Compositions and methods for treating HIV / aids with immunotherapy

Abstract

To provide novel compositions and methods for treating HIV / AIDS by using an approach capable of exhibiting specific and highly potent anti-HIV disease activity.SOLUTION: Chimeric antigen receptors (CARs) comprising HIV envelope antigen binding domains are disclosed. Further, disclosed are nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions relating to the CARs. Also, disclosed are methods of treating or preventing HIV infection in a subject, and methods of making CAR T cells. Further, disclosed are results of treating or preventing HIV infection, and results of making CAR T cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority pursuant to 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 608,479, filed December 20, 2017, and U.S. Provisional Patent Application No. 62 / 660,819, filed April 20, 2018, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy (created December 18, 2018) has the filename "SequenceListing.txt" and is 232 kilobytes in size.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable Field of the Disclosure This application relates to the field of diseases associated with infection with the human immunodeficiency virus / acquired immune deficiency syndrome (HIV, HIV / AIDS), and in particular to protein binding domains and chimeric antigen receptors (CARs) containing such binding domains, and methods of use thereof. [Background technology]

[0004] background Infection with the human immunodeficiency virus (HIV) remains a major threat to human health. In 2016, an estimated 1 million people died from HIV-related causes worldwide, and 36.7 million people are living with HIV. 54% of adults and 43% of children living with HIV are currently receiving lifelong antiretroviral drug-based therapy (ART). While there is no cure for HIV infection, ART has enabled many people to live longer, more productive lives. (All data from WHO fact sheets, updated July 2017.) Long-term antiretroviral therapy (ART) can increase the risk of severe toxicity and comorbidities, such as lipodystrophy, insulin resistance, cardiovascular disease, and organ failure. Additionally, long-term ART can lead to HIV drug resistance and reduced efficacy. While ART has been successful in controlling infection, it is not a cure.

[0005] HIV infection can be blocked by inhibiting its ability to enter and replicate in the host. As an alternative to ART, several HIV therapies have been attempted to inhibit and control HIV infection based on passive immunotherapy using broadly neutralizing antibodies specific to HIV-1 (reviewed in Margolis et al., Immunol Rev 2017;275:313-323). These therapies have excellent in vitro breadth and efficacy against a wide range of HIV clades. However, these monotherapies have not been shown to be effective in vivo. When applied in vivo, efficacy is poor and there are several issues that can lead to side effects, such as antibody stability (reviewed in Boesch et al., Current Opinion in HIV and AIDS 2015, 10(3):160-169), lack of virologic control (Bar et al., N Engl J Med 2016;375:2037-2050), HIV drug resistance (Wu et al., Journal of Virology 2012;86:5844-5853), and persistence of latent HIV reservoirs (reviewed in Margolis et al., Immunol Rev 2017;275:313-323). Several problems arise.

[0006] Chimeric antigen receptors (CARs) are hybrid molecules consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a binding / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is typically modeled after a single-chain fragment variable fragment (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs, such as receptor ligands (i.e., IL-13 was engineered to bind to the IL-13 receptor expressed in tumors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D or CD4), have also been engineered into CARs. Other cellular targets for expressing CARs, such as NK or gamma-delta T cells, are also under development (Brown CE et al., Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al., PLoS One. 2012;7(2):e31210). Substantial further effort remains to be expended in identifying the most active T cell populations to transduce with CAR vectors, in identifying optimal culture and expansion techniques, and in elucidating the molecular details of the CAR protein structure itself.

[0007] The binding motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed as a long, flexible linker. Structural motifs, such as those derived from the IgG constant domain, can be used to extend the ScFv binding domain farther from the T cell membrane surface. This may be important for some cellular targets (e.g., disialoganglioside GD2; Orentas et al., this observation unpublished) whose binding domains are particularly close to the tumor cell surface membrane. All signaling motifs used in CARs to date include the CD3-zeta chain, as this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was also used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the advent of new technologies, activation of T cells by beads coupled to anti-CD3 and anti-CD28 antibodies, as well as the presence of the classic "signal 2" from CD28, is no longer required for the CAR itself to encode. Third-generation vectors using bead activation have not been shown to be superior to second-generation vectors in in vitro assays, and furthermore, they offer no clear advantage over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-CARs targeting B cells precursor ALL, Blood. 2013;121(7):1165-74; Kochenderfer JN et al., Blood 2012;119(12):2709-20). In addition to CD137, other members of the tumor necrosis factor receptor superfamily, such as OX40, can provide important sustained signals in CAR-transduced T cells (Yvon E et al., Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which CAR T cell populations are cultured, such as the inclusion of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al., Cancer Gene Ther. 2015;22(2):72-78).

[0008] T cell-based immunotherapy featuring CARs has become a new frontier in synthetic biology for both cancer and infectious disease therapy. Genetic engineering techniques, such as the use of lentivirus-based gene vectors (LVs), can introduce multiple promoters and gene products into primary lymphocytes. It has been envisioned that T cells can be engineered with LVs expressing CARs and the resulting highly potent cells can be directed to sites of HIV infection and HIV reactivation from latency. Here, CAR-engineered T cells can circumvent HIV infection by inhibiting fusion with HIV viral particles or infected cells, and can also mediate the effective killing of cells expressing viral envelope proteins, thereby eliminating HIV-infected cells and, consequently, the viral reservoir.

[0009] The "Berlin patient" (an HIV-infected patient who underwent a bone marrow transplant of hematopoietic stem cells (HSCs) from a CCR5-variant donor) is the only reported case of HIV cure, essentially demonstrating that HIV eradication is feasible. One major obstacle in the search for an HIV cure is the ability of HIV to persist in a latent state within cellular reservoirs in the host. A "shock and kill" approach involves reactivating the latent HIV reservoir using latency-reactivating agents (LRAs). These agents shock latently infected cells, reactivating the virus at the epigenetic level. A combination approach, combining this with immunotherapy to effectively "kill" HIV-infected cells, has been proposed as a way to achieve an HIV cure.

[0010] Previously tested HIV immunotherapies using CAR therapy involve fusing the CD4 receptor or broadly neutralizing antibody domain (bnAb) to the transmembrane and intracellular domains of the CD3 zeta chain. In the late 1990s, the first generation of anti-HIV CARs was constructed using the extracellular domain of the CD4 receptor (CD4-zeta) and was shown to effectively eliminate HIV-infected cells in vitro (Tran et al., The Journal of Immunology 1995;155:(2)1000-1009; Yang et al., PNAS 1997;94(21):11478-11483). However, in vivo, they did not significantly inhibit HIV infection, possibly resulting in susceptibility to HIV infection (discussed in Lam et al., Immunotherapy 2013,5(4):404-414). CARs designed using bnAb also showed promising results in vitro. In 2015, one group reported that an anti-HIV CAR consisting of a CD4 receptor fused to bnAb(17b) effectively killed HIV-infected cells while simultaneously resisting infection (Liu et al., J. Virol. 2015;89:13 10 6685-6694). One major drawback to developing bnAb-based CARs is that further engineering is required to prevent therapeutic efficacy loss (Bar et al., N Engl J Med 2016;375:2037-2050; Sievers et al., Current Opinion in HIV and AIDS;2015:10(3),151-159). Furthermore, this approach may result in unwanted viral escape (Wu et al., Journal of Virology 2012;86:5844-5853; Barr et al. N Engl J Med 2016;375:2037-2050; Lynch et al., 2015 Journal of Virology;89(8):4201-4213).

[0011] Regarding clinical experience with CAR-transduced T cells for HIV, the first clinical trial was conducted using CAR-T for HIV almost 20 years ago. This clinical trial showed that anti-HIV CAR-T therapy was safe and had excellent in vivo persistence (Mitsuyasu et al., Blood 2000;96(3):785-793; Deeks et al., Mol Ther. 2002;5:788). One notable finding was that although the modified CAR T cells did not significantly control HIV viremia, they were transported to tissues harboring latent HIV reservoirs (Mits (Uyasu et al., Blood 2000;96(3):785-793). This represents a major advantage over non-CAR approaches and suggests that CAR-T is actively involved in immune surveillance. Subsequent studies have suggested that CAR-T may also be able to reactivate latent HIV reservoirs in chronically infected cells through cytokine release (Sahu et al., Virology 2013;446(0):268-275). These findings strongly suggest an HIV cure and support the rationale for developing improved anti-HIV CAR-T therapies.

[0012] HIV-1 entry inhibitors or fusion inhibitors combined with chimeric antigen receptors (anti-HIV New HIV therapies, including the creation of CARs (anti-HIV CARs), offer another attractive approach for effectively targeting and killing HIV-infected cells. The advantage of this approach is that anti-HIV CARs may intervene in immune surveillance of latent HIV reservoirs and simultaneously effectively eliminate HIV-infected cells, even in the absence of ART. Furthermore, combining multiple entry and / or fusion inhibitors makes it more difficult for HIV to infect CAR-modified T cells, further replicate, and develop resistance. Therefore, newly developed anti-HIV CAR therapies, especially those containing more than one inhibitor, offer strong indications of functional cure. Summary of the Invention [Problem to be solved by the invention]

[0013] There is an urgent and long-felt need in the art to discover new compositions and methods for treating HIV / AIDS using approaches that can exhibit specific and highly potent anti-HIV disease activity without exhibiting the problems described above. [Means for solving the problem]

[0014] The present invention addresses the above-mentioned needs by providing CAR compositions and therapeutic methods that can be used to treat HIV and other diseases and / or conditions. In particular, the invention disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with expression of HIV envelope proteins, which contain multiple HIV antigen-binding domains that have high surface expression on transduced T cells, a high degree of cytolysis of HIV-infected cells, and in vivo proliferation and persistence of the transduced T cells.

[0015] overview Provided herein are novel anti-HIV envelope protein antibodies, or antigen-binding domains thereof, chimeric antigen receptors (CARs) containing such anti-HIV envelope protein antigen-binding domains, host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. The CARs exhibit high levels of surface expression on transduced T cells, high degrees of cytolysis, and enable the transduced T cells to proliferate and persist in vivo. Additionally, methods of using the disclosed CARs, host cells, and nucleic acid molecules are provided, for example, to treat HIV infection or AIDS or HIV-associated cancer in patients.

[0016] In one embodiment, provided herein are improved second-generation CARs that contain three unique classes of HIV peptide inhibitors (mD1.22, m36.4, and C46 peptides). These domains, when engineered into specific orientations in the context of an HIV chimeric antigen receptor, form a new series of highly potent bispecific and trispecific anti-HIV CARs (combinations of two HIV inhibitors) and trispecific anti-HIV CARs (combinations of three HIV inhibitors). The anti-HIV CARs provided herein are designed to destroy HIV-infected cells while simultaneously providing protection to CAR T cells. .

[0017] Thus, in one aspect, there is provided an isolated polynucleotide encoding an anti-HIV envelope protein (anti-HIV binder, or simply anti-HIV), or a fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5.

[0018] In another embodiment, there is provided an isolated polynucleotide encoding an anti-HIV envelope protein linked to a second anti-HIV envelope protein, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 75, 79, 83, and 87, or a fragment thereof.

[0019] In another aspect, there is provided an isolated polynucleotide encoding an anti-HIV envelope protein, or a fragment thereof, expressed in a cell that encodes a second anti-HIV envelope protein, comprising the nucleic acid sequence of SEQ ID NO:69.

[0020] In yet another embodiment, there is provided an isolated polynucleotide encoding an anti-HIV envelope protein linked to two additional anti-HIV envelope proteins, or a fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 91, 95, and 99.

[0021] In another embodiment, an isolated polynucleotide encoding an anti-HIV envelope protein bound to another anti-HIV envelope protein expressed in a cell in which the other anti-HIV envelope protein is expressed, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 103, 111, 115, and 119.

[0022] In one embodiment, an isolated polynucleotide encoding a fully human anti-HIV antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

[0023] In one embodiment, an isolated polynucleotide encoding an anti-HIV antibody or fragment thereof or another anti-HIV binding protein is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

[0024] In another embodiment, an isolated polynucleotide is provided encoding an anti-HIV antibody or fragment thereof or other anti-HIV binding protein, wherein the antibody or fragment thereof or other anti-HIV binding protein is bound to a second such anti-HIV binder consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 50, 54, 58, 62, 66, 76, 80, 84, and 88.

[0025] In yet another embodiment, an isolated polynucleotide is provided that encodes an anti-HIV antibody or fragment thereof or other anti-HIV binding protein, wherein the antibody or fragment thereof or other anti-HIV binding protein is expressed in a cell that contains a second anti-HIV binder consisting of an amino acid sequence comprising SEQ ID NO: 70. In yet another embodiment, an isolated polynucleotide is provided that encodes an anti-HIV antibody or fragment thereof or other anti-HIV binding protein, wherein the antibody or fragment thereof or other anti-HIV binding protein is bound to two additional such anti-HIV binders consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 92, 96, and 100.

[0026] In another embodiment, an isolated polynucleotide encoding an anti-HIV envelope protein bound to another anti-HIV envelope protein expressed in a cell in which the other anti-HIV envelope protein is expressed, the polynucleotide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 104, 112, 116, and 120.

[0027] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising, from N-terminus to C-terminus, at least one HIV antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5.

[0028] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular HIV antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to an HIV envelope protein or a minimized single antibody domain (e.g., VH only).

[0029] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular HIV antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to HIV.

[0030] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen-binding domain of the encoded CAR further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to HIV.

[0031] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen-binding domain of the encoded CAR consists of one immunoglobulin domain, such as a VH-only domain, or an analogous single-chain Ig-like binding site.

[0032] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen binding domain is connected to the transmembrane domain by a linker domain.

[0033] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded HIV extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0034] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one HIV antigen binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5, wherein the CAR further encodes an extracellular antigen binding domain that targets an antigen including (but not limited to) an HIV latency-associated antigen (e.g., CD32a), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) E2 protein, cytomegalovirus (CMV) glycoprotein B, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0035] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the further encoded extracellular antigen-binding domain is an anti-CD32a ScFv antigen-binding domain, an anti-HBsAg ScFv antigen-binding domain, an anti-HCV E2 ScFv antigen-binding domain, or an anti-CD32b ScFv antigen-binding domain. domain, anti-CMV glycoprotein B ScFv antigen binding domain, anti-CD19 ScFv antigen binding domain, anti-CD20 ScFv antigen binding domain, anti-ROR1 ScFv antigen binding domain, anti-mesothelin ScFv antigen binding domain, anti-CD33 ScFv antigen binding domain, anti-CD38 ScFv antigen binding domain, anti-CD123 (IL3RA) ScFv antigen binding domain, anti-CD138 ScFv antigen binding domain, anti-BCMA (CD269) ScFv antigen binding domain, anti-GPC2 ScFv antigen binding domain, anti-GPC3 ScFv antigen binding domain, anti-FGFR4 ScFv antigen binding domain, anti-TSLPR ScFv antigen binding domain, anti-c-Met ScFv antigen binding domain, anti-PMSA ScFv antigen binding domain, anti-glycolipid F77 ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2 The antigen-binding domain of the ScFv includes an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0036] In one embodiment, the CAR provided herein further comprises one linker or spacer domain.

[0037] In another embodiment, a CAR provided herein that contains more than one HIV antigen binder may contain two, three, or four linker or spacer domains.

[0038] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein an extracellular HIV antigen binding domain, an intracellular signaling domain, or both, is linked to a transmembrane domain by a linker or spacer domain.

[0039] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen binding domains are linked to each other or to the transmembrane domains by a nucleic acid sequence encoding a linker or spacer domain selected from the group consisting of SEQ ID NOs: 9, 23, 25, 27, 29, and 31.

[0040] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen binding domains are linked to each other or to the transmembrane domain by a linker or spacer domain according to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 24, 26, 28, 30, and 32.

[0041] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen binding domains are linked to each other or to the transmembrane domain by a nucleic acid sequence encoding a linker or spacer domain containing a furin cleavage site upstream and downstream of a translational skip site, such as exemplified in SEQ ID NO: 33.

[0042] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen binding domains are linked to each other or to the transmembrane domain by a linker or spacer domain containing a furin cleavage site upstream and downstream of a translational skip site consisting of an amino acid sequence as exemplified in SEQ ID NO: 34.

[0043] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is linked to the transmembrane domain.

[0044] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0045] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0046] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0047] In yet another embodiment, where more than one HIV antigen binder may be expressed on two different transmembrane proteins in the same cell, these two proteins may express the same intracellular signaling domain, or may express different signaling domains, or one may not express a signaling domain.

[0048] In a further embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0049] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided that further contains a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO:35 or SEQ ID NO:37.

[0050] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:38.

[0051] In one embodiment, provided herein is a CAR comprising, from N-terminus to C-terminus, at least one HIV antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0052] In one embodiment, a CAR is provided, wherein the extracellular HIV antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0053] In another embodiment, a CAR is provided, wherein at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0054] In some embodiments, a CAR is provided, wherein the CAR is an HIV latency-associated The antibody further encodes an extracellular antigen-binding domain comprising an antigen (e.g., CD32a), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) E2 protein, cytomegalovirus (CMV) glycoprotein B, CD19, CD20, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0055] In one embodiment, a CAR is provided, wherein the extracellular antigen-binding domain is an anti-CD32a ScFv antigen-binding domain, an anti-HBsAg ScFv antigen-binding domain, an anti-HCV E2 ScFv antigen-binding domain, an anti-CMV glycoprotein B ScFv antigen-binding domain, an anti-CD19 ScFv antigen-binding domain, an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-ROR1 ScFv antigen-binding domain, an anti-mesothelin ScFv antigen-binding domain, an anti-CD33 ScFv antigen-binding domain, an anti-CD38 ScFv antigen-binding domain, an anti-CD123 (IL3RA) ScFv antigen-binding domain, an anti-CD138 ScFv antigen-binding domain, an anti-BCMA (CD269) ScFv antigen-binding domain, an anti-GPC2 ScFv antigen-binding domain, an anti-GPC3 ScFv antigen-binding domain, an anti-FGFR4 ScFv antigen-binding domain, an anti-TSLPR The ScFv antigen-binding domain comprises an ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0056] In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0057] In yet another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0058] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 39 (LTG1944, LP-mD1.22-CD8TM-41BB-CD3zeta nucleic acid sequence (Figure 2A)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 40 (LTG1944, LP-mD1.22-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2A)).

[0059] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 41 (LTG1945, LP-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2B)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 42 (LTG1945, LP-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2B)).

[0060] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 43 (LTG2328, LP-C46-CD8TM-41BB-CD3 zeta CAR nucleoside In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 44 (LTG2328, LP-C46-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2C)).

[0061] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 47 (LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2D)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 48 (LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2D)).

[0062] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 51 (LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2E)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 52 (LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2E)).

[0063] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 55 (LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2F)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 56 (LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2F)).

[0064] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 59 (LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2G)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 60 (LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2G)).

[0065] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 63 (LTG1947, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2H)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 64 (LTG1947, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2H)).

[0066] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 67 (LTG1948, LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 2I)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 68 (LTG1948, LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 2I)).

[0067] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 71 (LTG2303, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR nucleic acid sequence (Figure 2J)). In one embodiment, the nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 72 (LTG2303 , encoding a CAR comprising the LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR amino acid sequence (Figure 2J).

[0068] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73 (LTG2322, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR nucleic acid sequence (Figure 2K)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 (LTG2322, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR amino acid sequence (Figure 2K)).

[0069] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77 (LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2L)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78 (LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2L)).

[0070] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 81 (LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2M)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 82 (LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2M)).

[0071] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 85 (LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2N)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 86 (LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2N)).

[0072] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89 (LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (Figure 20)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 90 (LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta CAR amino acid sequence (Figure 20)).

[0073] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93 (LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2P)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94 (LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2P)).

[0074] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 97 (LTG2319, LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2Q)). In one embodiment, the nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 98 (LTG2319, LP-mD1.22-L3- C46-L3-m36.4-CD8TM-41BB-CD3zeta encodes a CAR containing the CAR amino acid sequence (Figure 2Q).

[0075] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 101 (LTG2320, LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2R)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 102 (LTG2320, LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2R)).

[0076] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 105 (LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CAR nucleic acid sequence (Figure 2S)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 106 (LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CAR amino acid sequence (Figure 2S)).

[0077] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 107 (LTG2329, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR nucleic acid sequence (Figure 2T)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 108 (LTG2329, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR amino acid sequence (Figure 2T)).

[0078] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 109 (LTG2330, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR nucleic acid sequence (Figure 2U)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 110 (LTG2330, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR amino acid sequence (Figure 2U)).

[0079] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113 (LTG2331, LP-C46-L3-mD1.22-CD8™-CD3 zeta-F2AF-m36.4-TNFRSF19™ CAR nucleic acid sequence (Figure 2V)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114 (LTG2331, LP-C46-L3-mD1.22-CD8™-CD3 zeta-F2AF-m36.4-TNFRSF19™ CAR amino acid sequence (Figure 2V)).

[0080] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 117 (LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR nucleic acid sequence (Figure 2W)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 118 (LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR amino acid sequence (Figure 2W)).

[0081] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 121. (LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CAR nucleic acid sequence (Figure 2X)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 122 (LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CAR amino acid sequence (Figure 2X)).

[0082] In one embodiment, a CAR disclosed herein is modified to express or contain a detectable marker for use in diagnosing, monitoring, and / or predicting treatment outcome, e.g., to monitor the progress of such treatment.

[0083] In one embodiment, a nucleic acid molecule encoding a disclosed CAR can be contained in a vector, such as a viral vector, which can be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0084] In certain embodiments, the vector further comprises a promoter, which is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, a synthetic promoter, or any combination thereof.

[0085] In yet another embodiment, the CAR-expressing vector may be further modified to include one or more operable elements for controlling the expression or function of CAR T cells (e.g., inducible homo / heterodimerized CARs) or for deleting CAR-T cells by a suicide switch. The suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the CAR-expressing vector may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD). In another aspect, a host cell is further provided that comprises a nucleic acid molecule encoding a CAR. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0086] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-HIV effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one extracellular antigen-binding domain comprising an anti-HIV antigen-binding domain or domain combination comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or a domain combination comprising the amino acid sequence of SEQ ID NO: 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the T cells are T cells of a human with HIV infection or T cells intended for administration to a patient with HIV / AIDS.

[0087] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0088] In another embodiment, a pharmaceutical composition is provided, wherein the human disease is HIV / A IDS infection, or oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), cancers related to Kaposi's sarcoma virus (HHV8) infection, gastrointestinal cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct) duct, gallbladder, pancreas), respiratory tract (larynx, lung, and bronchus), bone and joint cancer, soft tissue cancer, adult cancers, including skin cancer (melanoma, basal cell carcinoma, and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers associated with HIV, including cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicles, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous system, or any combination thereof.

[0089] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-HIV effective amount of a population of human T cells from a human with HIV / AIDS and an HIV-associated malignancy, such as leukemia, CNS leukemia, sarcoma, Kaposi's sarcoma, or infectious sequelae associated with HIV / AIDS, where the infection is resistant or unresponsive to one or more highly active antiretroviral therapies (ART) and / or chemotherapy that have failed to completely eradicate HIV / AIDS or the HIV-associated malignancy, respectively. Uses of CAR-modified T cells further include situations in which the viral reservoir is reactivated or exacerbated in order to reduce the ratio of latently infected cells to actively infected cells (including by discontinuing ART (therapeutic holiday), or by inducing activity with methylating or demethylating agents of promoter elements, or cellular activation pathway activators, or mimetics of epigenetic signals such as those that target CARs by activating latent virus to express envelope proteins).

[0090] In another embodiment, a method of producing CAR-containing T cells (hereinafter "CAR-T cells") is provided, which method comprises transducing T cells with a vector or nucleic acid molecule encoding a CAR (as disclosed) that specifically binds to an HIV envelope protein, thereby producing the CAR-T cells.

[0091] In yet another embodiment, a method for generating a population of RNA-engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed CAR into cells of a subject, thereby generating CAR cells.

[0092] In yet another aspect, a method for diagnosing a disease, disorder, or condition associated with expression of HIV envelope in a cell is provided, comprising: a) contacting the cell with a human anti-HIV antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120; and b) detecting the presence of HIV envelope, wherein the presence of the HIV envelope protein diagnoses the disease, disorder, or condition associated with HIV / AIDS.

[0093] In one embodiment, the disease, disorder, or condition associated with HIV manifestations is candidiasis of the esophagus, bronchi, trachea, or lungs, and mouth (thrush), cervical cancer, invasive, coccidioidomycosis, disseminated or extrapulmonary, cryptococcosis, extrapulmonary, cryptosporidiosis, chronic intestinal (lasting longer than 1 month), cytomegalovirus disease (other than liver, spleen, or nodules), cytomegalovirus retinitis (associated with vision loss), encephalopathy, HIV-associated, herpes simplex: chronic ulcers (lasting longer than 1 month); or bronchitis, interstitial pneumonia, or esophagitis, histoplasmosis, disseminated or extrapulmonary, isosporosis, chronic intestinal (lasting longer than 1 month), Kaposi's sarcoma, lymphoma, Burkitt (or equivalent term) lymphoma, immunosuppressant. leukoencephalopathy, Salmonella septicemia, recurrent, cerebral toxoplasmosis, HIV-associated wasting syndrome (Source: Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents) and adults. Morbidity and Mortality Weekly Report, December 18, 1992 / 1993, 41 (RR-17)). These may occur concomitantly with cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, adult B-cell malignancies including ALL, minimal residual disease (MRD) in AML, CLL, CML, NHL, pediatric B-cell malignancies (including B-lineage ALL), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematologic and solid tumors, or any combination thereof.

[0094] In another embodiment, a method for diagnosing or prognosing or risk assessment for HIV-related disease in a mammal is provided, comprising detecting expression of HIV envelope in a sample from the mammal, the method comprising: a) contacting the sample with a human anti-HIV antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120; and b) detecting the presence of HIV, wherein if HIV is present, diagnosing HIV-related disease in the mammal.

[0095] In another embodiment, a method of inhibiting HIV-dependent T cell inhibition is provided, comprising contacting a cell with a human anti-HIV antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120. In one embodiment, the cell is selected from the group consisting of an HIV-expressing cell, an HIV-susceptible cell, and any combination thereof.

[0096] In another embodiment, a method is provided for blocking T cell inhibition mediated by HIV-expressing cells and altering infected tissue to inhibit HIV pathogenesis in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-HIV antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120. In one embodiment, the cell is selected from the group consisting of an HIV envelope-expressing cell, an HIV-infected cell or tissue, an HIV-susceptible cell or tissue, and any combination thereof.

[0097] In another embodiment, a method of inhibiting, suppressing, or preventing immunosuppression of an anti-HIV response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-HIV antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of an HIV-expressing cell, an HIV-infected cell or tissue, and any combination thereof.

[0098] In another aspect, a method for inducing anti-HIV immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed CAR.

[0099] In another embodiment, a method of treating or preventing HIV infection in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs in an amount effective to treat or prevent HIV infection in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing a CAR (disclosed) that specifically binds to HIV and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex in the subject consisting of the antigen-binding domain of the CAR, the extracellular domain of HIV, and / or one or more of the above-mentioned antigens.

[0100] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of an HIV antigen, the method comprising administering to the subject an anti-HIV effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one extracellular HIV antigen binding domain comprising the amino acid sequence of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with cancer.

[0101] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one HIV antigen binding domain comprising the amino acid sequence of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with HIV infection or T cells to be administered to such a patient. In some embodiments of the methods described above, at least one transmembrane domain comprises a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0102] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with HIV infection. In one embodiment, the method comprises administering to the human T cells engineered to express a CAR, wherein the CAR comprises at least one HIV antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the persistent population of engineered T cells, or a population of progeny of the T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0103] In one embodiment, the progeny T cells in the human include memory T cells. In another embodiment, the T cells are autologous T cells.

[0104] In all aspects and embodiments of the methods described herein, any of the infections, cancers, diseases, disorders, or conditions associated with elevated expression of HIV antigens described above can be treated or prevented or ameliorated using one or more of the CARs disclosed herein.

[0105] In yet another embodiment, the bispecific and / or trispecific anti-HIV Each of the CARs can be used in adoptive T cell immunotherapy in an amount effective to inhibit, suppress, or prevent immunosuppression of an anti-HIV response in a mammal, or to treat or prevent HIV infection in said mammal, wherein said mammal is receiving said adoptive T cell immunotherapy without the need for a prior antiretroviral therapy (ART) treatment regimen, or to effectively substantially reduce the number of ART treatment regimens required by about 10% to 99%.

[0106] In yet another embodiment, a kit is provided for generating the CAR T cells described above, or for preventing, treating, or ameliorating any of the infections, cancers, diseases, disorders, or conditions associated with elevated expression of HIV antigens in a subject described above, comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, or instructions for use of the kit.

[0107] While the disclosure provided herein has initially focused on the generation of CARs utilizing HIV envelope protein antigen-binding fragments thereof, it is anticipated that the CARs, T cell receptors (TCRs) or nucleic acid sequences, polypeptides, and methods of use thereof may be used with HIV proteins other than the HIV envelope protein antigen-binding fragments specifically described herein, and thus are specifically meant to include any HIV-derived protein associated with latent or productive HIV infection, including, but not limited to, Rev, Tat, Vif, Nef, Vpu, Vpr, Gag, Pol, protease, nucleocapsid, matrix, capsid, integrase, and / or reverse transcriptase, or any combination thereof.

[0108] It is understood that the above-described CARs, host cells, nucleic acids, and methods are useful beyond the scope of the specific aspects and embodiments described in detail herein. The features and advantages of the present disclosure described above will become more apparent from the following detailed description, which is provided with reference to the accompanying drawings. [Brief explanation of the drawings]

[0109] [Figure 1]Schematic diagram of the general domain structure of a CAR with the sequence of a novel extracellular HIV antigen-binding domain. The monospecific CAR-T (top construct) is composed of an extracellular HIV-binding domain (binder 1), a hinge and transmembrane domain (e.g., CD8 hinge / spacer and CD8 transmembrane domain (TM)), an intracellular signaling CD137 costimulatory domain (41BB), and a CD3 zeta signaling domain. Bispecific CAR-T (middle three constructs) consist of two binders joined by a linker (black lines) attached to the hinge and transmembrane domain and the intracellular signaling domain (41BB, CD3 zeta), or one binder (binder 1) attached to the hinge / TM and intracellular signaling domain followed by the 2A ribosomal skip site followed by a second binder (binder 2) attached either to the hinge / linker and transmembrane only or to the hinge / linker transmembrane domain attached to a second intracellular CD3 zeta sequence. Trispecific CAR-T (bottom three constructs) consist of three linked anti-HIV binders attached to one hinge / transmembrane sequence and intracellular signaling sequence (41BB and CD3 zeta), or two binders attached to each other then attached to the hinge / transmembrane sequence followed by the intracellular signaling motif followed by the 2A ribosomal skip site, and then a third binder (binder 3) attached to either the hinge / transmembrane sequence alone or the hinge / transmembrane followed by the CD3 zeta intracellular signaling sequence. [Figure 2A] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2A shows a lentiviral vector expressing a CAR containing the LTG1944 (LP-mD1.22-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 39) and the encoded amino acid sequence (SEQ ID NO: 40). [Figure 2B] Figure 2B shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2B shows a lentiviral vector expressing a CAR containing the LTG1945 (LP-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 41) and the encoded amino acid sequence (SEQ ID NO: 42). [Figure 2C] Figure 2C shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2C shows a lentiviral vector expressing a CAR containing the LTG2328 (LP-C46-CD8TM-41BB-CD3 zeta) nucleotide sequence (SEQ ID NO: 43) and the encoded amino acid sequence (SEQ ID NO: 44). [Figure 2D] Figure 2D shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2D shows a lentiviral vector expressing a CAR containing the LTG2325 (LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 47) and the encoded amino acid sequence (SEQ ID NO: 48). [Figure 2E]Figure 2E shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2E shows a lentiviral vector expressing a CAR containing the LTG2313 (LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 51) and the encoded amino acid sequence (SEQ ID NO: 52). [Figure 2F] Figure 2F shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2F shows a lentiviral vector expressing a CAR containing the LTG1946 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 55) and the encoded amino acid sequence (SEQ ID NO: 56). [Figure 2G] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2G shows a lentiviral vector expressing a CAR containing the LTG2326 (LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 59) and the encoded amino acid sequence (SEQ ID NO: 60). [Figure 2H]Figure 2H shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2H shows a lentiviral vector expressing a CAR containing the LTG1947 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 63) and the encoded amino acid sequence (SEQ ID NO: 64). [Figure 2I] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2I shows a lentiviral vector expressing a CAR containing the LTG1948 (LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 67) and the encoded amino acid sequence (SEQ ID NO: 68). [Figure 2J] Figure 2J shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2J shows a lentiviral vector expressing a CAR containing the LTG2303 (LP-mD1.22-CD8™-41BB-CD3 zeta-F2AF-m36.4-TNFRSF19™-CD3 zeta2) nucleic acid sequence (SEQ ID NO: 71) and the encoded amino acid sequence (SEQ ID NO: 72). [Figure 2K]Figure 2 shows several CARs containing novel extracellular HIV antigen-binding domain sequences. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2K shows a lentiviral vector expressing a CAR containing the LTG2322 (LP-mD1.22-CD8™-41BB-CD3 zeta-F2AF-m36.4-TNFRSF19™) nucleic acid sequence (SEQ ID NO: 73) and the encoded amino acid sequence (SEQ ID NO: 74). [Figure 2L] Figure 2B shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2L shows a lentiviral vector expressing a CAR containing the LTG2314 (LP-mD1.22-L3-C46-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 77) and the encoded amino acid sequence (SEQ ID NO: 78). [Figure 2M] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2M shows a lentiviral vector expressing a CAR containing the LTG2315 (LP-mD1.22-L5-C46-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 81) and the encoded amino acid sequence (SEQ ID NO: 82). [Figure 2N]Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2N shows a lentiviral vector expressing a CAR containing the LTG2316 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 85) and the encoded amino acid sequence (SEQ ID NO: 86). [Figure 2O] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2O shows a lentiviral vector expressing a CAR containing the LTG2317 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 89) and the encoded amino acid sequence (SEQ ID NO: 90). [Figure 2P] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2P shows a lentiviral vector expressing a CAR containing the LTG2318 (LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 93) and the encoded amino acid sequence (SEQ ID NO: 94). [Figure 2Q]Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2Q shows a lentiviral vector expressing a CAR containing the LTG2319 (LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 97) and the encoded amino acid sequence (SEQ ID NO: 98). [Figure 2R] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2R shows a lentiviral vector expressing a CAR containing the LTG2320 (LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 101) and the encoded amino acid sequence (SEQ ID NO: 102). [Figure 2S] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2S shows a lentiviral vector expressing a CAR containing the LTG2323 (LP-mD1.22-L3-m36.4-CD8™-41BB-CD3 zeta-F2AF-C46-TNFRSF19™) nucleic acid sequence (SEQ ID NO: 105) and the encoded amino acid sequence (SEQ ID NO: 106). [Figure 2T]Figure 2 shows several CARs containing novel extracellular HIV antigen-binding domain sequences. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2T shows a lentiviral vector expressing a CAR containing the LTG2329 (LP-C46-L3-mD1.22-CD8™-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19™-CD3 zeta2) nucleic acid sequence (SEQ ID NO: 107) and the encoded amino acid sequence (SEQ ID NO: 108). [Figure 2U] Figure 2C shows several CARs containing novel extracellular HIV antigen-binding domain sequences. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2U shows a lentiviral vector expressing a CAR containing the LTG2330 (LP-C46-L5-mD1.22-CD8™-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19™-CD3 zeta2) nucleic acid sequence (SEQ ID NO: 109) and the encoded amino acid sequence (SEQ ID NO: 110). [Figure 2V] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2V shows a lentiviral vector expressing a CAR containing the LTG2331 (LP-C46-L3-mD1.22-CD8™-41BB-CD3 zeta-F2AF-m36.4-TNFRSF19™) nucleic acid sequence (SEQ ID NO: 113) and the encoded amino acid sequence (SEQ ID NO: 114). [Figure 2W]Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2W shows a lentiviral vector expressing a CAR containing the LTG2332 (LP-C46-L5-mD1.22-CD8™-41BB-CD3 zeta-F2AF-m36.4-TNFRSF19™) nucleic acid sequence (SEQ ID NO: 117) and the encoded amino acid sequence (SEQ ID NO: 118). [Figure 2X] Figure 2 shows several CARs containing the sequence of a novel extracellular HIV antigen-binding domain. The schematic diagram of the CAR includes, from N- to C-terminus, a signal peptide, an anti-HIV binder, an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Additionally, bispecific and trispecific CAR constructs are also illustrated. Figure 2X shows a lentiviral vector expressing a CAR containing the LTG2334 (LP-mD1.22-L5-m36.4-CD8™-41BB-CD3 zeta-F2AF-C46-TNFRSF19™) nucleic acid sequence (SEQ ID NO: 121) and the encoded amino acid sequence (SEQ ID NO: 122). [Figure 3A]

[0033] Figure 3A shows the functional characterization of monospecific anti-HIV CARs. Figure 3A shows the expression of anti-HIV CARs containing the mD1.22 domain or the C46 peptide on the surface of LV-transduced human T cells. mD1.22-CARs are detected by staining with anti-CD8 antibodies (y-axis) and anti-CD4 antibodies (recognizing the mD1.22 domain, x-axis, left panel), or C46-CARs are detected by staining with 2F5 antibodies (recognizing the C46 peptide, x-axis, right panel). [Figure 3B]Functional characterization of monospecific anti-HIV CARs is shown. Figure 3B shows how m36.4 expression was detected on the surface of LV-transduced primary T cells by fusing an mCherry reporter immediately upstream of the CD3 zeta signaling domain. The percentage of mCherry-positive cells was detected in the total T cell population (left panel), on the surface of CD4+ T cells (middle panel), and on the surface of CD8+ T cells (right panel). [Figure 3C] Functional characterization of monospecific anti-HIV CARs is shown. Figure 3C illustrates the distinct cytotoxic functions of the monospecific CARs. Using target cell lines expressing HIV envelope as cytotoxic targets, untransduced T cells (UTD, no activity), LTG1732-transduced T cells (control mCherry vector, no activity), LTG1944-transduced T cells (expressing the mD1.22 binder), LTG1945-transduced T cells (expressing the m36.4 binder), and LTG2328-transduced T cells (expressing the C46 binder) each exhibited cytotoxicity at the effector-target cell ratio (E:T) indicated on the x-axis. [Figure 3D] Functional characterization of monospecific anti-HIV CARs is shown. Figure 3D shows that similar transduced T cell populations do not mediate cytolytic activity against cell lines that do not express HIV envelope proteins. [Figure 3E] Functional characterization of monospecific anti-HIV CARs is shown. Figure 3E shows that human T cells transduced with LVs expressing monospecific CARs secrete IFN-γ in the presence of HIV envelope-expressing cells (solid bars). Background levels are seen in cells not expressing the HIV envelope (checkered bars) or cells cultured alone (striped bars). [Figure 4A]Figure 4 shows the expression of bispecific anti-HIV CARs on the surface of activated T cells. Bispecific CARs were designed with several unique configurations to determine their precise functional structure. Figure 4A shows a bispecific anti-HIV CAR designed with two anti-gp120 binders (mD1.22 and m36.4) fused using a flexible glycine-serine linker consisting of at most five GGGGS (G4S) motifs. This bispecific CAR was detected by flow cytometry using an antibody directed against the D1 domain of the CD4 receptor (anti-CD4 VIT4 clone, Miltenyi Biotec) that also recognizes the mD1.22 domain of the bispecific CAR. [Figure 4B] Figure 4B shows the expression of bispecific anti-HIV CARs on the surface of activated T cells. The bispecific CARs were designed with several unique configurations to determine their exact functional structure. Figure 4B shows the expression of a bispecific CAR designed with the m36.4 domain, which is presented as a first gp120 binder, and then fused to the mD1.22 domain using three G4S motifs (LTG1948). [Figure 4C] Figure 4C shows the expression of a bispecific anti-HIV CAR on the surface of activated T cells. The bispecific CAR was designed with several unique configurations to determine its precise functional structure. Figure 4C shows the expression of a bispecific CAR (LTG2303) containing the mD1.22 and m36.4 domains fused to the autologous intracellular CD3 zeta chain via a bicistronic construct incorporating the self-cleaving P2A peptide from porcine teschovirus-1. Expression of the LTG2303 CAR on the surface of T cells was determined by detecting the mD1.22 domain using anti-CD4 flow cytometry. [Figure 4D]Figure 4D shows the expression of bispecific anti-HIV CARs on the surface of activated T cells. The bispecific CARs were designed with several unique configurations to determine their precise functional structure. Figure 4D shows Western blot detection of both mD1.22-CAR and m36.4-CAR using anti-CD3 zeta staining. Both CARs were fully cleaved using the P2A self-cleaving peptide. Endogenous CD3 zeta was used as a loading control. [Figure 4E] Figure 4D shows the expression of bispecific anti-HIV CARs on the surface of activated T cells. The bispecific CARs were designed with several unique configurations to determine their precise functional structure. Figure 4E shows a graphical representation of the percentage of CAR-modified T cells by anti-CD4 flow cytometry detection of mD1.22. [Figure 5] These results demonstrate the high killing ability of anti-HIV CARs against 293T cells expressing HIV envelope and a firefly luciferase reporter. Notably, a bispecific anti-HIV CAR (LTG2303), constructed with each anti-HIV binder on one CD3 zeta chain, significantly killed target cells compared with the monospecific CARs LTG1944 and LTG1945. Untransduced T cells (inactive) and LTG1732 (T cells transduced with an mCherry reporter, inactive) were used as negative controls to monitor nonspecific T cell-mediated cytotoxicity. Results are from three donors. Error bars represent ± standard deviation. Statistical analysis was performed by two-way ANOVA (***P<0.0001, **P<0.001, *P<0.01). [Figure 6]This shows the exceptional specificity of the anti-HIV CAR in the absence of HIV envelope-expressing cell lines. Raji cells are known to express MHC class II molecules and therefore may nonspecifically interact with the mD1.22 domain from the CD4 receptor. As shown for both cell lines, no off-target cell lysis was observed with the anti-HIV CAR. This is a representative image for three donors. Error bars represent + / - standard deviation. [Figure 7] This shows that activation of anti-HIV CAR-T triggers the release of IFN-gamma. Anti-HIV CARs were incubated with (Env+) or without (Env-) 293T cells expressing the envelope and co-expressing a firefly luciferase reporter. After 24 hours, cell culture supernatants were collected and assayed for IFN-γ using ELISA. All anti-HIV CARs were activated in the presence of Env+ cells and, to a lesser extent, in the presence of Env- cells. The bispecific CAR LTG2303 secreted more IFN-gamma than the monospecific CARs LTG1944 and LTG1945. Results shown are from two different donors. Error bars represent + / - standard deviation. [Figure 8A] Figure 8A shows that the function of bispecific anti-HIV CARs is governed by linker length. Bispecific CARs were constructed using up to five G4S motifs, and their functionality was examined by examining their ability to destroy envelope-expressing target cells (Env+). [Figure 8B] This shows that the function of bispecific anti-HIV CARs is governed by linker length. Figure 8B shows the specificity of bispecific anti-HIV CARs in the presence of envelope-free Raji cells (Env-). Bispecific CARs with one G4S motif were significantly more active than anti-HIV CARs containing five G4S motifs. Statistics were performed by two-way ANOVA (***p<0.0001, LTG2325 vs. LTG1947). Error bars represent + / - standard deviation. [Figure 9]This shows that bispecific anti-HIV CARs designed with increasing linker length are triggered to release IFN-gamma in the presence of envelope-expressing target cells (Env+), and to a lesser extent in the absence of envelope-expressing cells (Env-) or when cultured with effectors alone. The anti-HIV CAR with the longest linker (LTG1947) released IFN-gamma at the lowest background level. Error bars represent + / - standard deviation. [Figure 10A] This shows that bispecific anti-HIV CARs designed using mD1.22 and C46 peptides exhibit configuration-dependent activity. Figure 10A shows the cytotoxic activity of bispecific CARs based on C46. The cytotoxicity of these CARs is highly dependent on the configuration of the bispecific gp120 / gp41 binder. For example, CARs LTG2316 and LTG2317, which are engineered with the C46 peptide distal to the mD1.22 domain, exhibit similar functions to LTG1946 CAR, which contains both the mD1.22 and m36.4 domains. Meanwhile, CARs LTG2314 and LTG2315, which are engineered with the mD1.22 peptide distal to the C46 peptide, completely lose their cytolytic function. [Figure 10B] Figure 10B shows that bispecific anti-HIV CARs designed using mD1.22 and C46 peptides exhibit configuration-dependent activity. Figure 10B shows that C46-based bispecific CARs nonspecifically produce IFN-gamma in the presence of Env+ target cells, and in some cases, even in their absence (Env- or effector alone). [Figure 11A] This shows that the trispecific anti-HIV CAR potently destroys and releases cytokines in response to envelope-expressing target cells. Figure 11A shows the expression of the trispecific anti-HIV CAR on the surface of primary T cells using the broadly neutralizing 2F5 antibody against the C46 peptide. The 2F5 antibody recognizes adjacent epitopes (ELDKWA) in the C46 peptide. [Figure 11B]This shows that the trispecific anti-HIV CAR potently destroys and releases cytokines in response to envelope-expressing target cells. Figure 11B shows the detection of the mD1.22 domain in the bispecific arm of the trispecific anti-HIV CAR LTG2323. The mD1.22 domain was detected on the surface of T cells by anti-CD4 (recognizing the mD1.22 domain) and anti-CD8 flow cytometry. This construct contains a combination of a bispecific CAR (LTG1946) and a membrane-anchored C46 peptide, which are separately presented on the surface of T cells. [Figure 11C] Figure 11C shows that trispecific anti-HIV CARs potently destroy and release cytokines in response to envelope-expressing target cells. HIV envelope (Env+)-expressing cells were potently destroyed by all trispecific CARs. [Figure 11D] Figure 11D shows that the trispecific anti-HIV CAR potently destroys and releases cytokines in response to envelope-expressing target cells. Figure 11D shows the specificity of the trispecific CAR in the absence of envelope-expressing, but MHC class II-expressing, Raji cells (Env-). [Figure 11E] Figure 11E shows that the trispecific anti-HIV CARs potently destroy and release cytokines in response to envelope-expressing target cells. Figure 11E shows the release of IL-2 cytokine for all trispecific CARs in comparison with the highly potent LTG1946 bispecific CAR. [Figure 11F] Figure 11F shows that trispecific anti-HIV CARs potently destroy and release cytokines in response to envelope-expressing target cells. Figure 11F shows the release of IFN-γ cytokines for all trispecific CARs in comparison with the highly potent LTG1946 bispecific CAR. [Figure 12A] Figure 12A shows a comparison of the expression and cytotoxicity of the most potent trispecific and bispecific anti-HIV CARs. Figure 12A shows the percentage of bispecific CAR-modified T cells by detection of the mD1.22 domain using anti-CD4 flow cytometry. [Figure 12B] Figure 12B shows a comparison of the expression and cytotoxicity of the most potent trispecific and bispecific anti-HIV CARs. Figure 12B shows the cytotoxicity of the trispecific anti-HIV CARs. [Figure 12C] Figure 12C shows a comparison of the expression and cytotoxicity of the most potent trispecific and bispecific anti-HIV CARs. Figure 12C shows the percentage of trispecific CAR-modified T cells by detecting the C46 domain using 2F5 flow cytometry. The dotted line across the y-axis indicates 30% genetic modification. [Figure 12D] Figure 12D shows a comparison of the expression and cytotoxicity of the most potent trispecific and bispecific anti-HIV CARs. Figure 12D shows the specificity of the trispecific anti-HIV CARs, as measured by cytotoxicity against non-envelope cell lines. Experimental data shown in the figure are from at least three donors (error bars = + / - SD). [Figure 13] Figure 1 shows the CD4+ and CD8+ composition of donors used in in vitro and in vivo HIV-1 challenge studies. Donors were enriched, activated, and expanded for CD4+ and CD8+ effector T cells for up to 9 days. On day 9, donor T cell composition was determined by anti-CD4 and anti-CD8 flow cytometry as described in Example 1. The figure shows the percentages of CD4+ and CD8+ effectors on day 9 of culture. [Figure 14A]This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14A shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the laboratory-adapted NL4-3 env gene (clade B, X4 tropism). [Figure 14B] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14B shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the clade B HIV-1 env gene (BaL, R5 tropism) isolated from the United States. [Figure 14C]This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14C shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding a second clade B HIV-1 env gene (SF162, R5 tropism) isolated from the United States. [Figure 14D] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14D shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the VRC01- and 3BNC117-resistant clade C HIV-1 env gene (C.Du422.1) isolated from southern Africa. [Figure 14E] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14E shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding a VRC01-resistant clade C HIV-1 env gene (C.Du172.17) isolated from southern Africa. [Figure 14F] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14F shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the VRC01 partially resistant clade C HIV-1 env gene (C.Cap45) isolated from southeastern Africa. [Figure 14G] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14G shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the HIV-1 env gene of a representative clade AC isolated from eastern Africa (AC.246-F3.LucR). [Figure 14H]This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14H shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR, encoding the HIV-1 env gene of a representative clade BC isolated from China (BC.CH119.10.LucR). [Figure 14I] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14I shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding a representative clade G HIV-1 env gene (GX1632_S2_B10.LucR) isolated from Spain. [Figure 14J] This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14J shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the HIV-1 env gene of clade AE ​​(AE.CNE8.LucR), representative of those found in southern China and / or Thailand. [Figure 14K]This figure shows the in vitro killing efficacy of monospecific, bispecific, and trispecific anti-HIV CAR-T cells against PBMCs infected with a replication-competent infectious molecular clone of HIV-1 encoding a broad-spectrum envelope (env) gene and a Renilla luciferase reporter (Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells (effectors) were cocultured with autologous PBMCs (targets) infected with the indicated Env-IMC-LucR virus 1 day prior to coculture. After 7 days, cocultures were lysed and luciferase activity was quantified. Bispecific and trispecific anti-HIV duoCAR-T cells eliminate PBMCs infected with broad-spectrum Env-IMC-LucR viruses across donors (LTG2303, LTG2329, and LTG2330). Figure 14K shows the killing efficacy of CAR-T against PBMCs infected with Env-IMC-LucR virus encoding the HIV-1 env gene (AE.CNE55.LucR) of the second representative clade AE ​​found in southern China and / or Thailand. The x-axis shows the designated anti-HIV CAR, and "T" represents "target" or Env-IMC-LucR-infected PBMCs. Error bars represent + / - SD. Statistical analysis was performed by multiple comparison Student's t-test. The significance of the findings is indicated on the graph. [Figure 15] Figure 1 shows a summary of in vitro CAR-T killing efficacy for all donors tested, expressed as log inhibition of HIV-1 infection. Log inhibition is calculated relative to HIV-infected, untransduced T cells after background subtraction using uninfected PBMCs. Data represent the average of at least three independent donors. [Figure 16] Figure 1 shows a summary of the in vitro CAR-T killing efficacy for all donors tested, expressed as % inhibition of HIV-1 infection. Percent inhibition is calculated relative to HIV-infected, untransduced T cells after background subtraction using uninfected PBMCs. Data represent the average of at least three independent donors. [Figure 17A]Figure 17A shows that multispecific anti-HIV duoCAR T cells exhibit excellent in vitro killing efficacy at very low E:T ratios. Figure 17A shows PBMCs from the same donor infected with Du422.1-IMC-LucR virus on day -1 (T = target) followed by addition of the indicated anti-HIV CAR-T cells (E = effector) on day 0 at different E:T ratios (1:1, 0.5:1, 0.25:1, and 0.125:1). [Figure 17B] These results demonstrate that multispecific anti-HIV duoCAR T cells exhibit excellent in vitro killing efficacy at very low E:T ratios. Figure 17B shows PBMCs from the same donor infected with Du422.1-IMC-LucR virus on day -1, followed by addition of the indicated anti-HIV CAR-T cells at different E:T ratios (1:25, 1:50, and 1:100) on day 0. Seven days after addition of CAR-T cells, cocultures were harvested and assayed for Renilla luciferase activity. Statistical analysis was performed by multiple comparison Student's t-test. P<0.05 was considered statistically significant and determined by the Holm-Sidak method. P<0.00001****, P<0.0001***, P<0.001**, P<0.05*. [Figure 18A] Figure 18A shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18B shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding the NL4-3 env gene. [Figure 18B]Figure 18B shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18B shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding the BaL env gene. [Figure 18C] Figure 18C shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18D shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding the C.Du422.1 env gene. [Figure 18D] Figure 18D shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18C shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding the C.Du172.17 env gene. [Figure 18E]Figure 18B shows broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18E shows in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding AC.246-F3 env gene (clade AC). [Figure 18F] Figure 18F shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18F shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding a representative clade BC env gene (BC.CH119.10). [Figure 18G] Figure 18G shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18G shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding a representative clade AE ​​env gene (AE.CNE8). [Figure 18H]Figure 18H shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18H shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding a representative clade G env gene (GX1632_S2_B10). [Figure 18I] Figure 18I shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18I shows the in vitro protection of anti-HIV CAR-T cells exposed to Env-IMC-LucR virus encoding the env gene of the second representative clade AE ​​(AE.CNE55). [Figure 18J] Figure 18J shows the broad in vitro protection of anti-HIV CAR-T cells for individual donors. For some donors, mD1.22-CAR-T cells are more susceptible to HIV-1 infection. Conversely, the m36.4 domain protects bispecific and trispecific CAR T cells from HIV-1 infection, independent of its structure. Figure 18J plots all donors together for each Env-IMC-LucR virus tested. Error bars represent + / - SD. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. Significant findings are indicated on the graph. [Figure 19A]Figure 19A shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19A shows examples of humanized, intrasplenic, PBMC, NSG acute and chronic HIV-1 infection models (hu-spl-PBMC-NSG). [Figure 19B] Figure 19B shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19B shows the in vivo 7-day killing efficacy of bispecific and trispecific anti-HIV duoCAR-T cells against PBMCs infected with bNAb-resistant Du422.1-IMC-LucR virus (acute). [Figure 19C] Figure 19C shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19C shows the in vivo killing efficacy of bispecific and trispecific anti-HIV duoCAR-T cells against PBMCs infected with bNAb-resistant Du422.1-IMC-LucR virus over 30 days (chronic). Data show luciferase activity detected in the spleens of mice at day 7 (acute) or day 30 (chronic). To account for the loss of CD4+ T cells in the UTD control group caused by uncontrolled HIV-1 infection, luciferase activity (RLU) at day 30 was normalized to the percentage (%) of CD4+ T cells. [Figure 19D] Figure 19D shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19D shows the percentage of CD4+ T cells isolated from mouse spleens on day 7 (acute). [Figure 19E]Figure 19E shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19E shows the percentage of CD4+ T cells isolated from mouse spleens at day 30 (chronic). [Figure 19F] Figure 19F shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19F shows the percentage of CD8+ cells isolated from mouse spleens on day 7 (acute). [Figure 19G] Figure 19G shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19G shows the percentage of CD8+ cells isolated from mouse spleens at day 30 (chronic). [Figure 19H] Figure 19H shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19H shows CAR copy numbers / μg of splenic DNA at day 7 (acute). [Figure 19I] Figure 19 shows that bispecific and trispecific anti-HIV duoCAR-T cells potently eliminate PBMCs infected with bNAb-resistant virus (Du422.1-IMC-LucR) in vivo. Figure 19I shows CAR copy numbers / μg of splenic DNA at day 30 (chronic). Statistical analysis was performed by one-way ANOVA followed by Tukey's post-test. The significance of findings is indicated on the graph. DETAILED DESCRIPTION OF THE INVENTION

[0110] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly contradicts. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" without excluding other elements. The term "and / or" means "and" or "or." Furthermore, unless otherwise specified, it is understood that any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, particularly preferred methods and materials are described below. In the event of any conflict, the present specification (including explanations of terms) will control. Additionally, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. To facilitate identification of various embodiments, explanations of terms are provided below.

[0111] The term "about," when referring to measurable values ​​such as amounts and durations, is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for the practice of the disclosed methods.

[0112] Unless otherwise noted, scientific terms herein are used in their conventional usage. Definitions of common terms in molecular biology are found in Benjamin Lewin, Genes VII, Oxford University Press, 1999; Kendrew et al. (eds.), The En Cyclopedia of Molecular Biology, Blackwell Science, 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, 1995; and other similar reference works.

[0113] The present disclosure provides HIV antibodies or fragments thereof and CARs having such HIV antigen-binding domains. Improving the functional activity of the CAR directly correlates with improving the functional activity of CAR-expressing T cells. As a result of one or more of these modifications, the CAR exhibits high levels of both cytokine-induced cytolysis and cell surface expression in transduced T cells, as well as high levels of in vivo T cell proliferation and persistence of transduced CAR-expressing T cells.

[0114] The unique ability to combine functional moieties from different protein domains is an innovative feature of CARs. The choice of these protein domains, as well as the specific binding mode, are important design features. Individual design domains are essential components that can be used in any heterogeneous CAR platform to manipulate lymphocyte function. For example, the selection of extracellular binding domains can enable otherwise ineffective CARs.

[0115] The non-variable framework components of the immunoglobulin-derived protein sequence used to generate the extracellular antigen-binding domain of a CAR can be completely neutral, or they can be self-binding and drive T cells into a metabolically exhausted state, significantly reducing the efficacy of therapeutic T cells expressing the CAR. This phenomenon occurs independently of the antigen-binding function of the CAR domain. Furthermore, the selection of the intracellular signaling domain can also govern the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability to bind target antigens and transmit activation signals to T cells via the above-described extracellular and intracellular domains are important CAR design aspects, it has become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on CAR potency and therefore may play a crucial role in CAR function and clinical utility.

[0116] Surprisingly and unexpectedly, it was found that the use of a fully human antigen-binding domain in a CAR, rather than a mouse-derived antigen-binding fragment (which tends to induce an anti-mouse immune response and CAR T elimination in the host) (see: University of Pennsylvania-funded clinical trial using a mouse-derived SS1 ScFv sequence, NCT02159716), can determine the functional activity of CAR-expressing T cells.

[0117] The CARs disclosed herein are expressed at high levels in cells. Cells expressing these CARs have high in vivo proliferation rates, produce large amounts of cytokines, and exhibit high toxic activity against cells bearing the HIV envelope antigen to which the CAR binds. The use of a human extracellular HIV antigen-binding domain results in the creation of CARs with improved in vivo function while avoiding the induction of anti-CAR immunity and the extinction of the CAR T cell population in the host immune response. CARs expressing a fully human extracellular HIV antigen-binding domain exhibit superior activity and / or properties, including: i) preventing the poor persistence and poor function of CAR T (as seen with murine-derived binding sequences); ii) lack of CAR targeting to specific regions (i.e., intrapleural) for efficacy; and iii) the ability to design CAR T cells based on both high and low HIV affinity binders.

[0118] The CARs of the present invention are now described in detail, including a description of their extracellular HIV antigen-binding domains, transmembrane domains, and intracellular domains, as well as further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, treatment methods, compositions, and kits using the disclosed CARs.

[0119] A. Chimeric Antigen Receptors (CARs) The CARs disclosed herein comprise at least one HIV antigen-binding domain capable of binding to an HIV envelope protein, at least one transmembrane domain, and at least one intracellular domain.

[0120] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing the antigen-binding domain of an antibody or receptor (e.g., a single-chain variable fragment (ScFv) or human CD4 binding to the gp120 portion of the HIV envelope protein) linked via a transmembrane domain to a T cell signaling domain via a linker or hinge domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity to a selected target in a major histocompatibility complex (MHC)-independent manner, leveraging the antigen-binding properties of a monoclonal antibody or receptor. Because of their ability to recognize antigens without MHC restriction, CAR-expressing T cells can recognize antigens independently of antigen processing. CARs also advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs) when expressed in T cells.

[0121] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens.

[0122] 1. Extracellular domain In one embodiment, CAR comprises target-specific binding element, also referred to as antigen binding domain or site.The selection of domain depends on the type and number of ligands that define the surface of target cells.For example, antigen binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cells related to specific disease state.Therefore, examples of cell surface markers that can act as the ligand of antigen binding domain in CAR include those related to viral infection, bacterial infection, and parasitic infection, autoimmune disease, and cancer cell.

[0123] In one embodiment, CARs can be engineered to target a viral antigen of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on virally infected cells. Viral antigens are proteins produced by virally infected cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding domain may depend on the specific type of viral infection being treated. Viral antigens that can serve as CAR targets include those expressed on the surface of infected cells, such as HIV envelope protein glycoproteins (gp160, gp120 / gp41), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HBV) E2 glycoprotein, Epstein-Barr envelope protein, and cytomegalovirus (CMV) glycoprotein B antigen. The viral antigens disclosed herein are included solely by way of example. This list is not intended to be limiting, and other examples will be readily apparent to those skilled in the art.

[0124] In one embodiment, the HIV envelope protein contains one or more antigenic epitopes associated with the viral envelope protein. HIV-infected cells express envelope proteins that can serve as target antigens for immune attack. In this disclosure, anti-HIV CARs targeting HIV envelope proteins were created using one extracellular domain to create a monospecific CAR, two extracellular domains with different orientations and linker lengths to create a bispecific CAR, or all three extracellular domains to create a trispecific CAR. The CARs are composed of three functionally distinct extracellular domains that target non-overlapping epitopes on the HIV envelope to effectively block HIV entry (mD1.22), co-receptor usage (m36.4), and viral fusion (C46). Unlike CARs containing either a single bnAb and / or wild-type CD4 receptor, all three extracellular domains are precisely designed to have increased specificity, potency, and the ability to target emerging T20-resistant strains.

[0125] The mD1.22 domain is derived from the human CD4 receptor and targets a highly conserved epitope on HIV-1 gp120. Compared to the full-length CD4 receptor, mD1.22 is designed to be small and have high specificity, affinity, and potency against diverse HIV-1 clades (Chen et al., J. Virol. 2014;88:(2)1125-1139). The m36.4 domain is a human antibody domain composed exclusively of the heavy chain and has mature affinity (Chen et al., Antiviral Research 2010;88:(1)107-115). It binds to a discontinuous CD4-induced (CD4i) epitope located near the coreceptor binding site on gp120 (Wan et al., PLOS One 2013;8(6):e66638). When combined, these two domains act synergistically to potently neutralize HIV-1 and inhibit viral entry (Chen et al., J. Virol. 2014;88:(2)1125-1139).

[0126] Targeting multiple, non-overlapping epitopes is an attractive approach to prevent viral escape. As a third layer of T cell protection, we engineered CAR-T or T cells with the highly potent C46 fusion inhibitor. The C46 peptide belongs to a class of gp41-derived "C-peptide" fusion inhibitors that block HIV-1 infection at the level of viral fusion. The C46 peptide is a longer version of the FDA-approved enfuviritide, or T20, which is similar to T20 and can be used to suppress HIV variants that are multidrug-resistant to current cART. Notably, when expressed on the surface of T cells or secreted by T cells (SAVE peptide), the C-peptide potently inhibits HIV-1 fusion to the T cell membrane (van Lunzen et al., Molecular Therapy 2007, 15:(5)1024-1033; Kimpel et al., PLOS One 2010, 5:(8)e12357; Egerer et al., Molecular Therapy 2010, 19(7), 1236-1244). Therefore, CARs engineered with these three domains are designed to potently destroy HIV-infected cells while simultaneously providing protection to CAR T cells.

[0127] To reduce immunogenicity, the CAR used herein is constructed with a fully human sequence. This is more advantageous than using a mouse-based ScFv binding sequence, because the latter tends to induce immune responses and CAR-T elimination in human hosts, resulting in poor T cell maintenance. As described herein, CAR-transduced T cells are generated by transduction with a lentiviral vector construct encoding an anti-HIV CAR gene. It is expected that this CAR-expressing T cell can have long-lasting therapeutic effects in patients. It is important to note that therapeutic CAR-T cell products can be administered in one dose, rather than by repeated IV administration.

[0128] In general, mD1.22-based CARs (containing minimal domain human CD4 protein fragments) are highly potent and demonstrate potent immunoglobulin E1 expression in 293T cells stably expressing HIV gp120 (env + The mD1.22 domain was then fused to m36.4 (a binding domain derived from an anti-HIV antibody) using a linker domain such as those described in SEQ ID NOs: 24, 26, 28, 30, and 32, and these domains were then linked to the CD8 or TNFRSF19 transmembrane domain (TM) (SEQ ID NOs: 8 and 14) via a CD8-derived linker domain such as those described in SEQ ID NO: 10, such that the CD8 linker is linked to the CD8 transmembrane domain such as those described in SEQ ID NO: 12, or the CD8 linker is linked to the TNFRSF19TM such as those described in SEQ ID NO: 16, thereby enabling binding to the intracellular signaling domain. The intracellular signaling domain is either first-generation or second-generation. Second-generation CARs bind to the 41BB (CD137) intracellular signaling domain of SEQ ID NO: 18, close to the transmembrane region leading to the CD3 zeta signaling domain. The intracellular domain for the CD3 zeta chain is directly linked to the transmembrane domain or, in second-generation constructs, to the 41BB domain. In constructing lentiviral gene vectors, it is very important to avoid repeating sequences, as vectors often edit or rearrange these sequences, which can result in the loss of structure in the CAR construct. Another unique feature of the vector family presented here is the creation of two different CD3 zeta domains, both of which encode the same amino acid sequence (SEQ ID NO:20 and SEQ ID NO:22), but whose nucleic acid sequences (SEQ ID NO:19 and SEQ ID NO:21) are different.

[0129] In some cases, a single chain forms a bispecific or trispecific CAR that contains two or three extracellular domains and ultimately binds to and is expressed on the same cell surface protein. In other cases, two separate chains are encoded and expressed in the same construct, but are processed into two proteins due to the inclusion of a complex furin / ribosomal skipping site or a self-cleaving peptide, such as those described in SEQ ID NO: 33 and SEQ ID NO: 34. The importance of bispecific or trispecific CARs should be recognized when one binder cannot recognize HIV variants or mutations that have occurred, and the second and / or third domains can compensate for this failure. The effective use of such CAR constructs described herein can eliminate dependence on ART and move toward a cure for HIV using CAR-T technology.

[0130] In a preferred embodiment, the target antigen is HIV envelope protein and infected cells and tissues associated with expression of the HIV envelope, including infected epithelial tissues, lymphoid tissues, and lymphocytes, which comprise and essentially define the HIV-infected state.

[0131] In a preferred embodiment, the antigen binding domain portion of the CAR targets an extracellular HIV envelope antigen.

[0132] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0133] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0134] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 5, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6.

[0135] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 45, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0136] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 49, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0137] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 53, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0138] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 57, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58.

[0139] In a preferred embodiment, the linked extracellular HIV envelope antigen binding domains are The encoding isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:61, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0140] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 65, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0141] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 69, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0142] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 75, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0143] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 79, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0144] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 83, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0145] In a preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain has the nucleotide sequence of SEQ ID NO: 87, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO:88, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:88.

[0146] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 91, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0147] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 95, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 96, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0148] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 99, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 100.

[0149] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 103, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0150] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 111, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 112.

[0151] In one preferred embodiment, an isolated nucleic acid molecule encoding a linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 115, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV envelope antigen-binding domain has the amino acid sequence of SEQ ID NO: 116, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 6, and includes an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of

[0152] In one preferred embodiment, the isolated nucleic acid molecule encoding the linked extracellular HIV envelope antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 119, or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular HIV antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 120.

[0153] In various embodiments of the HIV-specific CARs disclosed herein, a general scheme is set forth in Figure 1, which includes, from N- to C-terminus, a signal or leader peptide, an anti-HIV binder, an extracellular linker, a CD8 transmembrane segment, 4-1BB, and CD3 zeta. Additionally, CARs incorporating multiple binders linked by specific binding domains (bispecific, trispecific) are also shown.

[0154] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 39 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 40 [LTG1944, LP-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2A)].

[0155] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 39, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 40, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1944, LP-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2A)].

[0156] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 41 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 42 [LTG1945, LP-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2B)].

[0157] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 41, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 42, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1945, LP-m36.4-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2B)].

[0158] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 43 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 44 [LTG2328, LP-C46-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2C)].

[0159] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the amino acid sequence of SEQ ID NO: 44, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. encoding [LTG2328, LP-C46-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2C)].

[0160] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 47, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 48 [LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2D)].

[0161] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 47, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 48, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2D)].

[0162] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 51 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 52 [LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2E)].

[0163] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 51, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 52, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2E)].

[0164] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 55 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 56 [LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2F)].

[0165] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 55, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 56, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2F)].

[0166] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 59 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 60 [LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2G)].

[0167] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 59, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 60, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2G)].

[0168] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 63 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 64 [LTG1947, LP-mD1.22-L5-m36.4-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2H)].

[0169] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 63, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 64, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1947, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2H)].

[0170] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 67 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 68 [LTG1948, LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2I)].

[0171] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 67, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 68, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1948, LP-m36.4-L3-mD1.22-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2I)].

[0172] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 71 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 72 [LTG2303, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 amino acid sequence (shown in Figure 2J)].

[0173] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 71, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 72, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2303, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 amino acid sequence (shown in Figure 2J)].

[0174] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 [LTG2322, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in Figure 2K)].

[0175] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2322, LP-mD1.22-CD8 TM-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (Figure 2K)].

[0176] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78 [LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2L)].

[0177] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2L)].

[0178] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 81 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 82 [LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2M)].

[0179] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 81, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 82, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2M)].

[0180] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 85 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 86 [LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2N)].

[0181] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 85, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 86, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2N)].

[0182] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 90 [LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2O)].

[0183] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 90, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2O)].

[0184] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94 [LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2P)].

[0185] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2P)].

[0186] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 97 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 98 [LTG2319, LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2Q)].

[0187] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 97, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 98, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2319, LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (shown in Figure 2Q)].

[0188] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 101 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 102 [LTG2320, LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2R)].

[0189] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 101, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 102, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2320, LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2R)].

[0190] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 105 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 106 [LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM amino acid sequence (shown in Figure 2S)].

[0191] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 105, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 106, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM amino acid sequence (shown in Figure 2S)].

[0192] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 107 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 108 [LTG2329, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 amino acid sequence (shown in Figure 2T)].

[0193] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 107, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 108, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2329, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 amino acid sequence (shown in Figure 2T)].

[0194] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 109 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 110 [LTG2330, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 amino acid sequence (shown in Figure 2U)].

[0195] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 109, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 110, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2330, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 amino acid sequence (shown in Figure 2U)].

[0196] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114 [LTG2331, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in Figure 2V)].

[0197] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2331, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in Figure 2V)].

[0198] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 117 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 118 [LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in Figure 2W)].

[0199] In yet another embodiment, the nucleic acid sequence encoding the CAR has 85%, 90%, 95%, 96%, 97%, 98%, or more percent identity to the nucleic acid sequence of SEQ ID NO: 117. and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 118 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto [LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in Figure 2W)].

[0200] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 121 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 122 [LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TM amino acid sequence (shown in Figure 2X)].

[0201] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 121, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 122, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TM amino acid sequence (shown in Figure 2X)].

[0202] Overall, anti-HIV CARs were highly expressed on the surface of primary T cells (Figures 3A-B, 4, 11A-B, 12A, and 12C). The mD1.22-based CARs were highly potent and expressed in 293T cells stably expressing HIV gp120 (referred to herein as env ), which provides an alternative to HIV-positive cell lines for quantifying CAR-mediated cytotoxicity. +To understand the contribution of each anti-HIV target site to cytotoxicity, anti-HIV CARs were designed to contain either the mD1.22 domain, the m36.4 domain, or the C46 peptide. These anti-HIV CARs specifically destroyed env + differentially killed cells (Fig. 3C, mD1.22>m36.4>C46, P<0.0001) without off-target cytotoxic effects (Fig. 3D), and env + IFN-γ production was triggered in the presence of, and to some extent in the absence of, IFN-γ (Fig. 3E).

[0203] To define the most optimal bispecific structure, the mD1.22 and m36.4 domains were fused using a series of flexible glycine-serine linkers, or each binder was placed on its CD3 zeta signaling domain. As shown in Figure 4, all linker-specific CARs and other design iterations were detected on the surface of primary T cells. Interestingly, bispecific CARs significantly enhanced anti-HIV cytotoxicity (Figure 5, LTG2303 vs. LTG1944 or LTG1945) while maintaining excellent specificity (Figure 6). Shortening the linker length between these two domains enhanced CAR-mediated cytotoxicity of bispecific CARs designed with a single CD3 zeta domain while maintaining specificity (Figure 8). However, cytokine release assays showed that env +In the absence of target cells, no significant increase in tonic signaling was observed (Figure 9). More importantly, no off-target killing was observed in the presence of Raji cells, which are known to contain MHC class II molecules that can interact with the mD1.22 domain from the CD4 receptor (Figure 6, lower panel). Furthermore, the bispecific anti-HIV CAR released IFN-γ upon encountering HIV envelope target cells, and to a lesser extent in their absence (Figures 7 and 9). Taken together, the bispecific CAR serves as a highly potent architecture for designing more sophisticated trispecific CARs.

[0204] To construct a trispecific CAR composed of mD1.22, m36.4, and C46 peptides, we created additional bispecific CAR iterations within the design using mD1.22 and C46 peptides. Unlike the bispecific CAR, replacing m36.4 with C46 significantly suppressed CAR function when the C46 peptide was located near the T cell membrane. Anti-HIV CAR-mediated cytotoxicity was restored only when the C46 domain was located distal to the mD1.22 domain (Figure 10A). Similar to the previously described CD22 CAR, binder accessibility, which correlates with distance from the T cell membrane, is essential for CAR function (Haso et al., Blood 2013;121:1165-1174). As expected, the binder configuration of mD1.22 and C46 resulted in suppressed CAR function, which also resulted in poor levels of IFN-γ secretion (Figure 10B). Remarkably, simply reversing the order of the domains restored high levels of IFN-γ secretion (LTG2316), while increasing the spacing between the two domains reduced tonic signaling (LTG2317). Taken together, these data clearly indicate the most optimal bispecific binder configuration for a C46-based bispecific CAR. More importantly, a set of rules governing bispecific CAR function was identified that can be applied to the rational design of trispecific anti-HIV CARs.

[0205] Targeting multiple epitopic determinants on the HIV envelope protein is an attractive approach for designing CARs with superior breadth, potency, and the ability to prevent the emergence of escape mutants. The rationale for creating trispecific CARs is that if one binder fails to recognize an HIV variant, a second and / or third domain can compensate for this failure. Therefore, to improve the breadth of the most potent bispecific CAR candidate, we designed a bispecific CAR with the third most potent fusion inhibitor (C46 peptide) or entry inhibitor (m36.4) to create a trispecific CAR. Compared to the bispecific CAR, the trispecific CAR maintained its ability to potently destroy HIV surrogate cell lines and initiate a strong Th1 cytokine response (Figure 11C and Figure 11E-F). Furthermore, the trispecific CAR exhibited exceptional specificity for its intended target and had no off-target effects on Raji cells (Figure 11D). As shown by the data, the most optimal trispecific CAR was LTG2323, followed by LTG2320. Two additional trispecific CARs, generated by combining the structures of LTG2303 and LTG2316 or LTG2317, were further evaluated and again demonstrated robust killing of surrogate HIV envelope cell lines (Figure 12B). Both LTG2329 and LTG2330 maintained their specificity, except for the highest E:T ratio (Figure 12D). Taken together, the precise structure of the trispecific CARs improved their anti-HIV function.

[0206] Next, we challenged anti-HIV CAR T cells against diverse and resistant HIV-1 strains, further confirming the importance of the anti-HIV CAR structure. +Starting with a rich T cell population (Figure 13), CAR T cell products were generated for a group of selected anti-HIV CARs. Using a modified in vitro HIV-1 Env-IMC-LucR exposure assay, the most potent anti-HIV CARs were identified as bispecific and trispecific CARs containing two CD3 zeta chains (hereafter referred to as duoCARs). As shown in Figures 14-17, bispecific and trispecific duoCARs outperformed conventional anti-HIV CARs containing a single CD3 zeta chain (LTG2303, LTG2329, and LTG2330), regardless of their valency. This is a key design feature for generating anti-HIV CARs to increase potency and breadth while avoiding viral escape. It is speculated that if one domain is lost due to mutational escape, other domains can compensate for this loss. More importantly, the anti-HIV targeting domain is a key design feature for duoCARs. Because of their structure, they can act independently, sequentially, or simultaneously to attack productive HIV-infected cells. As shown in Figure 18, primary T cells engineered with mD1.22-CAR were more susceptible to HIV-1 infection than bispecific or trispecific CAR-T cells (open red bars). The incorporation of the m36.4 domain, an entry inhibitor, was sufficient to protect CAR T cells and halt HIV-1 infection. To further evaluate bispecific and trispecific duoCAR-T cells in vivo, we used a humanized NSG mouse model of acute and chronic HIV-1 infection (hu-spl-PBMC-NSG) (Figure 19A). To further investigate the function of duoCAR-T cells, we selected the VRC01 / 3BNC117-resistant Env-IMC-LucR virus. As shown in Figures 19B and 19C, bispecific and trispecific duoCARs significantly reduced HIV-1 infection compared with UTD-treated HIV-infected cohorts. Both the bispecific and trispecific duoCARs showed similar efficacy (LTG2303 vs. LTG2330). +T cells were significantly depleted in the spleens of mice treated with control UTD T cells (Figure 19E). Conversely, mice treated with bispecific and trispecific duoCAR-T cells showed significantly reduced CD4 T cells in the spleens harvested from infected spleens. + There was a significant improvement in the percentage of CD4 T cells, which was higher or close to the level of uninfected mice (see HIV-PBMC, Figure 19E). + Concomitant with T cell depletion, CD8 in the spleens of infected UTD-treated mice showed uncontrolled HIV infection. + The percentage of T cells increased (Figure 19G). The robust control of HIV-1 infection is likely due to the persistence of CAR-T cells in the spleens of infected mice in vivo, as shown in Figures 19H, 19I. Taken together, the invention presented herein represents a potent and general multi-targeted HIV-1 immunotherapy with strong implications for a functional cure.

[0207] While not intending to be limited to any particular mechanism of action, reasons for the improved therapeutic function associated with exemplary CARs of the present invention may include, but are not limited to, for example, a) improved multispecific targeting of non-overlapping viral epitopes, b) rational CAR design using intracellular T cell signaling domains to exploit the function of the antigen binding domain, c) lateral movement in the plasma membrane resulting in more efficient signaling, d) superior location in plasma membrane microdomains (such as lipid rafts) resulting in improved ability to interact with transmembrane signaling cascades associated with T cell activation, e) superior location in the plasma membrane due to preferential movement away from reducing or down-regulating interactions, for example, greater distance from or less interaction with phosphatases such as CD45, and f) superior assembly into the T cell receptor signaling complex (i.e., immune synapse), or any combination thereof.

[0208] To this point, the present disclosure has been illustrated using precise combinations of three exemplary HIV envelope targeting domains (mD1.22, m36.4, and C46 peptides), but other nucleotide and / or amino acid variants in these binding domains can also be used to derive HIV envelope binding domains for use in the CARs described herein.

[0209] Depending on the desired antigen to be targeted, the CAR may be further engineered to contain an appropriate antigen-binding domain specific for the desired antigen target.

[0210] In one embodiment of the present invention, the virus is selected from, for example, Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, provided are CARs that can bind to non-TSAs or non-TAAs, including, but not limited to, antigens derived from viruses such as rabies, rabies virus ...

[0211] In another aspect of the present invention, CARs are provided that can bind to antigens derived from bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella, among others, e.g., Helicobacter pylori, Legionella pneumophila, and the like. pneumophilia), mycobacterial species (e.g., Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis The present invention provides CARs capable of binding to antigens derived from infectious bacteria such as species of Lactobacillus meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof.

[0212] In another embodiment of the present invention, a CAR in combination with advanced gene editing technology (e.g., CRISPR / Cas9, CRISPR / Cas13, riboswitch, RNA interference, or intracellular antibody) is provided, which uses advanced gene editing technology to remove and / or disrupt viral ribonucleic acid, integrated viral DNA (e.g., HIV provirus), and / or viral proteins (e.g., viral reverse transcriptase) from host infected cells, or a combination thereof, to regulate disease-associated host genetic factors such as (but not limited to) chemokine receptor G protein-coupled receptors (e.g., CXCR4, CCR5), host susceptibility factors (e.g., LEDGF / p75), virulence factors (e.g., DC-SIGN), natural host resistance factors (e.g., defensins), or a combination thereof.

[0213] In another aspect of the present invention, CARs are provided in combination with small molecule inhibitors, latent infection reactivators, antivirals, antimicrobials, or antibodies, and any derivatives thereof, that enhance and / or act synergistically with CAR-T function (e.g., TLR7 agonists), or target disease states relevant to CAR therapy, or combinations thereof (but are not limited to the combinations described above).

[0214] 2. Transmembrane domain With respect to the transmembrane domains, the CAR comprises one or more transmembrane domains fused to the extracellular mD1.22 and m36.4 antigen binding domains of the CAR.

[0215] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0216] Particularly useful transmembrane regions in the CARs described herein include the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD6 The transmembrane domain may be derived from (i.e., comprise at least the transmembrane region of) CD4, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. Alternatively, the transmembrane domain may be synthetic, in which case it may comprise primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0217] In one embodiment, a transmembrane domain originally associated with one of the domains in the CAR is used in addition to the transmembrane domain described above.

[0218] In some instances, the transmembrane domain can be selected or amino acid substituted to prevent the domain from binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interactions of the domain with other receptor complex components.

[0219] In one embodiment, the transmembrane domain in a CAR of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 7. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8.

[0220] In one embodiment, the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 8, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 8 with at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions).

[0221] In some examples, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 10 or a sequence having 95-99% identity thereto.

[0222] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane TNFRSF19 domain, or a combination thereof.

[0223] In one embodiment, the encoded transmembrane TNFRSF19 domain in combination with the CD8 linker / hinge domain comprises the amino acid sequence of SEQ ID NO: 16, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 16 with at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions).

[0224] 3. Spacer domain In CARs, a spacer domain may be located between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer domain refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0225] In some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,2 84, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, which are incorporated by reference in their entireties.

[0226] The spacer domain preferably has a sequence that promotes binding between the CAR and the antigen and increases signal transduction into the cell. Examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine at potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0227] The spacer domain can be the entire or a portion of amino acids 137-206 (SEQ ID NO: 10) of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). A portion of the constant region of an antibody heavy or light chain can also be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0228] Furthermore, a signal peptide sequence may be attached to the N-terminus of the CAR. This signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above as intracellular domains have a signal peptide sequence, this signal peptide can be used as the signal peptide for the CAR. In one embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 36. In another embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 38.

[0229] 4. Intracellular domain The cytoplasmic domain or intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of an immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a protein portion that transmits an effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, it can be used in place of the entire chain, as long as the truncated portion is capable of transmitting the effector function signal. Thus, the meaning of the term "intracellular signaling domain" includes any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0230] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that cooperate to initiate signal transduction following binding of an antigen to the receptor, as well as any derivatives or variants of these sequences, and any synthetic sequence with the same functional capability.

[0231] It is known that signals emitted through the TCR alone are insufficient to fully activate T cells, and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct types of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (first cytoplasmic signaling sequence), and one that acts in an antigen-independent manner to provide a secondary or costimulatory signal (second cytoplasmic signaling sequence).

[0232] The first cytoplasmic signaling sequence regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. A first cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM.

[0233] Examples of ITAMs containing a first cytoplasmic signaling sequence that are particularly useful in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP__932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP__004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP__000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP__000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP__000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP__000724.1), and CD5 (NCBI RefSeq: NP__000724.1). The amino acids 402 to 495 of CD79a (NCBI RefSeq:NP__055022.2), 707 to 847 of CD79a (NCBI RefSeq:NP__001762.2), 166 to 226 of CD79a (NCBI RefSeq:NP__001774.1), 182 to 229 of CD79b (NCBI RefSeq:NP__000617.1), and CD66d (NCBI RefSeq:NP__000617.1) were also included. RefSeq:NP_001806.2), as well as variants having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information in NCBI RefSeq ID or GenBank described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0234] In a preferred embodiment, the intracellular domain of the CAR may be designed to contain a CD3-zeta signaling domain by itself, or may be combined with any other desired cytoplasmic domain useful in the context of a CAR. For example, the intracellular domain of a CAR may contain a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, and lymphocyte receptors. These include function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. Specific examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP__001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP__000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP__055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP__006130.1), and CD137 (4-1BB, NCBI The costimulatory signaling elements of the present disclosure include, but are not limited to, peptides having amino acids 214-255 of CD134 (OX40, NCBI RefSeq:NP_001552.2), amino acids 241-277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acids 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants having the same function as these peptides. Thus, although the present disclosure has thus far been exemplified primarily using 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the present disclosure.

[0235] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form this linkage. A glycine and serine doublet provides a particularly suitable linker.

[0236] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0237] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence of SEQ ID NO: 17 and the signaling domain of CD3-zeta comprises the nucleic acid sequence of SEQ ID NO: 19.

[0238] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0239] In one embodiment, the intracellular domain in the CAR is designed to comprise the codon-degenerate signaling domain of CD3-zeta2, wherein the signaling domain of CD3-zeta2 comprises the nucleic acid sequence of SEQ ID NO:21 and the amino acid sequence of SEQ ID NO:22.

[0240] 5. Further explanation of CAR Functional portions of the CARs disclosed herein are also expressly included within the scope of the present invention. The term "functional portion," when used in reference to a CAR, refers to any one or more portions or fragments of the CARs disclosed herein, which retain the biological activity of the CAR (parent CAR). A functional portion can, for example, recognize target cells or inhibit disease to a similar extent as the parent CAR, to the same extent as the parent CAR, or to a greater extent than the parent CAR. With respect to a parent CAR, a functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0241] A functional portion may contain additional amino acids at the amino or carboxy terminus, or both, of the portion that are not found in the amino acid sequence of the parent CAR. Desirably, these additional amino acids do not interfere with the biological function of the functional portion, such as, for example, target cell recognition, cancer detection, cancer treatment, or prevention. More desirably, these additional amino acids improve such biological activity over the biological activity of the parent CAR.

[0242] Functional variants of the CARs disclosed herein are included within the scope of this disclosure. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with the parent CAR, and this functional variant retains the biological activity of the CAR from which it is derived. Functional variants include, for example, variants of the CARs (parent CARs) described herein that retain the ability to recognize target cells to a similar extent to, or to a greater extent than, the parent CAR. With respect to the parent CAR, functional variants may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more amino acid sequence identity with the parent CAR.

[0243] A functional variant may, for example, comprise at least one conservative amino acid substitution in the amino acid sequence of the parent CAR. Alternatively, or in addition, a functional variant may comprise at least one non-conservative amino acid substitution in the amino acid sequence of the parent CAR. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may improve the biological activity of the functional variant, such that the biological activity of the functional variant is superior to that of the parent CAR.

[0244] The amino acid substitutions in CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / charged polar amino acid (e.g., Asp or Glu) with another acidic / charged polar amino acid, the substitution of a non-polar side chain-containing amino acid (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another non-polar side chain-containing amino acid, the substitution of a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, the substitution of a polar side chain-containing uncharged amino acid (e.g., Asn, Gin, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, the substitution of a beta-branched side chain-containing amino acid (e.g., He, Thr, and Val) with another beta-branched side chain-containing amino acid, the substitution of an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid, and the like.

[0245] A CAR can consist essentially of one or more of the specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0246] CARs (including functional portions and functional variants) are intended to be useful in a variety of applications where the CAR (or a functional portion or functional variant thereof) has a biological function, such as, for example, the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent disease in a mammal. The CAR may be of any length, i.e., contain any number of amino acids, so long as it retains its targeting activity. For example, the CAR may be from about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more amino acids in length.

[0247] CARs (including functional portions and functional variants according to the invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid. acid), a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0248] CARs (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized or conjugated.

[0249] CAR (including its functional part and functional variant) can be obtained by methods well known in the art. CAR can be produced by any suitable polypeptide or protein production method. Suitable methods for de novo synthesis of polypeptides and proteins are described in prior art documents such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional portions and functional variants thereof) may be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Isolation and purification methods are well known in the art. Alternatively, the CARs (including functional portions and functional variants thereof) described herein may be commercially synthesized by companies. In this regard, In this regard, the CAR may be synthetic, recombinant, isolated, and / or purified.

[0250] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding domain or portion thereof, that specifically binds to one or more of the antigens disclosed herein. As used herein, "CAR-expressing T cell" or "CAR T cell" or "CAR-T" refers to a T cell that expresses a CAR and has antigen specificity, e.g., determined by the antibody-derived targeting domain of the CAR.

[0251] As used herein, an "antigen-binding domain" may include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Examples of antibodies include, but are not limited to, intact immunoglobulins and variants and fragments thereof well known in the art that retain binding affinity for an antigen.

[0252] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for possible natural mutations, which may be present in trace amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the property of the antibody being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some instances, a monoclonal antibody is produced by a single clone of B lymphocytes or by cells transfected with nucleic acid encoding the light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or their progeny. In some instances, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are well known and are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, 2nd Edition. Cold Spring Harbor Publications, New York (2013).

[0253] Typically, immunoglobulins have heavy (H) chains and light (L) chains linked together by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0254] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind to an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting only of heavy chains are functional and stable even in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH," or "VH," refer to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment, such as Fv, ScFv, dsFv, or Fab. References to "VL," or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, ScFv, dsFv or Fab.

[0255] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity-determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Vol. 1, No. 1, pp. 111-114, 2002). (See, for example, the "International Framework for Antibody Development" (International Standards Board of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved among species. The framework regions of an antibody, i.e., the framework regions of the constituent light and heavy chains, together position and align the CDRs in three-dimensional space.

[0256] CDRs are primarily responsible for binding to an antigenic epitope. The amino acid sequence boundaries of a given CDR are determined by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, USA). Al-Lazikani et al. (JMB 273,927-948,1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N- to C-terminus) and are further typically identified by the chain in which the CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody that contains it, and a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody that contains it. Light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3.

[0257] "Antigen-binding fragments" are portions of full-length antibodies or full-length proteins (e.g., the D1 domain of the CD4 receptor), and various combinations of such portions, that retain the ability to specifically recognize their cognate antigen. Examples of antigen-binding fragments include, but are not limited to, protein domains, full-length proteins, Fvs, Fabs, Fab's, Fab'-SH, F(ab')2; diabodies; nanobodies; linear antibodies; single-chain antibody molecules (e.g., ScFvs); and multispecific antibodies formed from antibody fragments or multispecific proteins formed from more than one protein domain or fragment. Antibody fragments include antigen-binding fragments generated by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010). Multispecific proteins and derivatives thereof include antigen-binding fragments created by fusing together in precise configuration multiple original fragments, or fragments modified from these fragments, or fragments synthesized de novo by recombinant DNA techniques.

[0258] Single-chain antibodies (ScFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies joined by a suitable polypeptide linker into a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423). (See, e.g., J. Immunol., 2012, pp. 426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains within an ScFv is typically not determinative of the ScFv. Thus, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0259] In dsFvs, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the bond between the two chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but are connected to complementary domains on another chain using a linker that is too short to connect the two domains into a single chain, thereby forming two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0260] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0261] Non-naturally occurring antibodies or antigen-binding fragments can be constructed using solid phase peptide synthesis, or can be produced recombinantly, or can be prepared by methods such as those described in, for example, Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These methods, as well as other methods for generating, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies or multifunctional binding domains, are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 14:243-246 (1993)). 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference.

[0262] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are well known, and exemplary competition assays are provided herein.

[0263] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Constant regions may be absent, but if present, may be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90% (e.g., about 95% or more). Thus, all parts of a humanized antibody or antigen-binding fragment (possibly except for the CDRs) are substantially identical to corresponding parts of natural human antibody sequences.

[0264] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and the CDRs and / or framework regions from another human antibody.

[0265] A "fully human antibody," or "human antibody," or humanized derivative thereof consisting of protein fragments, is an antibody or derivative thereof that contains sequences from (or derived from) the human genome and does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated by using antibody production techniques based on sequences derived from the human genome, for example, by phage display or the use of transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0266] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For example, a native immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, a bispecific or bifunctional antibody has two different binding sites, and a trispecific or trifunctional antibody has three different binding sites.

[0267] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are well known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929, below).

[0268] Additionally, the CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof may be modified to contain a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., a gold particle).

[0269] C.conjugates CARs, CAR-expressing T cells, or monoclonal antibodies, or antigen-binding fragments thereof, specific for one or more of the antigens disclosed herein may be conjugated to agents such as effector molecules or detectable markers using any of a number of means well known to those skilled in the art. Both covalent and non-covalent means may be used. Conjugates include, but are not limited to, molecules in which an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein is covalently linked to an effector molecule or detectable marker. Those skilled in the art will be familiar with the use of conjugates that specifically bind to one or more of the antigens disclosed herein, such as antiviral agents, antimicrobial agents, chemotherapeutic agents, antiangiogenic agents, toxins, 125 I, 32 P, 14 C. 3 H, and 35 A variety of effector molecules and detectable markers, including but not limited to radioactive agents such as S, as well as other labels, targeting moieties, and ligands, can be used. It is understood that it can be used.

[0270] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin used to cause the death of a particular target cell (such as a virus-infected cell).

[0271] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on an antibody, resulting in the attachment of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. The derivatization may involve the attachment of any of several well-known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). The linker may be any molecule used to couple an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker is capable of forming covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may be attached to constituent amino acids through their side chains (e.g., to cysteine ​​via a disulfide bond) or to the amino and carboxy groups of the alpha carbon of the terminal amino acid.

[0272] In some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,28 Nos. 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated by reference in its entirety.

[0273] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by degradation of the antibody. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolea). The linker can be, for example, a peptide linker cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids long, or at least three amino acids long. However, linkers can be cleaved from 4, 5, 6, or 7 amino acids long. The dipeptide derivatives may be 1, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids in length. Proteases may include cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin-B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0274] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, such pH-sensitive linkers are hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, or ketals, etc.) that can be hydrolyzed in lysosomes can be used. (See, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions (such as in blood), but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

[0275] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0276] In yet another specific embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0277] In yet another embodiment, the linker is not cleavable, and the effector molecule or detectable marker is released by degradation of the antibody (see US Publication No. 2005 / 0238649, incorporated herein by reference in its entirety).

[0278] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating a conjugate containing the desired linker with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of effector molecule or detectable marker free in the plasma. A variety of exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0279] In some embodiments, conjugates of a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof, with one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a dolastatin, an auristatin, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0280] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to well-known methods, produced using genetic engineering techniques (see Yu et al., PNAS 2002, 99:7968-7973), or maytansinol and maytansinol analogs can be synthetically prepared according to well-known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, certain microorganisms were further discovered to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, and 4, Nos. 313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for their preparation, and therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and 6,441,163, and European Patent No. EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0281] Additional toxins can be used in conjunction with CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Examples of toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A-F. Such toxins are well known in the art, and many are available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Toxins are readily available from the U.S. Patent Nos. 5,079,163 and 4,689,401. Toxins contemplated also include variants of such toxins (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401).

[0282] Saporin is a toxin derived from Saponaria officinalis that inhibits protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin lacks a mechanism for specific intracellular entry and therefore must bind to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein for efficient entry into cells.

[0283] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins has been mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0284] Ricin is the lectin RCA60 obtained from Ricinus communis (Castor bean). For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, with molecular weights of approximately 65 kD and 120 kD, respectively, and hence RCA. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds lysine to cell surface galactose residues, facilitating transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0285] Ribonucleases have also been used as immunotoxins by conjugating them to target molecules (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribonucleases, such as α-sarcin and restrictocin, are described, for example, in Rathore et al., Gene 190:31-5, 1997, and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin was originally isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes DNA double-strand breaks, leading to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). The drug is the toxic moiety of immunotoxins in clinical trials (see, e.g., Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0286] Abrin includes toxic lectins obtained from Abrus precatorius. The toxic components, abrins a, b, c, and d, have molecular weights of approximately 63-67 kD and consist of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis, while the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0287] CARs, CAR-expressing T cells, monoclonal antibodies, and antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can also be conjugated to a detectable marker, such as a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (such as computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy). Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioactive isotypes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, and lanthanide illuminators. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also useful. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, may also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof are conjugated to a detectable enzyme, they can be detected by adding additional reagents that produce a distinguishable reaction product when used with the enzyme. For example, in the presence of the agent horseradish peroxidase, addition of hydrogen peroxide and diaminobenzidine yields a colored reaction product that can be detected visually. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding.Of note, the avidin itself may be conjugated to an enzyme or fluorescent label.

[0288] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may also be conjugated with lanthanides (such as europium and dysprosium) and manganese. Antibodies or antigen-binding fragments may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a secondary antibody binding site, a metal binding domain, an epitope tag, etc.).

[0289] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may also be conjugated with radiolabeled amino acids. Radiolabels may be used for both diagnostic and therapeutic purposes. For example, radiolabels may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabels may be used in therapy as toxins to treat tumors in subjects, such as neuroblastoma. Examples of labels for polypeptides include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 These include, but are not limited to, radioisotopes such as I or radionucleotides.

[0290] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels may be detected using photographic film or a scintillation counter, fluorescent markers may be detected by detecting emitted light using a photodetector. Enzyme labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0291] D. Nucleotides, Expression, Vectors, and Host Cells

[0013] Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof described herein (including functional portions and functional variants thereof). The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0292] In some embodiments, the nucleotide sequence may be codon-modified.Without being bound by any theory, it is believed that the codon optimization of the nucleotide sequence can increase the translation efficiency of mRNA transcripts.The codon optimization of the nucleotide sequence may involve replacing natural codons with other codons that code for the same amino acid but can be translated by tRNAs that are more easily utilized in cells, thus increasing translation efficiency.The optimization of the nucleotide sequence may also reduce secondary mRNA structures that may interfere with translation, thus increasing translation efficiency.

[0293] In one embodiment of the invention, a nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid may comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0294] As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, may be synthetic or obtained from natural sources (e.g., by isolation and / or purification), may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages (such as phosphoramidate or phosphorothioate linkages instead of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides). In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be preferred for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0295] Recombinant nucleic acids may have sequences that do not occur in nature or sequences that are an artificial combination of two separate regions of a sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificially manipulating separate nucleic acid regions using genetic engineering techniques, such as those described in Sambrook et al., supra. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the duplex formed by hybridization. Examples of modified nucleotides that can be used in nucleic acid generation include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, and N6-isopentenyladenylidene. N6-substituted adenines, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isothiazolinone Examples of nucleic acids include, but are not limited to, pentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention may be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0296] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any of the above-described CARs, or a functional portion or variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence degenerate to any of the above-described sequences, or a combination of degenerate sequences.

[0297] One embodiment further provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0298] Nucleotide sequences that hybridize under stringent conditions may also hybridize under highly stringent conditions. "Highly stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount detectably greater than nonspecific hybridization. Highly stringent conditions include conditions under which polynucleotides with exactly complementary sequences or those with only a few scattered mismatches can be distinguished from random sequences that coincidentally contain a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more in length, and can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions include, for example, conditions under which a polynucleotide with an exactly complementary sequence or one with only a few scattered mismatches can be distinguished from a random sequence that coincidentally contains a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more in length, and can be easily distinguished by highly stringent hybridization. These conditions may include low salt and / or high temperature conditions, such as NaCl or its equivalent at a temperature of about 50-70°C. Such highly stringent conditions tolerate very little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0299] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical.

[0300] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In one embodiment, a recombinant expression vector comprises any of the nucleic acids described above. For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and that is capable of causing a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with a host cell under conditions sufficient for expression of the mRNA, protein, polypeptide, or peptide in the host cell. Such vectors generally do not occur in nature.

[0301] However, some of these vectors may be naturally occurring. The recombinant expression vector may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or derived in part from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain natural or non-natural internucleotide bonds, or both types of bonds. Preferably, the non-natural or altered nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0302] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those (such as plasmids and viruses) designed for propagation and propagation, or for expression, or both. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0303] Bacteriophage vectors such as λυTIO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, for example, LENTIVECTOR® gene transfer technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc. Non-clinical versions of lentiviral vectors are also available and will be known to those skilled in the art.

[0304] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

[0305] Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capec, chi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid transfer using high-velocity particle bombardment (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0306] In one embodiment, recombinant expression vectors may be prepared using standard recombinant DNA techniques, e.g., as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear constructs of expression vectors may be prepared to contain replication mechanisms that function in prokaryotic or eukaryotic host cells. Replication mechanisms may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, and bovine papilloma virus.

[0307] Recombinant expression vectors may contain regulatory sequences, such as transcription and translation initiation and termination codons, that are appropriate for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector is to be introduced, and taking into account whether the vector is DNA or RNA based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0308] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0309] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a CAR (including functional portions and functional variants thereof) or a nucleotide sequence complementary to or hybridizing to the CAR-encoding nucleotide sequence. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific, developmental-specific, etc.) is within the ordinary skill of those in the art. Similarly, the association of a nucleotide sequence with a promoter is also within the ordinary skill of those in the art. The promoter may be a non-viral promoter or a viral promoter, such as a murine stem cell virus (MSCV) promoter, an elongation factor 1 alpha (EF1α) promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, or an RSV promoter. The recombinant expression vector may be designed for either transient expression, stable expression, or both. The recombinant expression vector may also be constructed for constitutive or inducible expression.

[0310] Additionally, recombinant expression vectors may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene may be a gene that sensitizes a cell in which it is expressed to an agent, such as a drug, causing the death of the cell when the cell comes into contact with or is exposed to the agent. Suicide genes are well known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0311] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. A host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. A host cell may be a cultured cell or a primary cell (i.e., isolated directly from an organism, such as a human). A host cell may be an adherent cell or a suspension cell (i.e., a cell that grows in suspension). Suitable host cells are well known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is to amplify or replicate a recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. When the purpose is to produce a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be any cell type, may be derived from any type of tissue, and may be at any stage of development, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell. The host cell may be a natural killer cell (NK cell). The host cell may be a hematopoietic stem cell (HSC).

[0312] For purposes described herein, a T cell may be any T cell, including cultured T cells (e.g., primary T cells), or T cells from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or T cells obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a wide variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may be enriched or purified. The T cells may be human T cells. The T cells may be T cells isolated from a human. The T cells may be any type of T cell, at any stage of development, and may be CD4 + / CD8 +Double positive T cells, CD4 + Helper T cells, such as Th1 and Th2 cells, CD8 + T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., T scm , and naive T cells, etc.

[0313] In one embodiment, the CARs described herein can be used in suitable cells that are not T cells, such as those that have immune effector functions, such as NK cells and T-like cells developed from pluripotent stem cells.

[0314] One embodiment also provides a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population that includes, in addition to host cells comprising any of the described recombinant expression vectors, at least one other cell, e.g., a host cell that does not comprise any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a substantially homogeneous population that primarily comprises (e.g., consists essentially of) a host cell that comprises the recombinant expression vector. The population may also be a clonal cell population, where all cells in the population are clones of a single host cell that comprises the recombinant expression vector and therefore all cells in the population contain this recombinant expression vector. In one embodiment of the present invention, the cells The population is a clonal population comprising host cells containing a recombinant expression vector described herein.

[0315] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the host cells are more pure than they are in their natural environment in the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, or may be greater than about 60%, about 70%, or about 80%, or may be about 100%.

[0316] E. Treatment Method It is contemplated that the CARs disclosed herein may be used in methods of treating or preventing HIV / AIDS in a mammal. In this regard, one embodiment provides a method of treating or preventing HIV / AIDS in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent HIV-1 infection and / or AIDS in the mammal.

[0317] One embodiment further comprises lymphodepleting the mammal prior to administering a CAR disclosed herein. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0318] For purposes of this method, in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells may be autologous to the mammal. As used herein, allogeneic refers to any material derived from an animal of the same species as the individual into which the material is introduced, but from a different individual. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci for those individuals are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically different to be able to interact antigenically. As used herein, "autologous" refers to any material derived from the same individual into whom the material will later be reintroduced.

[0319] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, Rodentia mammals, such as mice and hamsters, and Logomorpha mammals, such as rabbits. The mammal may be of the Order Carnivora, which includes Felidae (cats) and Canidae (dogs). The mammal may be of the Order Artiodactyla, which includes Bovinae (cattle) and Porcinae (pigs), or Persodactyla, which includes Equidae (horses). The mammal may be of the Order Primates, Ceboids, or Simoids (monkeys), or Apes (humans and apes). Preferably, the mammal is a human.

[0320] With respect to the methods described above, the cancer associated with HIV infection may be ALL, AML, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, Cancer of the neck, gallbladder, or pleura; cancer of the nose, nasal cavity, or middle ear; cancer of the oral cavity; cancer of the vulva; chronic lymphocytic leukemia (CLL); chronic myeloid carcinoma (CML); colon cancer; esophageal cancer; cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumor; head and neck cancer (e.g., head and neck squamous cell carcinoma); Hodgkin's lymphoma; hypopharyngeal cancer; kidney cancer; laryngeal cancer; leukemia; liquid tumors; liver cancer; lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma); The cancer may be any cancer, including any of lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, NHL, B-chronic lymphocytic leukemia, hairy cell leukemia, Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0321] With respect to the methods described above, HIV can be any type (e.g., HIV-1 or HIV-2), group, clade, subtype, subsubtype, and / or recombinant pandemic strain (CRF), including HIV-1 groups M, N, O, and P. Within HIV-1 group M, HIV can be any clade, clade subsubtype, and / or recombinant pandemic strain, including but not limited to clades A, A1, A2, A3, A4, B, C, D, F, F1, F2, G, H, J, and K, and recombinant pandemic strains CRF01-CRF90 (see hiv.lanl.gov / content / sequence / HIV / CRFs / CRFs.html on the World Wide Web). Within HIV-2, HIV can be any subtype of non-recombinant or recombinant subtypes (including A, B, C, D, E, F, G, and HIV2_CRF01_AB).

[0322] The terms "treatment" and "prevention," and derivatives thereof, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as potentially beneficial or therapeutic. In this regard, the subject methods can provide any amount or level of HIV / AIDS treatment or prevention in a mammal.

[0323] Furthermore, the treatment or prevention provided by the present methods may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented (e.g., HIV-1 infection). For purposes herein, "prevention" may also encompass delaying the onset of the disease or its symptoms or conditions.

[0324] With respect to the methods described above, treatment or prevention by the methods described above may include, or may be administered in conjunction with, treatment or prevention of one or more conditions or symptoms found to coexist with the disease (e.g., HIV / AIDS-related comorbidities such as Kaposi's sarcoma or acute myeloid leukemia, viral co-infections such as HIV / HBV or HIV / HCV co-infection).

[0325] With respect to the methods described above, treatment or prevention by the methods described above may be used in conjunction with allogeneic or autologous transplantation of cells derived from a mammal lacking a disease-associated genetic factor (for example, but not limited to, bone marrow transplantation of Δ32-CCR5 cells or genetically modified CXCR4-free T cells that are resistant to HIV-1 infection).

[0326] Another embodiment provides a method for detecting the presence of an infectious disease in a mammal, comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody, and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex. If the complex is detected, it indicates the presence of an infection in the mammal.

[0327] The sample may be obtained by any suitable method, such as, for example, a blood draw or a biopsy. A blood draw or venipuncture is the process of taking venous blood from within an individual's veins. A biopsy is the removal of tissue and / or cells from an individual. Such removal may involve the collection of tissue and / or cells from the individual for subjecting the removed tissue and / or cells to an experimental procedure. The experimental procedure may include an experiment to determine whether the individual has and / or is suffering from a particular condition or disease state. The condition or disease may be, for example, HIV / AIDS.

[0328] For one embodiment of the method for detecting the presence of an infectious disease, e.g., HIV-1 infection, in a mammal, the sample containing mammalian cells may be a sample containing whole cells, a lysate thereof, or a whole cell lysate fraction, e.g., a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction. When the sample contains whole cells, the cells may be any cells of the mammal, e.g., cells of any organ or tissue (including blood cells or endothelial cells). The contacting described above may occur in vitro or in vivo with respect to the mammal.

[0329] In addition, detection of the complex may be performed by any of several methods known in the art. For example, the CAR disclosed herein, the polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or antibody described herein, or antigen-binding portion thereof, may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle), as disclosed above.

[0330] The method for testing the target cell recognition ability and antigen specificity of CAR is well known in the art.For example, Clay et al., J.Immunol,163:507-513(1999) teaches the method for measuring the release of cytokines (for example, interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α), or interleukin 2 (IL-2)).In addition, the function of CAR can be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J.Immunol,174:4415-4423(2005).

[0331] The assay method for the ability of CARs to inhibit HIV-1 infection includes, but is not limited to, an in vitro assay for measuring HIV-1 viral replication. For example, HIV-1 viral replication can be monitored using an in vitro PBMC-based assay using a replication-competent HIV-1 molecular clone expressing both heterologous HIV envelope proteins and Renilla luciferase (described in Edmonds et al., Virology, 408:1-13 (2010)). In this assay, the level of viral infection can be monitored by measuring the luciferase activity of infected PBMCs contacted with an anti-HIV agent and / or T cells expressing a chimeric antigen receptor (such as those described herein). In another assay, the level of HIV-1 infection can be assayed by determining the amount of p24 antigen present in the culture supernatant of infected cells using an ELISA assay. In another assay, the level of HIV-1 infection can be assayed by using a molecular-based qualitative or quantitative method to detect the presence or absence of viral nucleic acid. In another assay, infectious H is determined by inoculating an HIV-permissive cell line and monitoring cytotoxic changes indicative of viral infection, in combination with other quantitative indicators of HIV infection. The presence of IV-1 virus can be assayed.

[0332] Another embodiment provides the use of a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody, or antigen-binding portion thereof, and / or pharmaceutical composition of the invention to treat or prevent an infectious disease in a mammal, such as HIV-1 infection, which may be any of the viral, microbial, and / or parasitic diseases described herein.

[0333] Any administration method, including local and systemic administration, may be used for the disclosed therapeutic agents. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration may be used. The specific mode of administration and dosing regimen may be selected by the attending clinician, taking into consideration the specifics of the case (e.g., the subject, the disease, any associated disease states, and whether the treatment is prophylactic). When more than one agent or composition is administered, one or more routes of administration may be used; for example, an antiviral agent may be administered orally, and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic and pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue area where an infection is present or has isolated an infection, or to an area suspected of being prone to or supporting the development of an infection. In some embodiments, sustained intraorgan (or nearby organ) release of a pharmaceutical preparation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as an eye drop or intravitreously to the eye.

[0334] The disclosed therapeutic agents may be formulated in unit dosage forms suitable for administering precise dosage amounts one at a time. Additionally, the disclosed therapeutic agents may be administered in a single-dose or multiple-dose schedule. A multiple-dose schedule may involve an initial series of treatment in which more than one dose (e.g., 1-10 doses) may be administered separately, followed by subsequent doses at intervals, as needed, to maintain or enhance the effect of the composition. Treatment may involve administering the compound once daily or multiple times daily (multi-daily doses) for a period ranging from two to three days to several months or even years. Accordingly, the dosage regimen may be determined, at least in part, based on the specific requirements of the subject being treated and may be dependent on the judgment of the administering physician.

[0335] Typical dosages of the antibody or conjugate may range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0336] In a specific example, a subject is administered a therapeutic composition comprising one or more of a conjugate, antibody, composition, CAR, CAR T cell, or additional agent on a multiple daily dosing schedule, such as at least 2 consecutive days and at least 10 consecutive days, for a period of, e.g., weeks, months, or years. In one example, a subject is administered a conjugate, antibody, composition, or additional agent for a period of at least 30 days, for example, for a period of at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0337] In some embodiments, the disclosed methods include administering to a subject a therapeutically effective amount of the disclosed antibody, antigen-binding fragment, or antibody fragments. This includes providing the subject with surgery, radiation therapy, and / or chemotherapy in combination with (e.g., sequentially, substantially simultaneously, or simultaneously) the combination, CAR, or CAR-expressing T cells. Methods and therapeutic dosages for such agents and treatments are well known to those skilled in the art and can be determined by a skilled clinician. The preparation and administration schedule for the additional agent can be used according to the manufacturer's instructions or according to the experienced judgment of a skilled physician. The preparation and administration schedule for such chemotherapy can also be found in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

[0338] In some embodiments, the combination therapy may include administering to a subject a therapeutically effective amount of an additional HIV inhibitor, an immunomodulatory protein, and / or a protein that improves the function of the CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Examples of additional therapeutic agents that can be used with the combination therapy include, but are not limited to, microtubule-binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, reverse transcription inhibitors, viral protein inhibitors, immunomodulators, antibodies, enzymes, enzyme inhibitors, gene regulators, antiproliferative agents, and latent infection reactivation agents. These agents (administered in therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable antiviral or immunomodulatory agent may be administered in combination with the CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages for such agents are well known to those skilled in the art and can be determined by a skilled clinician.

[0339] Additional antiviral agents that can be combined with the CARs, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein include reverse transcriptase inhibitors (e.g., nucleoside reverse transcriptase inhibitors (NRTIs) such as tenofovir, adefovir, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and apricitabine); non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz, nevirapine, delavirdine, rilpivirine, and etravirine; protease inhibitors (e.g., ritonavir, indinavir, amprenavir, atazanavir, darunavir, tipranavir, saquinavir, nelfinavir, lopinavir, and fosamprenavir); and entry or fusion inhibitors (e.g., enfuviritide, maraviroc, gp41-derived C-peptides, and gp41-derived N-peptides); HIV integrase strand transfer inhibitors (e.g., raltegravir, dolutegravir, and elvitegravir), and any combination of these antiviral agents. In some examples, a latency reactivating agent may be combined with a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein to reactivate a latent HIV infection. Examples of such compounds include, but are not limited to, PKC agonists (e.g., bryostatin, prostratin, ingenol B), innate immune activators (e.g., TLR7 agonists, IL-15SA), histone deacetylase inhibitors (e.g., suberoylanilide hydroxamic acid (vorinostat), romidepsin, panobinostat), DNA demethylating agents, and other chromatin remodeling agents. The selection and therapeutic dosage of such agents are well known to those skilled in the art and may be determined by a skilled clinician.

[0340] Additional radiation or chemotherapy agents that can be combined with the methods described above for treatment, prevention, or synergy include nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), alkylating agents such as busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as dophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and anticancer drugs such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparagus, and steroids. Other agents include, but are not limited to, laginases, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are well known to those skilled in the art and can be determined by skilled clinicians.

[0341] Combination therapy can produce synergistic effects and can be proven to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the combined effect achieved when the same compounds are used separately. Synergistic effects can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit-dose preparation, (2) delivered alternately or in parallel as separate preparations, or (3) when some other regimen is used. In the case of alternate delivery, synergistic effects can occur when the compounds are administered or delivered sequentially, for example, by separate injections in separate syringes. Generally, in the case of alternate administration, an effective dosage of each active ingredient is administered sequentially, i.e., consecutively, while in combination therapy, effective dosages of two or more active ingredients are administered together.

[0342] In one embodiment, an effective amount of a CAR, CAR-T cell, antibody, or antigen-binding fragment, or conjugate thereof, that specifically binds to one or more of the antigens disclosed herein is administered to a subject with HIV / AIDS. After a sufficient time has passed for the administered antibody, antigen-binding fragment, or conjugate to form an immune complex with the antigen expressed on the respective HIV-1-infected cells, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control obtained before the treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control obtained before the treatment indicates that the treatment is effective.

[0343] F. Biopharmaceutical Compositions A biopharmaceutical or biological composition (hereinafter "composition") comprising one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells, that specifically bind to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier), is referred to herein as a gene therapy. The compositions are provided for use in immunotherapy, immunotherapy, and / or cell therapy. The compositions may be prepared in a unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome. The compositions may be formulated for systemic (e.g., intravenous) or local (e.g., intraorgan) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are useful, for example, for the treatment and detection of infectious diseases (e.g., but not limited to, HIV-1 infection). Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are also useful, for example, for the detection of immune dysfunction.

[0344] The administration composition may comprise a solution of the CAR, or CAR-expressing T cells, conjugate, antibody, or antigen-binding fragment, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. Such solutions are sterile and generally free of undesirable material. The composition may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and adjuvants, as needed to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the CAR, or CAR-expressing T cells, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected depending primarily on fluid volume, viscosity, and body weight, depending on the particular mode of administration selected and the requirements of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are well known or will be apparent to those skilled in the art.

[0345] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing compositions for administration will be well known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0346] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be provided in lyophilized form and administered by reconstitution with sterile water, or may be provided dissolved in a sterile solution of known concentration. The CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugate solutions are then loaded into infusion bags containing 0.9% sodium chloride (USP) and, in some cases, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art in administering antibody, antigen-binding fragment, and conjugate drugs; for example, antibody drugs have been available on the U.S. market since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and conjugates thereof, may be administered by slow infusion rather than intravenous push or bolus. In one example, a higher loading dose is administered, followed by a lower maintenance dose. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or the corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) may be infused over approximately 90 minutes, and if this initial dose is well tolerated, then weekly maintenance doses of 2 mg / kg may be infused over 30 minutes for 4-8 weeks.

[0347] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particulate systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries are about 5 μm in diameter, only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, edited by A. Kydonieus, Marcel Dekker, Inc., New York, NY, pp. 315-339, (1992).

[0348] Polymers may be used for ion-controlled release of the CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugate compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are well known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous, mobile liquid at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the preparation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous other systems for controlled delivery of therapeutic proteins are known (U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,957,735, U.S. Pat. No. 5,019,369, U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,514,670, U.S. Pat. No. 5,413,797, U.S. Pat. No. 5,268,164, U.S. Pat. No. 5,004,697, U.S. Pat. No. 4,902,505, U.S. Pat. No. 5,506,206, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,254,342, and U.S. Pat. No. 5,534,496).

[0349] G.Kit In one embodiment, kits using the CARs disclosed herein are further provided. For example, kits for treating HIV / AIDS in a subject or for generating CAR T cells expressing one or more of the CARs disclosed herein. Such kits may typically include the antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells may be included. may be included in the kit.

[0350] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used for treating a particular condition.

[0351] The label or package insert may typically further include instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells, for example, in methods for treating or preventing HIV / AIDS or in methods for generating CAR T cells. The package insert typically includes instructions customarily included in commercial packaging for therapeutic products, which include information about the indications, usage, dosage, administration, contraindications, and / or warnings associated with the use of the therapeutic product. The contents of the instructions may be written in electronic format (e.g., floppy disk or compact disk) or in visual format (e.g., video file). The kit may further include additional components to facilitate the particular use for which the kit is designed. Thus, for example, the kit may further include label detection means (e.g., enzyme substrates for enzymatic labels, filter sets for detecting fluorescent labels, or appropriate secondary labels such as secondary antibodies). The kit may further include buffers and other reagents routinely used in the practice of a particular method. Such kits and suitable contents are well known to those of skill in the art.

[0352] Example The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. On the contrary, it is clearly understood that reliance must be placed on various other embodiments, modifications, and equivalents, which may occur to those skilled in the art after reading the description herein without departing from the spirit of the invention and / or the scope of the appended claims. [Example]

[0353] Assembly of HIV-specific binders into functional CAR molecules This example describes the general construction of monospecific, bispecific, and trispecific anti-HIV CARs containing mD1.22, m36.4, and C46 peptides, and how these CARs are expressed on the surface of primary T cells.

[0354] material and method: Preparation of lentiviral vector constructs The CAR antigen-binding domain sequences were derived from published sequences (Chen et al., J. Virol. 2014;88:(2)1125-1139; Chen et al., Antiviral Research 2010;88:(1)107-115; Egerer et al., Molecular Therapy 2010;19:(7)1236-1244) and synthesized by ATUM (formerly DNA 2.0; Newark, CA) or IDT Technologies (Coralville, IA). The synthesized gene fragments were subcloned in frame into an MSCV promoter-based lentiviral backbone containing the CD8 linker / hinge, CD8 transmembrane domain, 41-BB, and CD3 zeta signaling domain. For the ronic bispecific and trispecific CAR constructs, a cleavable furin-P2A-furin site was placed downstream of the first CAR containing one or two linked antigen-binding domains, followed by the third antigen-binding domain, CD8 linker / hinge, TNFRSF19 transmembrane, and optionally, the CD3 zeta signaling domain. A detailed description of the sequence and CAR structure is shown in Table 1 below. The DNA construct was confirmed by Sanger sequencing (GeneWiz, South Plainfield, NJ). Plasmid mapping was performed using Clone Manager software (Denver, CO).

[0355] Lentiviral vector production A lentiviral vector carrying a CAR transgene was generated by transiently transfecting 293T suspension cells using a four-plasmid system in the presence of polyethyleneimine (PEI). Briefly, suspension 293T cells were co-transfected with the CAR transfer plasmid, VSVg envelope, gag / pol, and rev plasmids, followed by the addition of sodium butyrate to the culture 16 hours later. After 48 hours, the supernatant containing the lentiviral vector (up to 400 mL) was concentrated by ultracentrifugation at 10,000 × g overnight for at least 18 hours. The pellet was resuspended in 2 mL of filter-sterilized SEC buffer containing 5.96 mM HEPES, 5% trehalose, and 100 mM NaCl, and the pellet was easily dissolved by gentle agitation. The resuspended lentiviral particles were stored in multiple aliquots at -80 °C for further use.

[0356] Detection of anti-HIV CAR on the surface of transduced primary T cells cells approximately 1 x 10 6The cells were washed in MACS buffer (phosphate-buffered saline containing 10% bovine serum albumin, pH 7.2 or less). Vioblue-labeled CD4 antibody (clone VIT4) and FITC-labeled CD8 antibody were then added to the cells according to the manufacturer's instructions (Miltenyi Biotec). After 30 minutes of incubation at 4°C, the cells were washed twice in MACS buffer and resuspended in 0.2 mL of MACS buffer. To detect CAR containing the C46 peptide, recombinant human monoclonal antibody 2F5 (Polymun Scientific, Klosterneuburg, Austria) was added at a dilution of 1:1000 for 30 minutes at 4°C, followed by two washes in MACS buffer. 2F5 recognizes the epitope ELDKWA found in the C46 peptide. The cells were then incubated with FITC-labeled F(ab')2 anti-human IgG for 30 minutes at 4°C, washed twice, and then resuspended in 0.2 mL of MACS buffer. Flow cytometry was performed using a MACS Quant VYB1 hemocytometer (Miltenyi Biotec) and analysis was performed using FlowJo software (Tree Star, Ashland, OR).

[0357] result: Overall, anti-HIV CARs were highly expressed on the surface of primary T cells in up to 70% of cases after gene modification using lentiviral vectors. As shown in Figures 3A and 3B, monospecific CARs containing either the mD1.22 domain (LTG1944), the m36.4 domain (LTG1945), or the C46 peptide (LTG2328) were functionally expressed on the surface of T cells. The m36.4 CAR was indirectly detected by fusing an intracellular mCherry reporter upstream of the CD3 zeta signaling domain (Figure 3B).

[0358] To define the optimal bispecific binder structure of a CAR constructed using the mD1.22 and m36.4 domains, up to seven different bispecific CAR constructs were analyzed. These CARs were constructed with various orientations and different linker lengths to preserve domain modularity and functionality. Compared to untransduced T cells, bispecific CARs containing the shortest glycine-serine linker (LTG2325, one G4S motif) or the longest linker (LTG1947, five G4S motifs) to spatially separate the mD1.22 and m36.4 domains were equally expressed with similar transduction efficiencies (Figure 4A). Reversing the orientation of these two domains, placing m36.4 distal to mD1.22 (LTG1948), slightly reduced CAR transduction efficiency (Figure 4B). This phenomenon is likely due to the accessibility of the CD4 antibody epitope rather than the design of the construct. To maximize the functionality of these two domains, we created a bicistronic P2A construct containing two CARs (mD1.22-CAR and m36.4-CAR) to form LTG2303. The rationale for this construct is that m36.4 alone can bind and neutralize HIV-1 strains, albeit with reduced affinity in the absence of the CD4 receptor (Weizao Chen et al., Journal of Virology 2014, 88:2, 1125-1139). Therefore, engineering these two domains into a CAR may improve CAR-mediated cytotoxicity. As shown in Figure 4C, the mD1.22-CAR portion of the bicistronic construct LTG2303 was detected on the surface of T cells. Further evaluation of this construct using anti-CD3 zeta Western blot revealed that both CARs were highly expressed, cleaved well, and migrated to the predicted molecular weight (Figure 4D). Overall, bispecific CARs were highly expressed on the surface of T cells with similar transduction efficiencies across constructs (Figure 4E).

[0359] Next, we used the structures of LTG1946 and LTG2303 to generate trispecific CARs containing all three domains (mD1.22, m36.4, and C46 peptide). Specifically, we designed trispecific CARs to yield: (1) CARs containing all three domains on a single CD3 zeta domain (LTG2318, LTG2319, and LTG2320); (2) CARs containing the bispecific CAR LTG1946 or LTG1947 in combination with the C46 peptide anchored only to the T cell membrane (LTG2323 and LTG2334); and finally, (3) CARs containing the bispecific CAR LTG2303 with the C46 peptide distal to the mD1.22 domain (LTG2329 and LTG2330). All of these combinations resulted in unique trispecific CARs, allowing us to efficiently investigate the function of the trispecific CARs.

[0360] As shown in Figure 11A, 2F5 flow cytometry detected high expression (50–70%) of trispecific CARs containing all three domains on a single CD3 zeta chain (LTG2318, LTG2319, and LTG2320). In contrast, detection of the membrane-anchored C46 peptide in the bicistronic trispecific construct (LTG2323) was significantly lower (18% in Figure 11A). However, the mD1.22-CAR portion of the trispecific construct LTG2323 was highly expressed on the surface of T cells compared to non-transduced T cells (Figure 11B). Similarly, the trispecific CARs LTG2329 and LTG2330 could be detected by 2F5 flow cytometry (up to 40%, Figure 12C). Taken together, the trispecific CARs are functionally detected on the surface of T cells.

[0361] [Table 1] [Example]

[0362] Novel bispecific and trispecific anti-HIV CARs potently disrupt HIV envelope targets This example describes the functional characterization of anti-HIV CARs as determined by a highly sensitive luciferase-based cytotoxicity assay. In addition, T cell activation is determined by quantifying cytokine secretion in the presence and absence of HIV envelope-expressing target cells.

[0363] material and method: Cell lines used for functional characterization The 293T cell line, engineered to stably express a single-stranded full-length HIV envelope protein (293T-Env), was kindly provided by Dr. Dimiter Dimitrov (NCI, Fort Detrick, MD). Briefly, 293T-Env cells were grown in Dulbecco's modified Eagle's medium (DMEM) in the presence of 10% fetal bovine serum and 60 μg / ml Zeocin for maintenance selection. To generate luciferase-expressing cells, 293T-Env cells were transduced with a lentiviral vector containing the firefly luciferase gene, single-cell cloning was performed, and both gp120 / gp41 expression (by 2G12, b12, and 2F5 flow cytometry) and luciferase activity on the surface of 293T cells were tested. One highly expressing HIV-1 envelope and luciferase clone was isolated and used in the cytotoxicity assay described herein (293T-Env-Luc). Raji cells and 293T HEK cells were purchased from ATCC (Manassas, VA) as envelope-free cell lines and subsequently transduced with a lentiviral vector encoding the firefly luciferase gene to generate the Raji-Luc and 293T-Luc cell lines, respectively. Raji-Luc cells were maintained in RPMI medium containing 10% fetal bovine serum. 293T-Luc cells were maintained in DMEM medium containing 10% fetal bovine serum. Single-cell cloning was performed on both cell lines, and the absence of the HIV envelope was confirmed by flow cytometry analysis using 2G12, b12, and 2F5 (monoclonal antibodies against the HIV-1 envelope).

[0364] Purification and transduction of primary T cells Human PBMCs from healthy volunteers were purified from buffy coats using a combination of ficoll-paque gradient separation and leucosep tubes, followed by CD4 + and CD8 +Immunomagnetic bead selection of T cells was performed according to the manufacturer's protocol (Miltenyi Biotec, Bergisch-Gladbach, Germany). On day 0, T cells were activated using CD3 / CD28 MACS® Large-scale T Cell TransAct Reagent (Miltenyi Biotec) in the presence of 40 IU / mL IL-2 (Miltenyi Biotec). On day 3, activated T cells were transduced with a lentiviral vector encoding a CAR construct in the presence of 10 μg / mL protamine sulfate (Sigma-Aldrich, St. Louis, MO) and 200 IU / mL IL-2. Cultures were expanded in TexMACS medium supplemented with 200 IU / mL IL-2 and harvested on days 9-10 for functional analysis.

[0365] Cytotoxicity assay using luciferase detection Briefly, 5 x 10 target cells stably expressing firefly luciferase were cultured in a sterile 96-well plate. 3 The cells were incubated overnight at 37°C in the presence of 5% CO2 with or without CAR T cells at various effector-target ratios. After 24 hours, 100 μL of SteadyGlo reagent (Promega, Madison, WI) was added to each well and incubated at room temperature for 10 minutes, followed by quantification of luminescence using an Enspire plate reader (Perkin Elmer, Waltham, MA). Luminescence was recorded as counts per second (CPS) for each experimental well containing the designated E:T ratio (sample CPS) and target cells alone (target CPS). The percentage of specific lysis was calculated as 1 - (sample CPS / target CPS).

[0366] Cytokine release assay using sandwich ELISA Supernatants from the cytotoxicity assays were collected (E:T ratio 10:1), diluted 10-fold, and assayed for IFN-γ and IL-2 using Ready-Set-Go ELISA according to the manufacturer's instructions (eBioscience, San Diego, CA).

[0367] result: HIV envelope proteins are heterotrimeric glycoproteins expressed on the surface of HIV virus particles and are used to target and hijack T cells. During HIV infection, HIV envelope proteins (gp120) bind to the CD4 receptor of helper T cells, leading to a conformational change in the HIV envelope protein. This change promotes interaction with co-receptors (e.g., CCR5 or CXCR4) on the surface of T cells in a tropism-dependent manner, ultimately resulting in viral fusion and subsequent intracellular release of HIV viral RNA into the T cell. The viral RNA is then reverse transcribed and integrated into the host genome, where it can further produce infectious virus or enter a latent state. While T cells are actively producing HIV virus, portions of the HIV envelope proteins (gp120 / gp41) remain on the surface of these infected T cells as a result of viral budding. Therefore, CARs designed to recognize HIV envelope glycoproteins can specifically target and subsequently eliminate proliferating HIV-infected cells through their designed efficacy.

[0368] HIV envelope epitopes have been used as targets for passive immunotherapy using monoclonal antibodies, but have not met with widespread success (reviewed in Mascola JR, Haynes BF. Immunological reviews. 2013;254(1):225-244; Jaworski JP, Vendrell A, Chiavenna SM. Frontiers in Immunology. 2016;7:661). In the absence of ART, antibodies alone lead to rapid viral rebound and eventual viral escape. This loss of virologic control is primarily due to antibody instability and a gradual decline in antibody concentrations in the host. Thus, HIV therapies capable of providing durable responses have the potential to not only control HIV in the absence of ART, but potentially eradicate the HIV reservoir.

[0369] With the success of CD19 CARs, rethinking CAR-based HIV treatment (and potentially cure) strategies is a very attractive approach. When combined with novel anti-HIV domains and cutting-edge genome engineering tools (e.g., lentiviral vectors), CARs have the potential to address the limitations of passive immunization and overcome traditional pitfalls in HIV CAR design. More importantly, CARs can be incorporated into a new "kick and kill" paradigm focused on eliminating the HIV reservoir through continuous immune surveillance and specific drugs that reactivate latent HIV.

[0370] Described herein is a series of unique anti-HIV CARs that exhibit high cytotoxic potency and cytokine function against envelope-containing cells while maintaining exceptional specificity. As shown in Table 1 below, over 20 anti-HIV CARs were designed to fully explore their potency against HIV-1. These CARs were engineered by uniquely combining three different HIV envelope-binding sequences in a precise manner to render them highly potent and synergistic inhibitors of HIV-1 infection. These binders were functionally characterized as CARs and their iterations by cloning them into lentiviral expression vectors containing selected structural and signaling domains under the control of a constitutively active promoter, and were analyzed in vitro using an HIV envelope cell line model for transduction efficiency, killing function, and other properties. , and cytokine production. Table 1 provides an overview of the terminology used. In some examples, the CAR construct LTG1732 (mCherry reporter) was used as a negative control to assess changes in T cell function resulting from viral transduction.

[0371] In the early 2000s, CD4-zeta CARs entered clinical trials. Although these studies showed a lack of efficacy, these CARs continued to be used for decades. A possible reason for the lack of virologic control is the need to redesign the original first-generation CD4 zeta CARs using current understanding of CARs. Therefore, and similar to other researchers in the CAR field, we designed and engineered a CD4-CAR using the second-generation structural elements described herein and compared it with the more potent, specific, and small CD4-derived mD1.22-CAR (LTG1944) disclosed herein. Unlike the CD4-CAR, the mD1.22-CAR was highly potent, but it showed improved specificity and tended to produce lower levels of off-target cytokines (data not shown). Therefore, the novel CD4-like improved mD1.22-CAR described above was selected as the initial prototype for further iterative CAR design.

[0372] To fully understand the relative contribution of each binder in the context of CARs, we further designed and evaluated m36.4-CAR (LTG1945) and C46-CAR (LTG2328). As shown in Figure 3C, mD1.22-CAR (LTG1944) had the highest potency, followed by m36.4-CAR (LTG1945) and C46-CAR (LTG2328). All three CARs demonstrated excellent specificity and no off-target killing in the absence of HIV envelope (Figure 3D). Furthermore, these CARs, except for C46-CAR, were specifically triggered by HIV envelope-expressing cells to secrete IFN-γ, a marker of antigen-driven T cell activation (Figure 3E). Overall, mD1.22-CAR possessed the most optimal structure for constructing more advanced bispecific and trispecific HIV CARs.

[0373] In designing bispecific HIV CARs, we used an iterative approach to determine the best combination of mD1.22 and m36.4 for optimal CAR function. As shown in Figure 5, all bispecific CARs destroyed up to 80% of their envelope-expressing targets (Figure 5) and were highly specific (Figure 6). However, T cells engineered with both mD1.22-CAR and m36.4-CAR using a bicistronic P2A vector (LTG2303) were significantly more potent than cells expressing only one CAR (e.g., LTG1944 or LTG1945), as measured by cytotoxicity (Figure 5) and cytokine release (Figure 7). Reversing the order of these two domains, placing m36.4 distal to mD1.22, did not significantly affect CAR activity (LTG1948). As shown in Figure 8A, shortening the spatial distance between the mD1.22 and m36.4 domains to a single G4S motif significantly increased CAR-mediated cytotoxicity (LTG2325 vs. LTG1947). Furthermore, these linker-specific CARs did not exhibit cytotoxicity against non-envelope-containing 293T cells or Raji cells (Figure 8B). Further investigation of these linker-specific CARs revealed that all anti-HIV CARs specifically triggered IFN-γ production, and in some cases, increased potency was often accompanied by increased off-target IFN-γ production (Figure 9). Overall, a series of functional assays confirmed that LTG2303 possessed the most potent bispecific CAR structure for the design of further trispecific HIV CARs.

[0374] Then, starting from the basic mD1.22-CAR structure, a trispecific HIV CAR structure was constructed. We designed and evaluated several intermediate constructs to evaluate the optimal placement of a highly potent C-peptide, C46. C-peptides (e.g., T20 or C46) are derived from highly conserved regions in the C-terminal heptad repeat (CHR) region of gp41. In nature, hydrophobic interactions between the C-peptide and the N-terminal heptad repeat (NHR) region are involved in viral fusion. Interestingly, several unique methods using C-peptides as decoys have clearly demonstrated potent viral fusion inhibition, leading to the suppression of HIV-1 infection. For example, a few published approaches have shown that anchoring C-peptide to the T cell membrane potently blocks HIV-1 infection (van Lunzen et al., Molecular Therapy 2007, 15:(5)1024-1033; Kimpel et al., PLOS One 2010, 5:(8)e12357; Melikyan et al., Journal of Virology 2006, 80:(7)3249-3258). In a second approach, constitutive secretion of C-peptide (SAVE) by T cells also protected bystander T cells from HIV-1 infection (Egerer et al., Molecular Therapy 2010, 19(7), 1236-1244). In 2003, enfuviritide (T20) was approved by the FDA as a rescue therapy for treating multidrug-resistant HIV-1 patients. However, the emergence of T20-resistant mutants led to the development of improved C-peptide designs.

[0375] These new C-peptides, such as C46, ​​have been demonstrated to inhibit T20-resistant HIV-1 mutants. Like other C-peptides, C46 targets the long, overlapping T20 region, which is involved in gp41-mediated viral fusion. Furthermore, improved C-peptides neutralize HIV-1 strains with potent inhibition independent of viral tropism and improved breadth both alone and in combination with mD1.22-based antibodies (Yao et al., The Journal of Biological Chemistry 2012, 287:(9), 6788-6796; Qi et al., Emerging Microbes & Infections 2017, 6:(6)). Therefore, it is reasonable to consider the design of trispecific CARs using the C46 peptide as an attractive approach to protect T cells from HIV-1 infection and to broaden the breadth of CARs when encountering highly resistant HIV-1 viruses.

[0376] As shown in Figure 10, C46-based CARs were configuration-dependent and performed best when the C46 peptide was designed to be distal to the mD1.22 domain (LTG2316, LTG2317 vs. LTG2314, LTG2315). As previously shown using m36.4-based bispecific CARs, increasing the linker length between the C46 and mD1.22 domains also reduced cytotoxicity (Figure 10A, LTG2316 vs. LTG2317). However, this slightly less active CAR (LTG2317) showed improved specificity as measured by IFN-γ secretion (Figure 10B). Furthermore, mD1.22-CARs designed with either the m36.4 domain (LTG1946) or the C46 peptide (LTG2316) exhibited similar cytotoxic effects and released IFN-γ in the presence of envelope-expressing targets (Figures 10A and 10B). In conclusion, an iterative approach was used to determine optimal binder structures for bispecific HIV CARs. This approach revealed a set of rules governing HIV CAR design and identified two highly potent CAR structures to use as prototypes for trispecific HIV CAR design.

[0377] Herein, trispecific anti-HIV CARs are described and characterized for their cytotoxicity capabilities. Three unique trispecific CAR structures were constructed by (1) fusing all three domains onto one CD3 zeta (LTG2318, LTG2319, LTG2320) or (2) combining the bispecific CAR LTG1946 with a membrane-anchored CD3 zeta domain (LTG2318, LTG2319, LTG2320). (1) by combining the C46 peptide with the C46 peptide located distal to the mD1.22 domain (LTG2323, LTG2334), and finally (2) by using the bispecific CAR LTG2303 with the C46 peptide located distal to the mD1.22 domain (LTG2329, LTG2330). As described in Example 1, these trispecific CARs were expressed on the surface of primary T cells. Functionally, when using a surrogate HIV envelope-expressing cell line, the trispecific CARs were comparable to the bispecific CARs, exhibiting less off-target killing at very high effector-to-target ratios. As shown in Figure 11C, the position of C46 had a significant impact on the trispecific CARs designed with all three domains located on a single CD3 zeta chain. For example, placing the C46 peptide between the mD1.22 and m36.4 domains (LTG2319) or near the T cell membrane (LTG2318) inhibited its cytolytic function. However, when the C46 domain was presented first, followed by the other two domains (LTG2320), or when the C46 peptide was anchored to the T cell membrane independently of the CAR (LTG2323), they exhibited potent cytolytic function, similar in function to the bispecific CAR LTG1946 (Figure 11C). These trispecific CARs were highly specific, showed no off-target killing of non-envelope-containing cells (Figure 11D), and produced cytokines in response to antigen (Figures 11E-F). Of the first set of trispecific CARs, LTG2323 appeared to be the most potent.

[0378] Similarly, a third set of trispecific CARs, LTG2329 and LTG2330, also exhibited potent cytolytic activity (Figure 12B), with some increased nonspecific killing at very high E:T ratios (Figure 12D). These observations were only observed in T cells coexpressing two different CARs (LTG2303, LTG2329, and LTG2330). As previously observed for bispecific CARs (LTG2316 vs. LTG2317), increasing the linker length also reduced off-target cytotoxicity (Figure 12D, LTG2330 vs. LTG2329). Thus, further optimization of the CAR structure may improve CAR specificity. In conclusion, bispecific and trispecific anti-HIV CARs are highly potent and represent a new class of HIV / AIDS treatment therapies. [Example]

[0379] Bispecific and trispecific anti-HIV duoCAR-T cells broadly and potently deplete HIV-infected PBMCs in vitro and in vivo This example examines the killing efficacy of anti-HIV CAR-T cells and the susceptibility of CAR-T cells to HIV-1 infection in vitro and in vivo using PBMCs infected with different Env-IMC-LucR HIV-1 viruses.

[0380] material and method: In vitro anti-HIV CAR efficacy when infected with replication-competent Env-IMC-LucR molecular clones into PBMCs from the same donor Replication-competent HIV-1 molecular clones (Env-IMC-LucR) containing the desired heterologous HIV-1 envelope upstream of an in-frame Renilla luciferase ORF were used to test for inhibition of HIV-1 infection. Infectious clones were generated as previously described (Edmonds et al., Virology 2010, 408:1-13). PBMC or CD4 +For HIV-1 infection of T cells, Renilla luciferase expression is used as a sensitive and quantifiable indicator of HIV-1 viral replication for up to several weeks after inoculation. Briefly, autologous donor PBMCs (HIV-1 naive) were activated with PHA (4 μg / mL) and IL-2 (100 U / mL) and cultured at 37°C in R10 medium (RPMI supplemented with 10% heat-inactivated FBS, penicillin (100 U / mL), streptomycin (10 μg / mL), glutamine (2 mM), and HEPES (10 mM). One day later, T cells were cultured in 96-well round-bottom plates. Over 24 hours, 1 x 10 5 PBMCs were cultured in 1 × 10 PBS containing the indicated Env-IMC-LucR virus. 5 The next day, infected PBMCs were infected with 1 × 10 5 Effector cells (anti-HIV CAR T cells) were added and co-cultured for 7 days. For in vitro protection assays, anti-HIV CAR-T effectors were directly exposed to the indicated Env-IMC-LucR viruses at an MOI of 1 in the absence of PBMCs. Cell culture supernatants (60 μL) were collected from the cocultures on days 0, 3, 5, and 7 for further analysis, and the cultures were supplemented with R10 medium. After 7 days, cells were pelleted and lysed with 20 μL of lysis buffer, and luminescence was quantified (relative light units, RLU) according to the manufacturer's instructions (Promega, Madison, WI). Data were obtained using three independent donors (error bars = standard deviation). Luciferase activity was quantified according to the manufacturer's instructions. Data are expressed as relative light units (RLU). For the in vitro killing assay, the log inhibition of HIV-1 infection was calculated using the formula: (log inhibition of HIV-1 infection) = Log10(CARRLU / UTDRLU). The percentage of HIV-1 inhibition was calculated by 1 - (CARRLU / UTDRLU) x 100%. Statistical analysis was performed using multiple analysis Student's t-test for the in vitro killing efficacy assay and one-way ANOVA for the in vitro protection assay.

[0381] Multispecific anti-HIV duoCAR T cells potently deplete acutely and chronically HIV-infected PBMCs in a humanized NSG mouse model We investigated the in vivo efficacy of anti-HIV duoCAR-T cells using a humanized intrasplenic NOD-SCID-IL2Rγ mouse model (hu-spl-PBMC-NSG) in which rapid, potent, and easily quantifiable HIV-1 infection was established in the mouse spleen using human PBMCs infected with Env-IMC-LucR HIV-1 virus as previously described (Bardhi et al., J Virol 2017, 91:20; Thomas et al., Methods Mol Bio 2016, 1354:221-35). Briefly, 10 million PBMCs from the same donor were activated as described above and spinfected with 10 IU of Du422.1 per 10 PBMCs. HIV-infected PBMCs were injected intraspleenically with untransduced T cells (UTD) or either LTG2303 (bispecific duoCAR) or LTG2330 (trispecific duoCAR) containing 30–50% CAR-containing T cells at an E:T ratio of 0.5:1 (e.g., 5 million total CAR-T cells and 10 million HIV-infected PBMCs). The PBMCs and T cells containing CAR effector cells were mixed immediately before injection. After 1 week (acute) or 1 month (chronic) of infection, mice were sacrificed, and spleens were collected for further analysis. Total splenocytes were divided into three groups and subjected to the following assays: 1) luciferase assay to measure infection; 2) CD4 + and CD8 + 3) flow cytometry analysis to detect T cells, and 3) DNA extraction by qPCR at the end of the experiment to accurately detect CAR-T cell persistence. HIV-1 infection was quantified in a portion of splenocytes using a Renilla luciferase assay system (Promega, Madison, WI) as previously described (Seay et al., Journal of Virology 2015).

[0382] In vivo CAR-T cell persistence using real-time c-Frag qPCR Briefly, one million mouse splenocytes were harvested and genomic DNA was extracted by ACGT (Germantown, MD) using the Promega Maxwell® 16 LEV Blood DNA Kit. To determine CAR-T persistence, a unique fragment (c-frag) contained in the lentiviral vector backbone of the anti-HIV CAR, rather than the HIV-1 virus, was used to determine the copy number per 50 ng of genomic DNA isolated from mouse splenocytes. The following primers and probe were used to detect c-frag: forward primer 5'GGAGTTGAGACC The following primers and probe were used to detect the polypyrimidine tract binding protein 2 (PTBP2) gene to control for PCR inhibition: forward primer 5'-TCTCCATTCCCTATGTTCATGC-3', reverse primer 5'-GTTCCCGCAGAATGGTGAGGTG-3', and probe 5'-JOEATGTTCCTCGGACCAACTTG-BHQ-1-3'. The copy number of cFrag in spleen DNA was calculated by using a standard curve consisting of known amounts of plasmid DNA containing the c-Frag gene tag and subsequently normalizing to the input spleen DNA (0.05 µg) by multiplying by the sample volume (12.5 µL) per PCR reaction.

[0383] result: A major challenge in the field of HIV immunotherapy is the development of therapies that can precisely target and control HIV and prevent viral escape. Similar to combination antiretroviral therapy, multiple approaches targeting the HIV envelope using immunotherapeutic approaches offer the advantage of successfully controlling HIV infection while also targeting latent HIV reservoirs, a challenge posed by current drug therapies. To effectively develop such immunotherapies, it is likely necessary to target multiple, non-overlapping, highly conserved epitopes on the HIV envelope protein, which may predispose to reduced viral fitness during viral escape. To this end, we designed bispecific CARs using domains targeting highly conserved regions required for key steps involved in HIV-1 viral entry (mD1.22), coreceptor usage (m36.4), and fusion (C46). Because the function of bispecific CARs is related to the binder structure, the principles governing the former allowed for the rational design of trispecific CARs. Herein, we describe the in vitro and in vivo efficacy of bispecific and trispecific anti-HIV CARs against genetically diverse and resistant Env-IMC-LucR HIV-1 viruses encoding a comprehensive set of env genes. Additionally, the m36.4 domain and C46 peptide were evaluated for their ability to protect the anti-HIV CARs in vitro, and only the most potent anti-HIV CARs were evaluated for in vivo efficacy.

[0384] As shown in Figure 13, for donors used in the HIV-1 exposure study, CD4 +T cell effector activity was enhanced. As shown in Figures 14A-14K, the anti-HIV CARs exhibited excellent potency against all Env-IMC-LucR HIV-1 viruses tested. As expected, the monospecific CARs were least potent (LTG1944, mD1.22-CAR), followed by the bispecific CAR containing a single CD3 zeta chain (LTG1946). For several donors and HIV-1 strains tested, the trispecific LTG2323 CAR was slightly more potent than the corresponding bispecific LTG1946, possibly suggesting a C46-mediated effect. This enhanced antiviral activity was observed for the Env-IMC-LucR viruses encoding the env gene from SF162 (donor I), Du172.17 (donors H and I), and AE.CNE8 (donor H). Conversely, LTG2323 was the least potent of the trispecific CARs. As shown in Figure 14 for different donors, anti-HIV bispecific CARs (LTG2303) and trispecific CARs (LTG2329 and LTG2330) designed with two CD3 zeta chains (multispecific duoCARs) had very good potency and breadth against PBMCs infected with several Env-IMC-LucR viruses (see Figure 15, up to 3-log suppression compared to UTD control). Notably, bispecific and trispecific duoCAR-T cells also inhibited VRC01 and in some cases cleared PBMCs infected with several Env-IMC-LucR viruses encoding clade C env genes resistant to 3BNC117, two broadly neutralizing antibodies currently in clinical trials that target the CD4 binding site (Bar et al., N Engl J Med 2016, 375:2037-2050; Liu, Bai, Liu, Zhang, & Wang, Int Immunopharmacol 2017;52,44-50). As shown in Figures 15 and 16, bispecific and trispecific duoCAR-T cells were consistently the most potent CAR constructs, eliminating HIV-1 infection by up to 3 logs or 99%, respectively, for nearly all Env-IMC-LucR viruses tested. Further investigation of the multispecific duoCAR T cells revealed that, unlike the UTD control or conventional CAR-T cells such as the monospecific 1944 CAR or the bispecific 1946 CAR, the multispecific duoCAR-T cells significantly depleted PBMCs infected with the 3BNC117 / VRC01-resistant Du422.1-IMC-LucR virus, even at extremely low E:T ratios, e.g., 1:100, as shown in Figure 17A and Figure 17B.

[0385] In Figure 18, mD1.22-CAR T cells were clearly more susceptible to HIV-1 infection in an in vitro protection assay than the other CARs tested. The presence of the m36.4 domain was sufficient to confer protection to CAR T cells, cutting off HIV-1 infection to levels comparable to uninfected PBMCs. The conventional bispecific 1946 CAR and the bispecific 2303 CAR, which contain the m36.4 domain but have different CAR structures, were compared. No statistically significant differences were observed between m36.4 and duoCAR (BaL, P = 0.06; NL4-3, P = 0.09; SF162, P = 0.2; AE.CNE55, P = 0.9). These results indicate that the ability of m36.4 to protect CAR T cells is independent of its structure. Furthermore, the ability of m36.4 to sufficiently protect CAR T cells and its overlap with the C46 peptide appear t...

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, the extracellular antigen-binding domain comprising an anti-HIV envelope antigen-binding domain encoded by a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 45, 49, 53, 57, 61, 65, 69, 75, 79, 83, 87, 91, 95, 99, 103, 111, 115, or 119.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one anti-HIV envelope antigen-binding domain binds to an HIV-1 envelope protein.

3. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one anti-HIV antigen binding domain, at least one intracellular signaling domain, or both, is linked to the transmembrane domain by a linker or spacer domain.

4. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 and is linked to the transmembrane domain.

5. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular anti-HIV antigen-binding domain is located after a leader nucleotide sequence encoding a leader peptide.

6. 2. The isolated nucleic acid molecule of claim 1, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 35, which encodes the leader amino acid sequence of SEQ ID NO:

36.

7. 2. The isolated nucleic acid molecule of claim 1, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 37, which encodes the leader amino acid sequence of SEQ ID NO:

38.

8. 2. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

9. The nucleic acid sequence encoding the extracellular anti-HIV antigen-binding domain is a nucleotide sequence comprising SEQ ID NOs: 1, 3, 5, 45, 49, 53, 57, 61, 65, 69, 75, 79, 83, 87, 91, 95, 99, 103, 111, 115, and 119.

2. The isolated nucleic acid molecule of claim 1, comprising a sequence identical to or having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

10. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

11. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

12. The encoded at least one costimulatory domain may be OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any of these.

12. The isolated nucleic acid molecule of claim 11, comprising any combination of functional signaling domains.

13. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.

14. The CAR of claim 13, comprising at least one extracellular antigen-binding domain comprising an anti-HIV antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, at least one transmembrane domain, and at least one intracellular signaling domain.

15. The CAR of claim 14, wherein the anti-HIV antigen-binding domain comprises at least one antigen-binding domain that binds to an HIV-1 envelope protein.

16. 15. The CAR of claim 14, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

17. The CAR of claim 14, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

8.

18. The CAR of claim 14, wherein at least one extracellular antigen-binding domain, at least one intracellular signaling domain, or both, comprising an anti-HIV antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, is linked to the transmembrane domain by a linker or spacer domain.

19. The CAR of claim 14, wherein the linker or spacer domain is derived from the extracellular domain of CD8 and is linked to the transmembrane domain.

20. The CAR of claim 14, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

21. The CAR of claim 14, wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

22. A vector comprising the nucleic acid molecule of claim 1.

23. 14. The vector of claim 13, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, and a retrovirus vector, or a combination thereof.

24. The vector of claim 13 further comprising a promoter.

25. 25. The vector of claim 24, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a synthetic promoter, a suicide promoter, or any combination thereof.

26. A cell comprising the vector of claim 24.

27. 27. The cell of claim 26, wherein the cell is a T cell.

28. The T cells are CD4 ＋ 27. The cell of claim 26, which is a T cell.

29. The T cells are CD8 ＋ 27. The cell of claim 26, which is a T cell.

30. 27. The cell of claim 26, wherein the cell is a human cell.

31. A method for producing cells, comprising the step of transducing a T cell with the vector of claim 25.

32. 10. A method for generating a population of RNA-engineered cells, comprising the step of introducing in vitro transcribed or synthesized RNA into cells, said RNA comprising the nucleic acid molecule of claim 1.

33. 30. A method of providing anti-HIV immunity to a mammal, comprising administering to said mammal an effective amount of the cells of claim 27.

34. A method for treating or preventing HIV-1 in a mammal, comprising a step of administering to the mammal the CAR of claim 13 in an amount effective for treating or preventing HIV-1 in the mammal.

35. 1. A pharmaceutical composition comprising an anti-HIV effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, the anti-HIV antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a human with HIV / AIDS.

36. 36. The pharmaceutical composition of claim 35, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSF19, and CD154, or any combination thereof.

37. 36. The pharmaceutical composition of claim 35, wherein the T cells are T cells from a human with HIV / AIDS.

38. 1. A method of treating a mammal having a cancer, disease, disorder, or condition associated with elevated expression of an HIV-1 envelope antigen, the method comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, the T cells encoding a chimeric antigen receptor (CAR). the CAR comprises a nucleic acid sequence encoding the CAR, wherein the CAR comprises at least one extracellular antigen-binding domain, including an anti-HIV antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain; and the T cell is a T cell of the subject with HIV / AIDS.

39. 1. A method of treating HIV / AIDS in a subject in need thereof, the method comprising: administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, the extracellular antigen-binding domain comprising an anti-HIV antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, and wherein the T cells are T cells of the subject with HIV / AIDS.

40. 40. The method of claim 38 or 39, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

41. 10. A process for producing a chimeric antigen receptor (CAR)-expressing cell, the process comprising introducing the isolated nucleic acid of claim 1 into the cell.

42. The process for producing a chimeric antigen receptor (CAR)-expressing cell of claim 41, wherein the cell is a T cell or a cell population containing a T cell.

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