Compositions and methods for treating HIV / AIDS using immunotherapy

Abstract

To provide: a pharmaceutical composition comprising an anti-HIV effective amount of a population of human T cells; and a method of treating HIV-related cancers or HIV / AIDS.SOLUTION: The invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular anti-HIV antigen binding domain, where the isolated nucleic acid encoding the extracellular anti-HIV antigen binding domain comprises a specific nucleotide sequence, at least one transmembrane domain, and at least one intracellular signaling domain. The invention also provides: a vector comprising the nucleic acid molecule; a cell comprising the vector; a pharmaceutical composition comprising an anti-HIV effective amount of a population of human T cells; and a method of treating HIV-related cancers or HIV / AIDS in a subject, the method comprising administering the pharmaceutical composition to the subject.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 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 disclosure of each of the above - mentioned U.S. provisional patent applications is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format, and the entire sequence listing is incorporated herein by reference. The file name of this ASCII copy (created on December 18, 2018) is "SequenceListing.txt" and its size is 232 kilobytes.

[0003] Description of Research or Development Sponsored by the Federal Government Not applicable Field of the Disclosure This application relates to the field of diseases associated with human immunodeficiency virus infection / acquired immunodeficiency syndrome (HIV, HIV / AIDS), particularly protein - binding domains, chimeric antigen receptors (CARs) containing such binding domains, and methods of using the same.

Background Art

[0004] Background Infection with the human immunodeficiency virus (HIV) remains a major threat to human health. In 2016, 1 million people died worldwide from causes related to HIV, and an estimated 36.7 million people were living with HIV. 54% of adults and 43% of children living with HIV are currently receiving lifelong antiretroviral therapy (ART). Although there is no cure for HIV infection, ART has enabled many people to live productive lives for longer (all data from the July 2017 update of the WHO fact sheet). Long-term antiretroviral therapy (ART) may increase the risk of severe toxicity and the development of co-morbidities such as lipodystrophy, insulin resistance, cardiovascular disease, and organ failure. In addition, long-term ART may also lead to HIV drug resistance and reduced drug efficacy. Although ART is effective in controlling infection, it is not a cure.

[0005] HIV infection can be blocked by inhibiting its ability to enter and replicate within 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 for HIV-1 (discussed in Margolis et al., Immunol Rev 2017;275:313-323). These therapies have very good breadth and potency in vitro against a wide range of HIV clades. However, when such monotherapies are applied in vivo, the effects are insufficient, and several problems occur, such as antibody stability (discussed in Boesch et al., Current Opinion in HIV and AIDS 2015,10(3):160-169), lack of virological 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 the latent HIV reservoir (discussed in Margolis et al., Immunol Rev 2017;275:313-323). ​

[0006] Chimeric antigen receptors (CARs) are hybrid molecules consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a linker / 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 a CAR is generally engineered to mimic the single chain Fragment variable (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs such as receptor ligands (i.e., IL-13 engineered to bind to the IL-13 receptor expressed in tumors), full-length immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D or CD4) have also been engineered into CARs by design. Other cell targets for CAR expression (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). Considerable additional effort will be required to identify the most active T cell populations to transduce with CAR vectors, find optimal culture and expansion techniques, and elucidate in detail the structure of the CAR protein itself at the molecular level.

[0007] The CAR binding motif may be a relatively stable structural domain such as the constant domain of IgG, or it 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 to a position far from the T cell membrane surface. This may be important for some cell targets where the binding domain is particularly close to the surface membrane of tumor cells (such as disialoganglioside GD2; Orentas et al., this observation is unpublished). All of the signaling motifs used in CARs to date include the CD3-zeta chain. This is because this core motif is an important signal for T cell activation. The first reported second-generation CARs were characterized by the CD28 signaling domain and the 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). The emergence of new technologies has eliminated the need for the CAR itself to encode T cell activation by beads conjugated to anti-CD3 and anti-CD28 antibodies, as well as the presence of the classical "signal 2" from CD28. Third-generation vectors using bead activation were found not to outperform second-generation vectors in in vitro assays, and furthermore, did not provide a clear benefit over second-generation vectors in a leukemia mouse model (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 cell 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 also provide important persistence signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions for culturing CAR T cell populations, such as including 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 characterized by CAR has become a new cutting-edge area in synthetic biology for both cancer treatment and infectious disease treatment. Using genetic engineering techniques such as the use of lentivirus-based gene vectors (LVs), multiple promoters and gene products can be introduced into primary lymphocytes. It has been envisioned to manipulate T cells using LV expressing CAR and direct the resulting highly potent cells to sites of HIV infection and sites where HIV has reactivated from latency. Here, CAR-engineered T cells can avoid HIV infection by inhibiting fusion with HIV virus particles or infected cells, and can also remove HIV-infected cells by intervening in the effective killing of cells expressing viral envelope proteins, and as a result, can also remove the viral reservoir.

[0009] The "Berlin Patient" (an HIV-infected patient who received a bone marrow transplant of hematopoietic stem cells (HSCs) from a CCR5 variant donor) is the only reported case of HIV cure and basically indicates that HIV eradication is achievable. One major obstacle in research towards HIV cure is that HIV can persist in a latent state in cellular reservoirs in the host. In the approach called "shock and kill", latency reversing agents (LRAs) are used to reactivate latent HIV reservoirs. Such agents shock the latently infected cells to reactivate the virus at the epigenetic level. A combination approach by combining with immunotherapy that effectively "kills" HIV-infected cells has been proposed as a way to achieve HIV cure.

[0010] Previously tested HIV immunotherapies using CAR therapy involve fusing the CD4 receptor or broad neutralizing antibody domain (bnAb) to the transmembrane and intracellular domains of the CD3 zeta chain. In the late 1990s, the extracellular region of the CD4 receptor (CD4-zeta) was used to construct the first generation of anti-HIV CARs, which were shown to be effective in removing 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 show significant control of HIV infection, and it was reported that they were themselves susceptible to HIV infection (discussed in Lam et al., Immunotherapy 2013,5(4):404-414). Also, CARs designed using bnAbs showed promising results in vitro. In 2015, one group reported that an anti-HIV CAR consisting of the CD4 receptor fused to bnAb (17b) effectively killed HIV-infected cells and simultaneously showed resistance to infection (Liu et al., J.Virol.2015;89:13 10 6685-6694). One major drawback in the development of CARs using bnAbs is that further manipulation is required to suppress the decline in therapeutic efficacy (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 also 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 the clinical experience of CAR-transduced T cells for HIV, the first clinical trial conducted nearly 20 years ago was carried out using CAR-T for HIV. This clinical trial showed that anti-HIV CAR-T therapy is safe and has very good in vivo persistence (Mitsuyasu et al., Blood 2000;96(3):785-793; Deeks et al., Mol Ther. 2002;5:788). One remarkable finding is that although the modified CAR T cells did not significantly control HIV viremia, they were transported to tissues that harbor the latent HIV reservoir (Mits uyasu et al., Blood 2000;96(3):785-793). This is a major advantage over approaches without CAR and suggests that CAR-T is actively involved in immune surveillance activity. Subsequent studies have also suggested that CAR-T may further have the potential to reactivate the latent HIV reservoir in chronically infected cells by cytokine release (Sahu et al., Virology 2013;446(0):268-275). These findings strongly indicate a cure for HIV and support the basis for developing improved anti-HIV CAR-T therapies.

[0012] A new HIV therapy that combines an HIV-1 entry inhibitor or fusion inhibitor to create a chimeric antigen receptor (anti-HIV CAR) is another attractive approach for effectively targeting and killing HIV-infected cells. The advantage of this approach is that even without ART being implemented, anti-HIV CAR may intervene in the immune surveillance of the latent HIV reservoir and at the same time may be able to effectively remove HIV-infected cells. Furthermore, by combining multiple entry inhibitors and / or fusion inhibitors, it becomes more difficult for HIV to infect, further replicate, and acquire resistance in CAR-modified T cells. Therefore, newly developed anti-HIV CAR therapies, especially those containing more than one inhibitor, strongly indicate a functional cure.

Summary of the Invention

Problems to be Solved by the Invention

[0013] There is an urgent and long-standing need in the art to discover novel compositions and methods for treating HIV / AIDS by using an approach that can exhibit specific and highly potent anti-HIV disease activity without presenting the problems described above.

Means for Solving the Problems

[0014] The present invention addresses the above-described need by providing CAR compositions and treatment methods that can be used for treating HIV as well as other diseases and / or conditions. In particular, the present invention disclosed and described herein provides a CAR that can be used for treating a disease, disorder, or condition associated with the expression of an HIV envelope protein, the CAR containing a plurality of HIV antigen-binding domains with high surface expression on transduced T cells, having a high degree of cytolysis of HIV-infected cells, and enabling the transduced T cells to proliferate and persist in vivo.

[0015] Summary Novel anti-HIV envelope protein antibodies, or antigen-binding domains thereof, and chimeric antigen receptors (CARs) containing such anti-HIV envelope protein antigen-binding domains, and host cells (e.g., T cells) expressing the receptor, and nucleic acid molecules encoding the receptor are provided herein. The CAR has a high level of surface expression on transduced T cells, a high degree of cytolysis, and enables the transduced T cells to proliferate and persist in vivo. Further, methods of using the disclosed CAR, host cells, and nucleic acid molecules for treating, for example, HIV infection or AIDS or HIV-related cancer in a patient are provided.

[0016] In one aspect, an improved second-generation CAR comprising three unique classes of HIV peptide inhibitors (mD1.22, m36.4, and C46 peptide) is provided herein. When these domains are engineered to be in a specific orientation in the context of an HIV chimeric antigen receptor, a new series of highly potent bispecific anti-HIV CARs (combinations of two HIV inhibitors) and trispecific anti-HIV CARs (combinations of three HIV inhibitors) are formed. The anti-HIV CARs provided herein are designed to destroy HIV-infected cells while simultaneously providing protection to CAR T cells. 。

[0017] Accordingly, in one aspect, 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 NO: 1, 3, and 5, is provided.

[0018] In another aspect, an isolated polynucleotide encoding an anti-HIV envelope protein that binds to a second anti-HIV envelope protein, or a fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 45, 49, 53, 57, 61, 65, 75, 79, 83, and 87, is provided.

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

[0020] In yet another aspect, an isolated polynucleotide encoding an anti-HIV envelope protein that binds to two additional anti-HIV envelope proteins, or a fragment thereof, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 91, 95, and 99, is provided.

[0021] In another aspect, an isolated polynucleotide encoding an anti-HIV envelope protein that binds to another anti-HIV envelope protein expressed in a cell expressing the other anti-HIV envelope protein, the other anti-HIV envelope protein 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 a fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of Fab fragment, F(ab’) 2 fragment, Fv fragment, and single-chain Fv (ScFv).

[0023] In one embodiment, an isolated polynucleotide encoding an anti-HIV antibody or a 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 encoding an anti-HIV antibody or a fragment thereof or another anti-HIV binding protein is provided, wherein the antibody or fragment thereof or another anti-HIV binding protein binds 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 encoding an anti-HIV antibody or fragment thereof or other anti-HIV binding protein is provided, wherein the antibody or fragment thereof or other anti-HIV binding protein is expressed in a cell containing a second anti-HIV binder consisting of the amino acid sequence comprising SEQ ID NO: 70. In yet another embodiment, an isolated polynucleotide encoding an anti-HIV antibody or fragment thereof or other anti-HIV binding protein is provided, wherein the antibody or fragment thereof or other anti-HIV binding protein binds to two additional such anti-HIV binders consisting of amino acid sequences selected from the group consisting of SEQ ID NO: 92, 96, and 100.

[0026] In another embodiment, an isolated polynucleotide encoding an anti-HIV envelope protein that binds to another anti-HIV envelope protein expressed in a cell expressing another anti-HIV envelope protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 104, 112, 116, and 120.

[0027] In one aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, the 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 NO: 1, 3, and 5, at least one transmembrane domain, and at least one intracellular signaling domain, from N-terminus to C-terminus.

[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 or minimized one antibody domain (e.g., VH only) that binds to an HIV envelope protein.

[0029] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen-binding domain encoded 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 a similar single-chain Ig-like binding site.

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

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

[0034] In yet another embodiment, there is provided an isolated nucleic acid molecule encoding a chimeric antigen receptor (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 NO: 1, 3, and 5, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen comprising, 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, there is provided an isolated nucleic acid molecule encoding a CAR, 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 A domain, an anti-CMV glycoprotein B 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 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.

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

[0037] In another aspect, a CAR containing more than one HIV antigen binder provided herein may contain 2, 3, or 4 linker or spacer domains.

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

[0039] In one aspect, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen-binding domain is linked to each other or to the transmembrane domain 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 domain is linked to each other or to the transmembrane domain by a linker or spacer domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 24, 26, 28, 30, and 32.

[0041] In one aspect, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen-binding domain is 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 as exemplified by SEQ ID NO: 33.

[0042] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular HIV antigen-binding domain is 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 by 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 bound 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 combinations 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 at least one encoded intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0047] In yet another embodiment, more than one HIV antigen binder may be expressed on two different transmembrane proteins in the same cell. Further, these two proteins may express the same intracellular signaling domain, or 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 at least one encoded co-stimulatory 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 combinations thereof.

[0049] In one embodiment, an isolated nucleic acid molecule encoding a CAR further containing a leader sequence or a signal peptide is provided, 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 aspect, a CAR is provided herein that includes, from the N-terminus to the 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 the antigen, or at least one heavy-chain variable region of an antibody that binds the antigen, or a combination thereof.

[0053] In another embodiment, a CAR is provided, wherein the at least one transmembrane domain comprises the 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 HIV latency-related It 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 comprises 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 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 at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0057] In yet another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory 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 combinations 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta CAR amino acid sequence (Figure 2B)).

[0060] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 43 (LTG2328, LP-C46-CD8TM-41BB-CD3 zeta CAR nucleo It includes the chimeric antigen receptor (CAR) amino acid sequence of SEQ ID NO: 44 (LTG2328, LP-C46-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2C)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 44 (LTG2328, LP-C46-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2C)).

[0061] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 47 (LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2D)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 48 (LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2D)).

[0062] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 51 (LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2E)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 52 (LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2E)).

[0063] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 55 (LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2F)). In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 56 (LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta 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-CD3 zeta-F2AF-m36.4-TNFRSF19TM-CD3 zeta 2 CAR nucleic acid sequence (Figure 2J)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 72 (LTG2303, LP-mD1.22-CD8TM-41BB-CD3 zeta-F2AF-m36.4-TNFRSF19TM-CD3 zeta 2 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-CD3 zeta-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-CD3 zeta-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-CD3 zeta CAR nucleic acid sequence (Figure 2O)). 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-CD3 zeta CAR amino acid sequence (Figure 2O)).

[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 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 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-CD3 zeta-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-CD3 zeta-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-CD3 zeta-F2AF-m36.4-TNFRSF19TM-CD3 zeta2 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-CD3 zeta-F2AF-m36.4-TNFRSF19TM-CD3 zeta2 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-CD3 zeta-F2AF-m36.4-TNFRSF19TM-CD3 zeta2 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-CD3 zeta-F2AF-m36.4-TNFRSF19TM-CD3 zeta2 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-CD8TM-CD3 zeta-F2AF-m36.4-TNFRSF19TM 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-CD8TM-CD3 zeta-F2AF-m36.4-TNFRSF19TM 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-CD3 zeta-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-CD3 zeta-F2AF-m36.4-TNFRSF19TM CAR amino acid sequence (Figure 2W)).

[0081] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 121 (LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3 zeta-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-CD3 zeta-F2AF-C46-TNFRSF19TM CAR amino acid sequence (Figure 2X)).

[0082] In one aspect, the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring, and / or predicting treatment outcome, such as for monitoring the progress of such treatment.

[0083] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector such as a viral vector. The vector may 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 may be 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 vector expressing the CAR 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 / heterodimerizing CAR), or for removing 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 vector expressing the CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD). In another aspect, a host cell containing the nucleic acid molecule encoding the CAR is further provided. 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 cell.

[0086] In yet another aspect, there is provided a pharmaceutical composition comprising a population of human T cells in an anti-HIV effective amount, wherein the T cells comprise a nucleic acid sequence encoding a CAR, and the CAR comprises at least one extracellular antigen-binding domain comprising an anti-HIV antigen-binding domain or domain combination comprising the amino acid sequences of SEQ ID NOs: 2, 4, 6, or a domain combination comprising the amino acid sequences of SEQ ID NOs: 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 the T cells are human T cells having HIV infection or T cells intended for administration to a patient having HIV / AIDS.

[0087] In one embodiment, there is provided a pharmaceutical composition, wherein at least one transmembrane domain of the CAR 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, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or combinations thereof.

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

[0089] In yet another embodiment, there is provided a pharmaceutical composition comprising an anti-HIV effective amount of a population of human T cells of a human having HIV / AIDS and HIV-related malignancies such as leukemia associated with HIV / AIDS, CNS leukemia, sarcoma, Kaposi sarcoma, or infectious sequelae, where the infection is resistant to or non-responsive to one or more highly active antiretroviral therapies (ART) and / or chemotherapy that were each unable to completely eradicate the HIV / AIDS or HIV-related malignancy. The use of CAR-modified T cells further includes situations where the viral reservoir is reactivated or exacerbated for the purpose of reducing the ratio of latently infected cells to actively infected cells (including therapeutic holiday of ART, or by mimicking epigenetic signals such that the CAR targets are activated by methylating or demethylating agents of promoter elements, or cell activation pathway activators, or agents that activate latent virus to express envelope proteins).

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

[0091] In yet another aspect, provided is a method for generating a population of RNA-engineered cells, which includes the step of introducing an in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding the disclosed CAR into a cell of interest, thereby generating CAR cells.

[0092] In yet another aspect, provided is a method for diagnosing a disease, disorder, or condition associated with the expression of an HIV envelope in a cell, which includes: a) contacting the cell with a human anti-HIV antibody or a fragment thereof, wherein the antibody or the fragment thereof includes 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 the HIV envelope, and diagnosing that it is a disease, disorder, or condition associated with HIV / AIDS when the HIV envelope protein is present.

[0093] In one embodiment, the disease, disorder, or condition associated with the expression of HIV is candidiasis of the esophagus, bronchus, trachea, or lung, and the mouth (thrush), cervical cancer, invasive, coccidioidomycosis, disseminated or extrapulmonary, cryptococcosis, extrapulmonary, cryptosporidiosis, chronic and intestinal (lasting more than 1 month), cytomegalovirus disease (other than liver, spleen, or nodules), cytomegalovirus retinitis (with loss of vision), encephalopathy, HIV-related, herpes simplex: chronic ulcer (lasting more than 1 month); or bronchitis, interstitial pneumonia, or esophagitis, histoplasmosis, disseminated or extrapulmonary, isosporiasis, chronic and intestinal (lasting more than 1 month), Kaposi sarcoma, lymphoma, Burkitt (or equivalent term) lymphoma, immune Mycobacterial (or equivalent terms) lymphoma, primary, brain, Mycobacterium avium complex or M. kansasi, disseminated or extrapulmonary, Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary), Mycobacterium, other species or unidentified species, disseminated or extrapulmonary, Pneumocystis jirovecii pneumonia, pneumonia, recurrence, progressive multifocal leukoencephalopathy, Salmonella sepsis, recurrence, cerebral toxoplasmosis, wasting syndrome due to HIV (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 / 41(RR-17), 1993), is any AIDS-defining illness. These may occur concomitantly with cancer, including hematopoietic cancers, myelodysplastic syndromes, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in ALL, AML, adult B-cell malignancies including 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 blood cancers and solid tumors, or any combination thereof.

[0094] In another embodiment, a method for diagnosing or prognosticating or risk-assessing an HIV-related disease in a mammal is provided, which comprises detecting the expression of an HIV envelope in a sample derived from the mammal, the method comprising: a) contacting the sample with a human anti-HIV antibody or a fragment thereof, wherein the antibody or the 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, and diagnosing that the mammal has an HIV-related disease when HIV is present.

[0095] In another embodiment, a method of inhibiting HIV-dependent T cell inhibition is provided, which includes the step of contacting a cell with a human anti-HIV antibody or a fragment thereof, and the antibody or the fragment thereof includes an amino acid sequence selected from the group consisting 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. In one embodiment, the cell is selected from the group consisting of HIV-expressing cells, HIV-sensitive cells, and any combination thereof.

[0096] In another embodiment, a method of blocking T cell inhibition mediated by HIV-expressing cells and altering infected tissues to inhibit HIV pathogenesis in a mammal is provided, which includes the step of administering to the mammal, in an effective amount, a composition comprising an isolated anti-HIV antibody or a fragment thereof, and the antibody or the fragment thereof includes an amino acid sequence selected from the group consisting 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. In one embodiment, the cell is selected from the group consisting of HIV envelope-expressing cells, HIV-infected cells or tissues, HIV-sensitive cells and tissues, and any combination thereof.

[0097] In another embodiment, a method of inhibiting, suppressing, or preventing immunosuppression of the anti-HIV response in a mammal is provided, which includes the step of administering to the mammal, in an effective amount, a composition comprising an isolated anti-HIV antibody or a fragment thereof, and the antibody or the fragment thereof includes an amino acid sequence selected from the group consisting 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. In one embodiment, the antibody or the fragment thereof inhibits the interaction between a first cell and a T cell, and the first cell is selected from the group consisting of HIV-expressing cells, HIV-infected cells or tissues, and any combination thereof.

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

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

[0100] In yet another embodiment, a method for treating a mammal having a disease, disorder, or condition associated with increased expression of an HIV antigen is provided, the method including administering to the subject a pharmaceutical composition comprising a population of anti-HIV effective amount of T cells, the T cells comprising a nucleic acid sequence encoding a CAR, the CAR comprising at least one extracellular HIV antigen-binding domain comprising the amino acid sequences 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 the T cells are T cells of a subject having cancer.

[0101] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, which comprises administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, wherein the T cells comprise a nucleic acid sequence encoding a CAR, and the CAR comprises at least one HIV antigen-binding domain comprising the amino acid sequences 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, and at least one intracellular signaling domain, and the T cells are T cells of a subject having an HIV infection or T cells to be administered to such a patient. In some embodiments of the methods described above, the at least one transmembrane domain comprises the transmembrane 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 for generating a population of persistently genetically engineered T cells in a human diagnosed with an HIV infection is provided. In one embodiment, the method comprises administering to the human T cells genetically engineered to express a CAR, wherein the CAR comprises at least one HIV antigen-binding domain comprising the amino acid sequences 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 transmembrane domain, and at least one intracellular signaling domain, and the population of persistently genetically engineered T cells, or the 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 a 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 described above that are associated with increased expression of an HIV antigen can be treated or prevented or remitted using one or more of the CARs disclosed herein.

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

[0106] In yet another aspect, a kit is provided for making the CAR T cells described above or for preventing, treating, or remitting any of the infections, cancers, diseases, disorders, or conditions described above that are associated with increased expression of an HIV antigen in a subject, which kit includes any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, or a container containing instructions for use of the kit.

[0107] The disclosure provided herein initially focused on the generation of CARs utilizing HIV envelope protein antigen-binding fragments. However, 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. Thus, specifically, it is contemplated that this includes, but is not limited to, Rev, Tat, Vif, Nef, Vpu, Vpr, Gag, Pol, protease, nucleocapsid, matrix, capsid, integrase, and / or reverse transcriptase, or any combination thereof, any HIV-derived protein associated with latent or proliferative HIV infection.

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

Brief Description of the Drawings

[0109]

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Mode for Carrying Out the Invention

[0110] Detailed Description Definition As used herein, the singular forms "a", "an", and "the" refer to both the singular and plural forms unless the context clearly dictates otherwise. For example, the term "an antigen" can include 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 phrase "and / or" means "and" or "or". Further, unless otherwise specified, 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 values 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 case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and 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, means a variation 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. This is because such variations are appropriate for the practice of the disclosed methods.

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

[0113] The present disclosure provides an HIV antibody or a fragment thereof, and a CAR having such an HIV antigen-binding domain. Improving the functional activity of the CAR is directly related to improving the functional activity of CAR-expressing T cells. As a result of making one or more of such modifications, the CAR exhibits both cytokine-induced cytolysis and cell surface expression in transduced T cells at high levels, and the proliferation of T cells in vivo and the persistence of transduced CAR-expressing T cells are at high levels.

[0114] The unique ability to combine functional sites derived from different protein domains is an innovative feature of the CAR. The choice of which of these protein domains to select is as important a design feature as the manner in which they specifically bind. The individual design domains are essential components that can be used in any heterologous CAR platform for the purpose of manipulating lymphocyte function. For example, the selection of the extracellular binding domain can render a CAR that is otherwise ineffective effective.

[0115] The invariant framework components of the sequences of immunoglobulin-derived proteins used to generate the extracellular antigen-binding domain of a CAR may be completely neutral or may be such that they self-associate and significantly reduce the efficacy of therapeutic T cells expressing this CAR by metabolically exhausting T cells. This phenomenon occurs independently of the antigen-binding function of this CAR domain. Furthermore, the choice of intracellular signaling domain can also govern the activity and durability of the therapeutic lymphocyte population used in immunotherapy. Here, the ability to bind the target antigen and to transmit an activating signal to T cells via each of the extracellular and intracellular domains described above are important CAR design aspects, but it has also become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on the efficacy of the CAR and may therefore have a decisive role in the function and clinical utility of the CAR.

[0116] Surprisingly and unexpectedly, it has been found that using 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 cell clearance in the host) (see clinical trial using a mouse-derived SS1 ScFv sequence with funding from the University of Pennsylvania, NCT02159716), can determine the functional activity of CAR-expressing T cells.

[0117] The CARs disclosed herein are highly expressed in cells. The cells expressing this CAR have a high proliferation rate in vivo, produce a large amount of cytokines, and have high toxic activity against cells having the HIV envelope antigen to which the CAR binds on their surface. As a result of using a human extracellular HIV antigen-binding domain, CARs with improved functions in vivo were produced, and at the same time, induction of anti-CAR immunity and death of the CAR T cell population in the host immune response were avoided. CARs expressing a fully human extracellular HIV antigen-binding domain exhibit excellent activities and / or characteristics, including i) prevention of poor persistence and poor function of CAR T (seen with mouse-derived binding sequences), ii) lack of delivery of the CAR to specific regions (i.e., intrapleural) for efficacy, and iii) the ability to enable the design of CAR T cells based on both high- and low-affinity HIV binders.

[0118] Next, the CARs of the present invention will be described in detail. This description includes descriptions of its extracellular HIV antigen-binding domain, transmembrane domain, and intracellular domain, and further descriptions of CARs, antibodies, and their antigen-binding fragments, 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 include 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] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains an antigen-binding domain of an antibody or receptor (e.g., a single-chain variable fragment (ScFv), or human CD4 that binds 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. The characteristics of CAR include the ability to redirect the specificity and reactivity of T cells to a selected target in a manner not restricted by the major histocompatibility complex (MHC), and to utilize the antigen-binding properties of a monoclonal antibody or receptor. Since it can recognize antigens without MHC restriction, T cells expressing CAR can recognize antigens independently of antigen processing. Also, when expressed in T cells, CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0121] As disclosed herein, the intracellular T cell signaling domain of a CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a CAR that includes an intracellular domain of a T cell receptor, such as (but not limited to) the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain refers to a portion of a CAR that includes an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for lymphocytes to efficiently respond to an antigen.

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

[0123] In one embodiment, the CAR can be designed to target a desired viral antigen by designing an antigen-binding domain that specifically binds to an antigen on virus-infected cells. A viral antigen is a protein produced by virus-infected cells that elicits an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding domain can 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 the HIV envelope protein glycoprotein (gp160, gp120 / gp41), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) E2 glycoprotein, Epstein Barr envelope protein, and cytomegalovirus (CMV) glycoprotein B antigen. The viral antigens disclosed herein are included merely by way of example. The listing is not intended to be limiting, and other examples will be readily recognized by those skilled in the art.

[0124] In one embodiment, the HIV envelope protein comprises one or more antigenic epitopes related to the viral envelope protein. HIV-infected cells express an envelope protein that can serve as a target antigen for an immune attack. In the present disclosure, anti-HIV CARs targeting the HIV envelope protein were generated, where single-specificity CARs were generated by using one extracellular domain, bispecific CARs were generated by combining two extracellular domains with different orientations and different linker lengths, or trispecific CARs were generated by combining all three extracellular domains. The CAR is composed of three functionally distinct extracellular domains that target non-overlapping epitopes on the HIV envelope to effectively block HIV entry (mD1.22), coreceptor usage (m36.4), and viral fusion (C46). Unlike CARs containing either one bnAb and / or the wild-type CD4 receptor, all three extracellular domains are precisely designed to have increased specificity, potency, and the ability to target newly 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 smaller in size and is designed to 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 designed human antibody domain composed of only the heavy chain and has a mature affinity (Chen et al., Antiviral Research 2010; 88: (1) 107-115). It binds to a CD4-induced (CD4i) epitope that is discontinuously present near the coreceptor binding site on gp120 (Wan et al., PLOS One 2013; 8(6): e66638). When these two domains are combined, they 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 way to prevent viral escape. As a third layer of T cell protection, CAR-T or its T cells were engineered using a 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 lengthened version of enfuvirtide or T20, which is approved by the FDA, is similar to T20, and can be used to suppress HIV variants that are multi-drug resistant to current cART. In particular, when expressed on the surface of T cells or secreted from T cells (SAVE peptide), the C-peptide potently inhibits the fusion of HIV-1 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 designed with these three domains are designed to potently destroy HIV-infected cells while providing protection against CAR T cells.

[0127] To reduce immunogenicity, the CARs used here are constructed with fully human sequences. This is advantageous over using mouse-based ScFv binding sequences, as the latter tend to induce immune responses and CAR-T clearance in human hosts and result in poor T cell maintenance. As described herein, CAR-transduced T cells were generated by transduction using a lentiviral vector construct encoding an anti-HIV CAR gene. It is expected that these CAR-expressing T cells can provide a long-lasting therapeutic effect in patients. It is important to note that the therapeutic CAR-T cell product can be administered once rather than by repeated IV administration.

[0128] Generally, CARs based on mD1.22 (containing the minimal domain human CD4 protein fragment) are highly potent and specifically destroy 293T cells (designated as env + cells) that stably express HIV gp120 and are used as an alternative to HIV-infected cells to reproducibly quantify CAR-mediated cytotoxicity. Subsequently, the mD1.22 domain was fused to m36.4 (a binding domain derived from an anti-HIV antibody) using linker domains as described in SEQ ID NOs: 24, 26, 28, 30, 32, and then these domains were linked to the CD8 or TNFRSF19 transmembrane domain (TM) (SEQ ID NOs: 8 and SEQ ID NO: 14) by a CD8-derived linker domain as described in SEQ ID NO: 10, such that the CD8 linker was linked to the CD8 transmembrane domain as described in SEQ ID NO: 12 or the CD8 linker was linked to the TNFRSF19 TM as described in SEQ ID NO: 16, thus enabling binding to the intracellular signaling domain. The intracellular signaling domain is either of the first or second generation. The second generation CAR binds to the 41BB (CD137) intracellular signaling domain of SEQ ID NO: 18 near the transmembrane portion leading to the CD3 zeta signaling domain. The intracellular domain for the CD3 zeta chain binds directly to the transmembrane domain or, in the second generation construct, to the 41BB domain. In the production of lentiviral gene vectors, it is very important to avoid sequence repeats because the vectors often edit or recombine these sequences, thereby losing the structure of the CAR construct. Another feature unique to the vector family presented here is the generation of two different CD3 zeta domains. Both of these encode the same amino acid sequence (SEQ ID NOs: 20 and SEQ ID NO: 22), but their nucleic acid sequences (SEQ ID NOs: 19 and SEQ ID NO: 21) are different.

[0129] In some cases, a bispecific or trispecific CAR containing two or three extracellular domains and ultimately binding to and expressed on the same cell surface protein forms a single chain. 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 / ribosome skip site or self-cleaving peptide as described in SEQ ID NO: 33 and SEQ ID NO: 34. The importance of the bispecific or trispecific CAR should be recognized when one binder fails to recognize an HIV variant or an emerging mutation and the second and / or third domains can compensate for that failure. Through the effective use of such CAR constructs described herein, dependence on ART can be eliminated and a transition can be made to a cure for HIV using CAR-T technology.

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

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

[0132] In a 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 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: 2, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2.

[0133] In a 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 extracellular HIV antigen binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 4.

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

[0135] In a preferred embodiment, the isolated nucleic acid molecule encoding the conjugated 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 extracellular HIV antigen binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 46.

[0136] In a 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 to the amino acid sequence of SEQ ID NO: 50.

[0137] In a 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 to the amino acid sequence of SEQ ID NO: 54.

[0138] In a 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 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: 58, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 58.

[0139] In a preferred embodiment, the linked extracellular HIV envelope antigen binding domain The isolated nucleic acid molecule to be encoded 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 extracellular HIV antigen-binding domain encoded 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 to the amino acid sequence of SEQ ID NO: 62.

[0140] In a preferred embodiment, the isolated nucleic acid molecule encoding the bound 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 extracellular HIV antigen-binding domain encoded 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 to the amino acid sequence of SEQ ID NO: 66.

[0141] In a preferred embodiment, the isolated nucleic acid molecule encoding the bound 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 extracellular HIV antigen-binding domain encoded 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 to the amino acid sequence of SEQ ID NO: 70.

[0142] In a 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 extracellular HIV antigen binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 76.

[0143] In a 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 extracellular HIV antigen binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 80.

[0144] In a 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 extracellular HIV antigen binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 84.

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

[0146] In a preferred embodiment, the isolated nucleic acid molecule encoding the attached 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 extracellular HIV antigen-binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 92.

[0147] In a preferred embodiment, the isolated nucleic acid molecule encoding the attached 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 extracellular HIV antigen-binding domain encoded thereby 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 to the amino acid sequence of SEQ ID NO: 96.

[0148] In a 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 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: 100, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 100.

[0149] In a 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 to the amino acid sequence of SEQ ID NO: 104.

[0150] In a 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 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: 112, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 112.

[0151] In a preferred embodiment, the isolated nucleic acid molecule encoding the 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 extracellular HIV antigen binding domain encoded thereby comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 11 6 and comprises an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0152] In a 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the extracellular HIV antigen binding domain encoded thereby comprises the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 120.

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

[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-CD3 zeta amino acid sequence (shown in FIG. 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1944, LP-mD1.22-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 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 a CAR comprising 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-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG1947, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 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 FIG. 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-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3 zeta2 amino acid sequence (shown in FIG. 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2303, LP-mD1.22-CD8TM-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3 zeta2 amino acid sequence (shown in FIG. 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-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in FIG. 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 having 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 having 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 (shown in Fig. 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-CD3 zeta amino acid sequence (shown in Fig. 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3 zeta amino acid sequence (shown in Fig. 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-CD3 zeta amino acid sequence (shown in Fig. 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3 zeta 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-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3 zeta 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2319, LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3 zeta 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 having 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 having 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-CD3 zeta-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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3 zeta-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-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3 zeta2 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2329, LP-C46-L3-mD1.22-CD8TM-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3 zeta2 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-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3 zeta2 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2330, LP-C46-L5-mD1.22-CD8TM-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3 zeta2 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-CD3 zeta-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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2331, LP-C46-L3-mD1.22-CD8TM-41BB-CD3 zeta-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-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in Figure 2W)].

[0199] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 117 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 118 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3 zeta-F2AF-LP2-m36.4-TNFRSF19TM amino acid sequence (shown in FIG. 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-CD3 zeta-F2AF-LP2-C46-TNFRSF19TM amino acid sequence (shown in FIG. 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3 zeta-F2AF-LP2-C46-TNFRSF19TM amino acid sequence (shown in FIG. 2X)].

[0202] Generally, the anti-HIV CAR was highly expressed on the surface of primary T cells (FIGS. 3A-3B, FIG. 4, FIGS. 11A-11B, FIG. 12A, and FIG. 12C). The CAR based on mD1.22 is very potent and serves as an alternative to HIV-positive cell lines for quantifying CAR-mediated cytotoxicity, 293T cells stably expressing HIV gp120 (env herein +Specifically destroyed the cells (referred to as cells) (Figure 3C, Figure 5, Figure 8, Figure 10, Figure 11C, and Figure 12B). 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 + Differentially killed the cells (Figure 3C, mD1.22 > m36.4 > C46, P < 0.0001), had no off-target cytotoxic effects (Figure 3D), and + Were triggered to some extent in the presence and absence of env to produce IFN-γ (Figure 3E).

[0203] To define the most optimal bispecific structure, the mD1.22 domain and the m36.4 domain 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, the bispecific CAR significantly increased the anti-HIV cytotoxic effect (Figure 5, LTG2303 vs. LTG1944 or LTG1945), while at the same time maintaining very good specificity (Figure 6). Shortening the linker length between these two domains increased the CAR-mediated cytotoxicity of the bispecific CAR designed with one CD3 zeta while maintaining specificity (Figure 8). However, as measured by the cytokine release assay, env +In the absence of target cells, no significant increase in tonic signaling was observed (Figure 9). More importantly, in the presence of Raji cells known to contain MHC class II molecules that can interact with the CD4 receptor-derived mD1.22 domain, off-target killing was not observed (Figure 6, lower panel). Also, the bispecific anti-HIV CAR released IFN-γ when encountering HIV envelope target cells and released it to a lesser extent even in its absence (Figure 7, Figure 9). Collectively, the bispecific CAR serves as a highly potent structure for designing even more advanced trispecific CARs.

[0204] To construct a trispecific CAR composed of mD1.22, m36.4, and C46 peptides, additional bispecific CAR iterations were created among those designed using mD1.22 and C46 peptides. Different from the case of bispecific CARs, when m36.4 was replaced with C46, CAR function was significantly suppressed 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 more distally than the mD1.22 domain (Figure 10A). Similar to the previously described CD22 CAR, the accessibility of the binder, which correlates with the distance from the T cell membrane, is essential for CAR function (Haso et al., Blood 2013;121:1165-1174). As expected, CAR function was suppressed as a result of the binder arrangement of mD1.22 and C46, which also led to poor levels of IFN-γ secretion (Figure 10B). Notably, high levels of IFN-γ secretion were restored (LTG2316) simply by reversing the order of the domains, while widening the interval between the two domains decreased tonic signaling (LTG2317). Collectively, these data clearly indicate the most optimal bispecific binder arrangement for C46-based bispecific CARs. More importantly, a set of rules governing the function of bispecific CARs, which can be applied to the rational design of trispecific anti-HIV CARs, were identified.

[0205] Targeting multiple epitope determinants on the HIV envelope protein is an attractive approach for designing CARs with excellent 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, the second and / or third domains can compensate for this functional failure acquired by the CAR. Therefore, to improve the breadth of the most potent bispecific CAR candidates, a trispecific CAR was created by designing a bispecific CAR using the third most potent fusion inhibitor (C46 peptide) or entry inhibitor (m36.4). Compared to the bispecific CAR, the trispecific CAR maintained the ability to potently destroy alternative cell lines of HIV and initiated a strong Th1 cytokine response (Figures 11C and 11E - 11F). Also, the trispecific CAR exhibited very excellent specificity for the 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 created by combining the structures of LTG2303 and LTG2316 or LTG2317 were further evaluated, and again, strong killing of alternative HIV envelope cell lines was shown (Figure 12B). Except for the highest E:T ratio, both LTG2329 and LTG2330 maintained their specificity (Figure 12D). Overall, the exact structure of the trispecific CAR improved its anti - HIV function.

[0206] Next, exposing anti - HIV CAR T cells to diverse and resistant HIV - 1 strains further confirmed the importance of the anti - HIV CAR structure. CD4 from multiple HIV - naive donors +Starting from highly enriched T cell populations (Figure 13), CAR T cell products were generated for a selected group of 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 (hereinafter referred to as duoCARs). As shown in Figures 14 - 17, bispecific and trispecific duoCARs were superior to conventional anti-HIV CARs containing one CD3 zeta chain (LTG2303, LTG2329, and LTG2330), regardless of their valency. This is an important design feature for creating anti-HIV CARs with increased potency and breadth while avoiding viral escape. It is presumed that if one domain is lost due to mutational escape, the other domain can compensate for this loss. More importantly, the anti-HIV target domains can act independently, sequentially, or simultaneously to attack proliferating HIV-infected cells because the duoCAR has a structure. As shown in Figure 18, primary T cells engineered with mD1.22-CAR were more sensitive to HIV-1 infection than bispecific or trispecific CAR-T cells (white-outlined red bars). Incorporating the m36.4 domain, an entry inhibitor, was sufficient to protect CAR T cells and cut off HIV-1 infection. To further evaluate bispecific and trispecific duoCAR-T cells in vivo, humanized NSG mouse models of acute and chronic HIV-1 infection (hu-spl-PBMC-NSG) were used (Figure 19A). To further investigate the function of duoCAR-T cells, a VRC01 / 3BNC117-resistant Env-IMC-LucR virus was selected. As shown in Figures 19B and 19C, bispecific and trispecific duoCARs significantly reduced HIV-1 infection compared to the untreated HIV-infected cohort. Both bispecific and trispecific duoCARs showed similar potency (LTG2303 vs. LTG2330). In the study of chronic HIV infection, CD4 +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 a significant improvement in the percentage of CD4 + T cells from infected spleens, and this level was higher than or close to that of uninfected mice (see: HIV-PBMC, Figure 19E). Associated with the depletion of CD4 + T cells in chronic HIV infection, the percentage of CD8 + T cells in the spleens of infected UTD-treated mice with uncontrolled HIV infection increased (Figure 19G). Strong control of HIV-1 infection is presumably due to the persistence of CAR-T cells in the spleens of infected mice in vivo, as shown in Figures 19H and 19I. Collectively, the invention presented herein represents a potent and general multi-target HIV-1 immunotherapy with strong implications towards functional cure.

[0207] Although not intended to be limited to any particular mechanism of action, reasons for the improved therapeutic function associated with exemplary CARs according to the present invention include, for example, a) improved multi-specific targeting of non-overlapping viral epitopes, b) the function of the antigen-binding domain being exploited by a rational CAR design using an intracellular T cell signaling domain, c) more efficient signaling due to lateral movement in the cell membrane, d) improved ability to interact with transmembrane signaling cascades associated with T cell activation due to an excellent position in cell membrane microdomains (such as lipid rafts), e) an excellent position in the cell membrane due to a preferential movement away from inhibitory or downregulatory interactions, for example, a relatively large distance from or relatively few interactions with phosphatases such as CD45, and, f) excellent assembly into the T cell receptor signaling complex (i.e., immune synapse), or any combination thereof, although not limited thereto.

[0208] So far, the present disclosure has been illustrated using the exact combinations of three exemplary HIV envelope target domains (mD1.22, m36.4, and C46 peptide), 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 designed to include an appropriate antigen-binding domain specific for the desired antigen target.

[0210] In one aspect of the invention, for example, from the Retroviridae family (such as human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae family (such as poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae family, Herpesviridae family (such as herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, Epstein Barr virus (EBV), cytomegalovirus (CMV), and herpes virus), Poxviridae family (such as variola virus, vaccinia virus, and poxvirus), or hepatitis C virus, or antigens (but not limited to these) derived from any combination thereof are provided with CARs that can bind to targets other than TSA or TAA.

[0211] In another aspect of the invention, CARs are provided that can bind to antigens derived from bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, for example, Helicobacter pylori, Legionella pneumophila (Legionella pneumophilia), mycobacteria species (e.g., Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, group A Streptococcus, group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or antigens derived from infectious bacteria such as combinations thereof. CARs are provided that can bind to antigens derived from infectious bacteria such as species or combinations thereof, such as Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, group A Streptococcus, group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani.

[0212] In another aspect of the invention, CARs are provided in combination with advanced gene editing techniques (e.g., CRISPR / Cas9, CRISPR / Cas13, riboswitches, RNA interference, or intracellular antibodies), which use the advanced gene editing techniques to remove and / or interfere with viral ribonucleic acid, integrated viral DNA (e.g., HIV provirus), and / or viral proteins (e.g., viral reverse transcriptase), or combinations thereof, from host infected cells, and to regulate host genetic factors associated with disease, such as chemokine receptor G protein-coupled receptors (e.g., CXCR4, CCR5), host susceptibility factors (e.g., LEDGF / p75), pathogenic factors (e.g., DC-SIGN), natural host resistance factors (e.g., defensins), or combinations thereof (but not limited to the combinations described above).

[0213] In another aspect of the invention, CARs are provided in combination with small molecule inhibitors, latency reversing agents, antiviral agents, antimicrobial agents, or antibodies, and any derivatives thereof, which enhance CAR-T function and / or act synergistically therewith (e.g., TLR7 agonists), or target disease states associated with CAR therapy, or combinations thereof (but not limited to the combinations described above).

[0214] 2. Transmembrane domain With respect to the transmembrane domain, 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 a natural or synthetic source. If the source is natural, this domain may be derived from any membrane-bound or transmembrane protein.

[0216] Transmembrane regions particularly useful in the CARs described herein are those derived from 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 4, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19 (i.e., including at least their transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it may predominantly contain hydrophobic residues such as leucine and valine. Preferably, a triplet 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 to 10 amino acids in length, may form a linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain. A doublet of glycine and serine 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 domains described above.

[0218] In some examples, the transmembrane domain can be selected or amino acid-substituted to avoid the domain binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize the interaction of the domain with other receptor complex components.

[0219] In one embodiment, the transmembrane domain in the CAR according to the present invention is the 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 is the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 1, 2, or 3 modifications (e.g., substitutions) added to a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 8, provided that the modifications (e.g., substitutions) are 20, 10, or 5 or less, and comprises an amino acid sequence.

[0221] In some examples, the transmembrane domain of the CAR comprises the 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 therewith.

[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 bound to the transmembrane CD8 domain, the transmembrane TNFRSF19 domain, or a combination thereof.

[0223] In one embodiment, the encoded transmembrane TNFRSF19 domain, in combination with the CD8 linker / hinge domain, is the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 1, 2, or 3 modifications (e.g., substitutions) added to a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16, provided that the modifications (e.g., substitutions) are 20, 10, or 5 or less, and comprises an amino acid sequence.

[0224] 3. Spacer domain In a CAR, a spacer domain may be disposed 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 couple the transmembrane domain to the extracellular domain and / or couple the transmembrane domain to the intracellular domain. The spacer domain contains 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 include a spacer element. When a spacer element is present, the linker is enlarged by the spacer element, increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art and include those listed 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, 5,521,284, 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, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (the entireties of which are incorporated herein by reference).

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

[0227] As this spacer domain, the whole or a part of amino acid numbers 137 to 206 (SEQ ID NO: 10) of the hinge region of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acid numbers 135 to 195 of CD8 beta (GenBank: AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: NP__000607.1), or amino acid numbers 137 to 152 of CD28 (NCBI RefSeq: NP__006130.1) can be used. Also, as this spacer domain, a part of the constant region of the H chain or L chain of an antibody can be used. Furthermore, this spacer domain may be an artificially synthesized sequence.

[0228] Furthermore, in the CAR, a signal peptide sequence may be bound to the N-terminus. This signal peptide sequence is present at the N-terminus of many secreted proteins and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules described above as the intracellular domain have a signal peptide sequence, this signal peptide can be used as the signal peptide for the CAR. In one embodiment, the signal peptide contains the amino acid sequence of SEQ ID NO: 36. In another embodiment, the signal peptide contains 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 the immune cell into which the CAR has been introduced. The term "effector function" refers to the specialized functions of a cell. For example, the effector functions of T cells can be cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the protein portion that transmits effector function signals and directs the cell to perform specialized functions. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, this truncated portion may be used in place of the full chain as long as it can transmit effector function signals. Therefore, the meaning of the term "intracellular signaling domain" includes any truncated portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[0230] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that cooperate to initiate signaling after antigen-receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional ability.

[0231] It is known that signals emanating only through the TCR are insufficient to fully activate T cells and that a second or co-stimulatory signal is further required. Thus, it can be said that two separate types of cytoplasmic signaling sequences are involved in T cell activation: one that initiates antigen-dependent first activation via the TCR (the first cytoplasmic signaling sequence) and one that acts in an antigen-independent manner to provide a second or co-stimulatory signal (the second cytoplasmic signaling sequence).

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

[0233] Examples of ITAMs containing the first cytoplasmic signaling sequence 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 are amino acid numbers 51 - 164 of CD3 zeta (NCBI RefSeq:NP__932170.1), amino acid numbers 45 - 86 of Fc epsilon RI gamma (NCBI RefSeq:NP__004097.1), amino acid numbers 201 - 244 of Fc epsilon RI beta (NCBI RefSeq:NP__000130.1), amino acid numbers 139 - 182 of CD3 gamma (NCBI RefSeq:NP__000064.1), amino acid numbers 128 - 171 of CD3 delta (NCBI RefSeq:NP__000723.1), amino acid numbers 153 - 207 of CD3 epsilon (NCBI RefSeq:NP__000724.1), amino acid numbers 402 - 495 of CD5 (NCBI RefSeq:NP__055022.2), amino acid numbers 707 - 847 of 0022 (NCBI RefSeq:NP__001762.2), amino acid numbers 166 - 226 of CD79a (NCBI RefSeq:NP__001774.1), amino acid numbers 182 - 229 of CD79b (NCBI RefSeq:NP__000617.1), and CD66d (NCBI Peptides having the sequence of amino acid numbers 177 to 252 of RefSeq:NP__001806.2), and variants having the same function as these peptides, including but not limited to these. Amino acid numbers based on the NCBI RefSeq ID or amino acid sequence information of GenBank described in this specification are numbered based on the full length of the precursor of each protein (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling sequence derived from CD3 zeta.

[0234] In a preferred embodiment, the intracellular domain of the CAR may be designed to itself include a CD3-zeta signaling domain, or may be combined with any other desirable cytoplasmic domain useful in the context of the CAR. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands necessary for lymphocytes to efficiently respond to antigens. Examples of such co-stimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytes It includes functional related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, etc. Specific examples of such co-stimulatory molecules include the amino acid numbers 236 to 351 of CD2 (NCBI RefSeq: NP__001758.2), the amino acid numbers 421 to 458 of CD4 (NCBI RefSeq: NP__000607.1), the amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: NP__055022.2), the amino acid numbers 207 to 235 of CD8 alpha (NCBI RefSeq: NP__001759.3), the amino acid numbers 196 to 210 of CD83 (GenBank: AAA35664.1), the amino acid numbers 181 to 220 of CD28 (NCBI RefSeq: NP__006130.1), the amino acid numbers 214 to 255 of CD137 (4-1BB, NCBI RefSeq: NP__001552.2), the amino acid numbers 241 to 277 of CD134 (OX40, NCBI RefSeq: NP__003318.1), and peptides having the amino acid sequences of 166 to 199 of ICOS (NCBI RefSeq: NP__036224.1), and variants having the same functions as these peptides, but are not limited thereto. Thus, although the present disclosure has so far been exemplified mainly using 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present disclosure.

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

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

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

[0238] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, and the signaling domain of CD3-zeta includes the 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 include the codon-degenerate signaling domain of CD3-zeta2, wherein the signaling domain of CD3-zeta2 includes 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 The functional portions of the CARs disclosed herein are also clearly included within the scope of the present invention. When the term "functional portion" is used in reference to a CAR, it refers to any one or more portions or fragments of the CARs disclosed herein, which portions or fragments retain the biological activity of the CAR (parent CAR). Functional portions, for example, retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent as, to the same extent as, or to a greater extent than the parent CAR. With respect to a parent CAR, a functional portion may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR. This includes CAR portions that retain the ability to perform these functions to a similar extent as, to the same extent as, or to a greater extent than the parent CAR. With respect to a parent CAR, a functional portion may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0241] A functional portion may include additional amino acids not found in the amino acid sequence of the parent CAR at the amino terminus or carboxy terminus or both termini of the portion. Desirably, these additional amino acids do not interfere with the biological function of the functional portion, such as recognition of target cells, detection of cancer, treatment of cancer, or prevention of cancer. 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 the present disclosure. When the term "functional variant" is used herein, it refers to a CAR, polypeptide, or protein having a substantial or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR from which the variant 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 as, to the same extent as, or to a greater extent than the parent CAR. With respect to a parent CAR, a functional variant may have, for example, an amino acid sequence identity to the parent CAR of at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0243] Functional variants may include, for example, those with at least one conservative amino acid substitution added to the amino acid sequence of the parental CAR. Alternatively or additionally, functional variants may include those with at least one non-conservative amino acid substitution added to the amino acid sequence of the parental CAR. In this case, it is preferred that this non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. This 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 parental CAR.

[0244] The amino acid substitutions of the 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 certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include substituting an acidic / negatively charged polar amino acid (e.g., Asp or Glu) with another acidic / negatively charged polar amino acid, substituting a non-polar side chain-containing amino acid (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another non-polar side chain-containing amino acid, substituting a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, substituting a polar side chain-containing uncharged amino acid (e.g., Asn, Gln, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, substituting a beta-branched side chain-containing amino acid (e.g., Ile, Thr, and Val) with another beta-branched side chain-containing amino acid, substituting an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid, etc.

[0245] The CAR may essentially consist of one or more of the specified amino acid sequences described herein, such that the biological activity of the functional variant is not substantially changed by other components (e.g., other amino acids).

[0246] A CAR (including functional parts and functional variants) is of any length, i.e., may contain any number of amino acids, as long as the CAR (or its functional part or functional variant) retains a biological activity 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 a disease in a mammal. For example, the CAR may be of a length of about 50 to about 5000 amino acids, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids.

[0247] A CAR (including functional parts and functional variants according to the present invention) may contain synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-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-hydroxy-phenylalanine, 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, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. ​

[0248] A CAR (including functional parts and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridging), or converted to an acid addition salt, and / or optionally dimerized or polymerized or conjugated.

[0249] CAR (including its functional parts and functional variants) can be obtained by methods well known in the art. CAR may be produced by any suitable polypeptide or protein production method. Suitable methods for newly synthesizing 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. Also, polypeptides and proteins may be recombinantly produced using the nucleic acids described herein and standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 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. Further, some of the CARs (including their functional parts and functional variants) 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 their functional parts and functional variants) described herein may be commercially synthesized by a company. 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 the 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, "T cell expressing a CAR" or "CAR T cell" or "CAR-T" means a T cell expressing a CAR and has antigen specificity, for example, as determined by the antibody-derived targeting domain of the CAR.

[0251] As used herein, "antigen-binding domain" may include an antibody and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and includes diverse 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 well known in the art that retain binding affinity for an antigen, as well as variants and fragments thereof.

[0252] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up this population are identical except for natural mutations that may be present in very small amounts. Monoclonal antibodies are highly specific and are directed against a single antigen epitope. The modifier "monoclonal" indicates the property that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted to mean that the antibody must be produced by any particular method. In some instances, monoclonal antibodies are antibodies produced by a single clone of B lymphocytes or by cells transfected with nucleic acids encoding the light and heavy chain variable regions of the antibody (or antigen-binding fragments thereof) and their progeny. In some instances, monoclonal antibodies are isolated from a subject. Monoclonal antibodies 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, see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition Cold Spring Harbor Publications, New York (2013).

[0253] Typically, an immunoglobulin has heavy (H) and light (L) chains that are linked to each other by disulfide bonds. Immunoglobulin genes include 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 main classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule (IgM, IgD, IgG, IgA, and IgE).

[0254] The heavy and light chains each contain a constant region (or constant domain) and a variable region (or variable domain) (see, for example, Kindt et al., Kuby Immunology, 6th Edition, W.H. Freeman and Co., page 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting of only heavy chains are functional and stable without a light chain (see, for example, 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 the antibody heavy chain, including the variable region of the antigen-binding fragment, such as Fv, ScFv, dsFv, or Fab, etc. References to "VL", or "VL" refer to the variable domain of the antibody light chain, including those of Fv, ScFv, dsFv, or Fab.

[0255] The variable regions of the light and heavy chains contain "framework" regions and three hypervariable regions that interrupt them (also called "complementary determining regions" or "CDRs") (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department 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 the antibody, i.e., the framework regions of the constituent light and heavy chains together, position and align the CDRs in three-dimensional space.

[0256] The CDRs are mainly responsible for binding to antigen epitopes. The boundaries of the amino acid sequences of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), 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 (from the N-terminus towards the C-terminus), and are further typically identified by the chain in which the CDR is located. Thus, VH CDR3 is the CDR3 from the variable domain of the heavy chain of an antibody that contains it, and VL CDR1 is the CDR1 from the variable domain of the light chain of an antibody that contains it. The light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3.

[0257] An "antigen-binding fragment" is a part of a full-length antibody or a part of a full-length protein (e.g., the D1 domain of the CD4 receptor) that retains the ability to specifically recognize cognate antigens, and various combinations of such parts. Examples of antigen-binding fragments include protein domains, full-length proteins, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; nanobodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments, or multispecific proteins formed from more than one protein domain or fragment, but are not limited thereto. Antibody fragments include antigen-binding fragments made by modification of the whole antibody or newly synthesized using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010). Multispecific proteins and their derivatives include antigen-binding fragments made by fusing these fragments together in the correct arrangement using multiple original fragments, or fragments modified from these fragments, or fragments newly synthesized by recombinant DNA technology.

[0258] A single-chain antibody (ScFv) is a genetically engineered molecule containing the VH domain and VL domain of one or more antibodies linked by a suitable polypeptide linker to form a gene-fused single-chain molecule (see, e.g., Bird et al., Science, 242:423 426, 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 domain and VL domain within the ScFv typically does not determine the ScFv. Thus, ScFvs having both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0259] In dsFv, the variable chains of the heavy and light chains have disulfide bonds introduced by mutation to stabilize the binding of both chains. Also included are diabodies, which are bivalent bispecific antibodies in which the VH domain and the VL domain are expressed on a single polypeptide chain, but a linker that is too short is used to link the two domains into a single chain, so the two domains are linked to the complementary domains of another chain, forming two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0260] Antibodies further include genetically engineered forms such as chimeric antibodies (such as humanized mouse 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 produced recombinantly, or obtained, for example, by screening a combinatorial library consisting of variable heavy and variable light chains as described by Huse et al., Science 246:1275-1281 (1989) (incorporated herein by reference). These methods, as well as other methods for making, 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 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 and acts as a reference antibody in an antagonistic assay refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more. Conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in the antagonistic assay. Antibody antagonistic assays are well known, and exemplary antagonistic assays are provided herein.

[0263] A "humanized" antibody or antigen-binding fragment contains a human framework region and one or more CDRs from a non-human (such as mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment that provides this CDR is called the "donor", and the human antibody or antigen-binding fragment that provides the framework is called the "acceptor". In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region may or may not be present, but if present, it may be substantially identical to the human immunoglobulin constant region, for example, at least about 85 - 90% (or more than about 95% or so) identical. Thus, all parts of the humanized antibody or antigen-binding fragment (possibly excluding the CDRs) are substantially identical to the corresponding parts of the native human antibody sequence.

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

[0265] A "fully human antibody", or "human antibody", or a humanized derivative thereof consisting of a protein fragment, 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 (when present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated, for example, by using antibody production techniques based on sequences from the human genome, such as by phage display or the use of genetically engineered animals (see, for example, 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. When there is more than one binding site, these binding sites may be the same as or different from each other. For example, a natural 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 radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, for example, Janeway et al. below, US Patent Application Publication No. 2002 / 0197266 Al, and US Patent No. 7,338,929).

[0268] In addition, a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof may be modified to include 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 elemental particles (e.g., gold particles).

[0269] C. Conjugates A CAR, a T cell expressing a CAR, or a monoclonal antibody, or an antigen-binding fragment thereof, specific for one or more of the antigens disclosed herein may be bound to an agent such as an effector molecule or a detectable marker using any of a number of means well known to those of skill in the art. Either means by covalent bond or means by non-covalent bond 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 bound to an effector molecule or a detectable marker. Those of skill in the art will appreciate that a wide variety of effector molecules and detectable markers can be used, including, but not limited to, antiviral agents, antimicrobial agents, chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C, 3 H, and 35 radioactive agents such as S, as well as other labels, target sites, and ligands, etc. (but not limited to these). It is understood that they can be used.

[0270] The choice of a specific effector molecule or detectable marker depends on the specific 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 specific 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 2 ), or sulfhydryl (-SH) groups, which can be utilized in reactions with suitable functional groups on the antibody, resulting in the attachment of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is a derivative for exposing or attaching additional reactive functional groups. Derivatization may be associated with the attachment of any of a number of well-known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). A linker can be any molecule used to couple an antibody or antigen-binding fragment to an effector molecule or detectable marker. A linker can form covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those of skill 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 linker may be attached to the constituent amino acids via its side chain (e.g., to cysteine via a disulfide bond), or to the amino and carboxy groups of the alpha carbon of the terminal amino acids.

[0272] In some embodiments, the linker may include a spacer element, and when the spacer element is present, the linker is enlarged by the spacer element, and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Specific examples of spacers are well known to those skilled in the art and include those listed 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, 5,521,284, 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, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (each of which is incorporated herein by reference in its entirety).

[0273] In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet another embodiment, 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 within the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker may be, for example, a peptide linker cleaved by an intracellular peptidase or a protease enzyme including, but not limited to, lysosomal protease or endosomal protease. In some embodiments, the peptide linker is at least two amino acids in length, or at least three amino acids in length. However, the linker may be four, five, six amino acids from 1 to 15 in number, for example, having a length of 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, or 1 to 15 amino acids. The protease may include cathepsin B and D, as well as plasmin, all of which are known to hydrolyze the dipeptide drug derivative to release the active drug in the target cell (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. Patent No. 6,214,345, which is hereby incorporated by reference. In a specific embodiment, the peptide linker cleavable by the intracellular protease is a valine-citruline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citruline linker).

[0274] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, such pH-sensitive linkers hydrolyze under acidic conditions. For example, acid-labile linkers that can hydrolyze within lysosomes (such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amide, orthoesters, acetals, or ketals, etc.) can be used. (See, e.g., U.S. Pat. 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 unstable at pH 5.5 or less than 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether linked to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929, etc.)).

[0275] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are well known in the art and include, for example, those 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. 68:92809290, 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 U.S. Publication No. 2005 / 0238649, which is hereby incorporated by reference in its entirety).

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

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

[0280] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art, can be isolated from natural sources according to well-known methods, can be produced using genetic engineering techniques (see Yu et al., PNAS 2002, 99:7968 - 7973), or maytansinol and maytansinol analogs can be prepared synthetically according to well-known methods. Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from Maytenus serrata, a shrub native to East Africa (U.S. Patent No. 3,896,111). Subsequently, it was further discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent 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, 4,313,946, 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 hereby incorporated by reference herein. Conjugates containing maytansinoids, methods for their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163, and European Patent No. EP0425235 B1, the disclosures of which are hereby expressly incorporated by reference herein.

[0281] Additional toxins can be used with the CAR, T cells expressing the CAR, 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 readily available from commercial suppliers (e.g., Sigma Chemical Company, St. Louis , MO). The intended toxins 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 and inhibits protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405 - 412, 1992). However, this toxin does not have a mechanism for specifically entering cells and thus needs to be conjugated to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein in order to efficiently enter 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 has sufficient enzymatic activity but significantly reduced nonspecific toxicity and has been well known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and is 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). See, for example, U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) has two forms, which are referred to as RCA 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for the inactivation of protein synthesis and cell death. The B chain binds ricin to cell surface galactose residues and promotes the 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 binding to target molecules (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins 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 first isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes DNA double-strand breaks and induces apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug has been in clinical trials and is the toxic moiety of immunotoxins (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0286] Abrin contains a toxic lectin obtained from Abrus precatorius. Its toxic components, abrin a, b, c, and d, have a molecular weight of approximately 63 to 67 kD and are composed of two polypeptide chains, A and B, linked by a disulfide bond. The A chain inhibits protein synthesis, and 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, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof that are specific for one or more of the antigens disclosed herein may also be bound to a detectable marker, such as a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging diagnostic techniques (computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound diagnosis, fiber optic examination, and laparoscopic examination, etc.). Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, 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-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. CARs, T cells expressing CARs, antibodies, or antigen-binding portions thereof may also be bound to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a CAR, T cell expressing a CAR, antibody, or antigen-binding portion thereof is bound to a detectable enzyme, it can be detected by adding a further reagent that produces a reaction product distinguishable from that used for the enzyme. For example, in the presence of the agent horseradish peroxidase, a colored reaction product is obtained by the addition of hydrogen peroxide and diaminobenzidine, which can be detected visually. CARs, T cells expressing CARs, antibodies, or antigen-binding portions thereof may also be bound to biotin and may be detected by indirectly measuring the binding of avidin or streptavidin.It should be noted that avidin itself may be bound to an enzyme or a fluorescent label.

[0288] The CAR, the T cell expressing the CAR, the antibody, or its antigen-binding portion may be bound to a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. The antibody may also be bound to lanthanides (such as europium and dysprosium) and manganese. The antibody or antigen-binding fragment may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a binding site for a secondary antibody, a metal-binding domain, an epitope tag, etc.).

[0289] The CAR, the T cell expressing the CAR, the antibody, or its antigen-binding portion may also be bound to a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For example, the radiolabel may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, luminescence spectroscopy, or other diagnostic techniques. Further, the radiolabel may be used as a toxin in the treatment of treating tumors in a subject, for example, for treating neuroblastoma. Examples of labels for polypeptides are 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 radioisotopes or radiolabeled nucleotides such as I, but are not limited thereto.

[0290] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, a radioactive label may be detected using a photographic film or a scintillation counter, and a fluorescent marker may be detected by detecting the emitted light using a photodetector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product generated 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 According to one embodiment of the present invention, there is further provided a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein. The nucleic acid according to the present invention may include 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 have modified codons. Without being bound by any theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing natural codons with other codons that encode the same amino acid but can be translated by tRNAs that are more readily available in the cell, and thus, the translation efficiency may be increased. Optimization of the nucleotide sequence may also be one that can reduce secondary mRNA structures that can interfere with translation, and thus, the translation efficiency may be increased.

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

[0294] As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally may be single-stranded or double-stranded, may be obtained from synthetic or 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 linkages or phosphorothioate linkages instead of the phosphodiester found between nucleotides of unmodified oligonucleotides), and means a polymer of DNA or RNA. In some embodiments, the nucleic acid contains no insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0295] A recombinant nucleic acid may have a sequence that does not exist in nature or may have a sequence in which two regions that are distant in the sequence are artificially combined. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificially manipulating distant nucleic acid regions by genetic engineering techniques such as those described in the literature of Sambrook et al. mentioned above. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, the literature of Sambrook et al. and Ausubel et al. mentioned above. For example, nucleic acids may be chemically synthesized using natural nucleotides or nucleotides modified in various ways designed to increase the biological stability of the molecule or to increase the physical stability of the double strand formed by hybridization (e.g., phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used in nucleic acid production are 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, N6-isopentenylade Nin, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine, including but not limited to these. Alternatively, one or more of the nucleic acids according to the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

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

[0297] One embodiment further provides an isolated or purified nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein, or a nucleotide sequence 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 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), and the amount thereof is detectably more than non-specific hybridization. Highly stringent conditions include conditions that can distinguish a polynucleotide having a strictly complementary sequence or having only 2 to 3 scattered mismatches from a random sequence that accidentally has 2 to 3 small regions (e.g., 3 to 10 bases) that match the nucleotide sequence. Such small complementary regions are more easily melted than the full-length complementary regions of 14 to 17 bases or more in length, and these can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions can include, for example, low salt and / or high temperature conditions such as about 0.02 to 0.1 M NaCl or equivalent and a temperature of about 50 to 70 °C. Thus, highly stringent conditions allow for very low levels of mismatches, if any, between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any of the CARs of the present invention. In general, it is understood that the conditions can be made more stringent by increasing the amount of formamide added.

[0299] Also provided are nucleic acids comprising nucleotide sequences having at least about 70% or more identity, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any of the nucleic acids described herein.

[0300] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard Thus, one embodiment provides a recombinant expression vector comprising any of the nucleic acids described above. For the purposes described herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector and a host cell are contacted under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the host cell, the host cell is capable of expressing the mRNA, protein, polypeptide, or peptide. Such vectors are generally not found in nature.

[0301] However, some of these vectors may occur naturally. 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, may be synthetic or obtained in part from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain natural or non-natural nucleotide linkages, or both types of linkages. Preferably, the non-natural or altered nucleotides or nucleotide linkages 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 can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for purposes of propagation and growth, or for expression, or for both purposes (such as plasmids and viruses). 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 λΤΙΟ, λΤΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, for example, a retroviral vector or a 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, the LENTIVECTOR® gene delivery technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc. Non-clinical grade lentiviral vectors are also available and may be well known to those skilled in the art.

[0304] Multiple transfection techniques are generally well known in the art (see, for example, 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, for example, Graham et al. supra), direct microinjection into cultured cells (see, for example, Capec chi, Cell, 22:479-488 (1980)), electroporation (see, for example, Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, for example, Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transfection (see, for example, Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid introduction using a high-velocity microparticle gun (see, for example, Klein et al., Nature, 327:70-73 (1987)).

[0306] In one embodiment, the recombinant expression vector may be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al. supra and Ausubel et al. supra. The circular or linear construct of the expression vector may be prepared to contain a replication mechanism that functions in a prokaryotic or eukaryotic host cell. The replication mechanism may be derived from, for example, ColE1, 2μ plasmid, λ, SV40, and bovine papillomavirus, among others.

[0307] The recombinant expression vector is appropriately specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is introduced, and may contain regulatory sequences such as transcription codons, translation start codons, and stop codons, taking into account whether the vector is based on DNA or RNA. The recombinant expression vector may contain restriction sites for facilitating cloning.

[0308] The recombinant expression vector may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include, for example, biocide resistance such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Marker genes suitable 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 contain a natural or non-natural promoter operably linked to a nucleotide sequence encoding a CAR (including its functional parts and functional variants), or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a CAR. The selection of the promoter (e.g., strong, weak, inducible, tissue-specific, and developmental-specific, etc.) is within the scope of ordinary knowledge of those skilled in the art. Similarly, the binding of the nucleotide sequence and the promoter is also within the scope of ordinary knowledge of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, for example, the mouse stem cell virus (MSCV) promoter, the elongation factor 1 alpha (EF1α) promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, or the RSV promoter. The recombinant expression vector may be designed for either transient expression, or stable expression, or both. Also, the recombinant expression vector may be prepared for constitutive expression or inducible expression.

[0310] Furthermore, the recombinant expression vector may be constructed to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, on the cells expressing the gene, such that contact or exposure of the cells to the agent causes the death of the cells. 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, the 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 the recombinant expression vector according to the present invention. The host cell may be a eukaryotic cell such as, for example, a plant, animal, fungus, or alga, or may be a prokaryotic cell such as, for example, a bacterium or protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism such as, for example, a human). The 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 Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is amplification or replication of the recombinant expression vector, the host cell may be a prokaryotic cell, such as DH5a cells. When the purpose is production of the recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, may be derived from any type of tissue, and may be at any developmental stage, 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 the purposes described herein, the T cell may be any T cell, may be a cultured T cell (such as, for example, a primary T cell), or a T cell from a cultured T cell line (such as, for example, Jurkat, SupTl, etc.), or a T cell obtained from a mammal. When obtained from a mammal, the T cell may be obtained from a very diverse source including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell may be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be of any type and may be at any developmental stage, 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., and are not limited thereto.

[0313] In one embodiment, the CAR described herein can be used in suitable cells that are not T cells. Such cells are those having immune effector functions, such as, for example, NK cells and T-like cells generated from pluripotent stem cells.

[0314] Also, one embodiment provides a population of cells comprising at least one host cell described herein. This population of cells, in addition to host cells containing any of the recombinant expression vectors described, includes at least one other cell, for example, a host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, such as, for example, a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., and may be a heterogeneous population. Alternatively, the population of cells may be a substantially homogeneous population mainly comprising (e.g., consisting essentially of) host cells containing the recombinant expression vector. Also, the population may be a clonal cell population in which all cells of the population are clones of a single host cell containing the recombinant expression vector, and thus all cells of the population contain this recombinant expression vector. In one embodiment of the present invention, the cells population is a clonal population comprising host cells containing the recombinant expression vector described herein.

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

[0316] E. Method of treatment It is contemplated that the CARs disclosed herein may be used in a method 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, which comprises administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or its antigen-binding portion, 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 the step of lymphodepleting the mammal prior to administering the CARs disclosed herein. Examples of lymphodepletion include, but are not necessarily limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, and the like.

[0318] For the purposes of the methods in which host cells or a population of cells are administered, the cells may be allogeneic cells from the same species as the mammal, or autologous cells thereof. Preferably, the cells may be autologous cells of the mammal. As used herein, "allogeneic" means any material that is derived from an animal that is of the same species as, but a different individual from, the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some embodiments, allogeneic materials from individuals of the same species may be genetically different enough to interact antigenically with each other. As used herein, "autologous" means any material that is derived from the same individual as the individual into which the material will later be re-introduced again.

[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, rodent mammals such as mice and hamsters and lagomorph mammals such as rabbits. The mammal may be of the order Carnivora including the families Felidae (cats) and Canidae (dogs). The mammal may be of the order Artiodactyla including the subfamily Bovinae (cattle) and swine (pigs), or of the order Perissodactyla including the family Equidae (horses). The mammal may be of the order Primates, Ceboids, or Simoids (monkeys), or of the Hominoidea (humans and apes). Preferably, the mammal is a human.

[0320] With regard to the methods described above, cancers associated with HIV infection are ALL, AML, alveolar rhabdomyosarcoma, bladder cancer (e.g., urinary bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, Any cancer may be included, including 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 cancer (CML), colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, NHL, B-chronic lymphocytic leukemia, hairy cell leukemia, Burkitt lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, omentum, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer, and urinary tract cancer.

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

[0322] The terms "treatment" and "prevention" and their derivatives, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that would be recognized by one of ordinary skill in the art as having the potential for a benefit or therapeutic effect. In this regard, the method can provide any amount or level of HIV / AIDS treatment or prevention in a mammal.

[0323] Furthermore, the treatment or prevention provided by the method may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented (e.g., HIV-1 infection). Also, for the purposes described herein, "prevention" may include delaying the onset of a 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 combination with, treatment or prevention of one or more conditions or symptoms not limited to those found to coexist with the disease (e.g., HIV / AIDS-related coexisting diseases such as Kaposi's sarcoma or acute myeloid leukemia, viral coinfections such as HIV / HBV or HIV / HCV coinfection).

[0325] With respect to the methods described above, treatment or prevention by the methods described above may be used in combination with allogeneic or autologous transplantation of cells derived from a mammal lacking a disease-related genetic factor (including, but not limited to, bone marrow transplantation of Δ32-CCR5 cells or genetically modified CXCR4 non-containing 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, which comprises: (a) forming a complex by contacting a sample containing one or more cells from the mammal with a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody, and / or an antigen-binding portion thereof, or a pharmaceutical composition; and (b) detecting the complex. When a complex is detected, it is suggested that an infectious disease exists in the mammal.

[0327] This sample may be obtained by any suitable method, such as blood sampling or biopsy. Blood sampling or venipuncture is the process of collecting venous blood from within the vein of an individual. A biopsy is the removal of tissue and / or cells from an individual. Such removal may be for collecting tissue and / or cells from an individual for subjecting the removed tissue and / or cells to laboratory methods. These laboratory methods may include experiments for determining whether this individual has a specific condition or disease state and / or whether they are suffering from it. This condition or disease may be, for example, HIV / AIDS.

[0328] Regarding one embodiment of a method for detecting the presence of an infectious disease in a mammal, such as HIV-1 infection, a sample containing mammalian cells may be a sample containing whole cells, their lysates, or whole cell lysate fractions, such as nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions. When the sample contains whole cells, these cells may be any cells of a mammal, such as cells of any organ or tissue (including blood cells or endothelial cells). The contact described above may occur in vitro or in vivo for the mammal.

[0329] Also, the detection of the complex may be performed by any of a plurality of methods well-known in the art. For example, the CARs disclosed herein, the polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies, or antigen-binding portions thereof described herein may be labeled with detectable labels such as, for example, radioisotopes, fluorophores (such as fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (such as alkaline phosphatase, horseradish peroxidase), and elemental particles (such as gold particles) as disclosed above.

[0330] Methods for testing the target cell recognition ability and antigen specificity of CARs are well known in the art. For example, Clay et al., J. Immunol, 163:507-513 (1999) teach methods for measuring the release of cytokines (e.g., 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 CARs may be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J. Immunol, 174:4415-4423 (2005).

[0331] Assay methods for the HIV-1 infection inhibitory ability of CARs are in-vitro assays that measure the replication of the HIV-1 virus, but are not limited thereto. For example, an in-vitro PBMC-based assay using a replicable HIV-1 molecular clone expressing both a heterologous HIV envelope protein and Renilla luciferase can be used to monitor the replication of the HIV-1 virus (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 that have been contacted with T cells expressing an anti-HIV agent and a chimeric antigen receptor (such as those described herein), or either of these. 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 by 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 whether viral nucleic acid is present. In another assay, the presence of infectious HIV-1 virus can be assayed by inoculating an HIV-permissive cell line and monitoring cytotoxic changes indicative of viral infection, in combination with other HIV infection quantification indicators. IV-1 virus can be assayed.

[0332] Another embodiment provides for 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 according to the invention for treating or preventing an infectious disease in a mammal, such as HIV-1 infection. The infectious disease can be any of the viruses, microorganisms, and / or parasites described herein.

[0333] Any method of administration, including topical and systemic administration, may be used with the disclosed therapeutic agents. For example, administration may be used topically, orally, intravascularly such as intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, subarachnoidally, and subcutaneously. Specific modes of administration and dosing regimens may be selected by the attending clinician taking into account the details of the case (e.g., the subject, the disease, the associated disease state, and whether the treatment is prophylactic). If more than one agent or composition is administered, more than one route 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. The method of administration includes 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, a non-volatile oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue region where the infectious disease is present or has been isolated, or a region where there is a suspected tendency or support for the occurrence of the infectious disease. In some embodiments, sustained release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment into (or near) an organ may be beneficial. In other examples, the conjugate is applied topically to the cornea or intravitreally in the eye as an eye drop.

[0334] The disclosed therapeutic agents may be formulated in unit dosage forms suitable for administering the exact dosage once at a time. In addition, the disclosed therapeutic agents may be administered on a schedule of single or multiple administrations. The multiple administration schedule may be such that more than one administration (e.g., 1 to 10 administrations) is performed separately each time in the first series of treatments, and subsequently, thereafter, the remaining administrations may be performed at time intervals as necessary to maintain or increase the action of the composition. The treatment may involve administering the compound once or multiple times a day (multi-daily doses) over a period of 2 to 3 days to several months, or even several years. Thus, the dosing regimen may be determined based at least in part on the specific requirements of the subject to be treated and may depend 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, such as from about 0.1 to about 10 mg / kg.

[0336] In a specific example, the subject is administered a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or a further agent on a multiple daily dosing schedule, such as for a period of several weeks, several months, or several years, for at least 2 consecutive days and up to 10 consecutive days. In one example, the subject is administered a conjugate, an antibody, a composition, or a further agent for a period of at least 30 days, such as 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 method is the disclosed antibody, antigen-binding fragment, conjugate Comprising providing to a subject surgery, radiation therapy, and / or chemotherapy, in combination with (e.g., sequentially, substantially simultaneously, or simultaneously) a conjugate, a CAR, or a T cell expressing a CAR. Methods and therapeutic dosages for such agents and treatments are well known to those of skill in the art and may be determined by a skilled clinician. Preparations and dosing schedules for additional agents may be used according to the manufacturer's instructions or based on the judgment of a skilled physician's experience. Also, preparations and dosing schedules for such chemotherapy are described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

[0338] In some embodiments, the combination therapy may comprise administering to the subject, in a therapeutically effective amount, an additional HIV inhibitor, an immunomodulatory protein, and / or a protein that enhances the function of a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Examples of additional therapeutic agents that can be used in combination therapy include, but are not limited to, microtubule-binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, reverse transcriptase inhibitors, viral protein inhibitors, immunomodulators, antibodies, enzymes, enzyme inhibitors, gene regulators, antiproliferative agents, and latent infection reactivating 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 a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages for such agents are well known to those of skill in the art and may 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-peptide, and gp41-derived N-peptide); HIV integrase strand transfer inhibitors (e.g., raltegravir, dolutegravir, and elvitegravir), and any combination of these antiviral agents, but not limited thereto. In some instances, a latency reactivating agent may be combined with the CARs, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein to reactivate latent HIV infection. Examples of such compounds include PKC activators (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, but not limited thereto. The selective and therapeutic dosages of such agents are well known to those of skill in the art and may be determined by a skilled clinician.

[0340] For treatment, prevention, or synergistic effect, additional radiation or chemotherapeutic agents that can be combined with the methods described above 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 alkylating agents such as busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uracil mustard; 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 podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antineoplastic antibiotics such as 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 other agents including, but not limited to, alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, 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 a skilled clinician.

[0341] Combination therapy can produce a synergistic effect and can be demonstrated to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the combined effect obtained when the same compounds are used separately. The synergistic effect can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit dosage preparation, (2) delivered as separate preparations, alternately or in parallel, or (3) some other regimen is used. When delivered alternately, for example, by being injected separately with separate syringes, a synergistic effect can occur when the compounds are administered or delivered sequentially. Generally, in the case of alternation, the effective dosage of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the 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 a conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject having HIV / AIDS. After sufficient time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on each HIV-1 infected cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the above treatment. For example, if the immune complex has increased compared to a control obtained prior to the above treatment, it is indicated that the above treatment is ineffective, and if the immune complex has decreased compared to a control obtained prior to the above treatment, it is indicated that the above treatment is effective.

[0343] F. Biopharmaceutical Compositions A biopharmaceutical composition or biological composition (hereinafter, "composition") containing one or more of the disclosed CARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR that specifically bind to one or more of the antigens disclosed herein in a carrier (such as a pharmaceutically acceptable carrier) is, in this specification, gene therapy It is provided for use in gene therapy, immunotherapy, and / or cell therapy. This composition may be prepared in 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. This composition may be formulated for systemic (such as intravenous) or local (such as intra-organ) administration. In one example, the disclosed CAR, or T cells expressing the CAR, antibody, antigen-binding fragment, conjugate is formulated for parenteral administration such as intravenous administration. Compositions comprising a CAR, or T cells expressing the CAR, conjugate, antibody, or antigen-binding fragment disclosed herein are useful, for example, for the treatment and detection of infectious diseases (such as, but not limited to, HIV-1 infection). Compositions comprising a CAR, or T cells expressing the CAR, conjugate, antibody, or antigen-binding fragment disclosed herein are also useful, for example, for the detection of immune deficiency.

[0344] This composition for administration may comprise a solution in which a CAR, or T cells expressing the CAR, conjugate, antibody, or antigen-binding fragment is 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 substances. This composition may be sterilized by conventional well-known sterilization techniques. This composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, toxicity modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as necessary to approximate physiological conditions. The concentration of the CAR, or T cells expressing the CAR, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected according to the particular mode of administration selected and the requirements of the subject, mainly based on factors such as fluid volume, viscosity, and body weight. The actual methods of 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] Typical compositions for intravenous administration include from about 0.01 to about 30 mg / kg per day per subject of an antibody or antigen-binding fragment or conjugate (or a corresponding dosage of a CAR, or T cells expressing a CAR, a conjugate comprising an antibody or antigen-binding fragment). The actual method of preparing the composition for administration may be well known or apparent to those skilled in the art and is described in more detail in publications such as Remington’s Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0346] The CAR, or T cells expressing a CAR, antibody, antigen-binding fragment, or conjugate may be provided in lyophilized form and reconstituted with sterile water for administration, but are also provided in dissolved form in a sterile solution at a known concentration. The solution of the CAR, or T cells expressing a CAR, antibody, or antigen-binding fragment, or conjugate is then filled into an infusion bag containing 0.9% sodium chloride (USP) and, optionally, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art regarding the administration of drugs of antibodies or antigen-binding fragments and conjugates. For example, antibody drugs have been on the U.S. market since the approval of Rituxan® in 1997. The CAR, or T cells expressing a CAR, antibody, its antigen-binding fragment, and conjugate may be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher loading dosage is administered, followed by a maintenance dosage at a lower level. For example, an antibody or antigen-binding fragment may be infused at an initial loading dosage of 4 mg / kg (or a corresponding dosage of a conjugate comprising an antibody or antigen-binding fragment) over about 90 minutes, and if this initial dosage is well tolerated, subsequently, a maintenance dosage of 2 mg / kg once a week may be infused for 30 minutes each time over 4 to 8 weeks.

[0347] Controlled release parenteral preparations may be prepared as transplants, oily injections, or particle systems. A comprehensive overview of protein delivery systems can be found in Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain therapeutic proteins, such as cytotoxins or drugs, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since 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] The polymer may be used for ion-controlled release of the CAR, or T cells expressing the CAR, antibody, or antigen-binding fragment, 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 polaxamer 407 exists as a mobile liquid with viscosity at low temperatures but forms a semi-solid gel at body temperature. This 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 aspect, liposomes are used for the controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). In addition to these, a very large number of systems for the controlled delivery of therapeutic proteins are well known (U.S. Patent No. 5,055,303, U.S. Patent No. 5,188,837, U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028, U.S. Patent No. 4,957,735, U.S. Patent No. 5,019,369, U.S. Patent No. 5,055,303, U.S. Patent No. 5,514,670, U.S. Patent No. 5,413,797, U.S. Patent No. 5,268,164, U.S. Patent No. 5,004,697, U.S. Patent No. 4,902,505, U.S. Patent No. 5,506,206, U.S. Patent No. 5,271,961, U.S. Patent No. 5,254,342, and U.S. Patent No. 5,534,496).

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

[0350] The kit may include a container and a label or package insert attached to 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 contains a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous administration fluid bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is to be used for the treatment of a particular condition.

[0351] The label or accompanying document may typically further include, for example, a description of the use of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing the CAR in a method for treating or preventing HIV / AIDS, or a method for producing CAR T cells. The accompanying document typically includes the instructions customarily included in the commercial package of a therapeutic product, which includes information regarding indications, usage, dosage, administration, contraindications, and / or warnings related to the use of the therapeutic product. The content of the instructions may be described in electronic form (such as a floppy disk or compact disk) or visual form (such as a video file). The kit may further include additional components for facilitating a particular use for which the kit is designed. Thus, for example, the kit may further contain means for detecting labels (such as enzyme substrates for enzyme labels, filter sets for detecting fluorescent labels, or appropriate secondary labels such as secondary antibodies). The kit may further contain buffers and other reagents customarily used in the practice of a particular method. Such kits and appropriate contents are well known to those skilled in the art.

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

Examples

[0353] Assembly of HIV-Specific Binders into Functional CAR Molecules This example describes a general method for constructing single-specificity, dual-specificity, and triple-specificity anti-HIV CARs containing the mD1.22, m36.4, and C46 peptides, and how to express such CARs on the surface of primary T cells.

[0354] Materials and Methods: Generation of Lentiviral Vector Constructs The CAR antigen-binding domain sequences were either 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), or were 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 a CD8 linker / hinge, CD8 transmembrane domain, 41-BB, and CD3 zeta signaling domain. For bicistronic and triple-specific 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 placement of a third antigen-binding domain, CD8 linker / hinge, TNFRSF19 transmembrane, and optionally a CD3 zeta signaling domain. A detailed description of the sequences and CAR structures is shown in Table 1 below. This DNA construct was confirmed by Sanger sequencing (GeneWiz, South Plainfield, NJ). Plasmid mapping was generated using Clone Manager software (Denver, CO).

[0355] Generation of Lentiviral Vectors In the presence of polyethyleneimine (PEI), a lentiviral vector with a CAR transgene was produced by transiently transfecting 293T suspension cells using a system that uses four plasmids. Briefly, suspension 293T was co-transfected with a CAR transfer plasmid, VSVg envelope, gag / pol, and rev plasmids, and subsequently, sodium butyrate was added to the culture 16 hours later. After 48 hours, the supernatant (≤400 mL) containing the lentiviral vector was ultracentrifuged at 10,000×g overnight for at least 18 hours to concentrate it. The pellet was resuspended in 2 mL of sterile-filtered SEC buffer containing 5.96 mM HEPES, 5% trehalose, and 100 mM NaCl, and the pellet was readily dissolved by gentle stirring. The resuspended lentiviral particles were stored as multiple aliquots at -80 °C until later use.

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

[0357] Results: Generally, the anti-HIV CAR was highly expressed on the surface of primary T cells in the range of up to 70% gene modification using a lentiviral vector. As shown in FIGS. 3A and 3B, single-specificity 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 (FIG. 3B).

[0358] To define the optimal bispecific binder structure of the CARs constructed using the mD1.22 and m36.4 domains, up to seven different bispecific CAR constructs were These CARs were constructed and designed with various orientations and different linker lengths to preserve the modularity and functionality of the domains. Compared to non-transduced T cells, a bispecific CAR containing the shortest glycine-serine linker (LTG2325, one G4S motif) to spatially separate the mD1.22 domain and the m36.4 domain, or a bispecific CAR containing up to the longest linker (LTG1947, five G4S motifs), were equally expressed with similar transduction efficiencies (Figure 4A). When the orientation of these two domains was reversed with m36.4 distal to mD1.22 (LTG1948), the CAR transduction efficiency decreased slightly (Figure 4B). This phenomenon is more likely due to the accessibility to the epitope of the CD4 antibody rather than the construct design. To maximize the function of these two domains, a bicistronic P2A construct containing two CARs (mD1.22-CAR and m36.4-CAR) was generated to form LTG2303. The basis of this construct is that m36.4 alone can bind to and neutralize HIV-1 strains even with reduced affinity in the absence of the CD4 receptor (Weizao Chen et al., Journal of Virology 2014, 88:2 1125-1139). Thus, when these two domains are designed as CARs, CAR-mediated cytotoxicity can be improved. 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 a Western blot of anti-CD3 zeta revealed that both CARs were highly expressed, fully cleaved, and migrated to the predicted molecular weight (Figure 4D). Generally, bispecific CARs were highly expressed on the surface of T cells with similar transduction efficiencies across constructs (Figure 4E).

[0359] Next, using the structures of LTG1946 and LTG2303, a trispecific CAR containing all three domains (mD1.22, m36.4, and C46 peptide) was constructed. Specifically, trispecific CARs were designed such that (1) CARs containing all three domains on one CD3 zeta (LTG2318, LTG2319, and LTG2320), (2) CARs containing a combination of the bispecific CAR LTG1946 or LTG1947 and 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) could be obtained. All these combinations formed unique trispecific CARs, enabling efficient investigation of the functions of the trispecific CARs.

[0360] As shown in Fig. 11A, detection by 2F5 flow cytometry revealed high expression (50 - 70% or less) of the trispecific CARs containing all three domains on one CD3 zeta chain (LTG2318, LTG2319, LTG2320). On the other hand, detection of the C46 peptide anchored to the membrane in the bicistronic trispecific construct (LTG2323) was significantly low (Fig. 11A, 18%). 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 (Fig. 11B). Similarly, the trispecific CARs LTG2329 and LTG2330 could be detected by 2F5 flow cytometry (up to 40%, Fig. 12C). Overall, the trispecific CARs were 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] Materials and methods: Cell lines used for functional characterization The 293T cell line engineered to stably express the full-length HIV envelope protein (293T-Env) was provided through the kindness of Dr. Dimiter Dimitrov (NCI, Fort Detrick, MD). Briefly, 293T-Env cells were grown in Dulbecco's Modified Eagle Medium (DMEM) in the presence of 10% fetal bovine serum and 60 μg / ml zeocin for selection maintenance. 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 tested for both the expression of gp120 / gp41 on the surface of 293T cells (by 2G12, b12, and 2F5 flow cytometry) and luciferase activity. One highly-expressing HIV-1 envelope and luciferase clone was isolated and used in the cytotoxicity assays described herein (293T-Env-Luc). As envelope-free cell lines, Raji cells and 293T HEK cells were purchased from ATCC (Manassas, VA), and subsequently transduced with a lentiviral vector encoding the firefly luciferase gene to generate Raji-Luc cell line or 293T-Luc cell line, 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. For both cell lines, single cell cloning was performed and flow cytometry analysis using 2G12, b12, and 2F5 (monoclonal antibodies against HIV-1 envelope) confirmed the absence of the HIV envelope.

[0364] Purification and transduction of primary T cells Using a combination of ficoll-paque gradient separation and leucosep tubes, human PBMCs from healthy volunteers were purified from buffy coats, and subsequently, CD4 + and CD8 +Magnetic-activated cell sorting 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 using a lentiviral vector encoding the 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, in sterile 96-well plates, 5 × 10 3 target cells stably expressing firefly luciferase were combined with CAR T cells at various effector:target ratios or without combination and incubated overnight at 37 °C in 5% CO 2 2. After 24 h, 100 μL of SteadyGlo reagent (Promega, Madison WI) was added to each well, incubated for 10 min at room temperature, and luminescence was then quantified 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 The supernatant from the cytotoxicity assay was 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] Results: The HIV envelope protein is a heterotrimeric glycoprotein expressed on the surface of HIV virus particles and is used to target and hijack T cells. During HIV infection, the HIV envelope protein (gp120) binds to the CD4 receptor of helper T cells, leading to a conformational change in the HIV envelope protein. This change promotes the interaction with a co-receptor (e.g., CCR5 or CXCR4) on the surface of the T cell in a tropism-dependent manner, ultimately resulting in viral fusion and subsequent intracellular release of HIV viral RNA into the T cell. Subsequently, the viral RNA is reverse transcribed and integrated into the host genome, where it can further produce infectious virus or become latent. While T cells are actively producing HIV virus, a portion of the HIV envelope protein (gp120 / gp41) remains on the surface of such infected T cells as a result of viral budding. Therefore, a CAR designed to recognize the HIV envelope glycoprotein can specifically target proliferating HIV-infected cells and subsequently, by the efficacy of its design, eliminate such HIV-infected cells.

[0368] HIV envelope epitopes have been used as targets for passive immunotherapy with monoclonal antibodies, but have not met with widespread success (discussed 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 result in rapid viral rebound and ultimately viral escape. The loss of virological control is mainly due to the instability of the antibodies and the gradual decline of antibody concentration in the host. Therefore, an HIV therapy that can provide a durable response may not only be able to control HIV even without ART, but may also, according to some, have a high potential to eradicate the HIV reservoir.

[0369] With the success of CD19 CAR, rethinking CAR-based HIV treatment (and perhaps cure) approaches is a very attractive approach. When combined with novel anti-HIV domains and state-of-the-art genomic engineering tools (e.g., lentiviral vectors), CARs may be able to address the limitations of passive immunization and overcome conventional pitfalls in the design of HIV CARs. More importantly, CARs can be incorporated into a new "kick and kill" paradigm that focuses on continuous immune surveillance and the elimination of the HIV reservoir with special agents that reactivate latent HIV.

[0370] Described herein are a series of unique anti-HIV CARs that have high cytotoxic efficacy and cytokine function against envelope-containing cells and at the same time maintain very excellent specificity. As shown in Table 1 below, more than 20 anti-HIV CARs were designed to fully investigate the efficacy against HIV-1. These CARs were designed by uniquely combining three different HIV envelope binding sequences in an exact manner to make them highly potent and synergistic HIV-1 infection inhibitors. By cloning them into a lentiviral expression vector containing the selected structural domain and signaling domain under the control of a constitutive active promoter, the functional characterization of these binders as CARs and their iterations was performed, and in vitro, using an HIV envelope cell line model, transduction efficiency, killing function , and cytokine production were tested. Table 1 shows an overview of the academic terms used. In some examples, the CAR construct LTG1732 (mCherry reporter) was used as a negative control to evaluate the T cell function changes 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. The reason for the lack of viral control is thought to be that the first generation CD4 zeta CARs need to be redesigned using current understanding of CARs. Thus, like other researchers in the CAR field, CD4-CARs were designed and engineered using the second generation structural elements described herein and compared to the more potent, specific, and smaller CD4-derived mD1.22-CAR (LTG1944) disclosed herein. The mD1.22-CAR was more potent than the CD4-CAR, but the mD1.22-CAR showed improved specificity and, as a trend, lower levels of off-target cytokines (data not shown). Thus, the novel CD4-like improved mD1.22-CAR described above was selected as the first prototype for further iterative CAR design.

[0372] To fully understand the relative contribution of each binder in the context of the CAR, m36.4-CAR (LTG1945) and C46-CAR (LTG2328) were further designed and evaluated. As shown in Figure 3C, the mD1.22-CAR (LTG1944) was the most potent, followed by the m36.4-CAR (LTG1945) and the C46-CAR (LTG2328). All three CARs showed very good specificity and no off-target killing in the absence of the HIV envelope (Figure 3D). Also, these CARs, except for the 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, the mD1.22-CAR had the most optimal structure for constructing more advanced dual and triple specificity HIV CARs.

[0373] In the design of the bispecific HIV CAR, an iterative approach was used to determine the best combination of mD1.22 and m36.4 for optimal CAR function. As shown in Figure 5, all bispecific CARs destroyed the envelope-expressing target up to 80% (Figure 5) and were highly specific (Figure 6). However, when measuring cytotoxicity (Figure 5) and cytokine release (Figure 7), T cells engineered with both mD1.22-CAR and m36.4-CAR using the bicistronic P2A vector (LTG2303) were significantly more potent than cells expressing only one CAR (such as LTG1944 or LTG1945). Reversing the order of these two domains, with m36.4 more distal than mD1.22, had no significant effect on CAR activity (LTG1948). As shown in Figure 8A, shortening the spatial distance between the mD1.22 domain and the m36.4 domain to one G4S motif significantly increased CAR-mediated cytotoxicity (LTG2325 vs. LTG1947). Also, these linker-specific CARs showed no cytotoxicity against envelope-free 293T cells or Raji cells (Figure 8B). Further examination of these linker-specific CARs revealed that all anti-HIV CARs were specifically triggered to produce IFN-γ, and in some cases, with increased potency, there was often an increase in off-target IFN-γ production (Figure 9). Overall, a series of functional assays confirmed that LTG2303 has the most potent bispecific CAR structure for the design of further trispecific HIV CARs.

[0374] Then, starting from the basic mD1.22-CAR structure, the trispecific HIV CAR construct The construct was designed and evaluated, and several intermediate constructs were designed to evaluate the optimal placement of C46, a highly potent C-peptide. C-peptides (e.g., T20 or C46) are derived from highly conserved regions within the C-terminal heptad repeat (CHR) region of gp41. Naturally, hydrophobic interactions between the C-peptide and the N-terminal heptad repeat region (NHR) are involved in virus fusion. Interestingly, several unique ways of using C-peptides as decoys have clearly shown a strong virus fusion inhibitory capacity leading to the suppression of HIV-1 infection. For example, from the two to three published methods, it has been shown that the anchoring of C-peptides to the T-cell membrane potently cuts off 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 method, by enabling the structural secretion of C-peptide (SAVE) by T cells, even bystander T cells were protected from HIV-1 infection (Egerer et al., Molecular Therapy 2010, 19(7), 1236-1244). In 2003, enfuvirtide (T20) was approved by the FDA as a rescue therapy for treating multi-drug resistant HIV-1 patients. However, due to the emergence of T20-resistant mutants, improved C-peptide designs have been developed.

[0375] These new C-peptides, such as C46, have been demonstrated to inhibit T20-resistant HIV-1 mutants. Similar to other C-peptides, C46 also targets overlapping long T20 sites involved in gp41-mediated viral fusion. There are also findings that improved C-peptides show strong inhibition independent of virus tropism and neutralize HIV-1 strains, with 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 that the design of a trispecific CAR using the C46 peptide is an attractive way to protect T cells from HIV-1 infection and to broaden the breadth of the CAR in the face of encounter with highly resistant HIV-1 viruses.

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

[0377] In this specification, trispecific anti-HIV CARs are described and characterized in terms of their cytotoxic ability. Three distinct trispecific CAR constructs were designed by (1) fusing all three domains on a single CD3 zeta (LTG2318, LTG2319, LTG2320), or (2) using the bispecific CAR LTG1946 in combination with the membrane-anchored C46 peptide (LTG2323, LTG2334), and finally (3) using the bispecific CAR LTG2303 with the C46 peptide 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 an alternative to the HIV envelope-expressing cell line, the trispecific CARs were comparable to the bispecific CARs and showed little off-target killing at very high effector:target ratios. As shown in Figure 11C, the position of C46 had a major impact on the trispecific CARs designed by placing all three domains 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), suppressed its cytolytic function. However, strong cytolytic function was shown 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), and was similar in function to the bispecific CAR LTG1946 (Figure 11C). These trispecific CARs were highly specific, showed no off-target killing against envelope-lacking cells (Figure 11D), and produced cytokines in response to antigen (Figures 11E–11F). Among the first set of trispecific CARs, LTG2323 appeared to be the most potent.

[0378] Similarly, the third set of trispecific CARs, LTG2329 and LTG2330, also showed strong cytolytic activity (Figure 12B), and there was some increase in non-specific killing at very high E:T ratios (Figure 12D). These observations were only seen in T cells co-expressing two different CARs (LTG2303, LTG2329, and LTG2330). As previously observed for the 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 can 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 eliminate HIV-infected PBMCs in vitro and in vivo In this example, the killing efficacy of anti-HIV CAR-T cells and the HIV-1 infectivity susceptibility of CAR-T cells were examined in in vitro and in vivo exposures using PBMCs infected with different Env-IMC-LucR HIV-1 viruses.

[0380] Materials and methods: Using replicable Env-IMC-LucR molecular clones, the in vitro efficacy of anti-HIV CARs when infecting PBMCs with the same donor Replicable HIV-1 molecular clones (Env-IMC-LucR) containing the desired heterologous HIV-1 envelope upstream of the in-frame Renilla luciferase ORF were used to examine the inhibition of HIV-1 infection. Infectious clones were generated as previously described (Edmonds et al., Virology 2010, 408:1-13). PBMCs or CD4 +For HIV-1 infection of T cells, after inoculation, the expression of Renilla luciferase is used as a highly sensitive and quantifiable HIV-1 virus replication indicator for a long period of several weeks. Briefly, autologous donor PBMCs (HIV-1 naive) are activated using 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, in a 96-well round-bottom plate, over 24 hours, 1×10 5 PBMCs are infected with the designated Env-IMC-LucR virus at 1×10 5 IU / mL (MOI = 1). The next day, 1×10 5 effector cells (anti-HIV CAR T cells) are added to the infected PBMCs and co-cultured for 7 days. For the in vitro protection assay, in the absence of PBMCs, anti-HIV CAR-T effectors are directly exposed to the designated Env-IMC-LucR virus at MOI = 1. Cell culture supernatants (60 μL) are collected from the co-cultures on days 0, 3, 5, and 7 for further analysis, and the cultures are supplemented with R10 medium. After 7 days, the cells are pelleted and lysed using 20 μL of lysis buffer, and luminescence is quantified as per the manufacturer's instructions (Promega, Madison, WI) (relative light units, RLU). Data are obtained using three independent donors (error bars = standard deviation). Luciferase activity is quantified according to the manufacturer's instructions. The data are represented as relative light units (RLU). For the in vitro killing assay, the log inhibition of HIV-1 infection is calculated using the formula "(log inhibition of HIV-1 infection) = Log10(CARRLU / UTDRLU)". The percentage of HIV-1 inhibition is calculated by 1-(CARRLU / UTDRLU)×100%. Statistical analysis was performed using a 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 eliminate acute and chronic HIV-infected PBMCs in a humanized NSG mouse model As previously described (Bardhi et al., J Virol 2017, 91:20; Thomas et al., Methods Mol Bio 2016, 1354:221-35), we used a humanized spleen NOD-SCID-IL2Rγ- / - mouse model (hu-spl-PBMC-NSG) that established rapidly, potently, and easily quantifiable HIV-1 infection in the mouse spleen using human PBMCs infected with Env-IMC-LucR HIV-1 virus to examine the efficacy of anti-HIV duoCAR-T cells in vivo. Briefly, 10 million PBMCs from the same donor were activated as described above and spinfected with 106 IU of Du422.1 per 107 PBMCs. HIV-infected PBMCs were injected into the spleen at an E:T ratio of 0.5:1 (e.g., 5 million total CAR-T cells and 10 million HIV-infected PBMCs) either with non-transduced T cells (UTD) or with either LTG2303 (bispecific duoCAR) or LTG2330 (trispecific duoCAR) containing 30-50% CAR-containing T cells. PBMCs and T cells containing CAR effector cells were mixed immediately before injection. One week (acute) or one month (c...

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular anti-HIV antigen-binding domain, wherein the isolated nucleic acid encoding the extracellular anti-HIV antigen-binding domain comprises a nucleotide sequence comprising SEQ ID NO: 75, 79, 83, 87, 91, 95, 99, 103, 111, or 119, and at least one transmembrane domain and at least one intracellular signaling domain. An isolated nucleic acid molecule.

2. A vector comprising the nucleic acid molecule according to claim 1.

3. The vector according to claim 2, 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.

4. A cell comprising the vector according to claim 2.

5. A method for producing a cell, comprising the step of transducing the vector according to claim 2 into T cells.

6. A pharmaceutical composition comprising a population of human T cells in an anti-HIV effective amount, wherein the population of human T cells comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises SEQ ID NO: 6, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or at least one extracellular anti-HIV antigen-binding domain comprising the amino acid sequence of 120, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the population of human T cells is T cells of a human subject having HIV / AIDS. A pharmaceutical composition.

7. Use of a T cell population for the manufacture of a pharmaceutical composition for the treatment of HIV-related cancer or HIV / AIDS, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises SEQ ID NO: 6, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or at least one extracellular anti-HIV antigen-binding domain comprising the amino acid sequence of 120, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the T cells are T cells of the subject having HIV-related cancer or HIV / AIDS.

8. The use according to claim 7, wherein the 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, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

9. The use according to claim 7, wherein the at least one extracellular anti-HIV antigen-binding protein binds to an HIV-1 envelope protein.

10. The use according to claim 7, wherein the at least one extracellular anti-HIV antigen-binding domain, the at least one intracellular signaling domain, or both are linked to the at least one transmembrane domain by the at least one linker or spacer domain.

11. The use according to claim 7, wherein the at least one linker or spacer domain is derived from the extracellular domain of CD8 and is bound to the at least one transmembrane domain.

12. The use according to claim 7, wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

13. The use according to claim 7, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

14. The use according to claim 7, wherein the at least one intracellular signaling domain comprises a co-stimulatory 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).

15. The use according to claim 7, wherein the nucleic acid sequence encoding the extracellular anti-HIV antigen-binding domain comprises a nucleotide sequence comprising SEQ ID NO: 75, 79, 83, 87, 91, 95, 99, 103, 111, or 119.

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