CMV-HIV specific chimeric antigen receptor T cell

Abstract

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

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 330,726, filed on April 13, 2022. Technical Field

[0003] The present disclosure relates to T cells expressing a chimeric antigen receptor (CAR) targeting HIV and a T cell receptor targeting cytomegalovirus (CMV). Background Art

[0004] Combination antiretroviral therapy (ART) has been successful with undetectable plasma viremia 1 However, HIV infection cannot be cured due to the persistence of a latent viral reservoir containing replication-competent HIV-1. 2 Alternative cellular immune strategies for adoptive immunotherapy have been proposed, which use expansion of endogenous HIV-specific T cells or autologous T cells or natural killer (NK) cells redirected to HIV-infected cells. 3-5 The first generation of HIV-CAR T cells was developed nearly 25 years ago by engineering the extracellular domain of the CD4 receptor on the surface of T cells. 6,7 These CD4-based CAR T cells are being tested in three clinical trials in HIV-seropositive individuals. 4,8-10 , but this strategy was discontinued due to insignificant clinical effects. Multiple factors may explain the lack of or minimal antiviral efficacy, such as limited CAR activity in the absence of intracellular co-stimulatory signaling domains, low expansion and minimal persistence of CAR T cells due to insufficient exposure of CAR-T cells to HIV antigens (as CAR recipients remain on ART), or susceptibility of CD4-CAR-expressing T cells to HIV infection. Notably, low levels of these first-generation CD4-CAR T cells were detected in these recipients a decade later, suggesting that long-term persistence is possible. 11 Since then, CARs have been optimized by adding the co-stimulatory domains CD28 or 4-1BB and have shown promising results with improved efficacy and durability in the setting of B-cell malignancies. 12-15 Additionally, a series of broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein gp120 were identified in HIV-infected nonprogressors, and these antibodies were used to develop bNAb-derived CAR T cells that potently kill gp120-expressing cells in vitro 16,17 To limit the emergence of resistance to HIV, these bNAb-based CAR T cells should be effective against nearly all HIV strains. Notably, Huang et al.18 A bNA named N6 was isolated that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that had evolved to circumvent common resistance mechanisms.

[0005] It may be necessary to have high numbers of CAR T cells to achieve therapeutic benefit, as currently tested in CD4-based CAR T cell clinical trials [NCT03617198]. Another approach to enhance CAR T cell persistence involves engagement of viral antigens as stimulatory factors for CAR T cells. 19-23 . SUMMARY OF THE INVENTION

[0007] The present disclosure is based at least in part on the following discovery: In some cases, T cells expressing HIV-specific chimeric antigen receptors (CAR) and cytomegalovirus (CMV)-specific T cell receptors ("CMV-HIVT cells") can be used to treat subjects infected with HIV together with CMV vaccines (e.g., one or more CMV antigens or nucleic acids encoding one or more CMV antigens). Some of the main problems that HIV patients still face today are CAR T cell activation and persistence in vivo. HIV patients taking antiretroviral drugs face these problems, and so far, there is no data showing an effective method for amplifying and maintaining CAR T cells in such patients because these cells disappear functionally. In addition, for many reasons, there are additional difficulties in separating and amplifying CMV-specific T cell populations. For example, the specific T cell population is usually very low in the subject, so it may be very difficult to prepare enough CMV CAR T cells to be administered to the subject. In the present application, the compositions and methods described herein result in increased amplification and persistence of CAR T cells (including CMV-HIV T cells).

[0008] Described herein are, inter alia, nucleic acid molecules encoding chimeric antigen receptors, wherein the chimeric antigen receptor comprises: a scFv that binds HIV Env; a spacer domain; a transmembrane domain; a co-stimulatory domain; and a CD3 zeta signaling domain.

[0009] In various embodiments: the scFv comprises: a VL domain and a VH domain, wherein the VL domain comprises: a light chain CDR1 comprising QTSQGVGSDLH (SEQ ID NO: 1), a light chain CDR2 comprising HTSSVED (SEQ ID NO: 2), and a light chain CDR3 comprising QVLQF (SEQ ID NO: 3); the VH domain comprises: 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, which is conjugated to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7); ID NO:7) is at least 90%, 95% or 98% identical; and (b) a heavy chain variable domain that is at least 90%, 95% or 98% identical to RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO:8); the scFV comprises: a light chain variable domain that comprises YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:7); and a heavy chain variable domain comprising RALVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO:9);The scFv comprises a VL domain and a VH domain, wherein the VL domain is 95% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:7), and includes 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:4). NO:3); the VH domain is 95% identical to RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO:8), and includes the following CDR sequences: AHILF (VH-CDR1; SEQ ID NO:4), WIKPQYGAVNFGGGFRD (VH-CDR2; SEQ ID NO:5), and RSYGDSSWALDA (VH-CDR3; SEQ ID NO:6). ;

[0010] Nucleic acid molecules encoding chimeric antigen receptors are described herein, wherein the chimeric antigen receptor comprises:

[0011] RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSAGGGSGGGSGGGSGGGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:9) or consisting thereof; a spacer comprising a sequence selected from the group consisting of SEQ ID NOs:24-34; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15-23; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs:36-40, and a CD3 zeta signaling domain comprising SEQ ID NO:35.

[0012] In the case of any nucleic acid molecule: the spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-34 or variants thereof having 1, 2, 3, 4 or 5 amino acid substitutions; the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain and a CD3ζ transmembrane domain; the co-stimulatory domain is selected from the group consisting of a 28 co-stimulatory domain, a 41-BB co-stimulatory domain, an OX40 co-stimulatory domain and a 2B4 co-stimulatory domain; the chimeric antigen receptor comprises an amino acid sequence (SEQ ID NO: 10) having 0, 1, 2, 3, 4 of the 5 amino acid substitutions; in some embodiments, the amino acid substitutions are conservative; in some embodiments, the amino acid substitutions are not in the CDRs;In some embodiments, the nucleic acid molecule further comprises an interdomain linker consisting of 1-5 amino acids between one or more of the following: scFV and spacer domain, spacer domain and transmembrane domain, transmembrane domain and co-stimulatory domain, and / or co-stimulatory domain and CD3 zeta signaling domain; the interdomain linker may consist of 1-5 glycines; in some embodiments, the CAR further comprises an interdomain linker consisting of the sequence GGG, which is located between the co-stimulatory domain and the CD3 zeta signaling domain. ;

[0013] In some embodiments, the CAR comprises: a scFv comprising or consisting of SEQ ID NO: 9 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions (the amino acid substitutions are not in the CDR); a spacer comprising a sequence selected from the group consisting of SEQ ID NO: 24-34 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NO: 15-23 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions; a co-stimulatory domain comprising a sequence selected from the group consisting of SEQ ID NO: 36-40 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions; and a CD3 zeta signaling domain comprising SEQ ID NO: 35 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative.

[0014] Also described herein are immune cells containing any nucleic acid molecules described herein. Also described herein are immune cells expressing any CAR described herein.

[0015] In various embodiments, the immune cell is a T cell that expresses a T cell receptor specific for CMV (CMV-specific T cell).

[0016] Also described are cell populations comprising CMV-specific T cells containing any nucleic acid molecule described herein. Also described are CMV-specific T cell populations expressing any HIV CAR described herein.

[0017] In various embodiments: at least 20%, 30%, 40% or 50% of the CMV-specific T cells are CD8+ T cells.

[0018] Also described is a method for preparing a population of CMV-specific T cells expressing HIV CAR, the method comprising: obtaining a population of CMV-specific T cells from posA cell population comprising PBMCs is isolated from a blood sample of a subject; the cell population is contacted with a CMV antigen to stimulate CMV-specific T cells; a CMV-specific T cell subpopulation is isolated (e.g., by selecting T cells that secrete IFNγ from the cell population); and cells in the T cell subpopulation that secretes IFNγ are transduced with a vector comprising any of the nucleic acid molecules described herein.

[0019] In some embodiments, described herein is a method for preparing a T cell that is specific to cytomegalovirus (CMV) and expresses a chimeric antigen receptor (CAR). In some embodiments, the method includes: (a) providing a cell group (e.g., PBMC) comprising T cells from a human donor with positive cytomegalovirus CMV serotype; (b) exposing a cell group (e.g., PBMC) to at least one CMV antigen; (c) treating exposed cells to produce a cell group enriched for CMV-specific stimulated cells; (d) transducing at least a portion of the enriched cell group with a vector expressing CAR, thereby preparing T cells that are specific to CMV and express CAR. In various cases: the step of treating the exposed cells (e.g., using a selection step) to produce a cell population enriched for CMV-specific stimulated cells comprises: treating the stimulated cells to produce a cell population enriched for cells expressing an activation marker (e.g., IFN-γ or IL-13); culturing the PBMCs for less than 5 days (less than 4, 3, 2, 1 days) prior to exposure to the CMV antigen; exposing the cells to the CMV antigen for less than 3 days (less than 48 hrs, 36 hrs, 24 hrs), the CMV antigen is a pp65 protein or an antigenic portion thereof, the CMV antigen comprises two or more different antigenic CMV pp65 peptide; the step of transducing the enriched cell population does not include CD3 stimulation; the step of transducing the enriched cell population does not include CD28 stimulation; the step of transducing the enriched cell population does not include CD3 stimulation or CD28 stimulation; the enriched cell population 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; before the step of transducing the enriched cell population with a vector encoding CAR, the enriched cell population is cultured for less than 10 (less than 9, 8, 7, 5, 3, 2) days. In some cases, T cells are from CMV-positive donors and are exposed to a mixture of CMV antigens such as CMV pp65 or CMV protein peptides (e.g., a 10-20 amino acid peptide that is a fragment of pp65) in the presence of IL-2 to produce a stimulated cell population. In some cases, the stimulated cell population is treated to produce a cell population expressing IFN-γ.

[0020] In various embodiments: CMV 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. T cell subsets (e.g., IFNγ-secreting T cells expressing a T cell receptor specific for CMV); IL-2 is added at 50 U / mL and IL-15 is added at 1 ng / mL; the IFNγ-secreting T cell subsets 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 following group: 1) HIV protease inhibitors (e.g., tipranavir, atazanavir, indinavir, darunavir or fosamprenavir); and 2) HIV fusion inhibitors, HIV entry inhibitors, HIV attachment inhibitors, HIV post-attachment inhibitors (e.g., maraviroc, ibalizuma-buiyk, fostemsavir), and the cells are not cultured in the presence of reverse transcriptase inhibitors; the blood sample is from a subject infected with HIV (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 lentiviral replication or the replication of the viral vector carrying the sequence encoding the HIV-targeting chimeric antigen receptor.

[0021] Also described is a method for treating a subject infected with HIV, the method comprising administering: (a) CMV-specific T cells expressing a chimeric antigen receptor, the chimeric antigen receptor comprising: a scFv binding to HIV Env; a spacer domain; a transmembrane domain; a co-stimulatory 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 some embodiments, while administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, before administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, after administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, before or after administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered in a single or repeated administration. In some embodiments, CMV-HIV CAR T cells are administered in a single or repeated administration.

[0022] Useful doses of CMV HIV T cells include approximately 5x10 6 , 10x10 6 , 15x10 6 , 20x10 6 , 25x10 6 , 30x10 6 , 35x10 6 , 40x10 6 , 45x10 6 , 50x10 6 ,55x10 6 , 60x10 6 , 65x10 6 ,70x10 6 , 75x10 6 ,80x10 6 ,85x10 6 ,90x10 6 ,95x10 6 and 100×10 6 Cells. In some embodiments, a single dose of CMV-HIV CART 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, infusion, or ingestion. Injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion. In some embodiments, a CMVHIV T cell population is administered to a subject with a single intravenous (IV) infusion.

[0023] In some embodiments, before administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered to the subject. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten days before administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, 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 CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered about one, two, three, or four weeks prior to administration of the CMV-HIV CAR T cells.

[0024] In some embodiments, after administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered to the subject. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten days after administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, 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 administering CMV-HIV CAR T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered. In some embodiments, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered about one, two, three, or four weeks after the administration of CMV-HIV CAR T cells. In some embodiments, this can be in addition to the administration of at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen before the administration of CMV-HIV CART cells, so in some embodiments, the subject will be administered at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen before the administration of CMV-HIV CAR T cells, and at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen will be administered after the administration of CMV-HIV CAR T cells.

[0025] Useful CMV vaccines include one or more CMV antigens or one or more nucleic acids encoding one or more CMV antigens.CMV antigens can be any CMV protein, a fragment of a CMV protein, a modified CMV protein, a fragment of a modified CMV protein, a mutated CMV protein or its fragment or a fusion CMV protein or its fragment.In some embodiments, useful CVM vaccines include 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), its fusions and its antigenic fragments and its variants with 1, 2, 3, 4 or 5 amino acid modifications.In some embodiments, 1, 2, 3, 4 or 5 amino acid modifications include 1-2 amino acid replacements or 1-5 amino acid replacements.In some embodiments, amino acid replacements are conservative.In some embodiments, CMV antigens include sequences selected from SEQ ID NO:57-64 and its variants with 1, 2, 3, 4 or 5 amino acid modifications. In some embodiments, 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 one 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 one of SEQ ID NOs: 57-64.

[0026] In various embodiments, the CMV antigen comprises a CMV protein or a fragment thereof (e.g., a CMV pp65 peptide); the nucleic acid molecule comprises a viral vector encoding (a) a CMV pp65 peptide or protein and (b) a fusion protein containing 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 (BACs), vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors, and other vectors 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.

[0027] In some embodiments, before or after administration of CMV-specific T cells, at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered in a single or repeated dose. 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 a 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 cause an immune response in a subject.

[0028] In some embodiments, the subject is also being treated with antiretroviral therapy (ART). In some embodiments, the ART regimen is reduced or stopped after the administration of CMV CAR T cells. In some embodiments, the ART regimen is temporarily stopped for 4 days before leukocyte separation to collect peripheral blood mononuclear cells (PBMC). In some embodiments, the subject resumes their ART regimen immediately after leukocyte separation.

[0029] After leukapheresis, the separation product can be incubated overnight with one or more CMV antigens and / or CMV peptides. In some embodiments, CMV-specific T cells are based on interferon gamma (IFNγ) positive enrichment. In some embodiments, the cells are then transduced with a self-inactivating lentiviral vector (e.g., vHIVR (N6) (EQ) BBζ-T2A-EGFRt_epHIV7 encoding CAR described herein; SEQ ID NO: 44). In some embodiments, in the presence of IL-2, IL-15 and ART mixture inhibitors, CMV / HIV-CAR T cell groups are expanded in vitro for about 2 weeks. In some embodiments, the CMV / HIV-CAR T cell groups after amplification are cryopreserved.

[0030] Amino acid modification refers to amino acid replacement, insertion and / or deletion in a protein or peptide sequence. "Amino acid replacement" or "replacement" refers to replacing an amino acid at a specific position in a parent peptide or protein sequence with another amino acid. The replacement can be carried out in a non-conservative manner (i.e., by changing a codon from an amino acid belonging to an amino acid grouping with a specific size or feature to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing a codon from an amino acid belonging to an amino acid grouping with a specific size or feature to an amino acid belonging to the same grouping) to change the amino acid in the resulting protein. This conservative change usually results in less changes in the structure and function of the resulting protein. The following are examples of various groupings 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 (at pH 6.0). Another grouping would be those amino acids with a phenyl group: phenylalanine, tryptophan, and tyrosine.

[0031] In order to more fully understand the invention, examples are listed. The materials and methods used in the following examples are described in detail after the examples. The examples described in this application are provided to illustrate the methods and compositions provided herein, and should not be interpreted in any way as limiting the scope thereof.

[0032] 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.

[0033] Other features and advantages of the described compositions and methods will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1AD. Functional characterization of N6-CAR T cells. (A) Schematic diagram of the N6-CAR construct. The construct contains the GM-CSF receptor-α chain signal sequence (GMCSFRss) under the control of the EF1α promoter, a single-chain variable fragment (scFv) of the anti-gp120bNAb N6 connected to the CD4 transmembrane (tm) and 4-1BB co-stimulatory domains via an IgG4 (EQ) spacer, followed by a truncated human EGFR (EGFRt) linked to CD3ζ and T2A. (B) Primary T cells were activated with CD3 / CD28 microbeads and transduced with a lentiviral vector encoding N6-CAR. After ~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. Four HIV neg Representative FACS plots of donor-derived CAR T cell products. (C) neg Donor's N6-CAR T cells or mock T cells were co-cultured with 8E5-gpl20 cells expressing eGFP at different total T cell: target cell (E:T) ratios for 96 hours, followed by immunostaining for CD3 and eGFP. Cytotoxicity was calculated as follows: 100% - (% of tumor cells remaining in the CAR T cell group / % of tumor cells remaining in the mock T cells or negative target cells). n = 3 donors. (D) CellTrace TM N6-CAR T cell products were labeled with violet dye (CTV) and stimulated with 8,000 rad irradiated 8E5-gp120 at a 1:1 E:T ratio for 8 days before CTV analysis (blue line). Lymphoblastoid cells expressing the CD3 agonist OKT3 (LCL-OKT3) irradiated with 8,000 rad were used as a positive control (red line), and culture medium was used as a negative control (black line). + Gated cells (lower panel) and EGFR - CTV dilutions of gated cells (top). Five HIV strains are presented neg Representative data of donor-derived CAR T-cell products.

[0035] Figure 2 AB: Development of the 8E5-gp120 cell line. Parental 8e5 cells are derived from HIV-infected lymphoblastoid cells and carry a single reverse transcriptase (RT)-deficient copy of the integrated HIV genome. (A) Flow cytometric analysis using anti-gp120 monoclonal antibody staining shows that ~30% of the parental cells express gp120. (B) Cells were transduced with a lentiviral vector encoding eGFP and firefly luciferase (ffLuc), and then eGFP and gpl20 double-positive cells were sorted and expanded for in vitro experiments.

[0036] Figure 3 : Specific cytotoxicity of N6-CAR T cells against gp120-positive cells.

[0037] N6-CAR T cells derived from HIV-neg donors were co-cultured with eGPF+8E5 cells sorted for gp120 expression at different E:T ratios (1:1, 1:2, or 1:5). Residual eGFP+ tumor cells were measured by flow cytometry after 96 hours.

[0038] Figure 4 AB: Phenotypic characterization of N6-CAR T cells after stimulation with 8E5-gp120 cells. N6-CAR T cells derived from three HIVneg donors were co-cultured with 8E5-gp120 cells, LCL-OKT3 cells, or medium at a 1:1 E:T ratio for 96 hours and then analyzed by flow cytometry for the expression of (A) memory markers (CD62L, CD127, and CD27), or (B) exhaustion (LAG-3, PD-1, and Tim-3) markers.

[0039] Figure 5 : Specific binding of N6-scFv-Fc on cells expressing gp120.

[0040] 8E5-gp120 cells were stained with soluble N6 scFv-Fc at the indicated dilutions. Positive cells were quantified by flow cytometry. Staining with anti-gp120 bNAb VRC01 was used as a positive control, and gp120-negative KG-1a cells or staining with secondary antibody alone was used as a negative control.

[0041] Figure 6 AF: Derived from HIV neg and HIV pos Clinical scale production of CMV-HIV CAR T cells from donors. (A) Production workflow for generating CMV-HIV CAR T cells as described in the methods. (B) Production of CMV-HIV CAR T cells using CliniMACS IFN-γ + Cells before and after enrichment from HIV pos Representative FACS plots of CMV-specific T cells isolated from donors. (C) Flow cytometric analysis of the percentage of enriched IFN-γ+CMV-specific T cells and their relative CD4 and CD8 expression. n = 6-7 donors per group. (D) HIV derived from amplification over approximately 15 days neg (n=6) and HIV pos(n=7) Growth curves of total cell numbers in the final product for each donor. Amplification curves in the presence of antiretroviral drugs (ARV, darunavir, and enfuvirtide) are indicated by red dashed lines. (C) Flow cytometric analysis of the percentage of CMV-HIV CAR T cells in the final cell product and their relative CD4 and CD8 expression. n=6-7 donors per group. (F) Total number of CMV-HIV CAR T cells in each final cell product.

[0042] Figure 7 : Memory cell subsets in CMV-specific T cells isolated from HIVneg and HIVpos donors. Using CliniMACS CMV-specific T cells (IFN-γ+CD3+) isolated from HIVneg and HIVpos donors were systematically enriched and immunostained with anti-CD27 and anti-CD45RA antibodies. Flow cytometry analysis showed the percentages of the following CMV-specific T cells: 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 represent mean ± SD; n = 5 donors per group.

[0043] Figure 8 AC: In vitro HIV replication, lentiviral transduction, and cell expansion in the presence of antiretroviral drugs (ARVs). (A) HIV replication was prevented when the HIV protease inhibitor darunavir (D, EC50 = 4.3 nM) and the HIV fusion inhibitor enfuvirtide (E, EC50 = 27.9 nM) were added to CD8+ depleted PBMCs infected with HIV-1BaL (left panel) or HIV-1NL4-3 (right panel) strains. (B) Darunavir (43 nM) and enfuvirtide (279 nM) were added on the day (day 0) or 2 days (day 2) after PBMCs were transduced with a lentiviral vector expressing eGFP. Flow cytometric analysis of eGFP expression 8 and 14 days after lentiviral transduction is shown. (C) Cell expansion of untransduced or transduced PBMCs in the presence or absence of antiretroviral drugs.

[0044] Fig. 9 AD: Derived from HIV neg and HIV pos Phenotypic characterization of donor CMV-HIV CAR T cell products. T cell memory markers: CD27 were analyzed by flow cytometry after INF-r enrichment (A) and in the final CAR T cell product (B). + 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). 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 represent mean ± SD; n = 2-6 donors per group.

[0045] Fig.10 : Derived from HIV neg Effector function of donor CMV-HIV CAR T cells. (A) CMV-HIV CAR T cell products were co-cultured with eGFP at different E:T ratios (2:1, 1:1, 1:2, or 1:5). + 8E5-gp120 or eGFP + KG-1a cells were co-cultured for 96 hours and then immunostained for CD3 and eGFP to determine specific cytotoxicity against target cells expressing gp120. + The percentage of tumor cells was measured by flow cytometry and cytotoxicity was calculated. (B) CMV-HIV CAR T cell products were labeled with CTV and cultured for 8 days with CMVpp65 peptide pulsed and 3,500rad irradiated PBMC (CMVpp65-PBMC), 8,000rad irradiated LCL-OKT3 or KG-1a cells or culture medium. CMV-HIVCAR T cell proliferation was determined by CTV dilution. Representative data of four donors are shown. (C) Autologous PBMC (CMVpp65-PBMC), LCL-OKT3, 8E5-gpl20, KG-1a cells or culture medium pulsed with CMVpp65 peptide derived from the same donor were stimulated overnight. Co-cultures were stained with surface CD8 and then intracellular IFN-γ expression was performed. Representative data of three different donors are shown.

[0046] Fig.11 : Derived from HIV pos Effector function of donor CMV-HIV CAR T cells. (A) From HIV posRepresentative FACS images of CMV-specific T cells enriched by donors and transduced with lentiviral vectors expressing N6-CAR (n=7). Transduction efficiency was assessed by measuring EGFR expression in T cells on day 7. The CMV-HIV CAR T cell product was then stimulated overnight with autologous PBMCs pulsed with CMVpp65 peptide, and the expression of IFN-γ, CD3, CD4, and CD8 was analyzed by flow cytometry. Representative data from three different donors are shown (n=3). (BC) CMV-HIV CAR T cells (n=3) were transduced with eGFP+8E5-gp120 or eGFP + KG-1a cells were co-cultured at different E:T ratios (2:1, 1:1, or 1:5) for 24 h (n=2) and 96 h (n=3), and then immunostained for CD3, EGFR, and LAG-3, PD-1, and Tim-3 depletion markers to determine specific cytotoxicity. The percentage of remaining eGFP+ tumor cells was measured by flow cytometry, and cytotoxicity was calculated as described in the methods. The figure presents 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-CD19CAR T cells from the same HIV pos prepared from donors and with HIV NL4-3 Infection with eGFP + Jurkat cells were cultured at different E:T ratios (1:1, 1:2, and 1:4) for 7 days. NL4-3 Infection with eGFP + Cytotoxicity of Jurkat cells was normalized to untreated control wells. HIV p24 levels in cell supernatants at 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 isolated from HIV on ART. pos Donor preparation. After 20 days of expansion, p24 levels in culture supernatants were measured by ELISA and normalized to p24 levels in supernatants of CMV-CD19 CAR T cells. (D), (E), and (F) show p24 levels in supernatants from an HIV- pos Donor data.

[0047] Fig.12: CMVpp65-driven expansion of CMV-HIV CAR T cells and dose-dependent control of HIV viremia in hu-PBMC-NSG mice receiving ART. (A) NSG humanized peripheral blood mononuclear cell (hu-PBMC) HIV mouse model and experimental design receiving ART. On day 28, CMV-HIV CAR T cells (0.1×10 6 EGFR+ T cells) (with or without CMVpp65 vaccine), or with high-dose CMV-HIV CAR T cells (1×10 6 EGFR+T cells) (using CMVpp65 vaccine) to treat HIV-infected mice receiving oral ART. neg Donor's CMV-negative T cells (1×10 6 Mice treated with (100 cells / mL) (with or without CMVpp65 vaccine) were used as controls. (B) HIV viral load in peripheral blood on day 28. Baseline HIV viral load (day 21), log 10 CD3, log 10 An additive model of CD4 and treatment group was used, and the best model was selected based on the Akaike information criterion. This model included only the treatment group, so analysis of variance was used, followed by Tukey's method for all possible one-sided comparisons (family-wise error rate (FWER) = 0.05) to assess whether there were treatment differences between the 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 cytometric analysis of EGFR+CART cell expansion in peripheral blood between days 33 and 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 to include log 10 CD3, log 10 Analysis of covariance between CD4 and treatment groups was followed by Tukey's method for all possible one-sided comparisons. ** P value < 0.01; * P value = 0.03; n = 6-8 / group. (E) EGFR in bone marrow at the time of sacrifice +Flow cytometric analysis of the frequency of CAR T cells. Data were transformed using logit transformation. ANOVA followed by Tukey's method was used for all possible one-sided comparisons (family-wise error rate (FWER) = 0.05) to assess whether there were treatment differences between the CAR T cell-treated groups; ** P value < 0.01; * P value = 0.02; n = 7-8 / group. (F) The percentage of EGFR+CART cells in the bone marrow is a graph of the percentage of p24+T cells in the bone marrow. Surface antibody (CD45, CD3 and EGFR) staining was performed as shown in Figure (C), while the intracellular p24HIV-1 antigen was stained with KC57-FITC antibody after fixation and permeabilization. The simple least squares model using only the % of EGFR+CAR T cells was optimal, and the % of EGFR+CAR T cells and the % of p24+T cells were transformed using logit. Box plots are used to present the data in (B), (D) and (E). Black boxes represent quartiles, black lines represent quartiles and medians, plus signs represent mean values, and values ​​outside the box plots are considered outliers.

[0048] Fig.13 : Body weight and temperature of HIV-infected hu-PBMC-NSG mice treated with ART, CMV-HIV CAR T cells (with or without CMVpp65 vaccine). Body weight (A) and body temperature (B) were monitored weekly in the hu-PBMC mouse model after transplantation with HIVneg donor-derived PBMCs (day 0). Mice began an oral ART regimen on day 12 and received a single IV dose of CMV-HIV CAR T cells (low [0.1×106] or high [1×10 6 ]) and received 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).

[0049] Fig.14: EGFR+CAR T cell expansion in peripheral blood between days 33 and 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 average slope of the linear regression line of the log10-transformed EGFR+CAR T cell number / μL from days 33 and 42. Statistical significance was determined using one-sided Tukey contrasts, * P value = 0.03; ** P value = 0.02. n = 8 / group, and Figure 6 The groups in C are the same.

[0050] Fig.15 : HIV in a humanized PBMC-NSG mouse model pos Distribution and phenotype of donor-derived CMV-HIV CAR T cells. Flow cytometric analysis of EGFR+ CAR T cells 6 weeks after CAR T cell infusion. + CAR T cell fraction (A) CD4 + and CD8 + T cells, (B) CD62L + and (C)CD27 + The lines represent mean ± SD; n = 5.

[0051] Fig.16 : Schematic representation of the clinical trial timeline. Following screening and enrolling patients in the trial, participants will interrupt their ART regimen for 4 days prior to leukapheresis to prevent lentiviral transduction from suppressing T cells during CAR T cell preparation. Participants will resume their ART regimen immediately after leukapheresis. Once the CMV-HIV T cell population is prepared, participants will receive 5x10 6 cells, 25x10 6 cells or 50x10 6 cells (Day 0). Dose-limiting toxicity (DLT) was assessed starting from the day before T cell infusion (Day -1) until 60 days after infusion. Blood was drawn and assessed on the dates indicated on the timeline, followed by long-term follow-up (LTFU).

[0052] Fig.17A- E. Schematic diagram of SHIVR(N6)(EQ)BBZ-T2A-EGFRt_epHIV7 plasmid. Map (A) and sequence (D-E) of HIVscFv(N6)-IgG4(L235E,N297Q)-41BB-Zeta(CO)-T2A-EGFRt_epHIV7 (10008 bp; SEQ ID NO: 44) lentiviral vector. DETAILED DESCRIPTION OF THE INVENTION

[0054] The studies described herein demonstrate that CMV-specific T cells expressing a CAR targeting HIV (CMV-CAR T cells) exhibit dual effector functions after in vitro stimulation via their endogenous CMV-specific T cell receptor or an introduced CAR. Using a humanized HIV mouse model, the studies described herein demonstrate that CMV vaccination during ART accelerates CMV-HIV CAR T cell expansion in the peripheral blood and that higher numbers of CMV-HIV CAR T cells are associated with better control of HIV viral load and fewer HIV antigen p24+ cells in the bone marrow after ART interruption. CMV-CAR T cells and CMV antigens can be used to treat subjects infected with HIV.

[0055] I. Chimeric Antigen Receptor

[0056] Chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to surface antigens. T cells expressing CAR polypeptides are called CAR T cells. CAR has the ability to redirect 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 independently of antigen processing, thereby bypassing the main mechanism of tumor escape. CAR can also be expressed by other immune effector cells, including but not limited to natural killer CAR ("NK CAR") and directed NK cell killing of cells expressing CAR targets.

[0057] There are multiple generations of CAR, each containing different components. The first generation of CAR connects the antibody-derived scFv to the CD3 ζ intracellular signaling domain of the T cell receptor through a spacer (also referred to as a hinge domain) and a transmembrane domain. The second generation of CAR incorporates additional costimulatory domains (e.g., CD28, 4-BB or ICOS) to provide costimulatory signals. The third generation of CAR contains two costimulatory domains fused to the TCR CD3 ζ chain (e.g., a combination of CD27, CD28, 4-1BB, ICOS or OX40).

[0058] There may be a spacer between the co-stimulatory domain and the CD3ζ domain, but this is optional. CAR is usually 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 be used as a marker, such as a truncated EGFR receptor that lacks signaling function or a truncated CD19 receptor that lacks signaling function.

[0059] The embodiments described herein relate to HIV CARs having the following sequence:

[0060] RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRV TLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA GGGSGGGSGGGSGGGS YQ SPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADD IATYYCQVLQFFGRGSRLHIK ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK MALIVLGGVAGLLLFIGLGIFF KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL GGG RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10)

[0061] From amino-terminus to carboxyl-terminus, CAR includes a VH domain having the following sequence:

[0062] RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA(SEQ ID NO:8);

[0063] A linker with the following sequence:

[0064] GGGSGGGSGGGSGGGS (SEQ ID NO: 11);

[0065] A VL domain having the following sequence:

[0066] YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK(SEQ ID NO:7);

[0067] A spacer domain having the following sequence:

[0068] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:33);

[0069] The CD4 transmembrane domain has the following sequence:

[0070] MALIVLGGVAGLLLFIGLGIFF(SEQ ID NO:18);

[0071] 41-BB co-stimulatory domain with the following sequence:

[0072] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:38);

[0073] A linker with the following sequence:

[0074] GGG; and

[0075] A CD3ζ domain having the following sequence:

[0076] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 35).

[0077] In some embodiments, the CAR sequence may be preceded by a GMCSFRa signal peptide having the following sequence:

[0078] MLLLVTSLLLCELPHPAFLLIP.

[0079] In some embodiments, the CAR sequence may be followed by a T2A skipping sequence having the following sequence:

[0080] LEGGGEGRGSLLTCGDVEENPGPR (SEQ ID NO:45);

[0081] The GMCSFRa signal peptide has the following sequence:

[0082] MLLLVTSLLLCELPHPAFLLIP; and

[0083] A truncated EGFR receptor lacking signaling activity and having the following sequence:

[0084] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKAT GQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:46).

[0085] (a) Extracellular binding domain

[0086] Useful HIV CARs described herein are fusion proteins comprising an extracellular binding domain that recognizes HIV. The extracellular domain is typically a single-chain fragment (scFv) of an antibody or other antibody fragment, but it can also be a ligand that binds to an HIV protein.

[0087] In the case where the binding domain is a scFv, there are heavy chain variable regions and light chain variable regions, which can be ordered and connected together via a flexible linker of, for example, 5-25 amino acids. In some embodiments, a useful flexible linker is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repetitions of the sequence GGGS (SEQ ID NO: 13). In some embodiments, a useful flexible linker is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repetitions of the sequence GGGGS (SEQ ID NO: 14). In some embodiments, the light chain variable domain is at the amino terminus of the heavy chain variable domain, and in other cases, the light chain variable domain is at the carboxyl terminus of the heavy chain variable domain. In some cases, the linker comprises the sequence SSGGGGSGGGGGSGGGGS (SEQ ID NO: 12).

[0088] The studies described here used HIV CARs that included 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 20 that have evolved to circumvent common resistance mechanisms. 18 The CDRs in the VL and VH domains are underlined in the following sequences.

[0089]

[0090] Therefore, the svFv in HIV CAR can include a VL domain and a VH domain, wherein the VL domain is 95% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 7) and includes 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: 4). NO:3), the VH domain is 95% identical to RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO:8) and includes 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).

[0091] The N6 scFv used in the HIV CAR described herein has the following sequence:

[0092] RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA GGGSGGGSGGGSGGGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK(SEQ ID NO:9)

[0093] (b) Transmembrane domain

[0094] The CAR polypeptide disclosed herein may contain a transmembrane domain, which may 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.

[0095] The transmembrane domain of the HIV CAR used in the example has a CD4 transmembrane domain having the following sequence: MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 18). Other transmembrane domains can be used, including those shown below.

[0096] Table 1: Examples of transmembrane domains

[0097]

[0098]

[0099] (c) Spacer domain

[0100] CAR or polypeptide described herein can include a spacer domain between HIV targeting domain (i.e. HIV targeting scFv or its variant) and transmembrane domain. The spacer can play a role in providing flexibility for CAR or its domain, or prevent the steric hindrance of CAR or its domain. Various different spacers can be used. Some of them include at least a portion of human Fc region, such as the hinge portion or CH3 domain or its variant in human Fc region. Table 2 below provides a variety of spacer domains that can be used in CAR described herein.

[0101] Table 2: Examples of spacer domains

[0102]

[0103]

[0104]

[0105] Some spacer domains include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., a sequence falling between the CH1 and CH2 domains of an immunoglobulin, such as an IgG4 Fc hinge or a CD8 hinge. Some spacer domains include an immunoglobulin CH3 domain (referred to as CH3 or ΔCH2) or both a CH3 domain and a CH2 domain. Immunoglobulin-derived sequences 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.

[0106] The spacer domain may also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 26) or ESKYGPPCPPCP (SEQ ID NO: 25). The hinge / linker region may also comprise the sequence ESKYGPPCPPCP (SEQ ID NO: 3), 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 can 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) compared to SEQ ID NO: 31. In some cases, the IgG4 Fc hinge / linker region is mutated at two positions (L235E; N297Q) to reduce binding of an Fc receptor (FcR).

[0107] (d) Intracellular signaling domain

[0108] Any CAR construct described herein contains one or more intracellular signaling domains (e.g., CD3 ζ and optionally one or more costimulatory domains), which are the functional ends of the receptors. After antigen recognition, the receptors aggregate and transmit signals to cells.

[0109] CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex. CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that deliver activation signals to T cells after they engage with cognate antigens. In some cases, CD3ζ provides primary T cell activation signals, but does not provide fully competent activation signals, which require costimulatory signals.

[0110] Therefore, in some examples, in addition to CD3ζ, the CAR polypeptide disclosed herein can further include one or more costimulatory signaling domains. For example, costimulatory domains CD28 and / or 4-1BB can be used to transmit proliferation / survival signals together with CD3ζ-mediated primary signaling.

[0111] The co-stimulatory domain is located between the transmembrane domain and the CD3 zeta signaling domain. Table 3 includes examples of suitable co-stimulatory domains and sequences of the CD3 zeta signaling domain.

[0112] Table 3: Examples of CD3 zeta domains and co-stimulatory domains

[0113]

[0114]

[0115]

[0116] 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 these 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.

[0117] In various embodiments: the co-stimulatory domain is selected from the group consisting of a co-stimulatory domain shown in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, and an OX40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications is present in a CAR polypeptide described herein.

[0118] In some embodiments, there are two costimulatory domains, for example, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments, 1-5 (e.g., 1 or 2) amino acid modifications are substitutions. In various embodiments, the costimulatory domain is at the amino terminus of the CD3 ζ signaling domain, and a short linker consisting of 2-10, for example, 3 amino acids (e.g., GGG) can be located between the costimulatory domain and the CD3 ζ signaling domain.

[0119] In some cases, CAR can be produced using a vector, wherein the CAR open reading frame is followed by a T2A ribosomal skipping sequence and a truncated EGFR (EGFRt) or truncated CD19R (also referred to as CD19t) lacking a cytoplasmic signaling tail. In this arrangement, the co-expression of EGFRt or CD19t provides an inert, non-immunogenic surface marker that allows accurate measurement of genetically modified cells, and enables positive selection of genetically modified cells, as well as effective cell tracking of therapeutic T cells in vivo after adoptive transfer. Effectively controlling proliferation to avoid cytokine storms and off-target toxicity is an important obstacle to the success of T cell immunotherapy. In the case of treatment-related toxicity, EGFRt or CD19t incorporated into a CAR lentiviral vector can act as a suicide gene to ablate CAR+T cells.

[0120] The CD3ζ signaling domain may be followed by a ribosomal skipping sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: NO:45) and a truncated EGFR having a sequence at least 90%, at least 95%, at least 98% identical to or identical to LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO:46). In some cases, the truncated EGFR has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative) compared to SEQ ID NO:46.

[0121] Alternatively, the CD3 zeta signaling domain may be followed by a ribosomal skipping sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:45) and a truncated CD19R (also known as CD19t) having a sequence at least 90%, at least 95%, at least 98% identical to or identical to MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO:47).

[0122] CAR described herein can be produced by any means known in the art, although preferably produced using recombinant DNA technology. Nucleic acids encoding several regions of chimeric receptors can be easily prepared and assembled into complete coding sequences by standard molecular cloning techniques known in the art (genomic library screening, overlapping PCR, primer-assisted connection, site-directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably T lymphocytes, and is optimally selected from in vivo T lymphocytes.

[0123] Various T cell subsets isolated from patients can be transduced with vectors for CAR or peptide expression. Central memory T cells are a useful T cell subset. This can be accomplished by selecting CD45RO+ / CD62L+ cells using, for example, The device is used to immunomagnetically select cells expressing the desired receptor and separate central memory T cells from peripheral blood mononuclear cells (PBMC). Cells enriched in central memory T cells can be activated with anti-CD3 / CD28, transduced with, for example, a lentiviral vector that directs CAR expression, or used as non-immunogenic surface markers for in vivo detection, ablation, and potential ex vivo selection. Activated / genetically modified central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. Other methods for preparing CART cells can be found in PCT / US2016 / 043392.

[0124] Methods for preparing useful T cell populations are described in, for example, 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, NK cells can be derived from human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC).

[0125] In some embodiments, compositions comprising iPSC-derived CAR T cells or CAR NK cells are described herein. In some embodiments, compositions comprising iPSC-derived CAR T cells or CAR NK cells have enhanced therapeutic properties. In some embodiments, iPSC-derived CAR T cells or CAR NK cells exhibit enhanced functional activity, including effective cytokine production, cytotoxicity, and cell inhibitory inhibition of tumor growth, for example, as an activity that reduces tumor burden.

[0126] CAR can be transiently expressed in a T cell population via mRNA encoding CAR. mRNA can be introduced into T cells via electroporation (Wiesinger et al. 2019 Cancers (Basel) 11: 1198).

[0127] In some embodiments, the composition comprising 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.

[0128] II. CMV-specific T cells

[0129] In some cases, the method includes the step of preparing T cells that are specific for cytomegalovirus (CMV) and express a chimeric antigen receptor (CAR), the method comprising: (a) providing T cells (e.g., PBMC) from a human donor that is seropositive for cytomegalovirus (CMV); (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a cell population enriched in CMV-specific stimulated cells; (d) transducing at least a portion of the enriched cell population with a vector expressing CAR, thereby preparing T cells that are specific for CMV and express CAR. In various cases: the step of treating the exposed cells (e.g., using a selection step) to produce a cell population enriched for CMV-specific stimulated cells comprises: treating the stimulated cells to produce a cell population enriched for cells expressing an activation marker (e.g., IFN-γ or IL-13); culturing the PBMCs for less than 5 days (less than 4, 3, 2, 1 days) prior to exposure to the CMV antigen; exposing the cells to the CMV antigen for less than 3 days (less than 48 hrs, 36 hrs, 24 hrs), the CMV antigen is a pp65 protein or an antigenic portion thereof, the CMV antigen comprises two or more different antigenic CMV pp65 peptide; the step of transducing the enriched cell population does not include CD3 stimulation; the step of transducing the enriched cell population does not include CD28 stimulation; the step of transducing the enriched cell population does not include CD3 stimulation or CD28 stimulation; the enriched cell population 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; before the step of transducing the enriched cell population with a vector encoding CAR, the enriched cell population is cultured for less than 10 (less than 9, 8, 7, 5, 3, 2) days. In some cases, T cells are from CMV-positive donors and are exposed to a mixture of CMV antigens such as CMV pp65 or CMV protein peptides (e.g., a 10-20 amino acid peptide that is a fragment of pp65) in the presence of IL-2 to produce a stimulated cell population. In some cases, the stimulated cell population is processed to prepare a cell population expressing IFN-γ. In some cases, the CMV / CAR T cells do not recognize antigens from a second virus. For example, they do not recognize Epstein-Barr virus antigens or influenza virus antigens or adenovirus antigens.

[0130] III. Treatment of HIV-infected patients

[0131] Aspects of the present disclosure provide methods for treating infected subjects by administering immune cells (e.g., CMV-specific T cells expressing HIV-CAR) and CMV vaccines.

[0132] (a) Subjects

[0133] The subject treated by the method can be a human subject infected with HIV, including a subject receiving antiretroviral therapy (ART). It can be applied to subjects with a viral load higher than 200 copies / ml or lower than 200 copies / ml, and subjects with undetectable viral load. The subject can be treated with one or more of the following: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), entry or fusion inhibitors, and integrase inhibitors (INSTIs). For example, the subject can be treated with two NRTIs together with INSTIs, NNRTIs or PIs, and in some cases with ritonavir or cobicistat.

[0134] (b) Application

[0135] An effective amount of therapy (e.g., CMV-CAR T cells and CMV vaccines) can be administered to a subject (e.g., a human) in need of treatment via any suitable route (e.g., local or systemic administration to a subject). Suitable modes of administration include injection, infusion, infusion, or ingestion. Injection includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion. CMV-CAR T cells and CMV vaccines can be administered simultaneously or sequentially.

[0136] An effective amount refers to each active agent alone or in combination with one or more other active agents, and the amount required for the treatment effect is given to the subject. As recognized by those skilled in the art, the effective amount depends on the specific condition treated, the severity of the condition, individual patient parameters (including age, physical condition, body shape, sex and weight), duration of treatment, the nature of concurrent treatment (if any), specific route of administration and similar factors. The effective amount can be administered once or more, applied or dosed. Compositions described herein (e.g., CMV HIV CAR T cells and CMV vaccines) can be administered once or more per day to once or more per week; including administration every other day. It will be understood by those skilled in the art that certain factors may affect the dosage and time required for the effective treatment of the subject, including but not limited to the severity of the disease or illness, previous treatment, the general health and / or age of the subject, and other diseases present. In addition, treating a subject with a therapeutic compound described herein in a therapeutically effective amount may include a single treatment or a series of treatments.

[0137] Useful doses of CMV HIV T cells include approximately 5x10 6 , 10x10 6 , 15x10 6 , 20x10 6 , 25x10 6, 30x10 6 , 35x10 6 , 40x10 6 , 45x10 6 , 50x10 6 ,55x10 6 , 60x10 6 , 65x10 6 ,70x10 6 , 75x10 6 ,80x10 6 ,85x10 6 ,90x10 6 ,95x10 6 and 100x10 6 cells. A healthcare professional can provide dose escalation or decrement to a patient as needed. In some embodiments, a single dose of CMV-HIV CAR T cells is administered to a patient. In some embodiments, a second dose of CMV-HIV CAR T cells is administered to a patient.

[0138] In some embodiments, 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. In some embodiments, a 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 a subject.

[0139] In some embodiments, a CMV vaccine is administered while administering CMV-HIV CAR T cells. In some embodiments, a CMV vaccine is administered before administering CMV-HIV CAR T cells. In some embodiments, a CMV vaccine is administered after administering CMV-HIV CAR T cells. In some embodiments, a CMV vaccine is administered before or after administering CMV-HIV CART cells. In some embodiments, a CMV vaccine is administered in a single or repeated dose. In some embodiments, CMV-HIV CAR T cells are administered in a single or repeated dose.

[0140] In some embodiments, before administering CMV-HIV CAR T cells, the subject is administered a CMV vaccine. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten days before administering CMV-HIV CAR T cells, a CMV vaccine is administered. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36 or 48 hours before administering CMV-HIV CAR T cells, a CMV vaccine is administered. In some embodiments, about one, two, three or four weeks before administering CMV-HIV CAR T cells, a CMV vaccine is administered.

[0141] In some embodiments, after administering CMV-HIV CAR T cells, the subject is administered a CMV vaccine. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten days after administering CMV-HIV CAR T cells, a CMV vaccine is administered. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36 or 48 hours after administering CMV-HIV CAR T cells, a CMV vaccine is administered. In some embodiments, about one, two, three or four weeks after administering CMV-HIV CAR T cells, a CMV vaccine is administered. In some embodiments, this can be in addition to administering a CMV vaccine before administering CMV-HIV CAR T cells, so in some embodiments, at least one CMV vaccine is administered to a subject before administering CMV-HIV CAR T cells, and at least one CMV vaccine is administered to a subject after administering CMV-HIV CAR T cells.

[0142] In some embodiments, at least one CMV vaccine is administered in a single or repeated dose prior to or after administration of CMV-specific T cells.

[0143] (c) CMV vaccine: CMV antigen or nucleic acid encoding CMV antigen

[0144] Useful CMV vaccines can include one or more CMV antigens or one or more nucleic acids encoding one or more CMV antigens.CMV antigens can be CMV proteins, fragments of CMV proteins, modified CMV proteins, fragments of modified CMV proteins, mutated CMV proteins or fragments thereof or fused CMV proteins or fragments thereof.In some embodiments, useful CVM vaccines include one or more nucleic acids encoding one or more CMV antigens.The example of CMV antigens includes pp65, IE1 exon 4 (IE1 / e4), IE2 exon 5 (IE2 / e5), its fusions and its antigenic fragments and its each with 1,2,3,4 or 5 amino acid modified variants.In some embodiments, variants include 1-2 amino acid replacements or 1-5 amino acid replacements.In some embodiments, amino acid replacements are conservative.The example of modified CMV protein antigens and fragments thereof can be found in U.S. Patent No. 7,163,685.

[0145] In some embodiments, the CMV antigen comprises a sequence selected from SEQ ID NO: 57-64 and a variant thereof with 1, 2, 3, 4 or 5 amino acid modifications. In some embodiments, the variant comprises 1-2 amino acid replacements or 1-5 amino acid replacements. In some embodiments, the amino acid replacements are conservative. In some embodiments, the CMV antigen may comprise a fragment of any one of SEQ ID NO: 57-64. The fragment may comprise 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 one of SEQ ID NO: 57-64 or consist of it.

[0146] Some examples of nucleic acids that can encode CMV antigens are DNA, RNA, mRNA, carriers, viral vectors, slow virus vectors, MVA carriers, bacterial artificial chromosomes (BAC), vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors and other carriers 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.

[0147] The fusion CMV protein antigen may comprise two or more CMV proteins, modified CMV proteins, mutated CMV proteins, or any antigenic fragment thereof. In some embodiments, a useful fusion protein is a fusion of IE1 exon 4 (IE1 / e4) and IE2 exon 5 (IE2 / e5), IE1 / e4-IE2 / e5 (IEfusion; e.g., SEQ ID NO: 58). In some embodiments, a useful fusion protein comprises SEQ ID NO: 58 or a variant thereof having 1-5 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.

[0148] Useful CMV vaccines can be rMVA vaccines, which are formed by combining the modified Ankara vaccinia (MVA) vaccine platform with bacterial artificial chromosome (BAC) technology. Modified Ankara vaccinia (MVA) is a genetically engineered, highly attenuated vaccinia virus strain that does not proliferate in most mammalian cells. This characteristic has minimal impact on viral or exogenous gene expression, because the ability of MVA to proliferate in mammalian cells is blocked in the late stage of viral assembly. However, DNA continues to replicate, thus serving as an effective template for RNA biosynthesis, resulting in high-level protein synthesis. MVA also has a large exogenous gene capacity and multiple integration sites, which make it an ideal carrier for expressing vaccine antigens. MVA has a good safety record and versatility in producing heterologous proteins. In fact, vaccines based on MVA for the treatment of infectious diseases and cancer have been developed and reached Phase I / II clinical trials. MVA is attractive as a vaccine carrier for CMV antigens in individuals who are severely immunosuppressed and are experiencing other complications (such as malignancies or organ failure) and need transplantation.

[0149] The CMV three-chain vaccine is a recombinant MVA expressing three CMV antigens, i.e., at least a portion of the immediate early gene-1 (IE1), at least a portion of the immediate early gene-2 ​​(IE2), and at least a portion of pp65. The IE1 antigen and the IE2 antigen can be expressed as a fusion protein, and the expression of the CMV antigen can be under the control of a modified H5 (mH5) promoter. The CMV three-chain vaccine is fully described in US8,580,276 and Wang et al. (Vaccine 28:1547, 2010.). The CMV three-chain vaccine can express CMVpp65 and CMV IE fusion protein (IEfusion). IEfusion can include an antigenic portion (e.g., exon 4) of IE1 and an antigenic portion (e.g., exon 5) of 1E2, wherein the antigenic portion triggers an immune response when expressed by the vaccine.

[0150] Various modifications and / or insertion sites can be made with the goal of increasing the stability of triplexes simultaneously expressing IE1, IE2, and pp65 in a single MVA vector (see, e.g., WO 2019 / 217922).

[0151] As explained in US 8,580,276, the CMV three-chain vaccine includes three best-recognized antigens in the CD8 subgroup: pp65, IE1 and IE2. There is no homology region greater than 5 amino acids between the major exons of the two proteins. Individually, both antigens are widely recognized by almost 70% of the general population.

[0152] Any vaccine composition disclosed in US 7,163,685, US 8,580,276, US 9,675,689 US20170246292 A1 can be used in the methods and compositions provided herein and are incorporated herein by reference in their entirety and for all purposes.

[0153] Selected CMV antigen amino acid sequences :

[0154] CMV pp65 protein (UniProt ID: P06725; SEQ ID NO: 57)

[0155] MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIHASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIGDQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIMLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG

[0156] lEfusion sequence (IE1- IE2 ; SEQ ID NO:58):

[0157] MVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEEMKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAKKDELRRKMMYMCYRNIEFFTKNSAFPKTTNGCSQAMAALQNLPQCSPDEIMAYAQKIFKILDEERDKVLTHIDHIFMDILTTCCVETMCNEYKVTSDACMMTMYGGISLLSEFCRVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSPSVDDLRAIAESDEEEAIVAYTLATAGVSSSDLVSPPESPVPATIPLSSVIVAENSDQEESEQSDEEEEEEGAQEEREDTVSVKSEPVSEIEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ GDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNT PLPGASATPELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQ GEEVETRGATASSSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSSSSSASDSESESEEMKCSSGGG ASVTSHHGRGGFGGAASSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKI DEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLALS TPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKF PKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ

[0158] lE1 sequence (SEQ ID NO:59):

[0159] MVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEEMKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAKKDELRRKMMYMCYRNIEFFTKNSAFPKTTNGCSQAMAALQNLPQCSPDEIMAYAQKIFKILDEERDKVLTHIDHIFMDILTTCCVETMCNEYKVTSDACMMTMYGGISLLSEFCRVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSPSVDDLRAIAESDEEEAIVAYTLATAGVSSSDLVSPPESPVPATIPLSSVIVAENSDQEESEQSDEEEEEEGAQEEREDTVSVKSEPVSEIEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ

[0160] IE2 sequence (SEQ ID NO:61):

[0161] MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPELSPRKKPRKKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTLPSQMNHPPLPDPLGRPDEDSSSSSSSSSCSSASDSESEEMKCSSGGGASVTSSHHGRGGFGGAASSSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLSTPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ

[0162] IE2 H363A sequence (SEQ ID NO:62):

[0163] MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPELSPRKKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTPLSQMNHPPLPDLGRPDEDSSSSSSSSSSCSSADSSESEEMKCSSGGGASVTSSHHGRGGFGGAASSSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLSTPFLME ATMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ

[0164] IE2 H369A sequence (SEQ ID NO:63):

[0165] MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSSSSSSCSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLALSTPFLMEHTMPVT A PPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ

[0166] IE2 H363A / H369A sequence (SEQ ID NO:64):

[0167] MGDILAQAVNHAGIDSSSTGPTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASATPELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQGEEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLGRPDEDSSSSSSSS CSSASDSESESEEMKCSSGGGASVTSSHHGRGGFGGAASSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRGRVKIDEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLALSTPFLME A TMPVT A PPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKFPKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ

[0168] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example

[0169] Specifically described below are the design and preparation of CMV-HIVCAR T cells using cells obtained from patients receiving ART, and studies using such cells and CMV vaccines in mouse models of HIV infection.

[0170] Example 1: N6-CAR T cells exhibit potent effector function in vitro

[0171] The cells were transduced from HIV by lentiviral vector (LV) encoding CAR containing scFv of bNAb N6. neg Primary T cells isolated from donors, preparation of N6-based CAR T cell products ( Figure 1 Error! Reference source not found. A) The CAR includes a variant IgG4 spacer between the scFv extracellular domain and the transmembrane domain 24 , CD4 transmembrane domain, 4-1BB co-stimulatory domain 25-29Truncated human epidermal growth factor receptor (EGFRt) was added to the CAR construct to serve as an element for immunomagnetic purification, cell tracking by flow cytometry and immunohistochemistry, and potential in vivo cell ablation with the anti-EGFR antibody cetuximab. 30 After ~15 days of in vitro expansion, the final T cell product contained an average of 38.16% ± 8.87% EGFR + CAR T cells, of which 69.63% ± 16.63% were CD4 + T cells, 31.57% ± 17.76% were CD8 + T cells (mean ± SD, n = 4, Figure 1 B shows a representative FACS graph). N6-CAR T cells were examined for cytotoxicity by performing a 96-hour killing assay targeting 8E5-gpl20 cells. These 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 cytometric analysis of the remaining target cells showed that N6-CAR T cells (normalized relative to mock T cells) effectively killed 8E5-gp120 cells ( Figure 1 C). In separate experiments, N6-CAR T cells were co-cultured with purified gp120-negative or gp120-positive 8E5 cells at various E:T ratios. Efficient and gp120-specific killing of gp120-positive 8E5 cells was observed, but not of gp120-negative 8E5 cells ( Figure 3 Finally, after stimulation with 8E5-gp120 cells, only gated EGFR + CAR T cells showed proliferation capacity, while the gated CAR-negative T cell fraction did not ( Figure 1 D). Flow cytometry analysis showed that these stimulated N6-CAR T cells maintained persistent memory (CD62L = 66.07%, CD127 = 51.42% and CD27 = 87.02%, the 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%, the average of three donors) ( Figure 4 ).

[0172] Example: N6 scFv-Fc does not cross-react with normal human tissues

[0173] To evaluate potential off-target effects of N6-CAR, normal human tissues were immunostained with soluble N6 scFv-Fc. As expected, concentration-dependent immunostaining was observed on 8E5-gp120 cells using N6 scFv-Fc or the positive control (anti-gp120 bNAb VRC01 obtained from the NIH HIV Reagent Program), but not on gp120-negative leukemia KG-1a cells ( Figure 5 ). N6 scFv-Fc was then used to pan-immunostained 37 frozen human tissues from three unrelated normal donors [Charles River Labs, CRL Study No.: 20182940]. Immunopathological analysis did not show membrane signals on these tissues. However, cytoplasmic staining was observed in epithelial cells in the esophagus (mucosal), kidney (renal pelvis), pituitary (adenohypophysis), salivary glands (ducts), skin (sweat glands), thymus (epithelial reticular), and ureter (mucosal), as well as in colloids in the thyroid gland. Binding to cytoplasmic sites is considered to be of little toxicological significance due to the limited ability of antibody-based therapeutics to enter the cytoplasmic compartment in vivo 31,32 Overall, immunohistochemical staining analysis supported the low risk of clinically relevant off-target tissue cross-reactivity for N6-CAR.

[0174] Example 3: CMV-HIV CAR T cells can be prepared at clinical scale from HIVneg and HIVpos donors

[0175] As mentioned before 33,34 , using GMP-compliant CliniMACS CMV-specific T cells were isolated by an automated closed system and transduced with a lentiviral vector encoding N6-CAR ( Figure 2 A). Briefly, PBMCs were collected from CMVposHIVneg or HIVpos donors receiving ART (Table 1) and cultured in a CliniMACS Cytokine capture system (CCS) Overlapping CMVpp65 peptide pools were stimulated and processed, followed by labeling with Catchmatrix reagent and anti-IFN-γ beads. The system separates IFN-γ+ cells by magnetic separation ( Figure 6 B shows a representative FACS plot). CMV-specific T cells (IFN-γ+CD3+) from HIV-neg donors were enriched from 4.33%±3.46% of total viable T cells to 74.71%±9.17% (mean±SD, n=6, Figure 6C). Similarly, IFN-γ+ T cells from HIVpos donors were enriched from 1.91%±0.94% of total live T cells to 67.28%±17.04% (mean±SD, n=7, Figure 2 C). Unlike CMV-specific T cells isolated from HIVneg donors, which had similar proportions of CD4+ and CD8+ T cells (42.34%±20.30% and 46.18%±18.86%, respectively), CMV-specific T cells from HIVpos donors had a higher proportion of CD8+ cells (78.04%±9.21%) compared with CD4+ cells (25.59%±12.19%) (mean±SD, Figure 6 C). This observation is consistent with previous reports showing that PLWH have a higher proportion of CD8+CMV-specific T cells compared with HIVneg individuals35,36. Notably, the overall composition of memory T cell subsets was similar between CMV-specific T cells isolated from HIVneg and HIVpos donors (Figure S5). Recovered IFN-γ+ T cells (~1×10 6 ) were transduced with N6-CAR lentiviral vector at MOI 3 to generate CMV-HIV CART cells and expanded in the presence of IL-2 (50 U / mL) and IL-15 (1 ng / mL) for ~15 days. To evaluate the effect of endogenous reactivation of HIV, CAR T cells were first expanded in the absence of antiretroviral drugs (ARVs). At the end of the culture, the total number of cells from HIV-negative donors was 209.5×10 6 ±97.62×10 6 , and the total number of cells of HIVpos donors was 13.49×10 6 ±12.17×10 6 (mean ± SD, Figure 6 D). During the expansion of CAR T cells from three HIVpos donors, the culture medium was supplemented with ARV (43nM darunavir and 279nM enfuvirtide) to inhibit HIV replication. This mixture was shown to prevent HIV replication in vitro ( Figure 8 A), does not affect the lentiviral transduction efficiency ( Figure 8 B) and cell expansion ( Figure 8 C). Interestingly, the three best cell expansions from HIVpos donors (64.1×10 6 ,237.2×10 6 and 269.53×10 6 ) occurs in the presence of ARVs ( Figure 6D, red dashed line). Therefore, using ARV, HIVpos donor cells can be expanded like HIVneg donor cells. To further explore the differences between products prepared from HIVneg and HIVpos donors, transduction efficiency was assessed by measuring EGFR expression by flow cytometry in the final cell products. Similar EGFR expression levels were observed in cell products derived from HIVneg donors (24.68% ± 18.34%) and HIVpos donors (21.45% ± 12.65%) (mean ± SD, Figure 96E). As expected, CMV-HIV CAR T cells from HIVpos donors were composed of a higher percentage of CD8+ cells (86.46%±16.52%) compared with CD4+ cells (10.49%±6.62%), whereas the proportions of CD8+ and CD4+ cells in CMV-HIV CAR T cells prepared from HIVneg donors were 61.35%±40.63% and 43.07%±42.56%, respectively (mean±SD, Figure 6 E). Finally, we evaluated the final number of CAR T cells prepared per activity ( Figure 6 F) The average number of CAR T cells in HIVpos-derived cell products expanded in the presence of ARVs was 34.19 × 10 6 .

[0176] Analysis of T cell memory subsets in the final T cell product showed that the HIV-neg donor-derived cell product had 19.03% ± 24.52% CD27+CD45RA+ stem cell memory T cells (Tscm), 18.92% ± 27.40% CD27+CD45RA- central memory (Tcm), 18.78% ± 22.41% CD27-CD45RA+ effector memory RA (TEMRA), and 43.26% ± 37.37% CD27-CD45RA- effector memory T cells (Tem) (mean ± SD, Fig. 9 A). In contrast, HIVpos donor-derived cell products contained less Tscm (1.71% ± 2.39%) than Tem (65.02% ± 21.76%). Similar observations were made when looking at the composition of cellular memory subsets within EGFR+CAR T cells ( Fig. 9 B). Finally, in the final T cell product ( Fig. 9 C) or in EGFR+CAR T cells derived from HIVpos or HIVneg donors ( Fig. 9 D) Only low expression levels of the exhaustion markers LAG-3, PD-1, and Tim-3 were observed.

[0177] Table 4: HIV pos Donor information

[0178]

[0179] N / A: Information not available

[0180] Example 4: CMV-HIV CAR T cells exhibit HIV and CMV antigen-specific effector functions

[0181] First, a 96-hour long-term killing assay was performed using 8E5-gp120 cells as target cells, demonstrating that neg The donor's CMV-HIV CAR T cells have specific cytotoxicity against cells expressing gp120 ( Fig.10 A). 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 by CTV dye dilution showed that CMV-HIV CAR T cells proliferated only when co-cultured with autologous PBMCs pulsed with CMVpp65 peptide as antigen presenting cells (APCs) (CMVpp65-PBMCs) or with LCL-OKT3 cells engaging all TCRs, but not when exposed to KG-1a cells or culture medium ( Fig.10 B). Thus, higher IFN-γ expression was measured in CMV-HIV CAR T cells after overnight stimulation with LCL-OKT3, CMVpp65-PBMC or 8E5-gp120 expressing cells compared to stimulation with KG-1a cells or medium ( Fig.10 C). As expected, CMV-specific T cells expressed IFN-γ only after stimulation with LCL-OKT3 cells and CMVpp65-PBMCs, but not after stimulation with 8E5-gp120 cells, KG-1a cells, or medium ( Fig.10 C) Relatively low IFN-γ expression in CMV-HIV CAR T cell products after overnight stimulation with 8E5-gp120 suggests that CAR T cells are slowly killing their target cells.

[0182] Similarly, HIV-derived pos Do the donor's CMV-HIV CAR T cells maintain their effector function? The CMV-HIV CAR T cell products are mainly CD8 + , and were responsive to CMVpp65 antigen stimulation, as shown by their high IFN-γ expression after overnight stimulation with CMVpp65-PBMCs ( Fig.11In addition, dose-dependent cytotoxicity against 8E5-gp120 cells was observed after short-term (24 hr, left panel) and long-term (96 hr, right panel) co-culture. Fig.11 B). Better cytotoxicity was observed for 96 hr co-culture than for 24 hr, supporting optimal killing kinetics for 96 hr in the context of HIV CAR and gp120 target. After 24 and 96 h of co-culture, T cell products and CAR T cells had similar low exhaustion phenotypes ( Fig.11 C), similar to its final product, indicating that this CMV-HIV CAR T cell retains function and efficacy after target engagement ( Fig. 9 C and 9D). CMV-HIV CAR T cells or CMV-CD19 CAR T cells derived from the same donor were combined with HIV NL4-3 Infection with eGFP + Jurkat cells were co-cultured at various E:T ratios for 7 days to further evaluate the cytotoxicity of the final cell product against HIV-infected cells ( Fig.11 D). CMV-HIV CAR T cells are cytotoxic against HIV-infected cells compared to CMV-CD19 CAR T cells. In the same experiment, HIV-1 p24 levels in the cell supernatant were measured by ELISA and showed a reduction in p24 release in the presence of CMV-HIV CAR T cells compared to CMV-CD19 CAR T cells ( Fig.11 E). Finally, compared with CMV-HIV CAR T cells derived from the same donor, pos Higher levels of p24 were detected in the supernatant of donor-derived CMV-CD19 CAR T cells ( Fig.11 F) Because the only source of HIV in these cultures is from HIV pos The results showed that the CMV-HIV CAR T cells could eliminate detectable HIV after endogenous reactivation.

[0183] Example 5: CMV-HIV CAR T cells exhibit anti-HIV activity in HIV humanized mouse model

[0184] HIV neg The donor is used to generate large numbers of CMV-HIV CAR T cells. Fig.12As outlined in A, the HIV-infected NSG humanized PBMC (NSG hu-PBMC) mouse model was established by transplanting autologous PBMCs in 3-5 week-old NSG mice (day 0). On day 7, mice were challenged with HIV-1BaL via intraperitoneal (IP) injection, and a 3-week oral ART regimen (emtricitabine, tenofovir, raltegravir) was started on day 12, which reduced plasma viral load from low levels to undetectable levels. On day 21, during ART, both cohorts were treated with low-dose CMV-HIV CAR T cells (0.1×10 6 The patients were treated with a single infusion of 1×10 EGFR+ T cells with or without CMVpp65 immunization on day 28. 6 One group of mice was treated with 100 EGFR+ T cells and then received the CMVpp65 vaccine on day 28. Two control cohorts included mice treated with neg Donor's CMV-negative T cells (1×10 6 Mice treated with CMV-HIV CAR T cells were treated with or without CMVpp65 vaccine on day 28. CMV-HIV CAR T cells were well tolerated in all mice, and no differences in body weight and body temperature were observed between the groups ( Fig.13 At day 28, when mice received ART before vaccination, high-dose CMV-HIV CAR T cells were found to significantly control HIV plasma viremia compared with mice treated with control T cells and low-dose CAR T cells ( Fig.12 B) Then measure EGFR in peripheral blood + CAR T cells ( Fig.12 C) and converted to log for each mouse 10 EGFR between day 33 (ie, the last day of ART) and day 42 (ie, 9 days after ART interruption) of the scale + The slope of the linear regression line of CAR T cell number / μL ( Fig.14 ). The mean slope, which represents the rate of expansion of CAR T cells after ART interruption, was significantly higher in both vaccinated CAR T cell treatment groups compared with the unvaccinated low-dose CAR T cell treatment cohort. This suggests that the vaccine induced CAR T cell expansion even after ART interruption. At day 42, after ART interruption and viral rebound, mice receiving high-dose CMV-HIV CAR T cells and CMVpp65 vaccine were the only cohort with controlled plasma viremia compared with mice treated with vaccinated or unvaccinated low-dose CAR T cells ( Fig.12D). This indicates the importance of CAR T cell dose, such that when viremia is high, the expansion of low-dose CAR T cells driven by CMVpp65 vaccine is insufficient to achieve a therapeutic effect. CAR T cells were also detected in the bone marrow of sacrificed mice ( Fig.12 E), and notably, active HIV-1-infected T cells (i.e., p24 + The frequency of EGFR+CAR T cells was inversely correlated with the frequency of EGFR+CAR T cells (P value = 0.045), indicating that CAR-mediated reduction of HIV-infected cells ( Fig.12 F).

[0185] Finally, in separate experiments, we evaluated pos Whether the donor's CMV-HIV CAR T cells can migrate to the bone marrow, because memory T cells from the bone marrow are persistent and persist long after circulating antigen-specific memory T cells have dissipated 37 EGFR+CAR T cells (50×10 3 ) were infused into hu-PBMC-NSG mice 14 days after transplantation of HIV-challenged PBMC (day 0) and in the absence of ART. + CAR T cells were detected in peripheral blood and bone marrow. As expected, these CAR T cells were mainly CD8 + ( Fig.15 A). Importantly, they still expressed the memory cell markers CD62L and CD27 ( Fig.15 B and Fig.15 C) Therefore, these results suggest that it is derived from HIV pos Individual CMV-HIV CAR T cells established long-lasting 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), which may be due to the limited number of CAR T cells infused and the high level of active HIV infection at the time of CAR T cell infusion.

[0186] The results show the ability of CMV / HIV-CAR T cells to control viremia in lymphoid tissues, and it may help to eradicate reservoirs of persistent infection and latency, which is not possible with ART or other current antiviral approaches.

[0187] Example 6: CMV-HIV CAR T cells exhibit anti-HIV activity in human HIV patients

[0188] Fig.16Describes a schematic of the first-in-human single-arm pilot study using autologous CMV / HIV-CART cells to treat PLWH who are receiving long-term ART and are virologically stable. This trial is designed to study the safety of a single dose of CMV / HIV-CAR T cells at three dose levels for the first time. Each study participant will receive treatment only after close monitoring of previously treated study participants for at least 60 days. Dose escalation, reduction, or expansion will not occur until at least three evaluable participants have reached the current dose level.

[0189] Step 1 of the protocol consisted of screening and signed informed consent at UCSD and the ACTG clinics. In step 2 of study entry, eligible participants temporarily interrupted their ART regimen for 4 days prior to leukapheresis to prevent lentiviral transduction that suppresses T cells during CAR T-cell preparation. Participants will resume their ART regimen immediately after leukapheresis. During this 4-day ATI, subjects will restart their previous ART regimen if any of the following occurs: (1) if requested by the participant or their HIV healthcare provider, or (2) if ART is considered medically necessary for non-HIV-related reasons, or (3) for symptomatic HIV disease (acute viral syndrome). If preparation is unsuccessful, a second apheresis can be scheduled no earlier than 3 weeks later, with another 4-day interruption in ARV therapy.

[0190] Study participants underwent leukapheresis to collect peripheral blood mononuclear cells (PBMCs). The apheresis product was incubated with CMV peptide overnight and analyzed using the CliniMACS The system (Miltenyi Biotec) was used to enrich CMV-specific T cells based on interferon gamma (IFNγ) positivity. Then, the cells were treated with a self-inactivating lentiviral vector (vHIVR(N6)(EQ)BBζ-T2A-EGFRT_EphIV7; Fig.17A These cells were transduced with self-inactivating lentiviral vectors directing co-expression of gp120BBζ-CAR (using anti-gp120bNAb N6 scFv to target cells expressing HIV gp120) and truncated human epidermal growth factor receptor (EGFRt, used as a tracer marker). The resulting autologous CMV / HIV-CAR T cell product (i.e., study agent) was expanded in vitro in the presence of IL-2, IL-15, and ART cocktail inhibitors for ~2 weeks and cryopreserved ( Figure 6 A).

[0191] Once the final cell product was released, participants proceeded to step 3. Participants assigned to dose level 1 received a single intravenous (IV) infusion of 25 x 10 6Subjects assigned to dose level 2 received 50x10 6 The DLT evaluation period of the study was defined as day -1 before CMV-HIV CAR T infusion to day 60 after CAR T cell infusion.

[0192] Unless otherwise specified, dose-limiting toxicities (DLTs) were defined as events considered at least possibly related to CMV-HIVCAR T infusion, except those listed in the expected AEs (adverse events) below and occurring within the DLT assessment period. DLTs included:

[0193] Any grade 3 or higher organ toxicity (cardiac, skin, gastrointestinal, liver, lung, genitourinary, nervous, hematologic, renal, secondary malignancies, and endocrine system) designated as possibly, definitely, or probably related to CAR-T cell infusion (attribution level);

[0194] Any grade 3 or higher cytokine release syndrome possibly, probably, or definitely attributed to CAR T-cell infusion;

[0195] Any grade 3 or higher hypersensitivity reaction possibly, probably, or definitely attributed to the CAR T cell infusion;

[0196] Any grade 3 or higher autoimmune toxicity possibly, probably, or definitely attributable to the CAR T cell infusion; or

[0197] Any grade 5 toxicity possibly, probably, or definitely related to the CAR T cell infusion.

[0198] Study participants may experience a variety of “expected” AEs related to gene-modified T cell infusion (usually occurring within the first 48 hours) and in vivo expansion and CAR-directed therapy (usually occurring within the first 21 days after CAR T cell infusion). The following are the highest allowable AEs for CTCAE v5.0 grading in addition to the ASTCT consensus criteria for CRS / neurotoxicity grading: * List of “expected” AEs (including grade and duration):

[0199] Dyspnea: Grade 3 dyspnea, lasting up to 24 hours without intervention;

[0200] Fever: Grade 4 fever, lasting up to 72 hours;

[0201] Cough: Grade 4 cough, lasting up to 24 hours;

[0202] Headache: Grade 3 headache, lasting up to 72 hours after intervention;

[0203] Hypotension: Hypotension: Grade 3 (without CRS symptoms), responding to fluid resuscitation and resolving to Grade 2 or less within 24 hours;

[0204] Rash: Grade 3 rash, lasting up to 72 hours after intervention.

[0205] Once the safety of this approach has been shown in patients, a CMV vaccine (CMV antigen or nucleic acid encoding a CMV antigen; e.g., CMV pp65) is added to Fig.16 In the scheme described in . The CMV vaccine is administered to the patient before CMV-HIV CAR T cell infusion (on day -1). This helps to amplify CMV-HIV CAR T cells in vivo and increase the persistence of memory T cells expressing memory cell markers CD62L and CD27. Some patients also receive CMV vaccine or 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).

[0206] CMV / HIV-CAR T cells control viremia in lymphoid tissues and eradicate reservoirs of persistent infection and latency. A single infusion of CAR T cells is intended to replace lifelong ART regimens (as well as other current antiviral approaches to treat HIV).

[0207] Materials and methods

[0208] The following materials and methods were used in the examples.

[0209] DNA constructs

[0210] The N6-CAR construct was modified with a previously described CD19-specific scFvFc:ζ chimeric immune receptor 63 HIV:41BB:ζ / EGFRt-epHIV7 lentiviral vector contains GM-CSF receptor-α chain signal sequence (GMCSFRss), which enhances CAR surface expression. The CAR sequence consists of: V of N6 bNAb H and V L Gene fragment, IgG4 hinge with two site mutations in the CH2 region (L235E; N297Q), CD4 transmembrane and 4-1BB co-stimulatory domains, cytoplasmic domain of CD3ζ chain 24 , ribosomal skipping T2A sequence, and a truncated human EGFR (EGFRt) sequence as previously described to allow CAR T cell enrichment, tracking, and potential cell ablation via ADCC 64The 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 the P2A-ffLuc-stop cassette in frame with eGFP.

[0211] Clinical-scale production of CMV-HIV CAR T cells

[0212] Fresh blood products from CMV pos HIV neg Donors (StemCell Technologies, Vancouver, Canada) and HIV patients receiving ART pos Donors (Zen-Bio Inc, Research Triangle Park, NC, see Table 1) were obtained. All procedures were performed in accordance with the Helsinki protocol statement (KC15TISI0494). CMV-specific T cells were isolated on the CliniMACS Prodigy and 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). PBMCs (10 9 ) was added to the application bag of the connection tubing set, and the CliniMACS Prodigy device automatically performed a continuous process including sample washing, PepTivator CMVpp65 antigen stimulation, Catchmatrix labeling, anti-IFN-γ microbead labeling, magnetic enrichment and elution. After magnetic enrichment, CMV-specific cells and non-CMV-specific cells were eluted in separate bags. After standing overnight in RPMI medium containing 10% human AB serum (Gemini Bio Products, Sacramento, CA), IL-2 (50 U / mL) and IL-15 (1 ng / mL), the cells were treated with research grade (for HIV) neg Donors and HIV pos Donor #551 and #552) or GMP grade (for HIV pos Donors #553, #572, #573, IEQR#2, IEQR#3) lentiviral vector HIV:41BB:ζ / EGFRt-epHIV7 transduced at MOI 3 IFN-γ recovered + Cells (~1×10 6 ). Fresh medium and cytokines were added every other day for ~15 days. posDuring the expansion of #573, IEQR#2 and IEQR#3 derived CMV-HIV CAR T cells, antiretroviral drugs (43 nM darunavir and 279 nM enfuvirtide) were added twice a week. The cultures were maintained at 37°C, 5% (v / v) CO2.

[0213] Cell lines

[0214] 8E5 cells contain a single defective proviral genome of HIV and are therefore non-infectious, but express most 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). To generate an 8E5 cell line expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (ffLuc), 8E5 cells were transduced with a lentiviral vector encoding eGFP-ffLuc, and GFP was expressed in the 8E5 cell line. + gp120 + Cell sorting and expansion. Acute myeloid leukemia (AML) cell line KG-1a (CCL-246.1) cells were purchased from ATCC and maintained in 10% FCSIMDM medium and used as negative target cells. LCL-OKT3 cells were generated as previously described and used as positive T cell stimulators 23 Cells were grown in complete medium supplemented with 0.4 mg / mL hygromycin. eGFP + Jurkat cells with HIV NL4-3 Infected and maintained in culture medium for 2 weeks.By stably transfecting HEK-293T cell line with C97ZA012 gp160 construct to express cell surface gp160 (finally cut into gp120 and gp41 HIV-1 envelope protein), HEK293-eGFP-ffLuc-gp160 cells were obtained and used as positive control cell lines for tissue cross-reactivity studies.All cell line libraries were verified by flow cytometry for the expression of expected antigens / markers before cryopreservation, and the thawed cells were cultured for less than 3 months before being used for determination.

[0215] Cytotoxicity assay

[0216] CAR T cell products (2.5×10 5) and eGFP-positive target cells (8E5-gp120, 8E5, LCL-OKT3, or KG-1a) were co-cultured for 4 days at different effector cell to target cell (E:T) ratios (2:1, 1:1, 1:2, or 1:5) of total T cells: target cells. Co-cultures with LCL-OKT3 and 8E5 or KG-1a were used as positive and negative target controls. Cells were stained with anti-CD3 antibodies. Live eGFP was measured using multicolor flow cytometry + CD3 - Percentage of tumor cells. "% of cytotoxicity" was calculated as follows: 100% - (% of tumor cells remaining in the CAR T cell group / % of tumor cells remaining in the mock T or negative target group). eGFP + HIV NL4-3 -Infected Jurkat cells (2.5×10 5 ) were co-cultured with CMV-HIV CAR T cells at different E:T ratios (1:1, 1:2, 1:4) for 7 days. The cells were then stained with the viability dye eFluor450 (Miltenyi) for live eGFP + Flow cytometric analysis of cells.

[0217] Proliferation assay

[0218] CAR T cell products (2.5×10 5 ) with 0.5 μM CellTrace TM Violet dye labeled (CTV) and co-cultured with stimulated cells LCL-OKT3, 8E5-gp120 and KG-1a irradiated with 8,000 cGy or autologous CMVpp65 peptide pulsed PBMCs irradiated with 3,500 cGy for 8 days at a 1:1 ratio. Co-cultures with LCL-OKT3, KG-1a cells and culture medium were used as positive and negative controls. CD3 + and EGFR + The proliferation of the population was determined using multicolor flow cytometry.

[0219] Intracellular IFN-γ staining

[0220] CAR T cell products (10 5 ) were cultured in 96-well tissue culture plates with LCL-OKT3, 8E5-gp120 or KG-1a cells (10 5 ) overnight, or autologous PBMCs (10 5) overnight activation. The cell mixture was then stained with anti-CD8 antibody, anti-EGFR antibody cetuximab and streptavidin to analyze the surface expression of CD8 and CAR, respectively. 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 antibodies. Cells were then analyzed using multicolor flow cytometry on MACSQuant (Miltenyi Biotec Inc.).

[0221] Mouse

[0222] At the beginning of the study, male and female NOD.Cg-Prkdc were used at 3-5 weeks of age. scid Il2rgtm1Wjl / SzJ (NSG) mice (JAX stock number 005557) were studied. Mice were housed in groups on corn cob litter ("Bed-o"-Cobs 1 / 8 in., The Andersons, Maumee, OH) in individually ventilated cages (OptiCages, Animal Care Systems), and square nest mats and PVC tubes were provided for enrichment. Mice were allowed free access to rodent food (LabDiet 5350) and acidified reverse osmosis purified water (pH 2.4 to 2.8) sterilized in bottles. After inoculation with HIV, mice were housed under animal biosafety level-2 (ABSL-2) conditions and grouped in static disposable cages (Innocage, Innovive). Room temperature was maintained in the range of 68°F to 79°F, and indoor humidity ranged from 30% to 70%. Mice were designated as specific pathogen free (SPF) for the following pathogens: mouse rotavirus, Sendai virus, pneumonia virus of mice, mouse hepatitis virus, minute virus of mice, parvovirus of mice, Theiler's murine encephalomyelitis virus, mouse reovirus type 3, mouse norovirus, lymphocytic choriomeningitis virus, mouse thymomavirus, mouse adenovirus type 1 and type 2, mouse cytomegalovirus, polyomavirus, K virus, mousepox virus, Hantavirus, Prospect Hill virus, rodent mycobacterium, rabbit encephalitis protozoa and mycoplasma, Helicobacter spp., Clostridium difficile, and free of any endoparasites and ectoparasites. Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals in a facility accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). All experiments were performed in accordance with the guidelines of the Institutional Animal Committee (IACUC 16095) of the Beckman Research Institute at City of Hope.

[0223] Transplantation and HIV challenge of hu-PBMC-NSG mice

[0224] From HIV neg or HIVpos PBMCs were collected from donors for the preparation of CMV-HIV CAR T cells. The CMV-negative fraction of autologous PBMCs was cryopreserved as control T cells. Before transplantation on day 0 of each mouse, CMV-negative PBMCs (1×10 6 ) and CMV-negative resting PBMC (9×10 6 ) were mixed. The cells (1×10 7 Each huCD45 mouse was resuspended in sterile saline and injected intraperitoneally (IP) into NSG mice. Prior to treatment, mice were randomized to ensure similar engraftment and sex ratios between groups. On day 7, mice were challenged with HIV-1BaL via IP injection. Longitudinal blood collection was performed using retro-orbital bleeding on anesthetized mice, and peripheral blood cell populations and plasma viral load were regularly analyzed using flow cytometry and qRT-PCR. + Cells (defined as >30 cells / μL huCD45 + Mice with GHVD-positive cells were excluded from the analysis. Mice showing severe GHVD symptoms were immediately humanely euthanized.

[0225] Oral ART

[0226] Infected mice with detectable viral infection (defined as HIV >10 3 cp / mL) were treated orally for 3 weeks with ART containing drugs that block new infections without suppressing viral production in infected cells. [tenofovir disoproxil fumarate (TDF; 300 mg / tablet), emtricitabine (FTC; 200 mg / tablet) (Gilead Sciences)] and [Raltegravir (RAL; 400 mg / tablet) (Merck)], scaled down to equivalent mouse doses using appropriate conversion factors, was administered in drinking water formulations (sweetened hydrogel, Sucralose, ClearH20) is administered. For 400 mL 1 / 2 Truvada tablet and 1 / 2 Isentress tablet were crushed into powder and mixed into a homogenous solution by shake bottle; the drug-containing water was changed once a week. The dosage of ART drugs was calculated based on previous studies using the same delivery system 65 .

[0227] CMV-HIV CAR T cells and CMVpp65 stimulation in vivo

[0228] Mice received CMV-HIV CAR T cells (0.05 to 1×10 6 EGFR+T cells), CMV-negative T cells (1×10 6); or autologous PBMCs as control T cells were injected retro-orbitally under general isoflurane anesthesia. Autologous PBMCs were pulsed with CMVpp65 peptide mixture (#PM-PP65, JPT Peptide Technologies, Germany) as antigen presenting cells (APC) as "CMVpp65 vaccine". When indicated, mice received CMVpp65 peptide-pulsed and irradiated (3500 rad) autologous CMV-negative PBMCs (5×10 6 ).

[0229] Antibody

[0230] 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, CA). The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: anti-HIV-1 gp120 monoclonal antibody (VRC01) from Dr. John Mascola (Catalog No. 12033). Biotinylated anti-EGFR antibody (Cetuximab) was obtained from City of Hope Pharmacy. Antibodies against EGFR were obtained from eBioscience (San Diego, CA). CellTrace TM Contrast Violet dye (CTV) was purchased from Invitrogen (Carlsbad, CA). All monoclonal antibodies and CTV were used according to the manufacturer's instructions.

[0231] Reagents

[0232] CliniMACS TS500 piping set, MACS GMP HCMV pp65, CCS reagent, CliniMACS PBS / EDTA buffer, and TexMACS TMGMP culture media were all purchased from Miltenyi Biotec. CliniMACS PBS / EDTA (HSA; Grifols Therapeutics, Los Angeles, CA) containing 2.5% human serum albumin 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. GmbH for pulsing on 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 No. 11447), and enfuvirtide (Fuzeon, Genentech) was reconstituted in water.

[0233] Synthesis of N6 scFv-Fc

[0234] The anti-gp120 N6 monoclonal antibody (mAb) variable domains were reformatted into recombinant single-chain scFv-Fc antibody fragments. The cDNA encoding the N6 variable light chain and heavy chain domains (VL-linker-VH-direction) was synthesized with a (Gly4Ser)3 linker and fused to the IgG4 Fc domain. In brief, the scFv-Fc of N6 was cloned into the Lonza pEE12.4 vector and transiently transfected using the EXPI293 expression system. The culture was then clarified by centrifugation (1,000×g, 5 minutes) followed by 0.22μm sterile filtration. The clarified harvest was treated with Ag1-x8 strong anion exchange resin overnight and affinity purified by protein A chromatography (ProSep vA high capacity resin, EMD Millipore). Containing N6 scFv-Fc (V L -V H ) was dialyzed against PBS buffer using a Slide-A-Lyzer 20k MWCO cassette. The final dialyzed sample was sterile filtered using a 0.22 μm PES filter and stored at 4° C. The expression of the test agent was determined by SDS-PAGE and ELISA assays.

[0235] Tissue Cross-Reactivity Analysis

[0236] Charles River Laboratories, Inc. conducted cross-reactivity studies of N6 scFv-Fc. First, N6 scFv-Fc was tested for specific reactivity to the positive control (gp160-transfected HEK293T cells expressing gp120) and the negative control parental HEK293T cells (gp120 negative) at 5 μg / mL and 15 μg / mL. The test article was replaced with a human IgG4κ antibody, named HuIgG4 (control article), and other controls (assay controls) were generated by omitting the test article or control article from the assay. The tissue panel used as a test system for in vitro cross-reactivity studies included all tissues recommended in the FDA Center for Biologics Evaluation and Research (CBER) document Points to Consider in the Manufacture and Testing of Monoclonal Antibody Productsfor Human Use. Fresh, unfixed tissues were collected as surgical or autopsy specimens from humans and frozen at -85-70°C in In OCT. Sections were cut at approximately 5 μm and fixed in acetone for 10 minutes at room temperature. Just before staining, slides were fixed in 10% neutral buffered formalin (NBF) for 10 seconds at room temperature. The labeled secondary antibody was allowed to specifically attach to the unlabeled primary antibody (for 5 μg / mL and 15 μg / mL of the test or control) by incubating the primary / secondary antibody mixture overnight. The test or control was mixed with a biotinylated F(ab')2 donkey anti-human IgG, Fcγ fragment specific (DkαHuIgG) antibody at a concentration that achieved a primary antibody:secondary antibody ratio of 1:1.5. The pre-complexed antibodies were incubated overnight at 2°C to 8°C. Before using the antibodies the next day, human gamma globulin was added to each vial to a final concentration of 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°C to 8°C for at least 2 hours. On the day of staining, the slides were rinsed twice with Tris buffered saline, 0.15M NaCl, pH7.6 (TBS). Then, 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. The slides were then treated with protein blockers (TBS + 1% bovine serum albumin (BSA); 0.5% casein; and 1.5% normal donkey serum) designed to reduce nonspecific binding for 20 minutes. After protein blocking, the pre-complexed primary and secondary antibodies were applied to the slides for 2 hours. Next, the slides were rinsed twice with TBS, and then endogenous peroxidase was quenched by incubating the slides with Dako peroxidase blocking reagent for 5 minutes. Next, the slides were rinsed twice with TBS, treated with ABC Elite reagent for 30 minutes, rinsed twice with TBS, and then treated with DAB as a substrate for peroxidase reaction for 4 minutes. All slides were rinsed with tap water, counterstained, dehydrated and fixed. TBS + 1% BSA was used as a diluent for all antibodies and ABC reagents. Using a polyclonal rabbit antibody directed against human β2-microglobulin, individual frozen sections from each human test tissue were stained in parallel for the expression of human β2-microglobulin (a relatively ubiquitous epitope). All human test tissues evaluated were positive for β2 microglobulin staining, indicating that they were suitable for cross-reactivity assessment. After staining, the slides were observed and evaluated by a pathologist under an optical microscope.

[0237] Flow cytometry

[0238] Cells were stained with optimized antibody panels for 20 min at 4°C and then washed twice with PBS. Data acquisition for all experiments involving flow cytometry was performed on a MACSquant (Miltenyi Biotec) and analyzed using FCS Express V7 (De Novo Software, Glendale, CA).

[0239] Under general anesthesia, peripheral blood samples were collected by retroorbital bleeding and stained for 30 minutes with anti-human CD3 conjugated by BV711, anti-human CD4 conjugated by APC, anti-human CD8 conjugated by BB515, anti-human CD45 conjugated by BUV395 (BD Biosciences, San Jose, CA) and anti-human EGFR conjugated by BV421 (Biolegend, San Diego, CA). The 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 BD Fortessa II instrument (BD Biosciences) and analyzed using FlowJo software (TreeStar, BD before).

[0240] Tissue samples were collected at autopsy and immediately processed for cell isolation and flow cytometry analysis. Bone marrow mononuclear cell suspensions were first stained with amine-binding dyes for dead cell exclusion (Biolegend), then stained with anti-human-CD3 (BD ​​clone UCHT1), anti-EGFR (Miltenyi biotinylated clone REA688), anti-CD4 (Biolegend clone RPA-T4), anti-human CD8 (BD clone RPA-T8), anti-CD62L (Biolegend clone DREG-56) and anti-CD27 (Biolegend clone M-T271) in brilliant staining buffer (BD) containing 0.5% human serum albumin and 0.5% gamma globulin. Primary EGFR antibody staining was completed with streptavidin conjugate (eBioscience) and fixed in 4% PFA. Samples were collected on a BD Fortessa SORP cytometer the next day. Data were analyzed using FlowJo software (previously TreeStar by BD). Cell doublets and dead cells were excluded before evaluating T cell lineage and phenotypic markers.

[0241] Intracellular HIV p24 staining

[0242] Collect samples of single cell suspensions from peripheral blood and mouse bone marrow (femur + / - tibia) at the time of euthanasia. Prepare single cell suspensions according to previously established protocols. 1. Briefly, for bone marrow cells, femurs and tibias were dissected and collected from euthanized mice and placed in ice-cold PBS. The bones were thoroughly cleaned to remove all connective and muscle tissue, and the heads of the bones were then removed using a scalpel blade. The bones were placed in a 0.5 mL microcentrifuge tube with a premade hole using a 20 g needle. The bones were placed cut side down and the 0.5 mL tube was placed in a 1.5 mL microcentrifuge tube and centrifuged at >10,000×g for 15 sec. The cell pellet was 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 was used according to the manufacturer's protocol. TM Kit (BD Biosciences, San Jose, CA). After surface marker staining (CD45, CD3, CD4, CD8, EGFR), cells were permeabilized and intracellularly stained with KC57-FITC monoclonal antibody Fortessa II instrument (BD Biosciences) and analyzed with FlowJo software (formerly TreeStar by BD).

[0243] ELISA assay

[0244] Quantification of HIV-1 p24 was measured on supernatants according to the manufacturer's instructions (Alliance ELISA; Perkin-Elmer Life Sciences, Boston, MA), with an assay lower limit of quantitation (LLOQ) of 12.5 pg / mL.

[0245] Plasma HIV qRT-PCR

[0246] Plasma viremia was measured using one-step reverse transcriptase real-time PCR [TaqMan assay] and the automated CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad). The qPCR primer sets were taken from previously published studies. 2HIV-1 levels in peripheral blood were determined by extracting RNA from plasma using the QIAamp Viral RNA mini kit (Qiagen) and performing Taqman qPCR using primers and probe sets targeting the HIV-1 LTR region [F primer: GCCTCAATAAAGCTTGCCTTGA, R primer: GGCGCCACTGCTAGAGATTTT, probe: 5'FAM / AAGTAGTGTGTGCCCGTCTGTTGTGTGACT / 3IABkFQ] or the HIV-1 Pol region [F primer: GACTGTAGTCCAGGAATATG, R primer: TGTTTCCTGCCCTGTCTC, probe: 5'Cy5 / CTTGGTAGCAGTTCATGTAGCCAG / 3'IABkFQ] using TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). The protocol was designed to purify viral RNA from a minimum of 140 μL of 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 typically about 40 copies / mL when using viral RNA isolated from 140 μL plasma samples. In this animal study, plasma samples were amplified by dilution (typically 1 in 3 dilutions) because only a limited volume of plasma (20-40 μL) was available. Under the experimental conditions, the LOD for diluted samples was ~2,000 RNA copies / mL using HIV LTR primers and ~500 RNA copies / mL using HIV Pol primers. Therefore, values ​​below these LOD values ​​are defined as undetectable.

[0247] Statistical analysis

[0248] Analyses were performed using Prism (GraphPad Software Inc.) or R version 4.0.266 and are described in separate figure legends. The significance level for all analyses was 0.05.

[0249] 1.Günthard,H.F.,Saag,M.S.,Benson,C.A.,del Rio,C.,Eron,J.J.,Gallant,J.E.,Hoy,J.F.,Mugavero,M.J.,Sax,P.E.,Thompson,M.A.,Gandhi,R.T.,et al.(2016).Antiretroviral Drugs for Treatment and Prevention ofHIV Infection in Adults:2016Recommendations of the International Antiviral Society-USA Panel.Jama316,191-210.10.1001 / jama.2016.8900.

[0250] 2.Sadowski,I.,and Hashemi,F.B.(2019).Strategies to eradicate HIV frominfected patients:elimination of latent provirus reservoirs.Cellular andmolecular life sciences:CMLS 76,3583-3600.10.1007 / s00018-019-03156-8.

[0251] 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 ofuniversal receptor-modified bone marrow into SCID mice.The Journal ofexperimental medicine 184,2261-2269.10.1084 / jem.184.6.2261.

[0252] 4.Wagner,T.A.(2018).Quarter Century of Anti-HIV CAR T Cells.CurrentHIV / AIDS reports 15,147-154.10.1007 / s11904-018-0388-x.

[0253] 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-SpecificT Cells Can Be Generated against Non-escaped T Cell Epitopes with a GMP-Compliant Manufacturing Platform.Molecular therapy.Methods&clinicaldevelopment 16,11-20.10.1016 / j.omtm.2019.10.001.

[0254] 6.Romeo,C.,and Seed,B.(1991).Cellular immunity to HIV activated byCD4fused to T cell or Fc receptor polypeptides.Cell 64,1037-1046.

[0255] 7.Roberts,M.R.,Qin,L.,Zhang,D.,Smith,D.H.,Tran,A.,Duil,T.J.,Groopman,J.E.,Capon,D.J.,Byrn,R.A.,and Finer,M.H.(1994).Targeting of HumanImmunodeficiency Virus-Infected Cells by CD8+T Lymphocytes Armed WithUniversal T-Cells Receptors.Blood 84,2878-2889.

[0256] 8.Mitsuyasu,R.T.,Anton,P.A.,Deeks,S.G.,Scadden,D.T.,Connick,E.,Downs,M.T.,Bakker,A.,Roberts,M.R.,June,C.H.,Jalali,S.,Lin,A.A.,et al.(2000).Prolonged survival and tissue trafficking following adoptive transfer ofCD4zeta gene-modified autologous CD4(+)and CD8(+)T cells in humanimmunodeficiency virus-infected subjects.Blood 96,785-793.

[0257] 9.Deeks,S.G.,Wagner,B.,Anton,P.A.,Mitsuyasu,R.T.,Scadden,D.T.,Huang,C.,Macken,C.,Richman,D.D.,Christopherson,C.,June,C.H.,Lazar,R.,et al.(2002).Aphase II randomized study ofHIV-specific T-cell gene therapy in subjects withundetectable plasma viremia on combination antiretroviral therapy.Moleculartherapy:the journal of the American Society of Gene Therapy 5,788-797.

[0258] 10.Walker,R.E.,Bechtel,C.M.,Natarajan,V.,Baseler,M.,Hege,K.M.,Metcalf,J.A.,Stevens,R.,Hazen,A.,Blaese,R.M.,Chen,C.C.,Leitman,S.F.,et al.(2000).Long-term in vivo survival of receptor-modified syngeneic T cells inpatients with human immunodeficiency virus infection.Blood 96,467-474.

[0259] 11.Scholler,J.,Brady,T.L.,Binder-Scholl,G.,Hwang,W.T.,Plesa,G.,Hege,K.M.,Vogel,A.N.,Kalos,M.,Riley,J.L.,Deeks,S.G.,Mitsuyasu,R.T.,et al.(2012).Decade-long safety and function of retroviral-modified chimeric antigenreceptor T cells.Science translational medicine 4,132ra153.10.1126 / scitranslmed.3003761.

[0260] 12.van der Stegen,S.J.,Hamieh,M.,and Sadelain,M.(2015).Thepharmacology of second-generation chimeric antigen receptors.Nat Rev DrugDiscov 14,499-509.10.1038 / nrd4597.

[0261] 13.Kuhlmann,A.S.,Peterson,C.W.,and Kiem,H.P.(2018).Chimeric antigenreceptor T-cell approaches to HIV cure.Current opinion in HIV andAIDS.10.1097 / COH.0000000000000485.

[0262] 14.Ying,Z.,He,T.,Wang,X.,Zheng,W.,Lin,N.,Tu,M.,Xie,Y.,Ping,L.,Zhang,C.,Liu,W.,Deng,L.,et al.(2019).Parallel Comparison of 4-1BB or CD28 Co-stimulated CD19-Targeted CAR-T Cells for B Cell Non-Hodgkin'sLymphoma.Molecular therapy oncolytics 15,60-68.10.1016 / j.omto.2019.08.002.

[0263] 15.Zhong,Q.,Zhu,Y.M.,Zheng,L.L.,Shen,H.J.,Ou,R.M.,Liu,Z.,She,Y.L.,Chen,R.,Li,C.,Huang,J.,Yao,M.D.,et al.(2018).Chimeric Antigen Receptor-TCells with 4-1BB Co-Stimulatory Domain Present a Superior Treatment Outcomethan Those with CD28 Domain Based on Bioinformatics.Acta haematologica 140,131-140.10.1159 / 000492146.

[0264] 16.Hale,M.,Mesojednik,T.,Romano Ibarra,G.S.,Sahni,J.,Bernard,A.,Sommer,K.,Scharenberg,A.M.,Rawlings,D.J.,and Wagner,T.A.(2017).EngineeringHIV-Resistant,Anti-HIV Chimeric Antigen Receptor T Cells.Molecular therapy:the journal of the American Society of Gene Therapy25,570-579.10.1016 / j.ymthe.2016.12.023.

[0265] 17.Mu,W.,Carrillo,M.A.,and Kitchen,S.G.(2020).Engineering CAR T Cellsto Target the HIV Reservoir.Frontiers in cellular and infection microbiology10,410.10.3389 / fcimb.2020.00410.

[0266] 18.Huang,J.,Kang,B.H.,Ishida,E.,Zhou,T.,Griesman,T.,Sheng,Z.,Wu,F.,Doria-Rose,N.A.,Zhang,B.,McKee,K.,O'Dell,S.,et al.(2016).Identification of aCD4-Binding-Site Antibody to HIV that Evolved Near-Pan NeutralizationBreadth.Immunity 45,1108-1121.10.1016 / j.immuni.2016.10.027.

[0267] 19.Lapteva,N.,Gilbert,M.,Diaconu,I.,Rollins,L.A.,Al-Sabbagh,M.,Naik,S.,Krance,R.A.,Tripic,T.,Hiregange,M.,Raghavan,D.,Dakhova,O.,et al.(2019).T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19Chimeric Antigen Receptor-Expressing T Cells.Clin Cancer Res 25,7340-7350.10.1158 / 1078-0432.ccr-18-3199.

[0268] 20.Pule,M.A.,Savoldo,B.,Myers,G.D.,Rossig,C.,Russell,H.V.,Dotti,G.,Huls,M.H.,Liu,E.,Gee,A.P.,Mei,Z.,Yvon,E.,et al.(2008).Virus-specific T cellsengineered to coexpress tumor-specific receptors:persistence and antitumoractivity in individuals with neuroblastoma.Nat Med 14,1264-1270.10.1038 / nm.1882.

[0269] 21.Savoldo,B.,Rooney,C.M.,Di Stasi,A.,Abken,H.,Hombach,A.,Foster,A.E.,Zhang,L.,Heslop,H.E.,Brenner,M.K.,and Dotti,G.(2007).Epstein Barr virusspecific cytotoxic T lymphocytes expressing the anti-CD30zeta artificialchimeric T-cell receptor for immunotherapy ofHodgkin disease.Blood 110,2620-2630.10.1182 / blood-2006-11-059139.

[0270] 22.Cooper,L.J.,Al-Kadhimi,Z.,Serrano,L.M.,Pfeiffer,T.,Olivares,S.,Castro,A.,Chang,W.C.,Gonzalez,S.,Smith,D.,Forman,S.J.,and Jensen,M.C.(2005).Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specificCTLs by cotransfer of T cells modified to present influenza MP1.Blood 105,1622-1631.10.1182 / blood-2004-03-1208.

[0271] 23.Wang,X.,Wong,C.W.,Urak,R.,Mardiros,A.,Budde,L.E.,Chang,W.C.,Thomas,S.H.,Brown,C.E.,La Rosa,C.,Diamond,D.J.,Jensen,M.C.,et al.(2015).CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively TransferredCD19-Redirected CMV-Specific T Cells.Clinical cancer research:an officialjournal of the American Association for Cancer Research.10.1158 / 1078-0432.ccr-14-2920.

[0272] 24.Jonnalagadda,M.,Mardiros,A.,Urak,R.,Wang,X.,Hoffman,L.J.,Bernanke,A.,Chang,W.C.,Bretzlaff,W.,Starr,R.,Priceman,S.,Ostberg,J.R.,et al.(2015).Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptorbinding and improve T cell persistence and antitumor efficacy.Moleculartherapy:the journal of the American Society of Gene Therapy 23,757-768.10.1038 / mt.2014.208.

[0273] 25.Philipson,B.I.,O'Connor,R.S.,May,M.J.,June,C.H.,Albelda,S.M.,andMilone,M.C.(2020).4-1BB costimulation promotes CAR T cell survival throughnoncanonical NF-κB signaling.Science signaling 13.10.1126 / scisignal.aay8248.

[0274] 26.Frigault,M.J.,Lee,J.,Basil,M.C.,Carpenito,C.,Motohashi,S.,Scholler,J.,Kawalekar,O.U.,Guedan,S.,McGettigan,S.E.,Posey,A.D.,Jr.,Ang,S.,etal.(2015).Identification of chimeric antigen receptors that mediateconstitutive or inducible proliferation of T cells.Cancer immunology research3,356-367.10.1158 / 2326-6066.CIR-14-0186.

[0275] 27.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 Tcells in patients with relapsed or chemotherapy refractory B-cellleukemias.Blood 118,4817-4828.10.1182 / blood-2011-04-348540.

[0276] 28.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).Chimericantigen receptor T cells persist and induce sustained remissions in relapsedrefractory chronic lymphocytic leukemia.Science translational medicine 7,303ra139.10.1126 / scitranslmed.aac5415.

[0277] 29.Leibman,R.S.,Richardson,M.W.,Ellebrecht,C.T.,Maldini,C.R.,Glover,J.A.,Secreto,A.J.,Kulikovskaya,I.,Lacey,S.F.,Akkina,S.R.,Yi,Y.,Shaheen,F.,etal.(2017).Supraphysiologic control over HIV-1 replication mediated by CD8 Tcells expressing a re-engineered CD4-based chimeric antigen receptor.PLoSpathogens 13,e1006613.10.1371 / journal.ppat.1006613.

[0278] 30.Wang,X.,Chang,W.C.,Wong,C.W.,Colcher,D.,Sherman,M.,Ostberg,J.R.,Forman,S.J.,Riddell,S.R.,and Jensen,M.C.(2011).A transgene-encoded cellsurface polypeptide for selection,in vivo tracking,and ablation of engineeredcells.Blood 118,1255-1263.10.1182 / blood-2011-02-337360.

[0279] 31.Hall,W.C.,Price-Schiavi,S.A.,Wicks,J.,and Rojko,J.L.(2008).TissueCross-Reactivity Studies for Monoclonal Antibodies:Predictive Value and Usefor Selection ofRelevant Animal Species for Toxicity Testing.

[0280] 32.Leach,M.W.,Halpern,W.G.,Johnson,C.W.,Rojko,J.L.,MacLachlan,T.K.,Chan,C.M.,Galbreath,E.J.,Ndifor,A.M.,Blanset,D.L.,Polack,E.,and Cavagnaro,J.A.(2010).Use of tissue cross-reactivity studies in the development ofantibody-based biopharmaceuticals:history,experience,methodology,and futuredirections.Toxicologic pathology 38,1138-1166.10.1177 / 0192623310382559.

[0281] 33.Kumaresan,P.,Figliola,M.,Moyes,J.S.,Huls,M.H.,Tewari,P.,Shpall,E.J.,Champlin,R.,and Cooper,L.J.(2015).Automated Cell Enrichment ofCytomegalovirus-specific T cells for Clinical Applications using theCytokine-capture System.Journal of visualized experiments:JoVE.10.3791 / 52808.

[0282] 34.Wang,X.,Urak,R.,Walter,M.,Guan,M.,Han,T.,Vyas,V.,Chien,S.H.,Gittins,B.,Clark,M.C.,Mokhtari,S.,Cardoso,A.,et al.(2022).Large-scalemanufacturing and characterization of CMV-CD19CAR T cells.Journal forimmunotherapy of cancer 10.10.1136 / jitc-2021-003461.

[0283] 35.Naeger,D.M.,Martin,J.N.,Sinclair,E.,Hunt,P.W.,Bangsberg,D.R.,Hecht,F.,Hsue,P.,McCune,J.M.,and Deeks,S.G.(2010).Cytomegalovirus-specific Tcells persist at very high levels during long-term antiretroviral treatmentof HIV disease.PloS one 5,e8886.10.1371 / journal.pone.0008886.

[0284] 36.Stone,S.F.,Price,P.,Khan,N.,Moss,P.A.,and French,M.A.(2005).HIVpatients on antiretroviral therapy have high frequencies of CD8 T cellsspecific for Immediate Early protein-1 of cytomegalovirus.AIDS 19,555-562.

[0285] 37.Okhrimenko,A.,Grun,J.R.,Westendorf,K.,Fang,Z.,Reinke,S.,von Roth,P.,Wassilew,G.,Kuhl,A.A.,Kudernatsch,R.,Demski,S.,Scheibenbogen,C.,et al.(2014).Human memory T cells from the bone marrow are resting and maintainlong-lasting systemic memory.Proceedings of the National Academy of Sciencesof the United States of America 111,9229-9234.10.1073 / pnas.1318731111.

[0286] 38.Walker,B.,and McMichael,A.(2012).The T-cell response to HIV.ColdSpring Harbor perspectives in medicine 2.10.1101 / cshperspect.a007054.

[0287] 39.Okulicz,J.F.,and Lambotte,O.(2011).Epidemiology and clinicalcharacteristics of elite controllers.Current opinion in HIV and AIDS 6,163-168.10.1097 / COH.0b013e328344f35e.

[0288] 40.Loffredo,J.T.,Sidney,J.,Bean,A.T.,Beal,D.R.,Bardet,W.,Wahl,A.,Hawkins,O.E.,Piaskowski,S.,Wilson,N.A.,Hildebrand,W.H.,Watkins,D.I.,et al.(2009).Two MHC class I molecules associated with elite control ofimmunodeficiency virus replication,Mamu-B*08 and HLA-B*2705,bind peptideswith sequence similarity.Journal of immunology(Baltimore,Md.:1950)182,7763-7775.10.4049 / jimmunol.0900111.

[0289] 41.Saez-Cirion,A.,Lacabaratz,C.,Lambotte,O.,Versmisse,P.,Urrutia,A.,Boufassa,F.,Barre-Sinoussi,F.,Delfraissy,J.F.,Sinet,M.,Pancino,G.,and Venet,A.(2007).HIV controllers exhibit potent CD8 T cell capacity to suppress HIVinfection ex vivo and peculiar cytotoxic T lymphocyte activationphenotype.Proceedings of the National Academy of Sciences of the UnitedStates of America 104,6776-6781.

[0290] 42.Chomont,N.,El-Far,M.,Ancuta,P.,Trautmann,L.,Procopio,F.A.,Yassine-Diab,B.,Boucher,G.,Boulassel,M.R.,Ghattas,G.,Brenchley,J.M.,Schacker,T.W.,etal.(2009).HIV reservoir size and persistence are driven by T cell survivaland homeostatic proliferation.Nature Medicine 15,893-900.nm.1972[pii]

[0291] 43.Jiang,C.,Lian,X.,Gao,C.,Sun,X.,Einkauf,K.B.,Chevalier,J.M.,Chen,S.M.Y.,Hua,S.,Rhee,B.,Chang,K.,Blackmer,J.E.,et al.(2020).Distinct viralreservoirs in individuals with spontaneous control of HIV-1.Nature585,261-267.10.1038 / s41586-020-2651-8.

[0292] 44.Chomont,N.(2020).HIV enters deep sleep in people who naturallycontrol the virus.Nature 585,190-191.10.1038 / d41586-020-02438-7.

[0293] 45.Hua,C.K.,and Ackerman,M.E.(2016).Engineering broadly neutralizingantibodies for HIV prevention and therapy.Advanced drug delivery reviews103,157-173.10.1016 / j.addr.2016.01.013.

[0294] 46.Rust,B.J.,Kean,L.S.,Colonna,L.,Brandenstein,K.E.,Poole,N.H.,Obenza,W.,Enstrom,M.R.,Maldini,C.R.,Ellis,G.I.,Fennessey,C.M.,Huang,M.L.,etal.(2020).Robust expansion of HIV CAR T cells following antigen boosting inART-suppressed nonhuman primates.Blood 136,1722-1734.10.1182 / blood.2020006372.

[0295] 47.Hebeis,B.J.,Klenovsek,K.,Rohwer,P.,Ritter,U.,Schneider,A.,Mach,M.,and Winkler,T.H.(2004).Activation of virus-specific memory B cells in theabsence of T cell help.The Journal of experimental medicine 199,593-602.10.1084 / jem.20030091.

[0296] 48.Wang,X.,Wong,C.W.,Urak,R.,Mardiros,A.,Budde,L.E.,Chang,W.C.,Thomas,S.H.,Brown,C.E.,La Rosa,C.,Diamond,D.J.,Jensen,M.C.,et al.(2015).CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively TransferredCD19-Redirected CMV-Specific T Cells.Clinical cancer research:an officialjournal of the American Association for Cancer Research 21,2993-3002.10.1158 / 1078-0432.Ccr-14-2920.

[0297] 49.Holguin,L.,Echavarria,L.,and Burnett,J.C.(2022).Novel HumanizedPeripheral Blood Mononuclear Cell Mouse Model with Delayed Onset of Graft-versus-Host Disease for Preclinical HIV Research.J Virol 96,e0139421.10.1128 / jvi.01394-21.

[0298] 50.Pampusch,M.S.,Abdelaal,H.M.,Cartwright,E.K.,Molden,J.S.,Davey,B.C.,Sauve,J.D.,Sevcik,E.N.,Rendahl,A.K.,Rakasz,E.G.,Connick,E.,Berger,E.A.,et al.(2022).CAR / CXCR5-T cell immunotherapy is safe and potentiallyefficacious in promoting sustained remission of SIV infection.PLoS pathogens18,e1009831.10.1371 / journal.ppat.1009831.

[0299] 51.Zhen,A.,Carrillo,M.A.,Mu,W.,Rezek,V.,Martin,H.,Hamid,P.,Chen,I.S.Y.,Yang,O.O.,Zack,J.A.,and Kitchen,S.G.(2021).Robust CAR-T memoryformation and function via hematopoietic stem cell delivery.PLoS pathogens17,e1009404.10.1371 / journal.ppat.1009404.

[0300] 52.Maldini,C.R.,Gayout,K.,Leibman,R.S.,Dopkin,D.L.,Mills,J.P.,Shan,X.,Glover,J.A.,and Riley,J.L.(2020).HIV-Resistant and HIV-Specific CAR-Modified CD4(+)T Cells Mitigate HIV Disease Progression and Confer CD4(+)TCell Help In Vivo.Molecular therapy:the journal of the American Society ofGene Therapy 28,1585-1599.10.1016 / j.ymthe.2020.05.012.

[0301] 53.Maldini,C.R.,Claiborne,D.T.,Okawa,K.,Chen,T.,Dopkin,D.L.,Shan,X.,Power,K.A.,Trifonova,R.T.,Krupp,K.,Phelps,M.,Vrbanac,V.D.,et al.(2020).DualCD4-based CAR T cells with distinct costimulatory domains mitigate HIVpathogenesis in vivo.Nat Med 26,1776-1787.10.1038 / s41591-020-1039-5.

[0302] 54.Julg,B.,Pegu,A.,Abbink,P.,Liu,J.,Brinkman,A.,Molloy,K.,Mojta,S.,Chandrashekar,A.,Callow,K.,Wang,K.,Chen,X.,et al.(2017).Virological Controlby the CD4-Binding Site Antibody N6 in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys.J Virol 91.10.1128 / JVI.00498-17.

[0303] 55.Anthony-Gonda,K.,Bardhi,A.,Ray,A.,Flerin,N.,Li,M.,Chen,W.,Ochsenbauer,C.,Kappes,J.C.,Krueger,W.,Worden,A.,Schneider,D.,et al.(2019).Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviralactivity and potent in vivo elimination ofHIV-infected cells in a humanizedmouse model.Science translational medicine 11.10.1126 / scitranslmed.aav5685.

[0304] 56.de Larrea,C.F.,Staehr,M.,Lopez,A.V.,Ng,K.Y.,Chen,Y.,Godfrey,W.D.,Purdon,T.J.,Ponomarev,V.,Wendel,H.G.,Brentjens,R.J.,and Smith,E.L.(2020).Defining an Optimal Dual-Targeted CAR T-cell Therapy Approach SimultaneouslyTargeting BCMA and GPRC5D to Prevent BCMA Escape-Driven Relapse in MultipleMyeloma.Blood Cancer Discov 1,146-154.10.1158 / 2643-3230.bcd-20-0020.

[0305] 57.Qin,H.,Ramakrishna,S.,Nguyen,S.,Fountaine,T.J.,Ponduri,A.,Stetler-Stevenson,M.,Yuan,C.M.,Haso,W.,Shern,J.F.,Shah,N.N.,and Fry,T.J.(2018).Preclinical Development of Bivalent Chimeric Antigen Receptors TargetingBoth CD19 and CD22.Mol Ther Oncolytics 11,127-137.10.1016 / j.omto.2018.10.006.

[0306] 58.Kang,L.,Zhang,J.,Li,M.,Xu,N.,Qi,W.,Tan,J.,Lou,X.,Yu,Z.,Sun,J.,Wang,Z.,Fu,C.,et al.(2020).Characterization of novel dual tandem CD19 / BCMAchimeric antigen receptor T cells to potentially treat multiplemyeloma.Biomark Res 8,14.10.1186 / s40364-020-00192-6.

[0307] 59.Zah,E.,Nam,E.,Bhuvan,V.,Tran,U.,Ji,B.Y.,Gosliner,S.B.,Wang,X.,Brown,C.E.,and Chen,Y.Y.(2020).Systematically optimized BCMA / CS1 bispecificCAR-T cells robustly control heterogeneous multiple myeloma.Nat Commun 11,2283.10.1038 / s41467-020-16160-5.

[0308] 60.La Rosa,C.,Longmate,J.,Martinez,J.,Zhou,Q.,Kaltcheva,T.I.,Tsai,W.,Drake,J.,Carroll,M.,Wussow,F.,Chiuppesi,F.,Hardwick,N.,et al.(2017).MVAvaccine encoding CMV antigens safely induces durable expansion of CMV-specific T-cells in healthy adults.Blood.10.1182 / blood-2016-07-729756.

[0309] 61.Aldoss,I.,La Rosa,C.,Baden,L.R.,Longmate,J.,Ariza-Heredia,E.J.,Rida,W.N.,Lingaraju,C.R.,Zhou,Q.,Martinez,J.,Kaltcheva,T.,Dagis,A.,et al.(2020).Poxvirus Vectored Cytomegalovirus Vaccine to Prevent CytomegalovirusViremia in Transplant Recipients:A Phase 2,Randomized Clinical Trial.AnnIntern Med 172,306-316.10.7326 / M19-2511.

[0310] 62.Diamond,C.S.G.W.a.D.(A5355)Phase II,Double-Blind,Randomized,Placebo-Controlled Trial to Evaluate the Safety and Immunogenicity of aModified Vaccinia Ankara(MVA)-based anti-Cytomegalovirus(CMV)Vaccine in Adults with Both Human Immunodeficiency Virus(HIV)-1and CMV WhoAre on Potent Combination ART with Conserved Immune Function.NIAID.

[0311] 63.Kowolik,C.M.,Topp,M.S.,Gonzalez,S.,Pfeiffer,T.,Olivares,S.,Gonzalez,N.,Smith,D.D.,Forman,S.J.,Jensen,M.C.,and Cooper,L.J.(2006).CD28costimulation provided through a CD19-specific chimeric antigen receptorenhances in vivo persistence and antitumor efficacy of adoptively transferredT cells.Cancer Res 66,10995-11004.

[0312] 64.Wang,X.,Li,H.,Matte-Martone,C.,Cui,W.,Li,N.,Tan,H.S.,Roopenian,D.,and Shlomchik,W.D.(2011).Mechanisms of antigen presentation to T cells inmurine graft-versus-host disease:cross-presentation and the appearance ofcross-presentation.Blood 118,6426-6437.10.1182 / blood-2011-06-358747.

[0313] 65.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 HumanizedNSG Mouse Model during Suppressive Oral Combinational AntiretroviralTherapy.J Virol 92.10.1128 / JVI.02118-17.

[0314] 66.R Core Team,RFfSC(2020).R:A Language and Environment for Statistical Computing.

[0315] Other Implementations

[0316] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: (i) scFv binding to HIV Env; (ii) a spacer domain; (iii) transmembrane domain; (iv) a co-stimulatory domain; and (v) CD3ζ signaling domain.

2. The nucleic acid molecule of claim 1, wherein the scFv comprises: a VL domain and a VH domain, the VL domain comprising: a light chain CDR1 comprising QTSQGVGSDLH, a light chain CDR2 comprising HTSSVED, and a light chain CDR3 comprising QVLQF; the VH domain comprising: a heavy chain CDR1 comprising AHILF, a heavy chain CDR2 comprising WIKPQYGAVNFGGGFRD, and a heavy chain CDR3 comprising DRSYGDSSWALDA.

3. The nucleic acid molecule of claim 1, wherein the scFV comprises: (a) a light chain variable domain that is at least 90%, 95% or 98% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK; and (b) a heavy chain variable domain that is at least 90%, 95% or 98% identical to RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA.

4. The nucleic acid molecule of claim 3, wherein the scFV comprises: a light chain variable domain comprising YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHT SSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK; and a heavy chain variable domain comprising RALVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVS.

5. The nucleic acid molecule of claim 3, wherein the scFv comprises a VL domain and a VH domain, wherein the VL domain is at least 95% identical to YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHT SSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK and includes the following CDR sequences: QTSQGVGSDLH (VL-CDR1), HTSSVED (VL-CDR2) and QVLQF (VL-CDR3); and the VH domain is at least 95% identical to RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVG WIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA is at least 95% identical and includes the following CDR sequences: AHILF (VH-CDR1), WIKPQYGAVNFGGGFRD (VH-CDR2) and DRSYGDSSWALDA (VH-CDR3).

6. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: an scFv comprising or consisting of RALVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSAGGGSGGGSGGGSGGGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK; a spacer comprising a sequence selected from the group consisting of SEQ ID NOs: 24-34; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15-23; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 36-40, and a CD3ζ signaling domain of NO:

35.

7. The nucleic acid molecule of any one of claims 1-6, wherein the spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-34.

8. The nucleic acid molecule of any one of claims 1-6, wherein the transmembrane domain is selected from the group consisting of: CD4 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane domain, and CD3ζ transmembrane domain; 9. The nucleic acid molecule of any one of claims 1-6, wherein the co-stimulatory domain is selected from the group consisting of: CD28 co-stimulatory domain, 41-BB co-stimulatory domain, OX40 co-stimulatory domain, and 2B4 co-stimulatory domain.

10. The nucleic acid molecule of claim 1, wherein the chimeric antigen receptor comprises an amino acid sequence having 0, 1, 2, 3, 4 or 5 single amino acid substitutions.

11. The nucleic acid molecule of any one of claims 1-6, further comprising an inter-domain linker consisting of 1-5 amino acids between one or more of the following: the scFV and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and the co-stimulatory domain and the CD3ζ signaling domain.

12. The nucleic acid molecule of claim 11, wherein the interdomain linker consists of 1-5 glycines.

13. The nucleic acid molecule of any one of claims 1-6, wherein an interdomain linker consisting of the sequence GGG is located between the co-stimulatory domain and the CD3 zeta signaling domain.

14. An immune cell comprising the nucleic acid molecule according to any one of the preceding claims.

15. The immune cell of claim 14, wherein the cell is a T cell expressing a T cell receptor specific for CMV (CMV-specific T cell).

16. A cell population comprising CMV-specific T cells containing the nucleic acid molecule according to any one of claims 1 to 13.

17. The cell population of claim 16, wherein at least 50% of the CMV-specific T cells are CD8+ T cells.

18. A method for preparing a CMV-specific T cell population expressing HIV CAR, the method comprising: Obtained from CMV pos isolating a cell population comprising PBMCs from a blood sample from a subject; contacting the cell population with a CMV antigen to stimulate CMV-specific T cells; isolating a subpopulation of T cells (eg, CMV-specific cells) secreting IFNγ from the cell population; and transducing cells in the subpopulation of T cells secreting IFNγ with a vector comprising the nucleic acid molecule of any one of claims 1-14.

19. The method of claim 18, wherein the subpopulation of IFNγ-secreting T cells is cultured in the presence of one or both of exogenous IL-2 and exogenous IL-15 before, after, or before and after transduction.

20. The method of claim 19, wherein IL-2 is added at 50 U / mL and IL-15 is added at 1 ng / mL.

21. The method of claim 19, wherein the IFNγ-secreting T cell subset 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, after, or before and after transduction.

22. The method of claim 21, wherein the IFNγ-secreting T cell subset is cultured in the presence of darunavir and enfuvirtide.

23. The method of any one of claims 18-19, wherein the blood sample is from a subject infected with HIV.

24. A method for treating a subject infected with HIV, the method comprising administering: (a) a population of CMV-specific T cells expressing a chimeric antigen receptor, the chimeric antigen receptor comprising: a scFv that binds HIV Env; a spacer domain; a transmembrane domain; a co-stimulatory 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.

25. The method of claim 24, wherein the at least one CMV antigen comprises at least one of a CMV protein, a fragment of a CMV protein, a modified CMV protein, a fragment of a modified CMV protein, a mutated CMV protein or a fragment thereof, a fused CMV protein or a fragment thereof, and combinations thereof.

26. The method of claim 24, wherein the at least one CMV antigen comprises at least one of pp65, IE1 exon 4 (IE1 / e4), IE2 exon 5 (IE2 / e5), fusions thereof, antigenic fragments thereof, and variants thereof having 1, 2, 3, 4, or 5 amino acid modifications.

27. The method of claim 24, wherein the at least one CMV antigen comprises a CMV pp65 peptide.

28. The method of claim 24, wherein the nucleic acid molecule comprises a viral vector encoding (a) a CMV pp65 peptide or protein and (b) a fusion protein comprising exon 4 (e4) of CMV protein 1E1 and exon 5 (e5) of CMV protein 1E2.

29. The method of claim 24, wherein the at least one antigen comprises at least one sequence selected from SEQ ID NOs: 57-64 and variants thereof having 1, 2, 3, 4 or 5 amino acid modifications.

30. The method of claim 24, wherein the at least one CMV antigen comprises at least one fragment of any one of SEQ ID NOs: 57-64, wherein 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 one of SEQ ID NOs: 57-64.

31. The method of claim 24, wherein the at least one CMV antigen or a nucleic acid molecule encoding at least one CMV antigen is administered before or after administration of the CMV-specific T cell population.

32. The method of claim 31, wherein the 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 prior to administration of the CMV-specific T cell population.

33. The method of claim 31, wherein the 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 the CMV-specific T cell population.

34. The method of claim 24, wherein the 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.

35. The method of claim 24, wherein the CAR comprises: a scFv comprising or consisting of SEQ ID NO: 9 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions, wherein the amino acid 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 having 1, 2, 3, 4 or 5 amino acid substitutions; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15-23 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions; a co-stimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 36-40 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions; and a CD3 zeta signaling domain comprising SEQ ID NO: 35 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions.

36. The method of claim 24, wherein the CAR comprises SEQ ID NO: 10 or a variant thereof having 1, 2, 3, 4 or 5 amino acid substitutions, wherein the amino acid substitutions are not in the CDRs.

37. The method of claim 24, wherein the CMV-specific T cell population is at least 40%, 50%, 60% or 70% IFN-γ positive.

38. The method of claim 24, wherein the CMV-specific T cell population is at least 20%, 30%, 35% CD8 positive.

39. The method of claim 24, wherein the CMV-specific T cell population is at least 20%, 25%, 30% or 35% CD4 positive.

40. The method of claim 24, wherein the CMV-specific T cell population is at least 40% IFN-γ positive, at least 20% CD8 positive, and at least 20% CD4 positive.

41. The method of claim 24, wherein the CMV-specific T cell population is at least 20% CD8 positive and at least 20% CD4 positive.

42. The method of claim 41, wherein the CMV-specific T cell population is at least 30% CD8 positive and at least 30% CD4 positive.

Images