGFP and gp120 double positive cells were selected and expanded for in vitro experiments. [Diagram 3] Figure 1 shows the specific cytotoxicity of N6-CAR T cells against gp120 positive cells. N6-CAR T cells from HIVneg donors were co-cultured with eGPF+8E5 cells sorted for gp120 expression at various E:T ratios (1:1, 1:2 or 1:5). Residual eGFP+ tumor cells were measured by flow cytometry after 96 hours. [Figure 4] Graph showing phenotypic characterization of N6-CAR T cells after stimulation with 8E5-gp120 cells. N6-CAR T cells from three HIVneg donors were co-cultured with either 8E5-gp120 cells, LCL-OKT3 cells, or media at a 1:1 E:T ratio for 96 hours, followed by flow cytometry analysis of the expression of (A) memory markers (CD62L, CD127, and CD27) or (B) exhaustion markers (LAG-3, PD-1, and Tim-3). [Diagram 5] Figure 1 shows the specific binding of N6-scFv-Fc on gp120 expressing cells. 8E5-gp120 cells were stained with soluble N6 scFv-Fc at the indicated dilutions. Positive cells were quantified by flow cytometry. Staining with anti-gp120bNAb VRC01 was used as a positive control and gp120 negative KG-1a cells or secondary antibody alone were used as negative controls. [Figure 6]Figure 3: Clinical-scale generation of CMV-HIV CAR T cells from HIVneg and HIVpos donors. (A) Generation workflow for generating CMV-HIV CAR T cells as described in the methods section. (B) Representative FACS plots of CMV-specific T cells isolated from HIVpos donors before and after IFN-γ+ enrichment using the CliniMACS Prodigy® platform. (C) Flow cytometric analysis of the proportion of enriched IFN-γ+ CMV-specific T cells and their relative CD4 and CD8 expression. n=6-7 donors / group. (D) Growth curves of total cell numbers in the final product from HIVneg (n=6) and HIVpos (n=7) donors over approximately 15 days of growth. Growth curves in the presence of antiretroviral drugs (ARVs, darunavir and enfuvirtide) are shown as red dotted lines. (E) Flow cytometric analysis of the proportion of CMV-HIV CAR T cells in the final cell product and their relative CD4 and CD8 expression. n=6-7 donors / group. (F) Total number of CMV-HIV CAR T cells in each final cell product. [Figure 7] Graph showing memory cell subsets among CMV-specific T cells isolated from HIVneg and HIVpos donors. CMV-specific T cells (IFN-γ+CD3+) isolated from HIVneg and HIVpos donors were enriched using the CliniMACS Prodigy® system and immunostained with anti-Cd 27 and anti-CD45 RA antibodies. Flow cytometry analysis shows the percentage of CMV-specific T cells. CMV-specific T cells are CD27+CD45RA+ stem cell memory T cells (Tscm), CD27+CD45RA central memory (Tcm), CD27-CD45RA+ effector memory RA (TEMRA), and CD27-CD45RA effector memory T cells (Tem). Lines indicate mean ± SD; n=5 donors / group. [Figure 8]Graphs showing in vitro HIV replication, lentiviral transduction and cell proliferation in the presence of antiretroviral drugs (ARVs). (A) Addition of the HIV protease inhibitor darunavir (D, EC50=4.3 nM) and the HIV fusion inhibitor enfluvitide (E, EC50=27.9 nM) to CD8+ depleted PBMCs infected with HIV-1 BaL (left panel) or HIV-1NL4-3 (right panel) strains prevents HIV replication. (B) Darunavir (43 nM) and enfluvitide (279 nM) were added on the day (day 0) or 2 days (day 2) after transduction of PBMCs with lentiviral vectors expressing eGFP. Flow cytometry analysis of eGFP expression 8 and 14 days after lentiviral transduction is shown. (C) Cell proliferation of non-transduced or transduced PBMCs in the presence or absence of antiretroviral drugs. [Figure 9] Graphs showing phenotypic characterization of CMV-HIV CAR T cell products derived from HIVneg and HIVpos donors. T cell memory markers: T cell memory T cells (Tscm), T cell memory RA central (Tcm), T cell memory effector T cells (TEMRA), and T cell memory RA effector (Tem) were analyzed by flow cytometry after INF-γ enrichment (A) and in the final CAR T cell product (B). Exhaustion markers (LAG-3, PD-1, and Tim-3) were analyzed by flow cytometry in the final cell product (C) or in the EGFR+ CAR T cell fraction (D). Lines indicate mean ± SD; n = 2-6 donors / group. [Figure 10]Figure 1. Effector function of CMV-HIV CAR T cells from HIVneg donors. (A) Specific cytotoxicity against gp120-expressing target cells was determined by immunostaining for CD3 and eGFP after 96 h of culture of CMV-HIV CAR T cell products with eGFP+8E5-gp120 or eGFP+KG-1a cells at different E:T ratios (2:1, 1:1, 1:2 or 1:5). The percentage of remaining eGFP+ tumor cells was measured by flow cytometry and cytotoxicity was calculated. (B) CMV-HIV CAR T cell products were labeled with CTV and cultured with CMVpp65 peptide-pulsed and 3,500 rad irradiated PBMCs (CMVpp65-PBMCs), 8,000 rad irradiated LCL-OKT3 or KG-1a cells or medium for 8 days. CMV-HIV CAR T cell proliferation was determined by CTV dilution. Representative data from 4 donors are shown. (C) CMV-HIV CAR T cells or CMV-specific T cell products from the same donor were stimulated overnight with CMVpp65 peptide-pulsed autologous PBMCs (CMVpp65-PBMCs), LCL-OKT3, 8E5-gp120, KG-1a cells or media. Co-cultures were stained for surface CD8 followed by intracellular IFN-γ expression. Representative data from three different donors are shown. [Figure 11]Graphs showing effector function of CMV-HIV CAR T cells derived from HIVpos donors. (A) Representative FACS plots of CMV-specific T cells enriched from HIVpos donors and transduced with lentiviral vector expressing N6-CAR (n=7). Transduction efficiency was assessed on day 7 by measuring EGFR expression in T cells. CMV-HIV CAR T cell products were then stimulated overnight with CMVpp65 peptide-pulsed autologous PBMCs and IFN-γ, CD3, CD4 and CD8 expression was analyzed by flow cytometry. Representative data from 3 different donors (n=3) are shown. (B-C) Specific cytotoxicity was determined by immunostaining for CD3, EGFR, and LAG-3, PD-1, and Tim-3 depletion markers after co-culture of CMV-HIV CAR T cells (n=3) with eGFP+8E5-gp120 or eGFP+KG-1a cells at different E:T ratios (2:1, 1:1, or 1:5) for 24 (n=2) and 96 h (n=3). The percentage of remaining eGFP+ tumor cells was measured by flow cytometry, and cytotoxicity was calculated as described in Methods. Graphs show the cytotoxicity of CAR T cells from two or three donors against 8E5-gp120 target cells at different E:T ratios. (D) CMV-HIV CAR T cells or CMV-CD19 CAR T cells were generated from the same HIVpos donor and cultured with HIVNL4-3 infected eGFP+ Jurkat cells at different E:T ratios (1:1, 1:2 and 1:4) for 7 days. Cytotoxicity of CAR T cell products against HIVNL4-3 infected eGFP+ Jurkat cells was calculated and normalized to untreated control wells. Levels of HIV p24 in cell supernatants on day 7 were measured by ELISA and normalized to p24 levels in control conditions at an E:T ratio of 1:1 (E). (F) CMV-HIV CAR T cells and CMV-CD19 CAR T cells were generated from HIVpos donors on ART. Levels of p24 in culture supernatants were measured by ELISA after 20 days of expansion and normalized to p24 levels in the supernatants of CMV-CD19 CAR T cells. Data from one HIVpos donor are shown in (D), (E) and (F). [Figure 12]Figure 1: Dose-dependent control of CMVpp 65-driven proliferation of CMV-HIV CAR T cells and HIV viremia in hu-PBMC-NSG mice on ART. (A) NSG humanized peripheral blood mononuclear cell (hu-PBMC) mouse model of HIV on ART and experimental design. HIV-infected mice receiving oral ART were treated on day 28 with a low dose of CMV-HIV CAR T cells (0.1x106 EGFR+ T cells) with or without CMVpp65 vaccine, or with a high dose of CMV-HIV CAR T cells (1x106 EGFR+ T cells) with CMVpp65 vaccine. Mice treated with CMV-negative T cells (1x106 cells) from the same HIVneg donor with or without CMVpp65 vaccine served as controls. (B) HIV viral load in peripheral blood on day 28. Additive models using baseline HIV viral load (day 21), log10CD3, log10CD4 and treatment group were considered and the best model was selected based on Akaike's information criterion. As this model only included treatment group, analysis of variance for all possible one-sided comparisons (family-wise error rate (FWER) = 0.05) followed by Tukey's method was used to assess whether there were treatment differences between control (T cell treated mice), low dose and high dose CMV-HIV CAR T cell treated cohorts; ***P value < 0.001; **P value = 0.002; n = 8-17 / group. (C) Flow cytometry analysis of EGFR+CAR T cell expansion in peripheral blood from day 33 to day 42. n = 8 / group. Note that one female mouse in the "low dose CAR T + vaccine group" did not have a measurement on day 42 and was not included in this analysis. (D) HIV viral load in peripheral blood on day 42 after vaccine stimulation and ART interruption. The best model was the Tukey test for all possible one-sided comparisons after analysis of covariance including log10CD3, log10CD4, and treatment group. (E) Flow cytometry analysis of the frequency of EGFR+CAR T cells in bone marrow at sacrifice. Data were transformed using a logit transformation.To assess whether there were treatment differences between CAR T-cell treatment cohorts, ANOVA was used, followed by Tukey's method for all possible one-sided comparisons (Family wise error rate (FWER) = 0.05); **P value < 0.01; *P value = 0.02; n = 7-8 / group. (F) Percentage of EGFR+ CAR T cells in bone marrow plotted against percentage of p24+ T cells in bone marrow. Staining for surface antibodies (CD45, CD3, and EGFR) was performed as in panel (C), while intracellular p24 HIV-1 antigen was stained with KC57-FITC antibody after fixation and permeabilization. A simple least squares model with only % of EGFR+ CAR T cells was best, and both % of EGFR+ CAR T cells and % of p24+ T cells were transformed using logit. Box plots were used to present the data in (B), (D), and (E). Black boxes represent quartiles, black lines represent quartiles and median, plus signs represent means, values ​​outside the whiskers are considered outliers. [Figure 13] Graphs showing body weight and temperature in HIV-infected hu-PBMC-NSG mice treated with ART, CMV-HIV CAR T cells, with or without CMVpp65 vaccine. Body weight (A) and temperature (B) were monitored weekly (day 0) in the hu-PBMC mouse model engrafted with HIVneg donor-derived PBMCs. Mice started an oral ART regimen on day 12 and received a single IV dose of CMV-HIV CAR T cells (low [0.1x106] or high [1x106] dose) on day 21 and CMVpp65 vaccine on day 28. No statistical significance between groups was observed using ANOVA mixed effects analysis. Group sizes were as follows: "T cells" n=8 (4F4M), "T cells + vaccine" n=8 (4F4M), "Low dose CAR T cells" n=8 (4F4M), "Low dose CAR T cells + vaccine" n=8 (4F4M), "High dose CAR T cells + vaccine" n=9 (4F5M). [Figure 14]Graph showing EGFR+CAR T cell expansion in peripheral blood from day 33 to day 42 in HIV-infected hu-PBMC-NSG mice treated with ART, CMV-HIV CAR T cells, with or without CMVpp65 vaccine. EGFR+CAR T cell expansion in peripheral blood was assessed based on the mean slope of the linear regression line for EGFR+CAR T cell counts / μL using log10 transformation from days 33 and 42. Statistical significance was determined using one-sided Tukey contrast *P value=0.03; **P value=0.02. n=8 / group, same groups as in Figure 6C. [Figure 15] Graph showing distribution and phenotype of HIVpos donor-derived CMV-HIV CAR T cells in a humanized PBMC-NSG mouse model. Flow cytometry analysis of EGFR+ CAR T cells 6 weeks after CAR T cell infusion. Frequency of (A) CD4+ and CD8+ T cells, (B) CD62L+ and (C) CD27+ within the EGFR+ CAR T cell fraction. Lines represent mean ± SD; n=5. [Figure 16] Figure 1 shows a schematic diagram of the clinical trial timeline. Patients are screened and participants signed up for the trial are interrupted on ART regimen for 4 days prior to leukapheresis to prevent inhibition of lentiviral transduction of T cells during CAR T cell manufacturing. Participants resume their ART regimen immediately following leukapheresis. CMV-HIV T cell populations are prepared and participants are infused with 5x106 cells, 25x106 cells, or 50x106 cells (day 0). Dose-limiting toxicity (DLT) is assessed starting the day prior to T cell infusion (day -1) and for 60 days following infusion. Blood is drawn and evaluated on the designated days indicated on the timeline followed by long-term follow-up (LTFU). [Figure 17] Schematic diagram of the HIVR(N6)(EQ)BBZ-T2A-EGFRt_epHIV7 plasmid. Map (A) and sequence (D-E) of the HIVscFv(N6)-IgG4(L235E, N297Q)-41 BB-Zeta(C 0)-T2A-EGFRt_epHIV7 (10008 bp; SEQ ID NO: 44) lentiviral vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The studies disclosed herein relate to CMV-specific T cells expressing HIV-targeted CARs (CMV-CAR T cells) exhibiting dual effector functions upon in vitro stimulation via their endogenous CMV-specific T cell receptor or an introduced CAR. Studies disclosed herein using a humanized HIV mouse model show that CMV vaccination during ART accelerates the expansion of CMV-HIV CAR T cells in peripheral blood, and that higher numbers of CMV-HIV CAR T cells are associated with better control of HIV viral load upon ART interruption and fewer HIV antigen p24+ cells in the bone marrow. CMV-CAR T cells and CMV antigens can be used to treat HIV-infected subjects.

[0020] I. Chimeric Antigen Receptor Chimeric antigen receptor (CAR) refers to an artificial immune cell receptor engineered to recognize and bind to surface antigens. T cells expressing CAR polypeptides are called CAR T cells. CARs can direct T cell specificity and reactivity to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition gives CAR T cells the ability to recognize antigens independent of antigen processing, circumventing a major mechanism of tumor escape. CARs can also be expressed by other immune effector cells, including but not limited to natural killer CARs ("NK CARs"), to direct NK cell killing to cells expressing the CAR's target. There are different generations of CARs, each containing different components. First generation CARs link an antibody-derived scFv to the CD3ζ intracellular signaling domain of the T cell receptor via a spacer region (also called a hinge domain) and a transmembrane domain. Second generation CARs incorporate additional costimulatory domains (e.g., CD28, 4-BB, or ICOS) to provide a costimulatory signal. Third generation CARs contain two costimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused to the TCR CD3ζ chain. There may be a spacer between the costimulatory domain and the CD3ζ domain, but this is optional.CAR is often fused to a signal peptide at the N-terminus for surface expression.In some cases, CAR can be co-expressed with a polypeptide that can function as a marker, such as a truncated EGFR receptor that has no signaling function or a truncated CD19 receptor that has no signaling function.

[0021] An embodiment disclosed herein is a method for producing a medicament comprising the steps of: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA GGGSGGGSGGGSGGGS YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK MALIVLGGVAGLLLFIGLGIFF KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL GGG RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) Regarding HIV CAR, From the amino terminus to the carboxy terminus, CAR has the following sequence: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 8) There are The linker sequence includes the following sequence: GGGSGGGSGGGSGGGS (SEQ ID NO: 11) There are The VL domain contains the following sequence: YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7) There are; The spacer domain contains the following sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 33) There are; The CD4 transmembrane domain contains the following sequence: MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 18) There are; The 41-BB costimulatory domain contains the following sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 38) There are; The linker has the following sequence: GGG; There is, and The CD3 ζ domain contains the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 35) Examples include:

[0022] In one embodiment, the CAR sequence has the following sequence: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 65) It may be preceded by a GMCSFRa signal peptide. In one embodiment, the CAR sequence is followed by the following sequence: LEGGGEGRGSLLTCGDVEENPGPR (SEQ ID NO: 45) There is a T2A skip sequence, The following array: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 65) A GMCSFRa signal peptide having the structure has no signal transduction activity and has the following sequence: RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO: 74) A truncated EGFR receptor, in which

[0023] (a) Extracellular binding domain Useful HIV CARs disclosed herein are fusion proteins that contain an extracellular binding domain that recognizes HIV, which may be a single chain fragment of an antibody (scFv) or other antibody fragment, but which is a ligand that binds to an HIV protein. When the binding domain is an scFv, there are heavy and light chain variable regions which may be present in order, linked together via a flexible linker of, for example, 5-25 amino acids. In certain embodiments, useful flexible linkers repeat the sequence GGGS (SEQ ID NO: 13) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In certain embodiments, useful flexible linkers repeat the sequence GGGGS (SEQ ID NO: 14) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In certain embodiments, the light chain variable domain is amino-terminal to the heavy chain variable domain, and in other cases it is carboxy-terminal to the heavy chain variable domain. In certain cases the linker comprises the sequence SSGGGGSGGGGSGGGGGS (SEQ ID NO: 12). Described herein is a study using an HIV CAR comprising the VH and VL domains of N6, a broadly neutralizing antibody that binds to the CD4 binding site of HIV Env and potently neutralizes 98% of HIV-1 isolates, including 16 of the 20 that have evolved to circumvent common mechanisms of resistance (Non-Patent Document 18). The CDRs in the VL and VH domains are underlined in the sequence below.

[0024] [Table 1] Therefore, the svFv in the HIV CAR YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7) and a VL domain comprising the following CDR sequences: QTSQGVGSDLH (VL-CDR1; SEQ ID NO: 1), HTSSVED (VL-CDR2; SEQ ID NO: 2), and QVLQF (VL-CDR3; SEQ ID NO: 3); RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 8) and comprising the following CDR sequences: AHILF (VH-CDR1; SEQ ID NO: 4), WIKPQYGAVNFGGGFRD (VH-CDR2; SEQ ID NO: 5), and DRSYGDSSWALDA (VH-CDR3; SEQ ID NO: 6). The N6 scFv used in the HIV CARs disclosed herein has the sequence: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSAGGGSGGGSGGGSGGGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 9) There is.

[0025] (b) Transmembrane domain The CAR polypeptides disclosed herein can contain a transmembrane domain, which can be a hydrophobic alpha-helix that spans the membrane. As used herein, a transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain of the HIV CAR used in the examples is the CD4 transmembrane domain, which has the following sequence: MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 18). Other transmembrane domains may be used, including those shown below.

[0026] [Table 2] (c) Spacer domain The CAR or polypeptide disclosed herein may include a spacer domain located between the HIV targeting domain (i.e., the HIV targeting scFv or variant thereof) and the transmembrane domain. The spacer region may function to provide flexibility to the CAR or its domains or to prevent steric hindrance of the CAR or its domains. A variety of different spacers can be used. Some of them include at least a portion of a human Fc region, such as a hinge portion or a CH3 domain of a human Fc region or variants thereof. Table 2 below provides various spacer domains that can be used in the CARs disclosed herein.

[0027] [Table 3] Some spacer domains include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer domains include an immunoglobulin CH3 domain (referred to as CH3 or ΔCH2) or both the CH3 and CH2 domains. The immunoglobulin-derived sequence may include one or more amino acid modifications, e.g., 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding. The spacer domain may also include an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:26) or ESKYGPPCPPCP (SEQ ID NO:25). The hinge / linker region may also include the sequence ESKYGPPCPPCP (SEQ ID NO:25), followed by the linker sequence GGGSSGGGSG (SEQ ID NO:24), followed by the IgG4 CH3 sequence: GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 34) Thus, the spacer domain may comprise the following sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 31) In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) relative to SEQ ID NO: 31. In some cases, the IgG4 Fc hinge / linker region is mutated at two positions (L235E; N297Q) to reduce binding by the Fc receptor (FcR).

[0028] (d) Intracellular signaling domain Any of the CAR constructs disclosed herein contain one or more intracellular signaling domains (e.g., CD3ζ and, optionally, one or more costimulatory domains) that are functional termini of the receptor. After antigen recognition, the receptors cluster and a signal is transmitted to the cell. CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex. CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which transmit activation signals to T cells after they bind to cognate antigens. In some cases, CD3ζ provides the primary T cell activation signal, but does not provide a fully competent activation signal, and a costimulatory signal is required. Thus, in some examples, the CAR polypeptides disclosed herein can comprise CD3ζ and further one or more costimulatory signaling domains. For example, the costimulatory domains CD28 and / or 4-1BB can be used to transmit a growth / survival signal along with the primary signaling mediated by CD3ζ. The costimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable costimulatory domains, along with the sequence of the CD3ζ signaling domain.

[0029] [Table 4] In some examples, the CD3 zeta signaling domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 35. In such cases, the CD3 zeta signaling domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative substitutions) compared to SEQ ID NO: 35. In other examples, the CD3 zeta signaling domain is SEQ ID NO: 35.

[0030] In various embodiments, the costimulatory domain is selected from the group consisting of a costimulatory domain or variant thereof having one to five (e.g., one or two) amino acid modifications as set forth in Table 3, a CD28 costimulatory domain or variant thereof having one to five (e.g., one or two) amino acid modifications, a 4-1BB costimulatory domain or variant thereof having one to five (e.g., one or two) amino acid modifications, and an OX40 costimulatory domain or variant thereof having one to five (e.g., one or two) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or variant thereof having one to five (e.g., one or two) amino acid modifications is present in a CAR polypeptide disclosed herein. In some embodiments, there are two costimulatory domains, e.g., a CD28 costimulatory domain or variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory domain or variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In some embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions. In some embodiments, the costimulatory domain is amino-terminal to the CD3ζ signaling domain, and a short linker of 2-10, e.g., 3 amino acids (e.g., GGG) can be located between the costimulatory domain and the CD3ζ signaling domain. In some cases, CARs can be produced using vectors in which the CAR open reading frame is followed by a truncated EGFR (EGFRt) lacking a T2A ribosomal skip sequence and a cytoplasmic signaling tail, or a truncated CD19R (also called CD19t). Co-expression of EGFRt or CD19t in this configuration provides an inert, non-immunogenic surface marker that can accurately measure genetically modified cells, positively select genetically modified cells, and efficiently track therapeutic T cells in vivo after adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is a key hurdle for successful T cell immunotherapy. EGFRt or CD19t incorporated into CAR lentiviral vectors can function as suicide genes to eliminate CAR+ T cells in the event of treatment-related toxicity. The CD3 zeta signaling domain is followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:45) and the following sequence: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO: 46) This may be followed by a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical or identical to SEQ ID NO: 46. In some cases, the truncated EGFR has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) relative to SEQ ID NO: 46. Alternatively, the CD3 zeta signaling domain is followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:45) and the following sequence: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: 47) This may be followed by a truncated CD19R (also referred to as CD19t) which has a sequence that is at least 90%, at least 95%, at least 98% identical or identical to

[0031] The CAR disclosed herein can be produced by any means known in the art, but is preferably produced using recombinant DNA technology. The nucleic acid encoding a region of the chimeric receptor can be conveniently prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (such as genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and transformed into a suitable expression host cell line (preferably T lymphocytes, and most preferably autologous T lymphocytes). Various T cell subsets isolated from patients can be transduced with vectors for CAR or polypeptide expression. Central memory T cells are one useful T cell subset. Central memory T cells can be isolated from peripheral blood mononuclear cells (PBMCs) by selecting for CD45RO+ / CD62L+ cells, for example, using a CliniMACS® device to immunomagnetically select cells expressing the desired receptor. Cells enriched for central memory T cells are activated with anti-CD3 / CD28 and transduced with, for example, a lentiviral vector to induce expression of a CAR and a non-immunogenic surface marker for in vivo detection, elimination, and potential ex vivo selection. Activated / genetically modified central memory T cells can be cryopreserved after ex vivo expansion with IL-2 / IL-15. Further methods for preparing CAR T cells can be found in PCT / US2016 / 043392. Methods for preparing useful T cell populations are described, for example, in WO 2017 / 015490 and WO 2018 / 102761. In some cases, it may be useful to use natural killer (NK) cells, for example allogeneic NK cells derived from peripheral blood or umbilical cord blood. In other cases, the NK cells may be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).

[0032] In some embodiments, disclosed herein are compositions comprising IPSC-derived CAR T cells or CAR NK cells. In some embodiments, the compositions comprising IPSC-derived CAR T cells or CAR NK cells have enhanced therapeutic properties. In some embodiments, the IPSC-derived CAR T cells or CAR NK cells have enhanced functional activities including potent cytokine production, cytotoxicity, and cytostatic inhibition of tumor growth, e.g., activities that reduce tumor burden. CARs can be transiently expressed in T cell populations by mRNA encoding the CAR, which can be introduced into T cells by electroporation (Wiesinger et al. 2019 Cancers (Basel) 11:1198). In certain embodiments, the composition comprising the CAR T cells comprises one or more of helper T cells, cytotoxic T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, or a combination thereof.

[0033] II. CMV-specific T cells In some cases, the method includes preparing T cells that are specific for cytomegalovirus (CMV) and express a chimeric antigen receptor (CAR) and includes (a) providing T cells (e.g., PBMCs) from a cytomegalovirus (CMV)-seropositive human donor; (b) exposing the PBMCs to at least one CMV antigen; (c) treating the exposed cells to produce a cell population enriched for CMV-specific stimulator cells; and (d) transducing at least a portion of the enriched cell population with a vector expressing the CAR to prepare the T cells that are specific for CMV and express the CAR. In various cases, treating the exposed cells (e.g., using selection) to generate a population of cells enriched for stimulator cells specific for CMV includes treating the stimulated cells to generate a population of cells enriched for cells expressing an activation marker (e.g., IFN-γ or IL-13), the PBMCs are cultured for less than 5 days (less than 4, 3, 2, 1 day) prior to exposure to a CMV antigen, the cells are exposed to a CMV antigen for less than 3 days (less than 48 hours, 36 hours, 24 hours), the CMV antigen is a pp65 protein or an antigenic portion thereof, and the CMV antigen is selected from two or more different antigenic CMV antigens. comprising a pp65 peptide, transducing the enriched population of cells does not include CD3 stimulation, transducing the enriched population of cells does not include CD28 stimulation, transducing the enriched population of cells does not include CD3 or CD28 stimulation, the enriched population of cells is at least 40% (e.g., 50%, 60%, 70%) IFN-γ positive, at least 20% (e.g., 25%, 30%, 35%) CD8 positive, and at least 20% (e.g., 25%, 30%, 35%) CD4 positive; the enriched population of cells is cultured for less than 10 days (less than 9, 8, 7, 5, 3, 2 days) prior to transducing the enriched population of cells with a vector encoding a CAR. In some cases, the T cells are derived from a CMV positive donor and exposed to a CMV antigen, such as CMV pp65 or a mixture of CMV protein peptides (e.g., a 10-20 amino acid peptide that is a fragment of pp65) in the presence of IL-2 to generate a population of stimulated cells. In some cases, the population of stimulated cells is treated to prepare a population of cells that express IFN-γ. In some cases, the CMV / CAR T cells do not recognize antigens from the second virus.For example, it does not recognize Epstein-Barr virus antigens, influenza virus antigens, or adenovirus antigens.

[0034] III. Treatment of HIV-infected patients Embodiments of the disclosure provide methods for treating an infected subject by administering immune cells, e.g., T cells specific for CMV expressing an HIV-CAR, and a CMV vaccine. (a) Subject The subject treated with the disclosed method can be a human subject infected with HIV, including a subject on antiretroviral therapy (ART). It can be administered to subjects with viral loads of more than 200 copies / ml or less than 200 copies / ml, and to subjects with undetectable viral loads. The subject can be treated with one or more of a nucleoside reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), an entry or fusion inhibitor, and an integrase inhibitor (INSTI). For example, the subject can be treated with an INSTI, an NNRTI, or a PI and, optionally, two NRTIs, including ritonavir or cobicistat.

[0035] (b) Administration An effective amount of the therapy (e.g., CMV-CAR T cells and CMV vaccine) can be administered to a subject (e.g., a human) in need of treatment via any suitable route (e.g., administered locally or systemically to the subject). Suitable modes of administration include injection, infusion, instillation, or ingestion. Injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracerebroventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion. The CMV-CAR T cells and CMV vaccine can be administered simultaneously or sequentially. An effective amount refers to the amount of each active agent required to provide a therapeutic effect to a subject, alone or in combination with one or more other active agents. The effective amount will depend on factors such as the particular condition being treated, the severity of the condition, the parameters of each patient (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of the combination therapy (if any), the particular route of administration, etc., as will be recognized by those of skill in the art. An effective amount may be administered in one or more administrations, applications, or dosages. The compositions disclosed herein (e.g., CMV HIV CAR T cells and CMV vaccines) may be administered from once or more times per day to once or more times per week (including once every other day). Those of skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound disclosed herein may include a single treatment or a series of treatments. A useful dose of CMV HIV T cells is approximately 5 × 10 6 , 10×10 6 , 15×10 6 , 20×10 6 , 25×10 6 , 30×10 6 , 35×10 6 , 40×10 6 , 45×10 6 , 50×10 6 , 55×10 6 , 60×10 6 , 65×10 6 , 70×10 6 , 75×10 6 , 80×10 6 , 85×10 6 , 90×10 6 , 95×10 6 , and 100×10 6 The patient may receive a single dose of CMV-HIV CAR T cells. A medical professional may provide a patient with dose escalation or de-escalation as needed. In some embodiments, the patient is administered a single dose of CMV-HIV CAR T cells. In some embodiments, the patient is administered a second dose of CMV-HIV CAR T cells. In one embodiment, an effective amount of a CMV vaccine comprising at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered to a subject, hi one embodiment, the CMV vaccine comprising at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered in an amount sufficient to stimulate an immune response in the subject.

[0036] In some embodiments, the CMV vaccine is administered simultaneously with administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered prior to administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered after administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered before and after administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered in a single dose or multiple doses. In some embodiments, the CMV-HIV CAR T cells are administered in a single dose or multiple doses. In some embodiments, the subject is administered the CMV vaccine prior to administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36, or 48 hours prior to administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered about 1, 2, 3, or 4 weeks prior to administration of the CMV-HIV CAR T cells. In some embodiments, the subject is administered the CMV vaccine after administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36, or 48 hours after administration of the CMV-HIV CAR T cells. In some embodiments, the CMV vaccine is administered about 1, 2, 3, or 4 weeks after administration of the CMV-HIV CAR T cells. In an embodiment, the above may be done in addition to administering a CMV vaccine prior to administration of the CMV-HIV CAR T cells, thus in an embodiment, the subject is administered at least one CMV vaccine prior to administration of the CMV-HIV CAR T cells and at least one CMV vaccine after administration of the CMV-HIV CAR T cells. In certain embodiments, at least one CMV vaccine is administered in a single dose or multiple doses, either before or after administration of the CMV-specific T cells.

[0037] (c) CMV vaccine: a CMV antigen or a nucleic acid encoding a CMV antigen Useful CMV vaccines may include one or more CMV antigens or one or more nucleic acids encoding one or more CMV antigens. The CMV antigens may be CMV proteins, fragments of CMV proteins, modified CMV proteins, fragments of modified CMV proteins, mutant CMV proteins or fragments thereof, or fusion CMV proteins or fragments thereof. In an embodiment, a useful CMV vaccine includes one or more nucleic acids encoding one or more CMV antigens. Examples of CMV antigens include pp65, IE1 exon 4 (IE1 / e4), IE2 exon 5 (IE2 / e5), fusions thereof, and antigenic fragments thereof, as well as variants thereof with 1, 2, 3, 4, or 5 amino acid modifications. In an embodiment, the variants include 1-2 amino acid substitutions or 1-5 amino acid substitutions. In an embodiment, the amino acid substitutions are conservative. Examples of modified CMV protein antigens and fragments thereof can be found in U.S. Pat. No. 7,163,685. In some embodiments, the CMV antigen comprises a sequence selected from SEQ ID NOs: 57-64 and variants thereof having 1, 2, 3, 4, or 5 amino acid modifications. In some embodiments, the variant comprises 1-2 amino acid substitutions or 1-5 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative. In some embodiments, the CMV antigen may comprise a fragment of any of SEQ ID NOs: 57-64. The fragment may comprise or consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 consecutive amino acids of any of SEQ ID NOs: 57-64.

[0038] Examples of nucleic acids that can encode CMV antigens include DNA, rna, mRNA, vectors, viral vectors, lentiviral vectors, MVA vectors, bacterial artificial chromosomes (BAC), vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors, and others known in the art. Useful nucleic acids can encode one or more CMV antigens. In some embodiments, the nucleotide sequence of the CMV antigen is optimized. A fusion CMV protein antigen may comprise two or more CMV proteins, modified CMV proteins, mutated CMV proteins, or any antigenic fragments thereof. In one embodiment, a useful fusion protein is a fusion of IE1 exon 4 (IE1 / e4) and IE2 exon 5 (IE2 / e5), IE1 / e4-IE2 / e5 (lEfusion; e.g., SEQ ID NO: 58). In one embodiment, a useful fusion protein comprises SEQ ID NO: 58 or a variant thereof having 1-5 amino acid modifications. In one embodiment, the variant comprises 1-2 amino acid substitutions or 1-5 amino acid substitutions. In one embodiment, the amino acid substitutions are conservative.

[0039] A useful CMV vaccine could be a rMVA vaccine, which includes the modified vaccinia Ankara (MVA) vaccine platform combined with bacterial artificial chromosome (BAC) technology. Modified vaccinia Ankara (MVA) is a genetically engineered, highly attenuated vaccinia virus strain that does not grow in most mammalian cells. The ability of MVA to grow in mammalian cells is blocked at late viral assembly, so this property has minimal impact on viral or foreign gene expression. However, DNA continues to replicate, thus acting as an efficient template for RNA biogenesis, and proteins are synthesized at high levels. MVA also has a large foreign gene capacity and multiple integration sites, two features that make it a desirable vector for expressing vaccine antigens. MVA has a well-established safety record and versatility for the production of heterologous proteins. Indeed, MVA-based vaccines for the treatment of infectious diseases and cancer have been developed and have reached phase I / II clinical trials. MVA is attractive as a vaccine vector for CMV antigens in individuals who are severely immunosuppressed and who experience additional complications such as malignancy or organ failure and require transplantation.

[0040] A CMV triple vaccine is a recombinant MVA that expresses three CMV antigens, namely, at least a portion of immediate early gene-1 (IE1), at least a portion of immediate early gene-2 ​​(IE2), and at least a portion of pp65. The IE1 and IE2 antigens can be expressed as fusion proteins. The CMV antigens are expressed under the control of a modified H5 (mH5) promoter. The CMV triple vaccine is fully described in U.S. Pat. No. 8,580,276 and Wang et al. (Vaccine 28:1547, 2010). The CMV triple vaccine can express CMV pp65 and a CMV IE fusion protein (IEfusion). The IEfusion can include an antigenic portion of IE1 (e.g., exon 4) and an antigenic portion of IE2 (e.g., exon 5), which elicits an immune response when expressed by the vaccine. Various modifications and / or insertion sites can be made to increase the stability of the triplex simultaneously expressing IE1, IE2 and pp65 in a single MVA vector (see, for example, WO 2019 / 217922). As explained in U.S. Patent No. 8,580,276, the CMV triple vaccine contains three of the most highly recognized antigens in the CD8 subset: pp65, IE1, and IE2. There are no regions of homology greater than 5 amino acids between the major exons of both proteins. Both antigens, individually, are widely recognized by approximately 70% of the general population. Any of the vaccine compositions disclosed in U.S. Patent No. 7,163,685, U.S. Patent No. 8,580,276, U.S. Patent No. 9,675,689, U.S. Patent Application Publication No. 20170246292, the entireties of which are incorporated by reference herein for all purposes, may be used in the methods and compositions provided herein.

[0041] Selected CMV antigen amino acid sequences : CMV pp65 protein (UniProt ID: P06725; SEQ ID NO: 57) MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHR HLPVADAVIHASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIGDQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNM IIKPGKISHIMLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFDFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRV TGGGAMAGASTSAGRKRKSASSATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG lEfusion sequence(IE1-IE2; sequence no.58): MVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEE MKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAKKDE LRRKMMYMCYRNIEFFTKNSAFPKTTNGCSQAMAALQNLPQCSPDEIMAYAQKIFKIL DEERDKVLTHIDHIFMDILTTCVETMCNEYKVTSDACMMTMYGGISLLSEFCRVLCCYV LEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSPSVDDLRAIAEE SDEEEAIVAYTLATAGVSSDSLVSPPESPVPATIPLSSVIVAENSDQEESEQSDEEEEE GAQEEREDTVSVKSEPVSEIEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ GDIL AQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVVTNTPLPGASATPELSPRKK PRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQGE EVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSSSSSCSS ASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGASTGPR KKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSLEYKN LPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLALS TPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHA ATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTE TPPEDLDTLSLAIEAAIQDLrnKSQ 1E1 sequence (SEQ ID NO:59): MVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEE MKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAKKDE LRRKMMYMCYRNIEFFTKNSAFKPTTNGCSQAMAALQNLPQCSPDEIMAYAQKIFKIL DEERDKVLTHIDHIFMDILTTCVETMCNEYKVTSDACMMTMYGGISLLSEFCRVLCCYV LEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSPSPVDDLRAIAEE SDEEEAIVAYTLATAGVSSDSLVSPPESPVPATIPLSSVIVAENSDQEESEQSDEEEEE GAQEEREDTVSVKSEPVSEIEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ IE2 sequence (SEQ ID NO:61): MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPEL SPRKKPRKTTRPFKVIIKPPPPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSE EEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSS SSSCSSADSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGAS TGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSL EYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCN LALSTPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNM IIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQ PTETPPEDLDTLSLAIEAAIQDLRNKSQ IE2 H363A sequence (SEQ ID NO:62): MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPEL SPRKKPRKTTRPFKVIIKPPPPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSE EEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSS SSSCSSADSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGAS TGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSL EYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCN LALSTPFLME A TMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNM IIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQ PTETPPEDLDTLSLAIEAAIQDLRNKSQ IE2 H369A sequence (SEQ ID NO:63): MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPEL SPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSE EEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSS SSCSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGAS TGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSL EYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCN LALSTPFLMEHTMPVT A PPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNM IIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQ PTETPPEDLDTLSLAIEAAIQDLRNKSQ IE2 H363A / H369A sequence (SEQ ID NO:64): MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPEL SPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSE EEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSS SSCSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGAS TGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSL EYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCN LALSTPFLME A TMPVT A PPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNM IIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQ PTETPPEDLDTLSLAIEAAIQDLRNKSQ Working Example The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. In particular, described below is the design and preparation of CMV-HIV CAR T cells using cells obtained from patients on ART, and studies using these cells and a CMV vaccine in a mouse model of HIV infection. EXAMPLES

[0042] N6-CAR T cells exhibit potent effector function in vitro The N6 CAR T cell line is a novel HIV negPrimary T cells isolated from donors were transduced with a lentiviral vector (LV) encoding a CAR containing the scFv of bNAb N6 ( Fig. 1 A). CARs contain a mutant IgG4 spacer between the scFv ectodomain and the transmembrane domain (Jonnalagadda, M., Mardiros, A., Urak, R., Wang, X., Hoffman, LJ, Bernanke, A., Chang, WC, Bretzlaff, W., Starr, R., Priceman, S., Ostberg, JR, et al. (2015). Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. Molecular therapy : the journal of the American Society of Gene Therapy 23, 757-768. 10.1038 / mt.2014.208), a CD4 transmembrane domain, and a 4-1BB costimulatory domain (Philipson, BI, O'Connor, RS, May, MJ, June, CH, Albelda, SM, and Milone, MC (2020). 4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling. Science signaling 13. 10.1126 / scisignal.aay8248;Frigault, MJ, Lee, J., Basil, MC, Carpenito, C., Motohashi, S., Scholler, J., Kawalekar, OU, Guedan, S., McGettigan, SE, Posey, AD, Jr., Ang, S., et al. (2015).Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer immunology research 3, 356-367. 10.1158 / 2326-6066.CIR-14-0186;Brentjens, R.J., Riviere, I., Park, J.H., Davila, M.L., Wang, X., Stefanski, J., Taylor, C., Yeh, R., Bartido, S., Borquez-Ojeda, O., Olszewska, M., et al. (2011). Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118, 4817-4828. 10.1182 / blood-2011-04-348540;Porter, D.L., Hwang, W.T., Frey, N.V., Lacey, S.F., Shaw, P.A., Loren, A.W., Bagg, A., Marcucci, K.T., Shen, A., Gonzalez, V., Ambrose, D., et al. (2015). Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Science translational medicine 7, 303ra139. 10.1126 / scitranslmed.aac5415;Leibman, R.S., Richardson, M.W., Ellebrecht, C.T., Maldini, C.R., Glover, J.A., Secreto, A.J., Kulikovskaya, I., Lacey, SF, Akkina, SR, Yi, Y., Shaheen, F., et al. (2017). Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor. PLoS pathogens 13, e1006613. 10.1371 / journal.ppat.1006613) and a truncated human epidermal growth factor receptor (EGFRt) was added to the CAR construct to serve as an element for CD3 zeta domain immunomagnetic purification, cell tracking by flow cytometry and immunohistochemistry, and potential in vivo cell depletion using the anti-EGFR antibody cetuximab (Wang, X., Chang, WC, Wong, CW, Colcher, D., Sherman, M., Ostberg, JR, Forman, SJ, Riddell, SR, and Jensen, MC (2011). A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 118, 1255-1263. 10.1182 / blood-2011-02-337360). EGFR in the final T cell product after approximately 15 days of in vitro expansion. + CAR T cells averaged 38.16% ± 8.87%, of which 69.63% ± 16.63% were CD4+ and 31.57% ± 17.76% were CD8 +T cells (mean ± SD, n = 4, Figure 1B shows a representative FACS plot). We performed a 96-h killing assay targeting 8E5-gp120 cells to investigate the ability of N6-CAR T cells to induce cytotoxic function. The 8E5-gp120 cells were obtained by engineering 8E5 cells to express eGFP-ffLuc and then sorting for co-expression of eGFP and surface gp120 (Figure 2). N6-CAR T cell products and 8E5-gp120 cells were co-cultured at various effector to target (E:T) ratios. Flow cytometry analysis of the remaining target cells showed that N6-CAR T cells, normalized to mock T cells, efficiently killed 8E5-gp120 cells (Figure 1C). In a separate experiment, N6-CAR T cells were co-cultured with purified gp120-negative or gp120-positive 8E5 cells at various E:T ratios. We observed efficient and gp120-specific killing of gp120-positive 8E5 cells, but not gp120-negative 8E5 cells (Figure 3). Finally, we observed gated EGFR T cell killing from the mixed T cell population, but not from the gated CAR-negative T cell fraction. + Only CAR T cells showed proliferation capacity after stimulation with 8E5-gp120 cells (Figure 1D).Flow cytometry analysis showed that the stimulated N6-CAR T cells maintained persistent memory (CD62L=66.07%, CD127=51.42% and CD27=87.02%, average of three donors) and low exhaustion characteristics (programmed cell death-1 [PD-1]=10.84%, lymphocyte activation gene-3 [LAG-3]=0.26%, and T cell immunoglobulin and mucin domain-3 [Tim-3]=4.71%, average of three donors) (Figure 4). EXAMPLES

[0043] N6 scFv-Fc does not cross-react with normal human tissues Immunostaining with soluble N6 scFv-Fc was performed on normal human tissues to evaluate potential off-target effects of N6-CAR. As expected, concentration-dependent immunostaining was observed on 8E5-gp120 cells, but not on gp120-negative leukemia KG-1a cells, using either N6 scFv-Fc or a positive control (anti-gp120 bNAb VRC01 obtained from the NIH HIV Reagent Program) (Figure 5). N6 scFv-Fc was then used for pan-immunostaining on 37 frozen human tissues from three unrelated normal donors [Charles River Labs, CRL Study Number: 20182940]. Immunopathological analysis showed no membrane signal on the tissues. However, cytoplasmic staining was observed in epithelial cells of the esophagus (mucosa), kidney (renal pelvis), pituitary (adenohypophysis), salivary gland (duct), skin (sweat gland), thymus (epithelium-reticular), and ureter (mucosa), as well as in the colloid of the thyroid gland.Binding to cytoplasmic sites is likely to be of little toxicological significance due to the limited ability of antibody-based therapeutics to access the cytoplasmic compartment in vivo (Hall, WC, Price-Schiavi, SA, Wicks, J., and Rojko, JL (2008). Tissue Cross-Reactivity Studies for Monoclonal Antibodies: Predictive Value and Use for Selection of Relevant Animal Species for Toxicity Testing;Leach, MW, Halpern, WG, Johnson, CW, Rojko, JL, MacLachlan, TK, Chan, CM, Galbreath, EJ, Ndifor, AM, Blanset, DL, Polack, E., and Cavagnaro, JA (2010). Use of tissue cross-reactivity studies in the development of antibody-based biopharmaceuticals: history, experience, methodology, and future directions. Toxicologic pathology 38, 1138-1166. 10.1177 / 0192623310382559). Overall, immunohistochemical staining analyses supported a low risk of clinically relevant off-target tissue cross-reactivity for N6-CAR. EXAMPLES

[0044] CMV-HIV CAR T cells are neg and HIV pos Can be produced on a clinical scale from donors CMV-specific T cells were isolated using a GMP-compliant CliniMACS Prodigy® automated closed system as previously described (Kumaresan, P., Figliola, M., Moyes, JS, Huls, MH, Tewari, P., Shpall, EJ, Champlin, R., and Cooper, LJ (2015). Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System. Journal of visualized experiments : JoVE. 10.3791 / 52808;Wang, X., Urak, R., Walter, M., Guan, M., Han, T., Vyas, V., Chien, SH, Gittins, B., Clark, MC, Mokhtari, S., Cardoso, A., et al. (2022). Large-scale manufacturing and characterization of CMV-CD19CAR T cells. Journal for Immunotherapy of cancer 10. 10.1136 / jitc-2021-003461). A lentiviral vector encoding N6-CAR (Figure 2A). Briefly, PBMCs were transfected with 100% IgG4-positive mice treated with CMV CAR on ART. pos HIV neg or HIV pos IFN-γ+ cells were then isolated via magnetic selection in the CliniMACS Prodigy® system (Figure 6B shows a representative FACS plot). negDonor-derived CMV-specific T cells (IFN-γ+CD3+) were enriched from 4.33% ± 3.46% to 74.71% ± 9.17% of total viable T cells (mean ± SD, n = 6, T). Error! Reference source not found. C). Similarly, HIV pos Donor-derived IFN-γ+ T cells were enriched from 1.91% ± 0.94% to 67.28% ± 17.04% of total viable T cells (mean ± SD, n = 7, Fig. 2C). neg Unlike CMV-specific T cells isolated from donors (42.34% ± 20.30% and 46.18% ± 18.86%, respectively), HIV pos Donor-derived CMV-specific T cells were more abundant in T cells (78.04% ± 9.21%) compared with T cells (25.59% ± 12.19%) (mean ± SD, Fig. 6C). This observation is consistent with the finding that PLWH are more likely to be HIV positive than those who are HIV positive. neg Compared with the individual CD8 +This is consistent with previous reports showing a higher proportion of CMV-specific T cells (Naeger, DM, Martin, JN, Sinclair, E., Hunt, PW, Bangsberg, DR, Hecht, F., Hsue, P., McCune, JM, and Deeks, SG (2010). Cytomegalovirus-specific T cells persist at very high levels during long-term antiretroviral treatment of HIV disease. PloS one 5, e8886. 10.1371 / journal.pone.0008886;Stone, SF, Price, P., Khan, N., Moss, PA, and French, MA (2005). HIV patients on antiretroviral therapy have high frequencies of CD8 T cells specific for Immediate Early protein-1 of cytomegalovirus. AIDS 19, 555-562). Notably, the overall composition of memory T cell subsets was significantly different in HIV neg Donors and HIV pos The CMV-specific T cells isolated from the donors were similar (Figure S5). The harvested IFN-γ T cells (approximately 1 × 10 6 ) were transduced with N6-CAR lentiviral vector at MOI 3 to generate CMV-HIV CAR T cells and expanded in the presence of IL-2 (50 U / mL) and IL-15 (1 ng / mL) for approximately 15 days. To evaluate the effect of endogenous reactivation of HIV, we first expanded CAR T cells in the absence of antiretroviral drugs (ARVs). The total number of cells at the end of culture was 209.5 × 10 6 HIV neg For donors, ±97.62 × 10 6 , H.I.V. pos For donors, it was 13.49 × 106 ± 12.17 × 10 6(Mean ± SD, Fig. 6D). ARv (43 nM darunavir and 279 nM enfuvirtide) was administered to three HIV pos The cocktail was supplemented to the medium during donor CAR T cell expansion to inhibit HIV replication. This cocktail was shown to prevent HIV replication in vitro (Figure 8A) without affecting lentiviral transduction efficiency (Figure 8B) and cell proliferation (Figure 8C). Interestingly, HIV pos Donor (64.1 × 10 6 , 237.2×10 6 and 269.53 x 10 6 ) occurred in the presence of ARV (Fig. 6D, red dotted line). Thus, with ARV, HIVpos donor cells can proliferate similarly to HIVneg donor cells. To further explore the differences between products generated from HIVneg and HIVpos donors, EGFR expression in the final cell products was measured by flow cytometry to assess transduction efficiency. Similar EGFR expression levels were observed in cell products derived from HIVneg (24.68% ± 18.34%) and HIVpos donors (21.45% ± 12.65%) (mean ± SD, D, Fig. 6E). As expected, HIV pos Donor-derived CMV-HIV CAR T cells were CD8+ compared with CD4+ cells (10.49% ± 6.62%). + The proportion of cells (86.46% ± 16.52%) was higher, but HIV neg CD8 in CMV-HIV CAR T cells produced from donors + The percentage of CD4+ and CD5+ cells was 61.35% ± 40.63% and 43.07% ± 42.56%, respectively (mean ± SD, Fig. 6E). Finally, we evaluated the final number of CAR T cells produced per campaign (Fig. 6F). HIV grown in the presence of ARVs pos The average number of CAR T cells in the derived cell product was 34.19 × 10 6 It was. Analysis of T cell line memory subsets in the final T cell line product supports the HIV negThe donor-derived cell line products were Tscm, 19.03% ± 24.52%, Tcm, 18.92% ± 27.40%, TemRA, 18.78% ± 22.41%, and Tem, 43.26% ± 37.37% (mean ± SD, Fig. 9A). pos The donor-derived cell product was less abundant in Tscm (1.71% ± 2.39%) than in Tem (65.02% ± 21.76%). + Similar observations were made when looking at the composition of cellular memory subsets within CAR T cells (Figure 9B). Finally, only low expression levels of the exhaustion markers LAG-3, PD-1 and Tim-3 were observed in the final T cell product (Figure 9C) or in HIV pos Or HIV neg EGFR from any donor + This was observed in CAR T cells (Figure 9D). Table 4: HIV pos Donor Information

[0045] [Table 5] N / A: Information not available. EXAMPLES

[0046] CMV-HIV CAR T cells exhibit HIV- and CMV antigen-specific effector functions For the first time, we performed a 96-h long-term killing assay using 8E5-gp120 cells as target cells to determine the HIV negWe showed that donor-derived CMV-HIV CAR T cells were specifically cytotoxic against gp120-expressing cells (Figure 10A). We then assessed whether CMV-HIV CAR T cells were responsive to CMV antigen stimulation via signaling of their endogenous CMV-specific T cell receptor (TCR). Proliferation assays with CTV dye dilution showed that CMV-HIV CAR T cells proliferated only when co-cultured with CMVpp65 peptide-pulsed autologous PBMCs (CMVpp65-PBMCs) as antigen-presenting cells (APCs) or with LCL-OKT3 cells binding all TCRs, but not when exposed to KG-1a cells or medium (Figure 10B). Accordingly, IFN-γ expression was higher in CMV-HIV CAR T cells after overnight stimulation with either LCL-OKT3, CMVpp 65-PBMCs or 8E5-gp120-expressing cells compared to stimulation with KG-1a cells or medium (Figure 10C). As expected, CMV-specific T cells expressed IFN-γ only after stimulation with LCL-OKT3 cells and CMVpp 65-PBMC, but not with 8E5-gp120 cells, KG-1a cells, or medium (Figure 10C). IFN-γ expression was relatively low in CMV-HIV CAR T cell products after overnight stimulation with 8E5-gp120, suggesting that CAR T cells were slowly killing their target cells. Similarly, the present inventors have pos We assessed whether donor-derived CMV-HIV CAR T cells maintained their effector function. The CMV-HIV CAR T cell product was primarily CD8 +and were responsive to CMVpp65 antigen stimulation as evidenced by high IFN-γ expression after overnight stimulation with CMVpp65-PBMCs (Figure 11A). Furthermore, we observed dose-dependent cytotoxicity against 8E5-gp120 cells after short-term (24 h, left panel) and long-term (96 h, right panel) co-culture (Figure 11B). We observed better cytotoxicity after 96 h of co-culture than after 24 h, supporting optimal killing kinetics at 96 h in the context of HIV CAR and gp120 targets. After 24 and 96 h of co-culture, the T cell product and CAR T cells had a similar low exhaustion phenotype (Figure 11C), similar to their end products, and our CMV-HIV CAR T cells maintained function and strength after target engagement (Figures 9C and 9D). CMV-HIV CAR T cells or CMV-CD19 CAR T cells from the same donor were co-cultured with HIV NL4-3 The cytotoxicity of the final cell product against HIV-infected cells was further evaluated by co-culture with eGFP+ Jurkat cells infected with HIV at various E:T ratios for 7 days (Figure 11D). Compared to CMV-CD19 CAR T cells, CMV-HIV CAR T cells were cytotoxic against HIV-infected cells. In the same experiment, HIV-1 p24 levels were measured by ELISA in cell supernatants, and p24 release was reduced in the presence of CMV-HIV CAR T cells compared to CMV-CD19 CAR T cells (Figure 11E). Finally, HIV pos Higher levels of p24 were detected in the supernatants of donor-derived CMV-HIV CAR T cells compared to CMV-HIV CAR T cells from the same donor (Figure 11F). The only source of HIV in these cultures was HIV that was viremic at the time of blood draw. pos Donor-derived, these results suggest that the therapeutic product can eliminate detectable HIV following endogenous reactivation.We next tested whether CMV-HIV CAR T cells could control HIV viremia in a humanized mouse model of HIV and expand in vivo in response to a CMVpp65 vaccine. EXAMPLES

[0047] CMV-HIV CAR T cells exhibit anti-HIV activity in a humanized mouse model of HIV HIV neg Donors were used to generate large numbers of CMV-HIV CAR T cells. The HIV-infected NSG humanized PBMC (NSGhu-PBMC) mouse model, outlined in Figure 12A, was established by transplantation of 3- to 5-week-old NSG mice (day 0) with autologous PBMC. On day 7, mice were challenged with HIV-1 BaL via intraperitoneal (IP) injection, and on day 12, they were started on a 3-week oral ART regimen (emtricitabine, tenofovir, raltegravir) that reduced plasma viral load to low to undetectable levels. During day 21 of ART, two cohorts were challenged with low-dose CMV-HIV CAR T cells (0.1 × 10 6 EGFR + Groups of mice were treated with or without CMVpp65 immunization on day 28 with a single injection of CMV-HIV CAR T cells (1 × 10 6 EGFR + T cells) and then treated with CMVpp65 vaccine on day 28. Two control cohorts received the same HIV neg Donor-derived CMV-negative T cells (1 × 10 6 The study included mice treated with EGFR 100-fold increased viremia compared to control T cells and low-dose CAR T cells. CMV-HIV CAR T cells were well tolerated by all mice, and no differences in body weight and temperature were observed between groups (Figure 13). On day 28, high-dose CMV-HIV CAR T cells significantly controlled HIV plasma viremia compared to control T cells and low-dose CAR T cell-treated mice when mice received ART prior to vaccination (Figure 12B). Thereafter, EGFR 100-fold increased viremia compared to control T cells and low-dose CAR T cells-treated mice. + CAR T cells were measured in peripheral blood (Figure 12C) and EGFR T cells were expressed in the EGFR T cells between days 33 (i.e., the last day of ART) and 42 (i.e., 9 days after ART cessation) in each mouse. +The slope of the linear regression line for CAR T cell counts / μL was calculated by converting it to a log10 scale (Figure 14). The average slope, which represents the rate of CAR T cell proliferation after ART interruption, was significantly higher in the two vaccinated CAR T cell-treated groups compared to the non-vaccinated low-dose CAR T cell-treated cohort. This suggests that the vaccine induced CAR T cell proliferation even after ART interruption. On day 42 after ART interruption and viral rebound, mice that received both high-dose CMV-HIV CAR T cells and CMVpp65 vaccine were the only cohort with controlled plasma viremia compared to vaccinated or non-vaccinated low-dose CAR T cell-treated mice (Figure 12D). This suggests the significance of CAR T cell dose, in that the proliferation of low-dose CAR T cells driven by the CMVpp65 vaccine was not sufficient to reach a therapeutic effect when viremia was high. CAR T cells were also detected in the bone marrow of mice at sacrifice (Figure 12E), notably by EGFR in bone marrow. + An inverse correlation (P value = 0.045) was observed in the frequency of active HIV-1 infected T cells (i.e., p24+ T cells) relative to the frequency of CAR T cells, suggesting a CAR-mediated reduction of HIV-infected cells (Figure 12F).

[0048] Finally, in separate experiments, we demonstrated that memory T cells from bone marrow are long-lasting and persist long after the resolution of circulating antigen-specific memory T cells, thus supporting the HIV posWe assessed whether donor-derived CMV-HIV CAR T cells could migrate to bone marrow (Okhrimenko, A., Grun, JR, Westendorf, K., Fang, Z., Reinke, S., von Roth, P., Wassilew, G., Kuhl, AA, Kudernatsch, R., Demski, S., Scheibenbogen, C., et al. (2014). Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory. Proceedings of the National Academy of Sciences of the United States of America 111, 9229-9234. 10.1073 / pnas.1318731111). EGFR+CAR T cells (50 × 10 3 ) was injected into hu-PBMC-NSG mice in the absence of ART 14 days after engraftment of HIV-challenged PBMCs (day 0). + CAR T cells were detected in peripheral blood and bone marrow. As expected, the CAR T cells were mostly CD8 + (Figure 15A). Importantly, they still expressed the memory cell markers CD62L and CD27 (Figures 15B and 15C). Thus, the results suggest that HIV pos We demonstrate that individual-derived CMV-HIV CAR T cells established persistent T cell memory in the bone marrow of HIV-infected mice. No significant differences in HIV viral load were observed in peripheral blood (data not shown), likely due to the limited number of infused CAR T cells and the high level of active HIV infection at the time of CAR T cell infusion. The results demonstrate the ability of CMV / HIV-CAR T cells to control viremia in lymphoid tissues, which may help eradicate persistent infection and latent reservoirs that cannot be achieved by ART or other current antiviral approaches. EXAMPLES

[0049] CMV-HIV CAR T cells exhibit anti-HIV activity in human HIV patients Figure 16 shows a schematic of the first in-human single-arm pilot study with autologous CMV / HIV-CAR T cells in PLWH stably virologically suppressed on long-term ART. The study is designed to initially study the safety of a single administration of CMV / HIV-CAR T cells at three dose levels. Each study participant will not be treated until the previously treated study participant has been closely monitored for a minimum of 60 days. No dose escalation, de-escalation, or expansion will occur until at least three evaluable participants have reached the current dose level. The first part of the protocol consists of screening and signing of informed consent at UCSD and ACTG clinics. In the second part of the study, eligible participants will temporarily interrupt their ART regimen for 4 days prior to leukapheresis to prevent inhibition of lentiviral transduction of T cells during CAR T cell manufacturing. Participants will resume their ART regimen immediately after leukapheresis. During this 4-day ATI period, subjects will resume their previous ART regimen if either: (1) requested by the participant or their HIV healthcare provider, or (2) ART is deemed medically necessary for non-HIV-related causes, or (3) symptomatic HIV disease (acute viral syndrome) occurs. If production is not successful, a second apheresis may be scheduled after 3 weeks, again with an interruption of ARV treatment for 4 days. Study participants are leukapheresed to collect peripheral blood mononuclear cells (PBMCs). The apheresis product is incubated overnight with CMV peptide and enriched for CMV-specific T cells for interferon gamma (IFNγ) positivity using the CliniMACS Prodigy® System (Miltenyi Biotec). The cells are then transduced with a self-inactivating lentiviral vector (vHIVR(N6)(EQ)BBζ-T2A-EGFRt_epHIV7; Figure 17 A-E) directing the co-expression of gp120BBζ-CAR (to target HIV gp120-expressing cells using scFv of anti-gp120 bNAb N6) and truncated human epidermal growth factor receptor (EGFRt, used as a tracking marker). The resulting autologous CMV / HIV-CAR T cell product, i.e., the investigational drug, is expanded in vitro in the presence of IL-2, IL-15, and ART cocktail inhibitors for approximately 2 weeks and cryopreserved (Figure 6A). Once the final cell product is released, participants will enter Phase 3. Participants assigned to dose level 1 will receive 25 × 10 6 Participants assigned to dose level 2 will receive a single intravenous (IV) infusion of 50 × 10 autologous CMV / HIV-CAR T cells. 6 Each patient will receive autologous CMV / HIV-CAR T cells. The DLT assessment period for the study will be defined as day -1 prior to CMV-HIV CAR T infusion to day 60 after CAR T cell infusion. Dose-limiting toxicities (DLTs) are listed below under expected AEs (adverse events) unless otherwise specified and are defined as events that are at least potentially related to the CMV-HIV CAR T infusion, except those occurring within the DLT evaluation period. DLTs include: Any grade 3 or higher organ toxicity designated as possibly, definitely, or probably (attributed level) related to the CAR T cell infusion (cardiac, dermatological, gastrointestinal, hepatic, pulmonary, genitourinary, neurological, hematological, renal, secondary malignancy, and endocrine); Any grade 3 or greater cytokine release syndrome in patients with CAR T cell infusion, likely, or definite; Any grade 3 or greater allergic reaction to CAR T cell infusion, likely, or definite; Any grade 3 or greater autoimmune toxicity that may, likely, or definite be treated with CAR T cell infusion; or Any grade 5 toxicity, probable, likely, or definite, with CAR T cells. Study participants may experience certain "expected" AEs related to the infusion of genetically modified T cells (usually occurring within the first 48 hours) and expansion in vivo, as well as CAR-directed therapy (usually occurring within the first 21 days after CAR T cell infusion). Below is a list of the highest tolerable* "expected" AEs (including grade and duration) as graded by CTCAE v5.0, excluding CRS / neurotoxicity grading by ASTCT consensus criteria. Dyspnea: Grade 3 dyspnea lasting up to 24 hours with intervention Fever: Grade 4 fever lasting up to 72 hours Cough: Grade 4 cough lasting up to 24 hours Headache: Grade 3 headache lasting up to 72 hours with intervention Hypotension: Hypotension: Grade 3 (no CRS symptoms) that responds to fluid resuscitation and resolves to Grade 2 or less within 24 hours Rash: Grade 3 rash lasting up to 72 hours with intervention Once the patient has demonstrated the safety of the process, a CMV vaccine (CMV antigen or nucleic acid encoding a CMV antigen; e.g., CMV pp65) is added to the protocol shown in Figure 16. The CMV vaccine is administered to the patient prior to CMV-HIV CAR T cell infusion (on day -1). This promotes in vivo expansion of CMV-HIV CAR T cells and increases the persistence of memory T cells expressing memory cell markers CD62L and CD27. Some patients also receive a CMV vaccine or a CMV vaccine booster after CAR T cell infusion (e.g., on days 1, 7, 10, 14, 21, 27, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, and / or 360). CMV / HIV-CAR T cells control viremia in lymphoid tissues and eradicate persistent infection and latent reservoirs. A single infusion of CAR T cells is designed to replace a lifelong regimen of ART (and other current antiviral approaches to treat HIV).

[0050] Materials and Methods The following materials and methods were used in the examples. DNA constructs The N6-CAR construct was modified from a previously reported CD19-specific scFvFcζ chimeric immune receptor (Kowolik, CM, Topp, MS, Gonzalez, S., Pfeiffer, T., Olivares, S., Gonzalez, N., Smith, DD, Forman, SJ, Jensen, MC, and Cooper, LJ (2006). CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res 66, 10995-11004).The HIV:41BB:ζ / EGFRt-epHIV7 lentiviral vector enhances CAR surface expression and contains the GM-CSF receptor-α chain signal sequence (GMCSFRss), which is a CAR sequence consisting of the VH and VL gene segments of the N6 bNAb, an IgG4 hinge with two site mutations (L235E; N297Q) in the CH2 region, the CD4 transmembrane and 4-1BB costimulatory domains, and the cytoplasmic domain of the CD3 ζ chain (Jonnalagadda, M., Mardiros, A., Urak, R., Wang, X., Hoffman, LJ, Bernanke, A., Chang, WC, Bretzlaff, W., Starr, R., Priceman, S., Ostberg, JR, et al. (2015). Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. Molecular therapy : the journal of the American Society of Gene Therapy 23, 757-768. 10.1038 / mt.2014.208), a ribosomal skipping T2A sequence, and a truncated human EGFR (EGFRt) sequence, allowing CAR T cell enrichment, tracking, and potential cell elimination via ADCC as previously described (Wang, X., Li, H., Matte-Martone, C., Cui, W., Li, N., Tan, HS, Roopenian, D., and Shlomchik, WD (2011). Mechanisms of antigen presentation to T cells in murine graft-versus-host disease: cross-presentation and the appearance of cross-presentation. Blood 118, 6426-6437. 10.1182 / blood-2011-06-358747).The complete CAR sequence is available upon request. A lentiviral vector encoding eGFP and ffLuc was generated by removing the stop codon in the eGFP open reading frame from pFUGW (Addgene plasmid #14883) and inserting a P2A-ffLuc-STOP cassette in frame with eGFP.

[0051] Clinical-scale production of CMV-HIV CAR T cells Fresh blood products were obtained from a CMVpos HIVneg donor (StemCell Technologies, Vancouver, Canada) and an HIVpos donor on ART (Zen-Bio Inc, Research Triangle Park, NC, see Table 1). All procedures were performed in accordance with the Helsinki protocol declaration (KC15TISI0494). CMV-specific T cells were isolated on a CliniMACS Prodigy and a cytokine capture system (CCS) (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, PBMCs were isolated and purified by density gradient centrifugation on Ficoll-Paque (Pharmacia Biotech, Sweden). After adding PBMCs (109) to an application bag connected to a tubing set, the CliniMACS Prodigy instrument automatically performed a continuous process including sample washing, antibody stimulation with PepTivator CMVpp65, Catchmatrix labeling, anti-IFN-γ microbeads labeling, magnetic enrichment and elution. CMV-specific and non-CMV-specific cells were eluted into separate bags after magnetic enrichment. After overnight incubation in RPMI medium containing 10% human AB serum (Gemini Bio Products, Sacramento, CA), IL-2 (50 U / mL) and IL-15 (1 ng / mL), harvested IFN-γ+ cells (approximately 1×106) were transduced at MOI 3 with research grade (for HIVneg donor and HIVpos donor #551 and #552) or GMP grade (for HIVpos donor #553, #572, #573, IEQR#2, IEQR#3) lentiviral vector HIV:41BB:ζ / EGFRt-epHIV7. Fresh medium and cytokines were added every other day for approximately 15 days. Antiretroviral drugs (43 nM darunavir and 279 nM enfuvirtide) were added twice a week during the expansion of HIVpos#573, IEQR#2 and IEQR#3 derived CMV-HIV CAR T cells. Cultures were maintained at 37°C under 5% (v / v) CO2.

[0052] cell line 8E5 cells are non-infectious because they contain a single defective proviral genome of HIV, but express most of the HIV viral proteins, including gp120. 8E5 (CRL-8993) cells were purchased from ATCC and maintained in RPMI 1640 (Irvine Scientific) medium supplemented with 10% heat-inactivated FCS (Hyclone). 8E5 cells were transduced with a lentiviral vector encoding eGFP-ffLuc, and GFP+gp120+ cells were selected and expanded to generate 8E5 cell lines expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (ffLuc). Acute myeloid leukemia (AML) cell line KG-1a (CCL-246.1) cells were purchased from ATCC, maintained in 10% FCS IMDM medium, and used as negative target cells. LCL-OKT3 cells were prepared as previously reported and served as positive T cell stimulators (Non-Patent Document 23). Cells were grown in complete medium supplemented with 0.4 mg / mL hygromycin. eGFP+ Jurkat cells were infected with HIVNL4-3 and cultured for 2 weeks. Hek293-eGFP-ffLuc-gp160 cells, used as a positive control cell line for tissue cross-reactivity studies, were obtained by stably transfecting the C97ZA012 gp160 construct into the HEK-293T cell line to express cell surface gp120 (ultimately cleaved into gp160 and gp41 HIV-1 envelope proteins). Banks of all cell lines were authenticated for desired antigen / marker expression by flow cytometry before cryopreservation, and thawed cells were cultured for less than 3 months before use in the assay.

[0053] Cytotoxicity assay CAR T cell product (2.5 × 10 5) and eGFP-positive target cells (8E5-gp120, 8E5, LCL-OKT3 or KG-1a) were co-cultured for 4 days at various effector-to-target (E:T) ratios of total T cells:targets (2:1, 1:1, 1:2 or 1:5). Co-cultures with LCL-OKT3 and 8E5 or KG-1a were used as positive and negative target controls. Cells were stained with anti-CD3 antibody. The percentage of viable eGFP+CD3- tumor cells was measured using multicolor flow cytometry. "Cytotoxicity%" was calculated as follows: 100%-(% of remaining tumor cells in CAR T cell group / % of remaining tumor cells in mock T or negative target group). eGFP+HIVNL4-3-infected Jurkat cells (2.5×10 5 ) were co-cultured with CMV-HIV CAR T cells at various E:T ratios (1:1, 1:2, 1:4) for 7 days. Cells were then stained with Viability Dye eFluor 450 (Miltenyi) and subjected to flow cytometric analysis of viable eGFP+ cells.

[0054] Proliferation assay CAR T cell product (2.5 × 10 5 ) were labeled with 0.5 μM CellTrace™ Violet dye (CTV) and co-cultured at a 1:1 ratio with 8,000 cGy irradiated stimulator cells LCL-OKT3, 8E5-gp120 and KG-1a, or 3,500 cGy irradiated autologous CMVpp65 peptide-pulsed PBMCs for 8 days. Co-cultures with LCL-OKT3, KG-1a cells and medium served as positive and negative controls. CD3+ and EGFR + Population expansion was determined using multicolor flow cytometry.

[0055] Intracellular IFN-γ staining CAR T cell products (10 5 ) was added to LCL-OKT3, 8E5-gp120, or KG-1a cells (10 5 ) or in the presence of brefeldin A (BD Biosciences, Franklin Lakes, NJ) with CMVpp65 peptide-pulsed autologous PBMC cells (105 ) overnight. The cell mixture was then stained with anti-CD8 antibody, anti-EGFR antibody cetuximab, and streptavidin to analyze the surface expression of each CD8 and CAR. The cells were then fixed and permeabilized using the BD Cytofix / Cytoperm kit (BD Biosciences). After fixation, T cells were stained with anti-IFN-γ antibody. The cells were then analyzed using multicolor flow cytometry on a MACSQuant (Miltenyi Biotec Inc.).

[0056] mouse Studies were performed with male and female NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice (JAX stock #005557) aged 3–5 weeks at the start of the study. Mice were group-housed in individually ventilated cages (OptiCages, Animal Care Systems) on corncob bedding ("Bed-o-Cob 1 / 8 in.", The Anderson, Maumee, OH) with square nests and PVC tubing provided for enrichment. Mice were fed rodent chow (LabDiet 5350) in bottles and autoclaved acidified reverse osmosis purified water (pH 2.4–2.8) ad libitum. Following HIV inoculation, mice were maintained under Animal Biosafety Level-2 (ABSL-2) conditions and group-housed in stationary disposable cages (Innovage, Innovive). Room temperature was maintained in the range of 68-79°F, and indoor humidity ranged from 30%-70%. Mice were designated specific pathogen free (SPF) for mouse rotavirus, Sendai virus, pneumonia virus of mice, mouse hepatitis virus, mouse minute virus, mouse parvovirus, Theiler's murine encephalomyelitis virus, mouse reovirus type 3, mouse norovirus, lymphocytic choriomeningitis virus, mouse thymus virus, mouse adenovirus types 1 and 2, mouse cytomegalovirus, polyomavirus, K virus, ectromelia virus, hantavirus, Prospect Hill virus, Filobacterium rodentium, Encephalitozoon cuniculi, and Mycoplasma pulmonis, Helicobacter species, and Clostridium piliforme, and were free of internal and external parasites. Mice were maintained in a facility accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC) in accordance with the Guide for the Care and Use of Laboratory Animals. All experiments were performed in accordance with the guidelines of the Institutional Animal Committee of the Beckman Research Institute of the City of Hope, IACUC 16095.

[0057] Engraftment and HIV challenge of hu-PBMC-NSG mice HIV PBMCs neg or HIV pos CMV-HIV CAR T cells were generated by collecting from donors. The CMV-negative fraction of autologous PBMCs was cryopreserved as control T cells. CMV-negative PBMCs (1 × 10 6 ) to each mouse before transplantation on day 0 with CMV-negative resting PBMCs (9 × 10 6 ) were mixed with cells (1 × 10 7 (1000 cells) were resuspended in sterile saline and injected intraperitoneally (IP) into NSG mice. Prior to treatment, mice were randomized to ensure similar engraftment and sex ratios across groups. On day 7, mice were challenged with HIV-1 BaL by IP injection. Longitudinal blood sampling was performed on anesthetized mice using retro-orbital bleeding, and peripheral blood cell populations and plasma viral load were periodically analyzed using flow cytometry and qRT-PCR. Mice that did not engraft huCD45+ cells (defined as >30 cells / μL huCD45+ cells in peripheral blood) were excluded from analysis. Mice showing severe signs of GHVD were immediately and humanely euthanized.

[0058] Oral ART therapy Detectable viral infection (HIV in blood >10 3Infected mice with 100% HIV infection (defined as 100 cp / mL) were orally treated for 3 weeks with ART consisting of drugs that block new infections without inhibiting virus production in infected cells. ART regimens consisting of Truvada® [tenofovir disoproxil fumarate (TDF; 300 mg / tablet), emtricitabine (FTC; 200 mg / tablet) (Gilead Sciences)] and Isentress® [raltegravir (RAL; 400 mg / tablet) (Merck)], scaled down to equivalent mouse doses using appropriate conversion factors, were administered in drinking water formulations (sweetened water gel, Medidrop® sucralose, ClearH20). For 400 mL of Medidrop®, ½ Truvada tablet and ½ Isentress tablet were crushed to powder and mixed by shaking the bottle to obtain a homogenous solution; medicated water was changed weekly. Doses of ART agents were calculated based on previous studies using the same delivery system (Satheesan, S., Li, H., Burnett, JC, Takahashi, M., Li, S., Wu, SX, Synold, TW, Rossi, JJ, and Zhou, J. (2018). HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy. J Virol 92. 10.1128 / JVI.02118-17).

[0059] CMV-HIV CAR T cells and in vivo CMVpp65 stimulation Mice were inoculated with CMV-HIV CAR T cells (0.05–1 × 10) by retro-orbital injection under general isoflurane anesthesia. 6 EGFR + T cells), CMV-negative T cells (1 × 10 6or autologous PBMCs as control T cells. Autologous PBMCs were pulsed with CMVpp65 peptide mix (#PM-PP65, JPT Peptide Technologies, Germany) as antigen-presenting cells (APCs) as the "CMVpp65 vaccine". Where indicated, mice received CMVpp65 peptide-pulsed and irradiated (3500 rad) autologous CMV-negative PBMCs (5 × 10 6 ) was received.

[0060] antibody Fluorescent dye-conjugated isotype controls for CD3 (#563109), CD4 (#557582), CD8 (#348793), IFN-γ (#554701), CD27 (#555440), CD45RA (#550855), CD62L (#341012), CD127 (#560822), programmed cell death-1 (PD-1) (#551892), lymphocyte activation gene-3 (LAG-3, #565720), and T cell immunoglobulin and mucin domain-3 (Tim-3, #563422) were obtained from BD Biosciences (San Diego, Calif.). The following agents were obtained: anti-HIV-1 gp120 monoclonal (VRC01) (catalog number 12033) was obtained from Dr. Campylobacter Mascola, NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. Biotinylated anti-EGFR antibody Erbitux® (cetuximab) was obtained from City of Hope pharmacy. Antibody against EGFR was obtained from eBioscience (San Diego, CA). CellTrace™ Violet dye (CTV) was purchased from Invitrogen (Carlsbad, CA). All monoclonal antibodies and CTV were used according to the manufacturer's instructions.

[0061] reagent CliniMACS Prodigy® TS500 tubing set, MACS GMP PepTivator® HCMV pp65, CCS reagents, CliniMACS PBS / EDTA buffer, and TexMACS™ GMP medium were all purchased from Miltenyi Biotec. CliniMACS PBS / EDTA with 2.5% human serum albumin (HSA; Grifols Therapeutics, Los Angeles, CA) was used as elution buffer. GMP grade cell transfer bags and luer / spike adapters were purchased from BD Medical (Franklin Lakes, NJ). pepMix HCMVA (pp65; pp65pepmix) was purchased from JPT Peptide Technologies. Millipore from Millipore GmbH was used for pulsing PBMCs according to the manufacturer's instructions. The antiretroviral drug darunavir was obtained from Tibotec, Inc. through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH (catalog number 11447), and enfuvirtide (Fuzeon, Genentech) was reconstituted with water.

[0062] Synthesis of N6 scFv-Fc Anti-gp120 N6 monoclonal antibody (mAb) variable domains were reformatted into recombinant single-chain scFv-Fc antibody fragments. cDNAs encoding N6 variable light and heavy domains (in VL-linker-VHorientation) were synthesized with a (Gly4Ser)3 linker (SEQ ID NO: 66) and fused to an IgG4 Fc domain. Briefly, N6 scFv-Fc was cloned into a Lonza Pee12.4 vector and transiently transfected using the EXPI293 expression system. The culture was then clarified by centrifugation (1,000×g, 5 min) followed by 0.22 μm sterile filtration. The clarified harvest was treated overnight with AG1-X8 strong anion exchange resin and affinity purified by Protein A chromatography (ProSep Va high capacity resin, EMD Millipore). The pooled eluate containing N6 scFv-Fc (VL-VH) was dialyzed using a Slide-A-Lyzer 20k MWCO cassette versus PBS buffer. The final dialyzed sample was sterile filtered using a 0.22 μm PES filter membrane and stored at 4° C. Test reagents were assayed for expression by SDS-PAGE and ELISA assay.

[0063] Tissue cross-reactivity analysis Charles River Laboratories, Inc. conducted a cross-reactivity study of N6 scFv-Fc. First, N6 scFv-Fc was tested for specific reactivity at 5 μg / Ml and 15 μg / Ml against a positive control (gp120-transformed HEK293T cells expressing gp 160) and a negative control parental HEK293T cells (gp120 negative). The test article was replaced with a human IgG4 κ antibody named HuIgG4 (control), and other controls were generated by omitting the test article or control from the assay (assay control). The tissue panel used as the test system for the in vitro cross-reactivity study was that recommended by the FDA, Center for Biologics Evaluation and Research (CBER) in the document Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use. Fresh, unfixed tissues were collected from humans as surgical or autopsy specimens and frozen at -85 to 70°C in Tissue-Tek® OCT. Sections were cut at approximately 5 μm and fixed in acetone for 10 min at room temperature. Immediately prior to staining, slides were fixed in 10% neutral buffered formalin (NBF) for 10 s at room temperature. Labeled secondary antibodies were specifically attached to unlabeled primary antibodies (either test or control substances at 5 μg / Ml and 15 μg / Ml) by overnight incubation of the primary / secondary antibody mixture. The test or control articles were mixed with biotinylated F(ab')2 donkey anti-human IgG, Fcγ fragment specific (DkαHuIgG) antibodies at concentrations that achieved a primary:secondary antibody ratio of 1:1.5. The pre-complexed antibodies were incubated overnight at 2-8°C. Prior to using the antibodies the next day, human gamma globulin was added to each vial to achieve a final concentration of either 4.5 mg / Ml (higher concentration of secondary antibody) or 1.5 mg / Ml (lower concentration of secondary antibody), and the antibodies were incubated at 2-8°C for at least 2 h. On the day of staining, slides were rinsed twice with Tris-buffered saline, 0.15 M NaCl, pH 7.6 (TBS).Next, the slides were incubated with avidin solution for 15 minutes, rinsed once with TBS, incubated with biotin solution for 15 minutes, and rinsed once with TBS. Slides were then treated with a protein block designed to reduce non-specific binding (TBS + 1% bovine serum albumin (BSA); 0.5% casein; and 1.5% normal donkey serum) for 20 minutes. After protein blocking, pre-complexed primary and secondary antibodies were applied to the slides for 2 hours. Slides were then rinsed twice with TBS and the slides were incubated with Dako peroxidase blocking reagent for 5 minutes to quench endogenous peroxidase. Slides were then rinsed twice with TBS, treated with ABC Elite reagent for 30 minutes, rinsed twice with TBS, and then treated with DAB for 4 minutes as a substrate for the peroxidase reaction. All slides were rinsed with tap water, counterstained, dehydrated, and mounted. TBS + 1% BSA served as the diluent for all antibodies and the ABC reagent. Separate frozen sections from each human test tissue were stained in parallel for expression of human β2-microglobulin (a relatively ubiquitous epitope) with a polyclonal rabbit antibody against human β2-microglobulin. All human test tissues evaluated stained positive for β2-microglobulin, indicating compatibility in the cross-reactivity assessment. After staining, slides were visualized and evaluated under a light microscope by a pathologist.

[0064] Flow cytometry Cells were stained with the optimized antibody panel for 20 min at 4°C and then washed twice with PBS. Data acquisition for all experiments, including flow cytometry, was performed on a MACSquant (Miltenyi Biotec) and analyzed using FCS Express V7 (De Novo Software, Glendale, CA). Peripheral blood samples were collected by retro-orbital bleeding under general anesthesia and stained with BV 711-conjugated anti-human CD3, APC-conjugated anti-human CD4, BB515-conjugated anti-human CD8, BUV 395-conjugated anti-human CD45 (BD Biosciences, San Jose, CA), and BV 421-conjugated anti-human EGFR (Biolegend, San Diego, CA) for 30 minutes. Stained peripheral blood samples were then lysed with red blood cell lysis buffer and absolute cell counts were calculated using BD Liquid Counting Beads (BD Biosciences, San Jose, CA). Flow cytometry was performed using a BD Fortessa II instrument (BD Biosciences) and analyzed with FlowJo software (formerly TreeStar from BD). Tissue samples were collected at necropsy and immediately processed for cell isolation and flow cytometry analysis. Bone marrow mononuclear cell suspensions were first stained with a dead cell exclusion amine-conjugated dye (Biolegend) followed by anti-human CD3 (BD ​​clone UCHT1), anti-human EGFR (Miltenyi biotinylated clone REA688), anti-human CD4 (Biolegend clone RPA-T4), anti-human CD8 (BD clone RPA-T8), anti-human CD62L (Biolegend clone DREG-56), and anti-human CD 27 (Biolegend clone M-T271) in Brilliant Stain Buffer (BD) containing 0.5% human serum albumin and 0.5% gamma globulin. Primary EGFR antibody staining was terminated with streptavidin conjugate (eBioscience) and fixed in 4% PFA. Samples were acquired the next day on a BD Fortessa SORP cytometer. Data were analyzed using FlowJo Software (BD, formerly TreeStar). Cell doublets and dead cells were excluded prior to assessment of T cell lineage and phenotypic markers.

[0065] Intracellular HIVp24 staining Peripheral blood samples and single cell suspensions of mouse bone marrow (femur + / - tibia) were collected at the time of euthanasia. Single cell suspensions were made according to previously established protocols (Non-Patent Document 1). Briefly, for bone marrow cells, femurs and tibias were dissected from euthanized mice and placed in ice-cold PBS. After thoroughly cleaning the bones to remove all connective and muscle tissue, the femoral heads were removed using a scalpel blade. The bones were placed into pre-punctured 0.5 mL microcentrifuge tubes using a 20 g needle. The bones were placed cut-side down and the 0.5 mL tubes were placed into 1.5 Ml microcentrifuge tubes and centrifuged at >10,000 x g for 15 seconds. The cell pellets were resuspended in ACK lysis buffer, incubated for 5 minutes, and washed with PBS. The cells were resuspended in PBS + 2% FBS and then processed for FACS staining or frozen in 10% Cryostor (Stem Cell Technologies, Vancouver, BC). For intracellular staining, BD Cytofix / Cytoperm™ kits (BD Biosciences, San Jose, CA) were used according to the manufacturer's protocol. After surface marker staining (CD45, CD3, CD4, CD8, EGFR), cells were permeabilized and intracellular staining was performed with KC57-FITC monoclonal antibody on a Fortessa II instrument (BD Biosciences) and analyzed with FlowJo software (BD formerly TreeStar).

[0066] ELISA assay HIV-1p24 was quantified in the supernatants according to the manufacturer's instructions (Alliance ELISA; Perkin-Elmer Life Sciences, Boston, MA), and the lower limit of quantification (LLOQ) of the assay was 12.5 pg / Ml. plasmaHIV Qrt-PCR Plasma viremia was assayed using reverse transcriptase real-time PCR [TaqMan assay] using the automated CFX96 Touch™ Real Time PCR Detection System (Bio-Rad). QPCR primer sets were obtained from a previously published study (Non-Patent Document 2). RNA was extracted using a QIAamp Viral RNA mini kit (Qiagen), and HIV-1 levels in peripheral blood were determined by TaqMan QPCR using primer and probe sets targeting the HIV-1 LTR region [F primer: GCCTCAATAAGCTTGCCTTGA (sequence number 67), R primer: GGCGCCACTGCTAGAGATTTT (sequence number 68), probe: 5'FAM / AAGTAGTGTGTGCCCGTCTGTTGTGTGACT / 3IABkFQ] (sequence number 69) or the HIV-1 Pol region [F primer: GACTGTAGTCCAGGAATATG (sequence number 70), R primer: TGTTTCCTGCCCTGTCTC (sequence number 71), probe: 5'Cy5 / CTTGGTAGCAGTTCATGTAGCCAG / 3'IABkFQ] (sequence number 72) with TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). Design a protocol for purification of viral RNA from a minimum of 140 μL plasma according to the manufacturer's instructions (QIAamp Viral RNA mini kit [Qiagen]). In standard Taqman qPCR-based HIV-1 plasma viral load tests, the limit of detection (LOD) is usually about 40 copies / mL when viral RNA isolated from 140 μL plasma samples is applied. In our animal studies, only limited volumes of plasma (20-40 μL) were available, so plasma samples were diluted (generally 1- to 3-fold diluted) for amplification. The LOD of the diluted samples was about 2,000 RNA copies / mL using HIV LTR primers and about 500 RNA copies / mL using HIV Pol primers under our experimental conditions. Therefore, we defined values ​​lower than the LOD number as undetectable. statistical analysis Analyses were performed using Prism (GraphPad Software Inc.) or R version 4.0.266 and are described in the individual figure legends. A significance level of 0.05 was used for all analyses. [References]

[0067] [Table 6] TIFF2025512407000007.tif238170TIFF2025512407000008.tif228170TIFF2025512407000009.tif233170TIFF2025512407000010.tif52170Other embodiments Although the invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for preparing a population of CMV-specific T cells expressing an HIV chimeric antigen receptor (HIV CAR), the following: CMV pos involves isolating cell populations containing PBMCs from blood samples obtained from test subjects. The aforementioned cell population is brought into contact with the CMV antigen to stimulate CMV-specific T cells. To isolate a subpopulation of IFNγ-secreting T cells from the aforementioned cell population, and, Transduction of cells in a subpopulation of IFNγ-secreting T cells with a vector containing a nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor is (i) scFv attached to HIV Env; (ii) Spacer domain; (iii) Transmembrane domain; (iv) Co-stimulatory domain; and (v) CD3ζ signaling domain This includes, and furthermore, a subpopulation of IFNγ-secreting T cells is cultured in the presence of at least one antiretroviral drug before, after, or before and after transduction. Methods that include...

2. The method according to claim 1, wherein a subpopulation of IFNγ-secreting T cells is cultured in the presence of one or both of exogenous IL-2 and exogenous IL-15 before transduction, after transduction, or before and after transduction.

3. The method according to claim 2, wherein IL-2 is added up to 50 U / mL and IL-15 is added up to 1 ng / mL.

4. The method according to claim 2, wherein a subpopulation of IFNγ-secreting T cells is cultured in the presence of at least one or both of an HIV protease inhibitor and an HIV entry / fusion inhibitor, and in the absence of a reverse transcriptase inhibitor, before transduction, after transduction, or both before and after transduction.

5. The method according to claim 4, wherein a subpopulation of IFNγ-secreting T cells is cultured in the presence of darunavir and enfuvirtide.

6. A method for treating a subject infected with HIV, (a) A population of T cells expressing a T cell receptor that binds to the CMV antigen and a chimeric antigen receptor, wherein the chimeric antigen receptor includes: scFv that binds to HIV Env; a spacer domain; a transmembrane domain; a costimulatory domain; and a CD3ζ signaling domain, In some cases, (b) at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen. A method including administering [a substance].

7. The method according to claim 6, wherein at least one CMV antigen comprises at least one of the following: CMV protein, a fragment of CMV protein, a modified CMV protein, a fragment of a modified CMV protein, a mutant CMV protein or a fragment thereof, a fusion CMV protein or a fragment thereof, and a combination thereof.

8. The method according to claim 6, wherein at least one CMV antigen comprises at least one of pp65, IE1 exon 4 (IE1 / e4), and IE2 exon 5 (IE2 / e5).

9. The method according to claim 6, wherein the nucleic acid molecule comprises (a) CMV pp65 peptide or protein and (b) a viral vector encoding a fusion protein comprising exon 4 (e4) of CMV protein 1E1 and exon 5 (e5) of CMV protein 1E2.

10. The method according to claim 6, wherein at least one CMV antigen comprises at least one sequence selected from SEQ ID NOs. 57 to 64.

11. The method according to claim 6, wherein at least one CMV antigen comprises at least one fragment of any of SEQ ID NOs: 57 to 64, and the fragment comprises or consists of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 consecutive amino acids of any of SEQ ID NOs: 57 to 64.

12. The method according to claim 6, wherein at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered before or after administration of a population of CMV-specific T cells.

13. The method according to claim 12, wherein at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 28, 36, 48, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, and / or 360 hours before administering a population of CMV-specific T cells.

14. The method according to claim 12, wherein at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 28, 36, 48, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, and / or 360 days after administration of a population of CMV-specific T cells.

15. The method according to claim 6, wherein at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered to a subject in an amount sufficient to induce an immune response.

16. The method according to claim 6, wherein the chimeric antigen receptor comprises: an scFv comprising a variant thereof having one, two, three, four, or five amino acids substituted, but the substitutions not being present in the CDR; a spacer comprising a sequence selected from the group consisting of SEQ ID NOs: 24 to 34; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15 to 23; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 36 to 40; and a CD3ζ signaling domain comprising SEQ ID NO:

35.

17. The method according to claim 6, wherein the chimeric antigen receptor includes variants thereof in which one, two, three, four or five amino acids are substituted, but the substitutions are not present in the CDR.

18. The method according to claim 6, wherein at least 40%, 50%, 60%, or 70% of a population of CMV-specific T cells are IFN-γ positive.

19. The method according to claim 6, wherein at least 20%, 30%, and 35% of a population of CMV-specific T cells are CD8-positive.

20. The method according to claim 6, wherein at least 20%, 25%, 30%, and 35% of a population of CMV-specific T cells are CD4-positive.

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