Humanized anti-HLA-G chimeric antigen receptors and their uses

Abstract

Provided herein are humanized anti-HLA-G chimeric antigen receptors (CARs), genetically engineered immune effector cells, and the use of these compositions to effectively treat cancers that express HLA-G.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 385,142, filed November 28, 2022, which is incorporated herein by reference in its entirety for all purposes. Electronic Sequence Listing Reference

[0002] The contents of the electronic sequence listing (INVE_015_01WO_SeqList_ST26.xml; size: 84,578 bytes; and creation date: November 27, 2023) are incorporated herein by reference in their entirety.

[0003] Technical Field The present disclosure relates to the fields of immunology, cell biology, and molecular biology. More specifically, the present disclosure relates to humanized chimeric antigen receptors (CARs) that bind to human leukocyte antigen (HLA-G), cells expressing such CARs, and therapeutic uses of these compositions. [Background technology]

[0004] For many years, the cornerstones of cancer treatment have been surgery, chemotherapy, and radiation therapy. More recently, immunotherapy has emerged as an effective tool in cancer treatment.

[0005] Chimeric antigen receptors (CARs) are synthetic tumor-targeting receptors that can be introduced into human immune cells, such as T cells, to alter antigen specificity and enhance effector immune cell function. Many attempts have been made to successfully administer CAR T cells to solid tumors, but the results have sometimes been disappointing. Three major obstacles encountered in applying CAR T cell therapy to solid tumors are (1) identifying appropriate tumor-associated antigens (TAA), (2) limited transport of adoptively transferred cells to the tumor site, (3) the immunosuppressive effects of the tumor microenvironment, and (4) potential safety issues of unwanted or untransformed cells.

[0006] Human leukocyte antigen G (HLA-G) plays a key role in the generation of fetal-induced maternal immune tolerance. HLA-G has also been proposed as an immune checkpoint (ICP) molecule that inhibits the effector functions of infiltrating immune cell subsets through interaction with its specific receptor. HLA-G is expressed in numerous tumors of diverse origins, and its tissue expression is highly restricted. In some malignant transformations, HLA-G expression by tumor cells increases dramatically, rendering them potently immunosuppressive. Preclinical models have shown that HLA-G expression on cancer cells predisposes them to further metastasis and significantly reduces patient survival (Lin A. et al., Int. J. Cancer, 2012, Jul. 1;131(1):150-7; Lin A. et al. Hum. Immunol. 2013, Apr;74(4):439-46). Additional immunotherapeutic approaches targeting HLA-G are needed to treat cancer. Summary of the Invention

[0007] Provided herein is a humanized chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a humanized antigen-binding domain that specifically binds human leukocyte antigen G (HLA-G), (b) a transmembrane region, and (c) an intracellular domain. Humanized anti-HLA-G CARs as disclosed herein exhibit reduced immunogenicity in human patients.

[0008] In some embodiments, the humanized antigen-binding domain of the anti-HLA-G CAR is a humanized antigen-binding fragment of an anti-HLA-G CAR antibody. In some embodiments, the humanized antigen-binding fragment comprises a humanized heavy chain variable (VH) region comprising HCDR1, HCDR2, and HCDR3 of the anti-HLA-G antibody, and a humanized light chain variable (VL) region comprising LCDR1, LCDR2, and LCDR3. In some embodiments, the humanized antigen-binding fragment comprises HCDR1 of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 15, and comprises LCDR1 of SEQ ID NO: 37, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 41.

[0009] Further provided herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) (i) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 25, and (ii) a light chain variable region (VL) comprising the sequence of SEQ ID NO: 35; (b) (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 50; or (c) An anti-HLA-G CAR comprising a humanized antigen-binding fragment comprising (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 60.

[0010] Also provided herein are (a) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 25; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 35; (b) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 50; or (c) An anti-HLA-G CAR comprising a humanized antigen-binding fragment comprising: (i) a VH comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 60.

[0011] In some embodiments, the humanized antigen-binding fragment of the anti-HLA-G CAR is a single-chain Fv (scFv). In some embodiments, the scFv comprises a sequence selected from SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67.

[0012] In some embodiments, the CAR specifically binds to HLA-G isoforms bound to β2M, preferably HLA-G1 and HLA-G5, and preferably, such a CAR is capable of distinguishing between HLA-G isoforms, i.e., the CAR does not recognize or bind to all seven HLA-G isoforms.

[0013] In particular, the present disclosure provides a humanized anti-HLA-G CAR comprising, in order from N- to C-terminus: (a) a peptide signal sequence, b) a humanized anti-HLA-G antibody or humanized antigen-binding fragment thereof, optionally c) a spacer domain, optionally d) a hinge domain, e) a transmembrane domain, f) an intracellular domain, optionally g) a cleavable linker, and optionally g) a truncated human CD19 domain. In some embodiments, the HLA-G CAR comprises the sequence of SEQ ID NO: 68.

[0014] In one aspect, the spacer domain comprises (i) a human IgG4 hinge domain, (ii) a human IgG4 hinge domain and a CH3 human IgG4 domain, or (iii) a mutated CH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 human IgG4 hinge domain.

[0015] In another embodiment, the signal peptide is selected from the group consisting of CD8a signal peptide, mouse Ig kappa signal peptide, human IgG4 signal peptide, IL2 signal peptide, human IgG2 signal peptide, and Gaussia luc signal peptide.

[0016] In some embodiments, the transmembrane domain is selected from the transmembrane domains of CD28, CD3, and CD8, preferably the transmembrane domain is the transmembrane domain of CD28.

[0017] In some embodiments, a humanized anti-HLA-G CAR of the present disclosure comprises a signaling domain of CD3 zeta and at least one costimulatory domain selected from CD28, 41BB, CD28, CD134, ICOS, OX40, CD149, DAP10, CD30, IL2-R, IL7r6, IL21-R, NKp30, NKp44, CD27, CD137, and DNAM-1, preferably, the two costimulatory domains are a 41BB costimulatory domain and a CD28 costimulatory domain.

[0018] In one embodiment, the cleavable linker is selected from the group consisting of P2A, T2A, E2A, B2A, and F2A.

[0019] In another embodiment, the truncated human CD19 domain consists of the sequence shown in SEQ ID NO:76.

[0020] In particular, the CAR, anti-HLA-G antibody, or antigen-binding fragment thereof, preferably scFv, selectively binds to HLA-G isoforms bound to β2M, but does not bind to all HLA-G isoforms.

[0021] In another embodiment, the CAR, the anti-HLA-G antibody or antigen-binding fragment thereof, preferably an scFv, or the cell expressing the CAR specifically binds to the α1 domain of HLA-G.

[0022] Also provided herein is an anti-HLA-G antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is: (a) (i) a VH comprising the sequence of SEQ ID NO: 25, and (ii) a VL comprising the sequence of SEQ ID NO: 35; (b) (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 50; or (c) (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 60.

[0023] Also provided herein is an anti-HLA-G antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is: (a) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 25; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 35; (b) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 50; or (c) (i) a VH comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 60.

[0024] The present disclosure also contemplates nucleic acid molecules encoding a CAR of the present disclosure, expression vectors comprising the nucleic acid molecules, and cells comprising a CAR of the present disclosure or a nucleic acid molecule of the present disclosure, or an expression vector of the present disclosure, preferably wherein the cell is selected from the group consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a B cell, an NK cell, an NKT cell, a monocyte, and a dendritic cell, preferably wherein the cell is a T cell, a B cell, or an NK cell.

[0025] The present disclosure also relates to a pharmaceutical composition comprising a nucleic acid molecule, expression vector, or cell according to the present disclosure, and optionally a pharmaceutically acceptable carrier.

[0026] In some embodiments, the cells or pharmaceutical compositions of the present disclosure are for use in treating cancer or viral infections. The cells or pharmaceutical compositions for such uses can be administered in combination with CAR therapy that does not target HLA-G.

[0027] In one embodiment, the pharmaceutical composition comprises cells comprising a CAR that specifically binds to HLA-G that binds to β2M, preferably to both HLA-G1 and HLA-G5.

[0028] Also provided herein are methods of treating cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising cells comprising a CAR disclosed herein. In some embodiments, the cells are T cells, B cells, NK cells, NKT cells, monocytes, or dendritic cells. In some embodiments, the cancer is an HLA-G-expressing cancer. In some embodiments, the cancer is clear cell renal cell carcinoma, epithelial ovarian cancer, melanoma, kidney cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, renal cell carcinoma, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, hepatocellular carcinoma, cholangiocarcinoma, neuroblastoma, tongue cancer, oral and pharyngeal cancer, bronchogenic carcinoma, laryngeal cancer, osteosarcoma, prostate cancer, testicular cancer, or gastrointestinal stromal tumor. pancreatic cancer, kidney cancer, colorectal cancer, glioma, glioblastoma multiforme, medulloblastoma, thyroid cancer, adrenal cancer, acute myeloid leukemia, chronic lymphocytic leukemia, non-small cell lung cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, monocytic lymphoma, marginal zone lymphoma, Burkitt's lymphoma, T-cell lymphoma, B-cell lymphoma, plasmacytoma, prohemocytic leukemia, acute nonlymphocytic leukemia, acute lymphoblastic leukemia, erythroleukemia, myeloid leukemia, or lymphocytic leukemia.

[0029] Also provided herein are cells comprising an anti-HLA-G CAR disclosed herein or a composition disclosed herein, for use as a medicament. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic diagram of the HLA-G CAR protein. [Figure 2] The degree of humanization of the LFTT-1 humanized variants is indicated by homology (to the parent and human germline) and by counting back mutations. [Figure 3] 1 shows the degree of humanization plotted against the relative IC50 score for LFTT-1 humanized variants. [Figure 4] Shows the specific cytotoxicity of anti-HLA-G CAR-T cells against multiple chemoresistant SKOV-3-HLA-G+ cell lines. [Figure 5A] Figure 1 shows in vitro infiltration and activation of anti-HLA-G CAR-T cells in renal cell carcinoma tissue. [Figure 5B] Figure 1 shows in vitro infiltration and activation of anti-HLA-G CAR-T cells in renal cell carcinoma tissue. [Figure 5C] Figure 1 shows in vitro infiltration and activation of anti-HLA-G CAR-T cells in renal cell carcinoma tissue. [Figure 6A] Demonstrates the effect of anti-HLA-G CAR-T cells on primary tumor control and elimination in an in vivo PDX model. [Figure 6B] Demonstrates the effect of anti-HLA-G CAR-T cells on primary tumor control and elimination in an in vivo PDX model. [Figure 7A] Specific cytotoxicity of anti-HLA-G CAR-T cells derived from three donors against LCL-GFP-HLA-G cells is shown. [Figure 7B] Specific cytotoxicity of anti-HLA-G CAR-T cells derived from three donors against LCL-GFP-HLA-G cells is shown. [Figure 7C] Specific cytotoxicity of anti-HLA-G CAR-T cells derived from three donors against LCL-GFP-HLA-G cells is shown. [Figure 8A] Shown are the levels of IFN-γ secreted by anti-HLA-G CAR-T cells from three donors. [Figure 8B]Shown are the levels of IFN-γ secreted by anti-HLA-G CAR-T cells from three donors. [Figure 8C] Shown are the levels of IFN-γ secreted by anti-HLA-G CAR-T cells from three donors. [Figure 9A] Figure 1 shows the specific cytotoxicity of anti-HLA-G CAR-T cells derived from three donors against LCL-HLA-G cells in the presence of increasing concentrations of soluble HLA-G. [Figure 9B] Figure 1 shows the specific cytotoxicity of anti-HLA-G CAR-T cells derived from three donors against LCL-HLA-G cells in the presence of increasing concentrations of soluble HLA-G. [Figure 9C] Figure 1 shows the specific cytotoxicity of anti-HLA-G CAR-T cells derived from three donors against LCL-HLA-G cells in the presence of increasing concentrations of soluble HLA-G. DETAILED DESCRIPTION OF THE INVENTION

[0031] overview The present disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular domain composed mostly of a humanized antigen-binding domain of an anti-HLA-G specific antibody, and optionally a hinge domain comprising or consisting of: (i) a human IgG4 hinge domain, (ii) a human IgG4 hinge domain and a CH3 human IgG4 domain, or (iii) a mutated CH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 human IgG4 hinge domain, a transmembrane domain, and an intracellular domain comprising one, two or three costimulatory structures depending on the generation of the CAR design, optionally a cleavable linker, and optionally a reporter.

[0032] The humanized anti-HLA-G CARs disclosed herein exhibit reduced immunogenicity in human patients. When generating humanized anti-HLA-G antibodies whose antigen-binding domains are used in the CARs disclosed herein, both the degree of humanization and the relative IC50 score for each antibody had to be considered, as there is often a trade-off between binding efficiency and similarity to human germline. Generally, a more human-like design results in lower binding efficiency because fewer mouse residues are conserved in the design, increasing the likelihood that the antibody will lose affinity for its target. Generally, a more conservative design results in better binding efficiency. Provided herein are humanized anti-HLA-G CARs containing antigen-binding domains with lower relative IC50 scores and a higher degree of humanization, allowing for retention of antibody affinity for the target and tolerance by the human immune system.

[0033] In some embodiments, a CAR of the present disclosure specifically binds to an HLA-G isoform bound to β2M, preferably an HLA-G isoform selected from HLA-G1 and HLA-G5.

[0034] Provided herein are monoclonal antibodies or single-chain variable fragment (scFv) molecules that specifically bind to both HLA-G1 and HLA-G5. The present disclosure further relates to monoclonal antibodies or single-chain variable fragment (scFv) molecules that specifically bind to HLA-G isoforms bound to β2M, preferably the HLA-G1 and HLA-G5 isoforms.

[0035] The present disclosure also relates to nucleic acid constructs or vectors containing the nucleic acid constructs that can transduce cells, preferably immune cells such as T cells, thereby creating recombinant immune cells engineered to express the encoded CAR. Also provided are cells transduced to express the CARs of the present disclosure, cell populations, and pharmaceutical compositions containing cells expressing the CARs.

[0036] In some embodiments, the immune effector cells are engineered to express an anti-HLA-G CAR. In some embodiments, the engineered immune effector cells are administered to a subject with cancer cells that express HLA-G, including, but not limited to, solid tumors and hematological malignancies.

[0037] Provided herein are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are methods of preparing CAR-expressing cells and administering the cells and compositions to a subject, e.g., a patient.

[0038] Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may generally be performed as described in various general and more specific texts in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, as cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July, 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning:A Practical Approach,vol.I & II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober,2001,John Wiley & Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames & S. Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008,Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010,Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q.E. Coligigan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as field-specific research articles in journals such as Advances in Immunology.

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of the compositions, methods, and materials are described herein. For purposes of this disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0040] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or to more than one) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0041] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0042] The term "and / or" should be understood to mean either one or both of the alternatives.

[0043] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method used to determine the value, or the variation that exists between samples measured. Unless otherwise specified or clear from the context, the term "about" means within 10% of the reported numerical value (except when such number is greater than 100% or less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each recited value in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used synonymously.

[0044] Numerical ranges, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refer to each of the numbers encompassed by the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0045] As used herein, the term "substantially" refers to an amount, level, value, number, frequency, proportion, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference amount, level, value, number, frequency, proportion, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, proportion, dimension, size, amount, weight, or length that produces approximately the same effect, e.g., physiological effect, as the reference amount, level, value, number, frequency, proportion, dimension, size, amount, weight, or length.

[0046] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of these phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment serves as a basis for excluding the feature in certain embodiments.

[0047] HLA-G antigen "HLA-G" refers to human leukocyte antigen G. HLA-G is a non-classical HLA class I molecule first identified in choriocarcinoma cells. Unlike classical HLA class I molecules, HLA-G is characterized by limited polymorphism, and its expression, structure, and function are similarly distinct. Its expression is primarily restricted to the fetal-maternal interface of extravillous cytotrophoblasts; the placenta, amniotic membrane; a few healthy adult tissues such as the thymus, cornea, bronchial epithelial cells, and pancreas; and various cell types such as mesenchymal stem cells, a small number of activated monocytes, erythroid progenitor cells, and endothelial progenitor cells. Soluble HLA-G is also found in body fluids such as plasma, cerebrospinal fluid, malignant ascites, pleural effusion, and semen. Although the HLA-G gene is inactive in some tissues, its expression can be induced by certain molecules such as progesterone or anticancer drugs. Furthermore, this molecule can also be de novo expressed in pathological conditions such as cancer, multiple sclerosis, inflammatory diseases, and viral infections, or after allogeneic transplantation. Soluble HLA-G (sHLA-G) can be detected in the serum / plasma of individuals.

[0048] HLA-G differs from classical HLA class I molecules by its low genetic diversity, tissue-restricted expression, the existence of seven isoforms, and immunoinhibitory function. This molecule exerts its immunoinhibitory function through direct binding to three inhibitory receptors: leukocyte immunoglobulin-like receptor B1 (LILRB1 / ILT2 / CD85j), LILRB2 (ILT4 / CD85d), and KIR2DL4 (or CD158d). The LILRB receptors recognize HLA-G via the α3 domain and are unlikely to be affected by peptides. LILRB1 is expressed by B cells, some T cells, some NK cells, and all monocytes / dendritic cells, whereas LILRB2 is myeloid-specific and its expression is restricted to monocytes / dendritic cells. KIR2DL4 is a specific receptor for HLA-G that is expressed only by the CD56-positive subset of NK cells. LILRB1 and LILRB2 have been shown to bind a wide range of classical HLA molecules via their α3 domains and β2M, for which HLA-G is the highest affinity ligand, whereas HLA-G is the only known ligand for KIR2DL4. Furthermore, it has been demonstrated that LILRB1 and LILRB2 exhibit higher affinity for HLA-G multimers than for the monomeric form. It is important to note the differences in how LILRB1 and LILRB2 bind to their ligands: LILRB1 exhibits higher affinity for HLA-G heavy chains bound to β2M, whereas LILRB2 binds to the α3 domain rather than β2M, and exhibits significantly different MHC1 binding recognition, involving aromatic amino acids Phe-195 and Tyr-197. This explains the latter receptor's β2M-independent HLA-G binding and its higher affinity for β2M-free isoforms.

[0049] By engaging these receptors, HLA-G acts as a down-regulator of the immune system, and several of its functions have been described: inhibiting the cytolytic function of uterine and peripheral blood NK cells, the antigen-specific cytolytic function of cytotoxic T lymphocytes, the alloproliferative response of CD4+ T cells, the proliferation of T cells and peripheral blood NK cells, and the maturation and function of dendritic cells. Furthermore, HLA-G can induce the generation of suppressor cells. However, unlike classical HLA class I molecules, no stimulatory function for HLA-G has been reported to date, and none of the responses are directed against allogeneic HLA-G.

[0050] HLA-G can inhibit all immune cell subsets and thus block all stages of the anti-tumor response. This molecule is expressed on many types of primary tumors, metastases, and malignant effusions, and is also found on tumor cells and tumor-infiltrating cells. Expression of HLA-G by tumor cell lines has been shown to protect them from destruction by cytotoxic T lymphocytes and NK cells. Thus, expression of HLA-G by malignant cells may hinder tumor immune clearance by inhibiting the activity of tumor-infiltrating NK cells, cytotoxic T lymphocytes (CTLs), and antigen-presenting cells (APCs).

[0051] HLA-G expression is primarily regulated at the transcriptional level by its own gene promoter and at the post-transcriptional level by alternative splicing, mRNA stability, translation, and protein transport to the cell surface.

[0052] The primary transcript of HLA-G is alternatively spliced, resulting in the expression of seven isoforms, four of which are membrane-bound (HLA-G1, HLA-G2, HLA-G3, and HLA-G4) and three of which are soluble (HLA-G5, HLA-G6, and HLA-G7). HLA-G1 and HLA-G5 display the typical structure of classical HLA class I molecules, with the heavy chain consisting of three globular domains noncovalently bound to β2-microglobulin (β2M) and peptide, whereas other isoforms are shorter, lack one or two domains of the heavy chain, and should not bind to β2M.

[0053] HLA-G1 and HLA-G5 appear to be the most abundant isoforms, likely due to the lack of antibody diversity against other isoforms, particularly those that do not contain β2M.

[0054] The HLA-G1 isoform is a complete isoform with α1, α2, and α3 domains that bind to β2-microglobulin. The HLA-G2 isoform lacks the α2 domain, while HLA-G3 lacks the α2 and α3 domains, and HLA-G4 lacks the α3 domain. None of the isoforms, HLA-G2, HLA-G3, and HLA-G4, bind to β2M. The soluble HLA-G5 and HLA-G6 isoforms contain the same extra globular domain as HLA-G1 and HLA-G2, respectively. The HLA-G7 isoform has only the α1 domain linked to two amino acids encoded by intron 2. The HLA-G5 isoform binds to β2M, whereas the isoforms HLA-G6 and HLA-G7 do not.

[0055] Furthermore, HLA-G molecules can form dimers via disulfide bonds between two unique cysteine ​​residues at positions 42 (Cys42-Cys42 bond) and 147 (Cys42-Cys147 bond) of the HLA-G heavy chain. This dimerization exposes the HLA-G receptor-binding site in the α3 domain upward, with an oblique orientation that makes it more accessible to the receptor. As a result, HLA-G dimers bind to the receptor with higher affinity and a slower dissociation rate than monomers, and also signal more efficiently than monomers.

[0056] Another name is HLA-G histocompatibility antigen class I or G or MHC-G. HLA-G is listed in databases under the following accession number: Gene ID: 3135, UniGene Hs.512152. The protein is disclosed in UniProt under accession number P17693. The GenBank entries for the protein and mRNA sequences are NP_002118.1 and NM_002127.5, respectively. When comparing sequences of HLA-G isoforms, HLA-G1 is typically chosen as the reference sequence, i.e., the DNA, RNA, or amino acid sequence that reflects the most frequent nucleic acid, base, or amino acid at each position; therefore, databases generally refer to this isoform sequence as "HLA-G." HLA-G2 through HLA-G7 differ from HLA-G1 by amino acid deletion(s) and / or substitution(s). HLA-G human isoforms are listed under Uniprot accession numbers P17693-1 for HLA-G1, P17693-2 for HLA-G2, P17693-3 for HLA-G3, P17693-4 for HLA-G4, P17693-5 for HLA-G5, P17693-6 for HLA-G6, and P17693-7 for HLA-G7.

[0057] Antibodies against HLA-G isoforms The present disclosure provides antibodies that specifically bind to one to six, preferably two to five, of the seven HLA-G isoforms, but do not specifically bind to or recognize all HLA-G isoforms. For example, the antibodies can specifically bind to HLA-G1 and HLA-G5 isoforms.

[0058] For example, the antibody or antigen-binding domain of a CAR of the present disclosure can recognize HLA-G1 and HLA-G5 when the epitope recognized by the antibody or antigen-binding domain is on the β2M domain of HLA-G or on a domain specific for HLA-G bound to the β2M domain.

[0059] In one embodiment, the antibody can specifically bind to the HLA-G1 and HLA-G5 isoforms. The antibody then does not substantially bind to other HLA-G isoforms, particularly HLA-G2, HLA-G3, HLA-G4, HLA-G6, and HLA-G7. More specifically, the antibody is specific for HLA-G isoforms that bind to β2M. In this regard, the antibody does not substantially bind to the HLA-G1 and HLA-G5 isoforms that lack β2M.

[0060] The present specification provides a humanized antibody LFTT-1. In particular, the humanized LFTT-1 is a humanized scFv antibody. LFTT-1 is described in WO2020043899, which is incorporated herein by reference in its entirety. The CDR of the humanized antibody LFTT-1 is, according to Kabat, the following sequence: (a) heavy chain CDR1 of SEQ ID NO: 11; (b) heavy chain CDR2 of SEQ ID NO: 13; (c) a heavy chain CDR3 of SEQ ID NO: 15; (d) a light chain CDR1 of SEQ ID NO: 37; (e) a light chain CDR2 of SEQ ID NO: 3, and (f) having a light chain CDR3 of SEQ ID NO: 41; In this regard, each CDR may optionally contain 1, 2, 3, or 4 amino acid substitutions, deletions, or insertions.

[0061] Thus, the present disclosure: (a) (i) a VH comprising the sequence of SEQ ID NO: 25, and (ii) a VL comprising the sequence of SEQ ID NO: 35; (b) (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 50; or (c) An anti-HLA-G antibody or antigen-binding fragment thereof, which is an anti-HLA-G scFv comprising (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 60.

[0062] The antibody can be a chimeric, human, or humanized antibody. The antibody can be an antibody fragment selected from Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, or single-chain antibody fragments including multiple different antibody fragments. The antibody can be conjugated or covalently linked to a toxin or a detectable label.

[0063] In some embodiments, the antibody or fragment thereof has a nucleotide sequence of at least about 10 7 M -1 , preferably at least about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 It binds with high affinity to one or some, but not all, HLA-G isoform(s).

[0064] In some embodiments, the antibody or fragment thereof does not bind to or recognize the α1 domain of an HLA-G isoform. This means that such an antibody or fragment thereof can bind to or recognize an HLA-G isoform lacking the α1 domain, such as an HLA-G isoform containing the α2 and α3 domains, or containing only the α3 domain, as described in Tronik-Le Roux et al., Molecular Oncology, 11 (2017) 1561-1578. For example, such an antibody or fragment thereof can bind to the α2, α3, or β2M domain.

[0065] The antibody or antibody fragment sequences of the present disclosure may be used in methods for preparing CARs or in methods for preparing pharmaceutical compositions. Alternatively, the antibodies or antibody fragments of the present disclosure may be used to detect HLA-G isoform(s) in diagnostic tests such as immunoassays.

[0066] Antibodies or antibody fragments can be identified by immunoassays such as, for example, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) assays, or other techniques known to those skilled in the art.

[0067] Preferably, antibodies (including antibody fragments thereof) that specifically bind to one or more HLA-G isoform(s) do not significantly cross-react with other antigens (i.e., are undetectable in a given immunological assay). An antibody specifically binds to an antigen if it binds to the antigen with higher affinity than the antigen with which it cross-reacts, as determined using experimental techniques such as Western blot (WB), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and particularly competitive ELISA.

[0068] Chimeric Antigen Receptor In various embodiments, provided herein are engineered receptors that redirect the cytotoxicity of immune effector cells to cancer cells that express human leukocyte antigen G (HLA-G). These engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are artificially constructed hybrid proteins or polypeptides that combine binding specificity for a desired antigen (e.g., HLA-G) with a T cell receptor activation intracellular domain to generate chimeric proteins that exhibit specific anti-HLA-G cellular immune activity and activate T cells upon interaction with the target antigen (e.g., HLA-G).

[0069] The prototype single-chain CAR was first described in a study by Eshhar and colleagues in 1993, in which specific activation and targeting of T cells were mediated by a molecule consisting of a target antigen-specific antibody domain and the γ- or ζ-signaling subunit of the Fcε receptor or the T cell receptor-CD3 complex, respectively (Eshhar et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 720-4). In more recent versions, the binding domain of the CAR typically consists of the antigen-binding domain of a library-selected single-chain antibody (scFv) or antibody-binding fragment (Fab), containing the variable fragments of the light and heavy chains of monoclonal antibodies (mAbs) linked by a flexible linker. The scFv retains the same specificity and affinity as the intact antibody from which it is derived and is capable of specifically binding to the desired target. Thus, CARs combine antigen specificity and T cell activation properties in a single fusion molecule. Indeed, scFvs are linked to intracellular signaling modules containing CD3ζ, which induce T cell activation upon antigen binding. The modular architecture has expanded from first-generation CARs, which contain only the CD3ζ signaling domain in an attempt to mimic costimulation, to second- and third-generation CARs, which link signaling endodomains such as CD28, 4-1BB, and OX40 to CD3ζ. Generally, a spacer or hinge domain serves as a linker between the endodomain and the scFv. The incorporation of such a hinge domain not only improves flexibility, spatial organization, and / or proximity, but also improves CAR cell proliferation (Qin et al., Journal of Hematology & Oncology. 2017;10:68) or tumor localization (Watanabe et al., Oncoimmunology. 2016;5(12):e1253656).

[0070] In some embodiments, a CAR disclosed herein comprises an extracellular domain (including a binding domain or antigen-specific binding domain) that binds to HLA-G, a transmembrane domain, and an intracellular domain. In some embodiments, the CAR comprises, in order from amino terminus to carboxyl terminus, (a) an extracellular domain that binds to HLA-G, (b) a transmembrane domain, and (c) an intracellular domain. Binding of the anti-HLA-G antigen-binding domain of the CAR to HLA-G on the surface of a target cell delivers an activating stimulus to the CAR-expressing cell. In some embodiments, binding of the anti-HLA-G antigen-binding domain of the CAR to HLA-G on the surface of a target cell results in clustering of the CAR and subsequent activation of the CAR-expressing cell.

[0071] A key feature of CARs is their ability to harness the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors by redirecting the specificity of immune effector cells, thereby inducing proliferation, cytokine production, phagocytosis, and / or the production of molecules that mediate cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner. MHC-independent antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0072] Binding domain In some embodiments, the CAR comprises an extracellular domain comprising an antigen-binding domain that specifically binds to HLA-G (e.g., human HLA-G). For example, HLA-G can be a human HLA-G polypeptide expressed on target cells, e.g., cancer cells. In some embodiments, the antigen-binding domain of the CAR is an anti-HLA-G antibody or antigen-binding fragment thereof. As used herein, the terms "binding domain," "antigen-binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and provide the CAR with the ability to specifically bind to a target antigen of interest, e.g., HLA-G. The binding domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0073] As used herein, the term "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" describes that an anti-HLA-G antibody or antigen-binding fragment thereof (or a CAR comprising the same) binds to HLA-G with a binding affinity higher than background binding. For example, about 10 5 M -1 or greater affinity or K a A binding domain (or a CAR comprising the binding domain or a fusion protein containing the binding domain) "specifically binds" to an HLA-G polypeptide if it binds to or associates with HLA-G with a binding constant of about 10 (i.e., the equilibrium association constant of the particular binding interaction in units of 1 / M). In certain embodiments, the binding domain (or fusion protein thereof) 6 M -1 , 10 7 M -1 , 10 8 M- 1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 More than Ka A "high affinity" binding domain (or single-chain fusion protein thereof) binds to a target with at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or higher K a refers to a binding domain having the following structure:

[0074] Alternatively, affinity may be expressed in units of M (e.g., 10 -5 M~10 -13 M or less) of a specific binding interaction d The affinity of the binding domain polypeptides and CAR proteins of the present disclosure can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association or displacement assays using labeled ligands, or by using surface plasmon resonance instruments such as the BIACORE® T100 available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology such as the EPIC system or EnSpire available from Corning Corporation and Perkin Elmer, respectively (see, e.g., Scatchard et al. (1949), Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or equivalents thereof).

[0075] In some embodiments, the extracellular domain of the CAR comprises an antibody or an antigen-binding fragment thereof. "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, e.g., one recognized by immune cells. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. 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.

[0076] "Antigen (Ag)" refers to a compound, composition, or substance capable of stimulating antibody production or a T-cell response in an animal, including compositions (such as those containing cancer-specific proteins) that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens. In some embodiments, the target antigen is an epitope of an HLA-G polypeptide.

[0077] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous amino acids and from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3, more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0078] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0079] A "chimeric antibody" has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as mouse. In some embodiments, the CAR comprises an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0080] In some embodiments, the antibody is a human antibody (e.g., a human monoclonal antibody) or fragment thereof that specifically binds to a human HLA-G polypeptide. Human antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from a human-derived phage display library or yeast display library with known human constant domain sequence(s), as described above. Alternatively, human monoclonal antibodies can be produced by hybridoma technology. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, by Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991). Furthermore, transgenic animals (e.g., mice) can be used to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Gene shuffling can also be used to derive human antibodies from nonhuman, e.g., rodent, antibodies, where the human antibody has similar affinities and specificities as the starting nonhuman antibody. (See WO 93 / 06213.) Unlike traditional humanization of nonhuman antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of nonhuman origin.

[0081] In one embodiment, a CAR comprises a "humanized" antibody. A humanized antibody is an immunoglobulin containing a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all portions of a humanized immunoglobulin, excluding the CDRs, are substantially identical to corresponding portions of native human immunoglobulin sequences. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0082] In some embodiments, the CAR comprises one or more humanized framework regions (FRs). In some embodiments, the anti-HLA-G CAR comprises a heavy chain variable region comprising one, two, three, or four humanized heavy chain FRs. In some embodiments, the anti-HLA-G CAR comprises a heavy chain variable region comprising, in order from N-terminus to C-terminus, a humanized heavy chain FR1 set forth in SEQ ID NO: 10, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 10; a CDR1 set forth in SEQ ID NO: 11; a humanized heavy chain FR2 set forth in one of SEQ ID NOs: 12 and 26, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 12 or 26; a CDR2 set forth in SEQ ID NO: 13; a humanized heavy chain FR3 set forth in SEQ ID NO: 14, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 14; a CDR3 set forth in SEQ ID NO: 15; and a humanized heavy chain FR4 set forth in SEQ ID NO: 16, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 16. In some embodiments, the anti-HLA-G CAR comprises a light chain variable region comprising one, two, three, or four humanized light chain FRs. In some embodiments, the anti-HLA-G CAR comprises a light chain variable region comprising, in order from N-terminus to C-terminus: a humanized light chain FR1 set forth in SEQ ID NO: 36, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 36; a CDR1 set forth in SEQ ID NO: 37; a humanized light chain FR2 set forth in SEQ ID NO: 38, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 38; a CDR2 set forth in SEQ ID NO: 39; a humanized light chain FR3 set forth in one of SEQ ID NOs: 40 and 51, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 40 or 51; a CDR3 set forth in SEQ ID NO: 41; and a humanized light chain FR4 set forth in SEQ ID NO: 42, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 42.

[0083] Antigen-binding fragments include camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and the portion of a full-length antibody that is responsible for antigen binding. An "isolated antibody or antigen-binding fragment thereof" is one that has been identified and separated and / or recovered from a component of its natural environment.

[0084] As will be understood by those skilled in the art and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, and each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is made up of two linked heavy chains, the second and third constant regions (and the fourth constant region for IgE and IgM), and a disulfide bond (interchain bond) is formed at the hinge. Heavy chains γ, α, and δ have constant regions composed of three in-line Ig domains and a hinge region for added flexibility; heavy chains μ and ε have constant regions composed of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of the Y contains the variable and first constant regions of one heavy chain bound to the variable and constant regions of one light chain. The light and heavy chain variable regions are responsible for antigen binding.

[0085] Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." CDRs can be defined or identified by conventional methods, for example, by the sequences according to Kabat et al. (Wu, TT and Kabat, EA, J. Exp. Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987); (see Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991, which are incorporated herein by reference) or by the sequences according to Chothia et al. (Chothia, C. and Lesk, AM, J. Mol. Biol., 196(4):901-917 (1987), Chothia, C. et al., Nature, 342:877-883 (1989)).

[0086] The sequences of framework regions of different light or heavy chains are relatively conserved within species such as humans. The framework region of an antibody, consisting of the framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically numbered sequentially starting from the N-terminus and designated CDR1, CDR2, and CDR3. They are also typically identified by the chain in which a particular CDR is located. Thus, CDRs located in the variable domain of an antibody's heavy chain are designated HCDR1, HCDR2, and HCDR3, while CDRs located in the variable domain of an antibody's light chain are designated LCDR1, LCDR2, and LCDR3. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. While it is the CDRs that differ between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). Illustrative examples of light chain CDRs suitable for construction of anti-HLA-G CARs contemplated in some embodiments include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 37, 39, and 41. Illustrative examples of heavy chain CDRs suitable for construction of anti-HLA-G CARs contemplated in some embodiments include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 11, 13, and 15.

[0087] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment contemplated herein. Illustrative examples of heavy chain variable regions suitable for construction of anti-HLA-G CARs contemplated in some embodiments include, but are not limited to, the heavy chain variable region sequences set forth in SEQ ID NOs: 9 and 25.

[0088] References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment contemplated herein. Illustrative examples of light chain variable regions suitable for construction of anti-HLA-G CARs contemplated in some embodiments include, but are not limited to, the light chain variable region sequences set forth in SEQ ID NOs: 35, 50, and 60.

[0089] In some embodiments, the anti-HLA-G antibody or antigen-binding fragment thereof includes, but is not limited to, camelid Ig (camelid antibody (VHH)), Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), single domain antibody (sdAb, nanobody), and shark antibody domain.

[0090] As used herein, "camelid Ig" or "camelid VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte, et al., FASEB J., 21:3490-3498 (2007)). A "heavy chain antibody" or "camelid antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). In some embodiments, the antigen-binding domain is a camelid nanobody.

[0091] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0092] An "Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0093] Fab fragments contain the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. A bispecific Fab dimer (Fab2) has two Fab' fragments, each binding a different antigen. A trispecific Fab trimer (Fab3) has three Fab' fragments, each binding a different antigen.

[0094] The term "diabody" refers to an antibody fragment with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be paired with complementary domains on another chain, creating two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS. USA, 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0095] A "single domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11):484-490).

[0096] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in either orientation on a single polypeptide chain (e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0097] In some embodiments, the anti-HLA-G antigen-binding fragment is an scFv. In some embodiments, the scFv is a murine, human, or humanized scFv. Single-chain antibodies may be cloned from the V region genes of hybridomas specific for a desired target. The production of such hybridomas has become routine. Techniques that can be used to clone the variable heavy (VH) and variable light (VL) chains are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837.

[0098] In some embodiments, the antigen-binding domain of the CAR comprises a heavy chain variable (VH) region comprising HCDR1, HCDR2, and HCDR3 of an anti-HLA-G antibody, and a light chain variable (VL) region comprising LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-HLA-G antibody or antigen-binding fragment thereof comprises a variable heavy chain sequence comprising the HCDR1 sequence set forth in SEQ ID NO: 11, the HCDR2 sequence set forth in SEQ ID NO: 13, and the HCDR3 sequence set forth in SEQ ID NO: 15. In some embodiments, the anti-HLA-G antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the LCDR1 sequence set forth in SEQ ID NO: 37, the LCDR2 sequence set forth in SEQ ID NO: 39, and the LCDR3 sequence set forth in SEQ ID NO: 41. In some embodiments, the anti-HLA-G antibody or antigen-binding fragment thereof comprises the variable heavy chain sequence set forth in any one of SEQ ID NOs: 9 and 25 and / or the variable light chain sequence set forth in any one of SEQ ID NOs: 35, 50, and 60.

[0099] In some embodiments, the HLA-G binding domain may comprise an antibody mimic. The term "antibody mimic" can describe an organic compound that specifically binds to a target sequence and has a structure different from that of naturally occurring antibodies. Antibody mimics may include proteins, nucleic acids, or small molecules. The target sequence to which the antibody mimic specifically binds may be HLA-G. Antibody mimics may offer superior properties to antibodies, including, but not limited to, superior solubility, tissue permeability, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimics include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, and avimers (also known as avidity multimers), DARpins (designed ankyrin repeat proteins), finomers, Kunitz domain peptides, and monobodies.

[0100] Linker In certain embodiments, the anti-HLA-G CAR comprises linker residues between the various domains, e.g., added for proper spacing and conformation of the molecule. In some embodiments, the CAR comprises 1, 2, 3, 4, or 5 or more linkers. Particularly, some linkers are about 1 to about 25 amino acids in length, about 5 to about 20 amino acids in length, or about 10 to about 20 amino acids in length, or any intervening length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more amino acids in length.

[0101] Illustrative examples of linkers include glycine polymers (G) n ; glycine-serine polymer (G 1-5 S 1-5 ) n(where n is an integer of at least 1, 2, 3, 4, or 5); glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Because glycine and glycine-serine polymers are relatively unstructured, they may be able to function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine utilizes much more φ-ψ space than alanine and is much less restricted than residues with long side chains. (See Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that, because the design of a CAR in some embodiments can include a linker that is fully or partially flexible, the linker can include one or more moieties that confer less flexible structure, as well as a flexible linker, to provide the desired CAR structure. In some embodiments, the linker connecting the heavy chain variable region and the light chain variable region comprises the sequence of SEQ ID NO: 70.

[0102] In some embodiments, a CAR according to the present disclosure comprises a cleavable linker. The cleavable linker can be a peptide, polypeptide, or portion of a polypeptide that is cleaved after production of the protein or polypeptide, particularly after translation of the CAR according to the present disclosure.

[0103] In particular, a cleavable linker is a self-cleavable, self-cleaving, self-cleaving peptide or linker, and these terms are used interchangeably herein.

[0104] In one embodiment, the cleavable linker comprises a 2A peptide. "2A" or "2A-like" sequences are part of a large family of peptides that can cause peptide bond skipping. In particular, the mechanism of 2A-mediated "self-cleavage" has recently been found to be the ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide. 2A-peptide-mediated cleavage begins post-translationally. Successful translation skipping and resumption results in two "cleaved" proteins: the protein upstream of 2A is linked to the intact 2A peptide except for the C-terminal proline, and the protein downstream of 2A is linked to a single proline at the N-terminus. Successful skipping results in the ribosome dropping off and translation halting, leaving only the protein upstream of 2A. Multiple 2A peptides have been identified in picornaviruses, insect viruses, and type C rotaviruses.

[0105] Examples of cleavable linkers according to the present disclosure include, but are not limited to, porcine teschovirus-1 2A (P2A), FMDV 2A (F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flagella virus 2A (BmIFV2A), or combinations thereof, such as those described in Kim et al. (2011), PLoS ONE, 6(4): el8556 and Liu et al. (2017), Sci. Rep. 2017; 7: 2193.

[0106] Preferably, the cleavable linker is a P2A comprising or consisting of the sequence set forth in SEQ ID NO: 75, or a sequence having at least 80, 85, 90, or 95% identity thereto.

[0107] In one embodiment, the N-terminus of the cleavable linker is operably linked to the C-terminus of the CAR endodomain and / or the C-terminus of the cleavable linker is operably linked to the N-terminus of the reporter.

[0108] signal peptide In some embodiments, the extracellular domain of the anti-HLA-G CAR comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence encoding human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, CD37, CD45, 4-1BB, GM-CSFR, IL-2, CD33, human IgKVIII, human IgG2H, chymotrypsinogen, trypsinogen-2, HSA, insulin, or tPA signal peptide. In some embodiments, the signal peptide of the anti-HLA-G CAR comprises the sequence of SEQ ID NO: 69.

[0109] spacer domain In some embodiments, the extracellular domain of an anti-HLA-G CAR comprises one or more "spacer domains," which refers to a region that distances the antigen-binding domain from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). In some embodiments, a CAR comprises a spacer domain between the antigen-binding domain and the transmembrane (TM) domain. Spacer domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring or altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD.

[0110] Hinge domain In some embodiments, the extracellular domain of an anti-HLA-G CAR comprises one or more "hinge domains," which serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. Anti-HLA-G CARs generally comprise one or more hinge domains between the antigen-binding domain and the transmembrane (TM) domain. The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0111] In some embodiments, the hinge domain may be derived from or comprise at least a portion of an immunoglobulin Fc region, e.g., an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In specific embodiments, the hinge domain comprises at least a portion of an IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulin Fc region contained within its CH2 and CH3 domains. In some embodiments, the spacer domain may also comprise at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the hinge is derived from or comprises at least a portion of a modified immunoglobulin Fc region, e.g., a modified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The Fc region of the modified immunoglobulin may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) that result in the substitution, modification, or deletion of one or more amino acids that impair binding of the spacer domain to an Fc receptor (FcR). In some embodiments, the Fc region of the modified immunoglobulin may be designed with one or more mutations that result in the substitution, modification, or deletion of one or more amino acids that impair binding of the spacer domain to one or more FcRs, including, but not limited to, FcyRI, FcyR2A, FcyR2B1, FcyR2B2, FcyR3A, FcyR3B, FcsRI, FcsR2, FcaRI, Fca / μK, or FcRn.

[0112] Exemplary hinges include, but are not limited to, a CD8α hinge, a CD28 hinge, an IgG1 / IgG4 (hinge-Fc portion) sequence (CD4, CD7, and IgD in a single study), an IgG4 hinge alone, an IgG4 hinge linked to a CH2 and CH3 domain, or an IgG4 hinge linked to a CH3 domain, as described in Hudecek et al. (2013), Clin. Cancer Res., 19:3153, International Patent Application Publication No. WO2014031687, U.S. Patent No. 8,822,647, or Published Application No. US2014 / 0271635. The present disclosure relates to all or part of residues 118-178 of CD8a (GenBank Accession No. NP_001759.3), residues 135-195 of CD8 (GenBank Accession No. AAA35664), residues 315-396 of CD4 (GenBank Accession No. NP_000607.1), or residues 137-152 of CD28 (GenBank Accession No. NP_006130.1) as the hinge domain. Furthermore, a portion of the constant region of an antibody heavy or light chain (the CHI region or CL region) can be used as the spacer domain. Furthermore, the spacer domain may be an artificially synthesized sequence. In particular, the CAR according to the present disclosure comprises a hinge selected from CD8a, CD28, and IgG1 / IgG4 (hinge-Fc portion) sequences (CD4, CD7, and IgD in a single test). This selection is based on the fact that these sequences are relatively neutral, flexible, and structurally well characterized.

[0113] Preferably, the hinge domain comprises or consists of (i) a CD28 hinge, (ii) a CD8 alpha hinge, (iii) a human IgG4 hinge domain, (iv) a human IgG4 hinge domain and a CH3 human IgG4 domain, or (v) a mutated CH2 human IgG4 domain, a human IgG4 hinge domain, and a CH3 human IgG4 hinge domain. In one embodiment, the hinge domain comprises the sequence of SEQ ID NO: 71, or a sequence having at least 80, 85, 90, or 95% identity thereto.

[0114] In one embodiment, the hinge domain comprises or consists of (i) a human IgG4 hinge domain, (ii) a human IgG4 hinge domain and a CH3 human IgG4 domain, or (iii) a mutated CH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 human IgG4 hinge domain. In particular, the hinge domain comprises or consists, in order from N-terminus to C-terminus, of (i) a human IgG4 hinge domain and a CH3 human IgG4 domain, or (ii) a mutated CH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 human IgG4 hinge domain.

[0115] Transmembrane domain The "transmembrane (TM) domain" or "transmembrane (TM) region" is the portion of an anti-HLA-G CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The TM domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. TM domains may be derived from (i.e., include at least the transmembrane region(s) thereof) the alpha or beta chains of the T cell receptor, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD90, CD16, CD19, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, 4-1BB, GM-CSFR, PD1, or FcRIγ, and less frequently CD7, OX40, and MHC(H2-Kb), with the choice depending on adjacent spacer and intracellular sequences. Those skilled in the art are aware of numerous transmembrane regions and the structural elements (e.g., lipophilic amino acid regions) that create transmembrane domains in numerous membrane proteins and can substitute convenient sequences.

[0116] In embodiments of the present disclosure, the transmembrane domain comprises the transmembrane domain of CD8α or the transmembrane domain of CD28. In some embodiments, CD8 and CD28 are derived from the human CD8α sequence or CD28 sequence. The CD8α or CD28 may be less than the entire CD8α or CD28, respectively. In this regard, in some embodiments, the CAR comprises a CD28 transmembrane domain comprising, consisting of, or consisting essentially of SEQ ID NO: 72, or a sequence having at least 80, 85, 90, or 95% identity thereto.

[0117] Alternatively, the transmembrane domain may be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet will be found at both ends of the synthetic transmembrane domain. The transmembrane domain of the present disclosure is thermodynamically stable within the membrane. It may be a single alpha helix, a transmembrane beta barrel, a gramicidin A beta helix, or any other structure.

[0118] Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the bond between the transmembrane domain and the intracellular signaling domain(s) of the CAR. A glycine-serine duo may provide a suitable linker.

[0119] Intracellular domain In some embodiments, the anti-HLA-G CAR comprises an intracellular domain. The "intracellular domain" refers to the portion of the CAR that is involved in transmitting the message of effective anti-HLA-G CAR binding to a human HLA-G polypeptide inside an immune effector cell and eliciting effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors, toward CAR-bound target cells, or other cellular responses elicited by antigen binding to the extracellular CAR domain. The intracellular domain may comprise one or more signaling domains.

[0120] The term "effector function" refers to the specialized function of an immune effector cell. The effector function of a T cell may include, for example, cytolytic activity or support, or cytokine secretion. Thus, the term "signaling domain" refers to a portion of a protein that transduces an effector function signal and instructs the cell to perform a specialized function. While the entire signaling domain can usually be used, it is often not necessary to use the entire domain. When a truncated portion of a signaling domain is used, such a truncated portion may be used in place of the entire domain, so long as it transmits the effector function signal. The term signaling domain is meant to include any truncated portion of a signaling domain sufficient to transmit the effector function signal. In some embodiments, the signaling domain of a CAR comprises one or more of the intracellular signaling portions of human CD3 zeta, CD28, CD137, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, or CD3 epsilon.

[0121] In some cases, the signal generated via the TCR alone is insufficient for full activation of an effector cell (e.g., a T cell), and a secondary or costimulatory signal may be required. Thus, activation of effector cells (e.g., T cells) can be said to be mediated by two distinct classes of intracellular signaling domains: signaling domains that initiate antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex), and costimulatory domains that act antigen-independently to provide secondary or costimulatory signals. In some embodiments, a CAR comprises an intracellular domain comprising one or more "costimulatory domains" and "signaling domains."

[0122] The signaling domain regulates primary activation of the TCR complex, either stimulatory or inhibitory. Stimulatory signaling domains may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Illustrative examples of ITAM-containing signaling domains useful in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some embodiments, an anti-HLA-G CAR comprises a CD3ζ signaling domain and one or more costimulatory domains. The intracellular signaling domain and costimulatory domain may be tandemly linked to the carboxyl terminus of the transmembrane domain in any order. In some embodiments, the intracellular domain comprises the amino acid sequence of a CD3ζ signaling domain comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO: 74.

[0123] In some embodiments, the CAR comprises one or more costimulatory domains to enhance the efficacy and proliferation of T cells expressing the CAR receptor. As used herein, the term "costimulatory domain" refers to the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or an Fc receptor, that provides a second signal required for efficient activation and function of T lymphocytes upon antigen binding. Illustrative examples of such costimulatory molecules include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD40LG (CD40L), CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, DAP-12, ITGB2 (LFA-1), LAT, MyD88, NKD2C (KLRC2), SLP76, TNFRS18 (GITR), TNFRSF14 (HVEM), TRIM, and ZAP 70. In one embodiment, the CAR comprises one or more costimulatory domains selected from the signaling domains of CD28, CD137, and CD3ζ.

[0124] In some embodiments, the intracellular domain comprises one or more costimulatory domains selected from CD28 and 4-1BB. Signaling through CD28 is necessary for IL2 production and proliferation, but does not play a primary role in maintaining T cell function and activity. 4-1BB (a member of the tumor necrosis factor receptor family that is expressed after CD28 activation) and OX-40 are involved in promoting long-term T cell survival and T cell accumulation. Ligands for these receptors are typically expressed on professional antigen-presenting cells such as dendritic cells and activated macrophages, but not on tumor cells.

[0125] In some embodiments, the intracellular domain of the CAR comprises a CD28 costimulatory domain and a CD3ζ signaling domain. In some embodiments, the intracellular domain of the CAR comprises a 4-1BB costimulatory domain and a CD3ζ signaling domain. In some embodiments, the CD28, 4-1BB, and CD3ζ domains are human. In some embodiments, the CD4 + Expression of CARs incorporating CD28 and / or 4-1BB signaling domains in T cells enhances the activity and anti-tumor efficacy of these cells compared to those expressing CARs containing only the CD3ζ signaling domain. In some embodiments, an anti-HLA-G CAR contains both the CD28 and 4-1BB costimulatory domains.

[0126] In some embodiments, the intracellular domain comprises a CD28 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 72. In some embodiments, the intracellular domain comprises a 4-1BB sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 73. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain and a CD3ζ signaling domain, wherein the CD28 sequence comprises, consists of, or consists essentially of SEQ ID NO: 72 and the CD3ζ sequence comprises, consists of, or consists essentially of SEQ ID NO: 74. In some embodiments, the intracellular domain comprises a 4-1BB costimulatory domain and a CD3ζ signaling domain, wherein the 4-1BB sequence comprises, consists of, or consists essentially of SEQ ID NO: 73 and the CD3ζ sequence comprises, consists of, or consists essentially of SEQ ID NO: 74.

[0127] Third-generation CARs are based on combining two or more costimulatory sequences (e.g., 4-1BB·CD28·CD3ζ). These receptors secrete a broader range of cytokines (including TNFα, GM-CSF, and IFNγ), are less susceptible to activation-induced cell death, and exhibit greater efficacy in tumor elimination in mouse models. So-called third-generation CARs have at least two or three signaling domains fused to each other, e.g., for additive or synergistic effects, so more than one endodomain may be employed.

[0128] The CAR of the present disclosure may be a first-generation, second-generation, or third-generation CAR as described above. Preferably, the CAR is a second-generation or third-generation CAR. Even more preferably, the CAR is a third-generation CAR when expressed by T cells, and a first-generation CAR when expressed by NK or NKT cells.

[0129] Exemplary CAR Constructs In some embodiments, the anti-HLA-G CAR constructs provided herein comprise an anti-HLA-G binding domain, a hinge domain, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain comprises a signaling domain. In some embodiments, the intracellular domain comprises a signaling domain and a costimulatory domain. In some embodiments, the anti-HLA-G CAR constructs provided herein comprise, in order from amino terminus to carboxyl terminus, an scFv that specifically binds to human HLA-G, a spacer, a transmembrane domain, a costimulatory domain, and a signaling domain.

[0130] In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising HCDR1, HCDR2, and HCDR3, and an immunoglobulin VL chain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; HCDR2 comprises the amino acid sequence of SEQ ID NO: 13; HCDR3 comprises the amino acid sequence of SEQ ID NO: 15; LCDR1 comprises the amino acid sequence of SEQ ID NO: 37; LCDR2 comprises the amino acid sequence of SEQ ID NO: 39; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 41.

[0131] In some embodiments, the anti-HLA-G CAR comprises a heavy chain variable region VH comprising one, two, three, or four heavy chain framework regions (FRs) that are humanized. In some embodiments, the anti-HLA-G CAR comprises a VH that comprises, in order from N-terminus to C-terminus, a humanized heavy chain FR1 set forth in SEQ ID NO: 10, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 10; a CDR1 set forth in SEQ ID NO: 11; a humanized heavy chain FR2 set forth in one of SEQ ID NOs: 12 and 26, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 12 or 26; a CDR2 set forth in SEQ ID NO: 13; a humanized heavy chain FR3 set forth in SEQ ID NO: 14, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 14; a CDR3 set forth in SEQ ID NO: 15; and a humanized heavy chain FR4 set forth in SEQ ID NO: 16, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 16.

[0132] Exemplary humanized VH chains of the anti-HLA-G binding domains described herein are shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] In the variable domain, the CDR1, CDR2 and CDR3 sequences (from left to right) are underlined.

[0133] In some embodiments, the anti-HLA-G CAR comprises a light chain variable region comprising one, two, three, or four humanized light chain FRs. In some embodiments, the anti-HLA-G CAR comprises a light chain variable region comprising, in order from N-terminus to C-terminus: a humanized light chain FR1 set forth in SEQ ID NO: 36, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 36; a CDR1 set forth in SEQ ID NO: 37; a humanized light chain FR2 set forth in SEQ ID NO: 38, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 38; a CDR2 set forth in SEQ ID NO: 39; a humanized light chain FR3 set forth in one of SEQ ID NOs: 40 and 51, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 40 or 51; a CDR3 set forth in SEQ ID NO: 41; and a humanized light chain FR4 set forth in SEQ ID NO: 42, or that is at least 80, 85, 90, or 95% identical to SEQ ID NO: 42.

[0134] Exemplary humanized VL chains of the anti-HLA-G binding domains described herein are shown in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] In the variable domain, the CDR1, CDR2 and CDR3 sequences (from left to right) are underlined.

[0135] In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 25, and an immunoglobulin VL chain comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 35. In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising an amino acid sequence that is 100% identical to SEQ ID NO: 25, and an immunoglobulin VL chain comprising an amino acid sequence that is 100% identical to SEQ ID NO: 35. In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain consisting of, or consisting essentially of, the amino acid sequence of SEQ ID NO: 25, and an immunoglobulin VL chain consisting of, or consisting essentially of, the amino acid sequence of SEQ ID NO: 35.

[0136] In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9, and an immunoglobulin VL chain comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 50. In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising an amino acid sequence that is 100% identical to SEQ ID NO: 9, and an immunoglobulin VL chain comprising an amino acid sequence that is 100% identical to SEQ ID NO: 50. In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain consisting of, or consisting essentially of, the amino acid sequence of SEQ ID NO: 9, and an immunoglobulin VL chain consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 50.

[0137] In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9, and an immunoglobulin VL chain comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 60. In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain comprising an amino acid sequence that is 100% identical to SEQ ID NO: 9, and an immunoglobulin VL chain comprising an amino acid sequence that is 100% identical to SEQ ID NO: 60. In some embodiments, the anti-HLA-G binding domain comprises an immunoglobulin VH chain consisting of, or consisting essentially of, the amino acid sequence of SEQ ID NO: 9, and an immunoglobulin VL chain consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 60.

[0138] Exemplary VH and VL sequences for the anti-HLA-G binding domains described herein are provided in Tables 1 and 2.

[0139] In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 63. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv comprising an amino acid sequence that is 100% identical to SEQ ID NO: 63. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv that consists of or consists essentially of the amino acid sequence of SEQ ID NO: 63.

[0140] In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 65. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv comprising an amino acid sequence that is 100% identical to SEQ ID NO: 65. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv that consists of or consists essentially of the amino acid sequence of SEQ ID NO: 65.

[0141] In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 67. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv comprising an amino acid sequence that is 100% identical to SEQ ID NO: 67. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv that consists of or consists essentially of the amino acid sequence of SEQ ID NO: 67.

[0142] Exemplary scFv sequences of the anti-HLA-G binding domains described herein are provided below in Table 3. [Table 3-1] [Table 3-2] In the variable domains, the sequences of VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3 (from left to right) are underlined and the linker is in bold.

[0143] In some embodiments, the anti-HLA-G CAR constructs provided herein comprise an anti-HLA-G binding domain, a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular domain comprising a CD3ζ signaling domain. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence 100% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv consisting of, or consisting essentially of, the amino acid sequence of one of SEQ ID NOs: 63, 65, and 67.

[0144] In some embodiments, the anti-HLA-G CAR constructs provided herein comprise an anti-HLA-G binding domain, a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular domain comprising a CD3ζ signaling domain and a 4-1BB costimulatory domain. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence 100% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv consisting of, or consisting essentially of, the amino acid sequence of one of SEQ ID NOs: 63, 65, and 67.

[0145] In some embodiments, the anti-HLA-G CAR constructs provided herein comprise an anti-HLA-G binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular domain comprising a CD3ζ signaling domain. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence 100% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv consisting of, or consisting essentially of, the amino acid sequence of one of SEQ ID NOs: 63, 65, and 67.

[0146] In some embodiments, the anti-HLA-G CAR constructs provided herein comprise an anti-HLA-G binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular domain comprising a CD3ζ signaling domain and a CD28 costimulatory domain. In some embodiments, the anti-HLA-G binding domain is a humanized scFv domain comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an scFv domain comprising an amino acid sequence 100% identical to one of SEQ ID NOs: 63, 65, and 67. In some embodiments, the anti-HLA-G binding domain is an anti-HLA-G scFv consisting of, or consisting essentially of, the amino acid sequence of one of SEQ ID NOs: 63, 65, and 67.

[0147] Exemplary nucleotide sequences of CAR constructs comprising CD28 and 4-1BB costimulatory sequences and a CD3 signaling sequence are shown in Table 4. The general design of the constructs shown in Table 4 is as follows: Ig kappa signal peptide-humanized anti-HLA-G scFv-hIgG4 hinge domain-CD28 and 4-1BB costimulatory domain-CD3 zeta signaling domain. In one embodiment, the CAR construct comprises a truncated CD19 sequence linked to the C-terminus of the CAR construct via a P2A sequence. In some embodiments, the anti-HLA-G CAR construct comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 68. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] In the variable domains, the sequences of VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3 (from left to right) are underlined.

[0148] Figure 1 shows an exemplary schematic design of a CAR construct based on LFTT-1 with a cleavable linker P2A and truncated hCD19 as a reporter. LFTT-1 is described in WO2020043899, which is incorporated herein by reference in its entirety. The truncated CD19 marker is used to identify the CAR-T product. The costimulatory domains CD28 and 4-1BB are used to enhance anti-tumor activity. An internal EF1α promoter is used for optimal CAR expression.

[0149] Polypeptides In some embodiments, the present disclosure provides anti-HLA-G CAR polypeptides and fragments thereof. In some embodiments, the CAR is an anti-HLA-G CAR comprising the nucleotide sequence set forth in SEQ ID NO:68.

[0150] The terms "polypeptide," "peptide," and "protein" are used interchangeably unless specified to the contrary and according to their conventional meaning, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length and may include, for example, a full-length polypeptide or a polypeptide fragment, and may include one or more post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications, both naturally occurring and non-naturally occurring, known in the art. In some embodiments, a CAR polypeptide includes a signal (or leader) sequence at the N-terminus of the protein, which directs translocation of the protein co-translationally or post-translationally. Illustrative examples of suitable signal sequences useful in CARs contemplated in some embodiments include, but are not limited to, an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSFR-α signal polypeptide. Polypeptides may be prepared using any of a variety of well-known recombinant and / or synthetic techniques.

[0151] Polypeptide variants Polypeptides contemplated herein include CARs of the present disclosure, as well as functional variants thereof. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR, where the functional variant retains the biological activity of the CAR of the parent CAR. Functional variants include, for example, CAR variants that retain the ability to recognize target cells to a similar, identical, or even greater extent than the parent CAR. Such variants may be naturally occurring or synthetically produced, for example, by modifying one or more of the above polypeptide sequences. For example, in some embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a CAR by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge, TM domain, costimulatory domain, or signaling domain of the CAR polypeptide. In some embodiments, a CAR polypeptide comprises a polypeptide having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% identity to any of the CAR polypeptides described herein (e.g., SEQ ID NO: 68), typically wherein the variant maintains at least one biological activity of the reference sequence.

[0152] As noted above, in some embodiments, polypeptides may be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by DNA mutations. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, e.g., Kinkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492); Kinkel et al. (1987, Methods in Enzymol. 154:367-382); U.S. Patent No. 4,873,192; Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0153] A functional variant can, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. A "conservative substitution" refers to the substitution of an amino acid with another amino acid of similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure or hydropathicity of the polypeptide to remain substantially unchanged. Modifications can be made to the polynucleotide and polypeptide structures contemplated in some embodiments to result in functional molecules that encode mutant or derivative polypeptides with desirable properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid having a nonpolar side chain for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (Lys, His, Arg, etc.), substitutions of an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substitutions of an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (Ile, Thr, Val, etc.), substitutions of an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (His, Phe, Trp, Tyr, etc.), etc. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without losing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software.

[0154] Alternatively, or in addition, the functional variant can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, thereby increasing the biological activity of the functional variant compared to the parent CAR.

[0155] Polypeptide variants also include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., PEGylated molecules). Covalent variants can be prepared by linking functional groups to groups found in the amino acid chain or at the N- or C-terminal residues, as is known in the art. Variants also include allelic variants, interspecies variants, and mutant proteins. Truncation or deletion of regions that do not affect the functional activity of the protein is also a variant.

[0156] In one embodiment, when expression of two or more polypeptides is desired, the polynucleotide sequences encoding them can be separated by an IRES sequence, as discussed elsewhere herein, hi another embodiment, two or more polypeptides can be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences, e.g., 2A sequences.

[0157] Fusion Polypeptides In some embodiments, contemplated polypeptides include fusion polypeptides (e.g., CAR fusion proteins). In some embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides, such as CARs, are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. Fusion polypeptides are typically linked C-terminus to N-terminus, but can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein can be in any order or a specific order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired transcriptional activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by chemical synthesis or chemical ligation between two moieties, or generally prepared using other standard techniques. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements, as discussed elsewhere herein.

[0158] In one embodiment, the fusion partner contains sequences (expression enhancers) that aid in the expression of the protein in higher yields than the native recombinant protein. Other fusion partners may be selected to increase the solubility of the protein, or to enable the protein to be targeted to a desired intracellular compartment, or to facilitate transport of the fusion protein across the cell membrane.

[0159] In some embodiments, the fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. Additionally, polypeptide cleavage sites can be incorporated into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8);616-26).

[0160] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004), Nature, Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, biovirus NIa protease, biovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase.

[0161] In some embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence, or domain (Donney et al., 2001. J. Gen. Virol. 82:1027-1041). In particular, the mechanism of 2A-mediated "self-cleavage" has recently been found to be the ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide. 2A-peptide-mediated cleavage begins post-translationally. Successful translational skipping and resumption results in two "cleaved" proteins: the protein upstream of 2A is linked to the intact 2A peptide, excluding the C-terminal proline, and the protein downstream of 2A is linked to a single proline at its N-terminus. Successful skipping results in the ribosome being dropped, terminating translation and leaving only the protein upstream of 2A. Multiple 2A peptides have been identified in picornaviruses, insect viruses, and type C rotaviruses.

[0162] Examples of cleavable linkers according to the present disclosure include, but are not limited to, porcine teschovirus-1 2A (P2A), FMDV 2A (F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flagella virus 2A (BmIFV2A), or combinations thereof, such as those described in Kim et al. (2011), PLoS ONE, 6(4): el8556 and Liu et al. (2017), Sci. Rep. 2017; 7: 2193.

[0163] Preferably, the cleavable linker is a P2A comprising or consisting of the sequence set forth in SEQ ID NO: 75, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0164] In one embodiment, the N-terminus of the cleavable linker is operably linked to the C-terminus of the CAR endodomain and / or the C-terminus of the cleavable linker is operably linked to the N-terminus of the reporter.

[0165] Polynucleotides In some embodiments, the present disclosure provides polynucleotides or nucleic acid molecules encoding one or more CAR polypeptides. As used herein, the term "nucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded and may be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. A polynucleotide refers to a polymeric form of nucleotides of a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500 nucleotides, at least 1000, at least 5000, at least 10,000, or at least 15,000 or more nucleotides, either ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, it will be readily understood that "intermediate length" means 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc., any length between the recited values.

[0166] In some embodiments, a polynucleotide may be codon optimized. As used herein, the term "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to enhance expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of: (i) variation in codon bias between one or more organisms or genes or synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons with context; (iv) variation of codons according to their decoding tRNAs; (v) variation of codons with respect to GC % either throughout the triplet or in a single position; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence upon which the design of the codon substitution set is based; (x) systematic variation of the codon set for each amino acid; (xi) individual removal of false translation start sites; and / or (xii) elimination of inadvertent polyadenylation sites that result in truncated RNA transcripts.

[0167] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percent sequence identity," and "substantial identity." A "reference sequence" is at least 12, but frequently 15-18, and often at least 25, monomeric units in length, including both nucleotides and amino acid residues. Because two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) sequences that differ between the two polynucleotides, sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least six, usually about 50 to about 100, more usually about 100 to about 150 contiguous positions, over which the sequence is compared to the reference sequence for the same number of contiguous positions after the two sequences are optimally aligned. For optimal alignment of the two sequences, the comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). Optimal alignment of sequences for aligning the comparison window may be performed by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA) or by examining and selecting the best alignment (i.e., resulting in the highest percentage of homology across the comparison window) generated by any of a variety of methods. See also the BLAST family of programs, e.g., as disclosed in Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0168] As used herein, "sequence identity" or, for example, including "50% identical sequences" refers to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid over a comparison window.Therefore, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in both sequences where the nucleic acid base (e.g., A, T, C, G, I) is identical or the amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is identical to obtain the number of matched positions, and dividing the number of matched positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0169] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides, compared to the reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, such that the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0170] In some embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence.

[0171] Furthermore, those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein, or a variant fragment thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in some embodiments, e.g., polynucleotides optimized for human and / or primate codon preferences. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that vary as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions.

[0172] The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides or epitope tags, as disclosed elsewhere herein or known in the art. Their respective total lengths may vary considerably. Thus, in some embodiments, polynucleotide fragments of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0173] Provided herein is a nucleic acid construct comprising sequences encoding an ectodomain, an intracellular domain, a transmembrane domain, optionally a hinge domain, an optional cleavable linker, and a reporter and / or signal peptide, as described above. In one embodiment, the nucleic acid construct comprises the nucleotide sequence set forth in SEQ ID NO:68.

[0174] In one embodiment, the nucleic acid construct comprises or consists of, in N- to C-terminal order, an optional peptide signal sequence, an anti-HLA-G antibody or fragment thereof, preferably an anti-HLA-G scFv, a spacer domain, a transmembrane domain, at least one intracellular domain, optionally a cleavable linker, and a reporter. In yet a further embodiment, the nucleic acid construct further comprises a tag, preferably a Flag tag.

[0175] In some embodiments, the nucleic acid construct comprises: (a) a nucleic acid sequence encoding an anti-HLA-G scFv as described above; (b) optionally, a nucleic acid sequence encoding a hinge, preferably selected from the group consisting of: (i) a CD28 hinge, (ii) a CD8 alpha hinge, (iii) a human IgG4 hinge domain, (iv) a human IgG4 hinge domain and a CH3 human IgG4 domain, or (v) a mutated CH2 human IgG4 domain, a human IgG4 hinge domain, and a CH3 human IgG4 hinge domain; (c) a nucleic acid sequence encoding a transmembrane domain, preferably the transmembrane domain of CD28; (d) a nucleic acid sequence encoding an endodomain, preferably a 4-1BB domain and / or a CD3ζ domain; (e) an optional cleavable linker, preferably a P2A cleavable linker; (f) optionally a reporter, preferably a hCD19t reporter, and / or (g) optionally comprising a signal peptide, preferably selected from the group consisting of CD8a, mouse Ig kappa signal peptide, human IgG4 signal peptide, and IL2 signal peptide.

[0176] In some embodiments, the nucleic acid construct comprises: (i) a nucleic acid sequence encoding a VH of SEQ ID NO: 17 comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 21, and an HCDR3 of SEQ ID NO: 23; and (ii) comprises a nucleic acid sequence encoding a VL of SEQ ID NO: 27, which comprises an LCDR1 of SEQ ID NO: 29, an LCDR2 of SEQ ID NO: 31, and an LCDR3 of SEQ ID NO: 33.

[0177] In another embodiment, the nucleic acid construct comprises: (i) a nucleic acid sequence encoding a VH of SEQ ID NO: 1 comprising an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 5, and an HCDR3 of SEQ ID NO: 7; and (ii) comprises a nucleic acid sequence encoding a VL of SEQ ID NO: 43, which comprises an LCDR1 of SEQ ID NO: 45, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 33.

[0178] In another embodiment, the nucleic acid construct comprises: (i) a nucleic acid sequence encoding a VH of SEQ ID NO: 1 comprising an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 5, and an HCDR3 of SEQ ID NO: 7; and (ii) comprises a nucleic acid sequence encoding a VL of SEQ ID NO: 52, which comprises an LCDR1 of SEQ ID NO: 54, an LCDR2 of SEQ ID NO: 56, and an LCDR3 of SEQ ID NO: 58.

[0179] In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding an scFv of one of SEQ ID NOs: 62, 64, and 66.

[0180] In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding an anti-HLA-G CAR of SEQ ID NO: 68.

[0181] Also provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding an anti-HLA-G CAR described herein and a nucleotide sequence encoding one, two, or three costimulatory molecules. In some embodiments, the costimulatory molecules are one or both of CD40-L or 4-1BB-L.

[0182] Also provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding an anti-HLA-G CAR described herein and a nucleotide sequence encoding one, two, or three degradative enzymes.

[0183] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA, cDNA, PCR-generated cDNA, or other sources. Alternatively, it can be chemically synthesized, or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been shown to stabilize mRNA or provide immune cell, particularly T cell, specific expression (Barthel and Goldferd, 2003). It may also be advantageous to stabilize mRNA using endogenous or exogenous non-coding regions.

[0184] For expression of the chimeric antigen receptor of the present disclosure, the naturally occurring or endogenous transcription initiation region of the nucleic acid sequence encoding the N-terminal component of the chimeric receptor can be used to generate the chimeric receptor in the target host cell. Alternatively, an exogenous transcription initiation region that allows for constitutive or inducible expression can be used, where expression can be controlled depending on the target host, the desired expression level, the properties of the target host, etc.

[0185] Similarly, the signal sequence that directs chimeric receptor to the surface membrane can be the endogenous signal sequence of the N-terminal component of chimeric receptor.Optionally, in some cases, it may be desirable to replace this sequence with a different signal sequence.However, the signal sequence selected should be compatible with the secretory pathway of the immune cell that expresses CAR, so that the chimeric receptor is presented on the surface of the cell.

[0186] According to the present disclosure, the nucleic acid construct is transformed or introduced into a cell and transcribed and translated to produce a product (i.e., a chimeric receptor). Thus, the nucleic acid construct can further comprise at least one promoter to direct transcription of the CAR.

[0187] In one embodiment, promoters are operably linked to the nucleic acid sequence encoding the chimeric receptor of the present disclosure, i.e., they are positioned to promote transcription of messenger RNA from the DNA encoding the chimeric receptor. Promoters can be of genomic origin or synthetically produced. Various promoters for use in immune cells, particularly T cells, are well known in the art (e.g., the CD4 promoter disclosed by Marodon et al. (2003)). Promoters can be constitutive or inducible, with induction associated, for example, with a particular cell type or a particular maturation level, or with a drug (e.g., tetracycline or doxorubicin). Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Alternatively, several well-known viral promoters are also suitable. Promoters of interest include the β-actin promoter, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen focus-forming virus promoters, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukemia virus promoters, cytomegalovirus immediate early promoters, and Rous sarcoma virus promoters, as well as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Promoters may or may not be associated with enhancers, and enhancers may be naturally associated with a particular promoter or may be associated with a different promoter.

[0188] Similarly, the termination region may be provided by the naturally occurring or endogenous transcription termination region of the nucleic acid sequence encoding the C-terminal component of the chimeric receptor. Alternatively, the termination region may be derived from a different source. In most cases, the source of the termination region is generally not considered important for the expression of the recombinant protein, and a wide variety of termination regions can be employed without adversely affecting expression. As will be understood by those skilled in the art, sometimes a small number of amino acids, typically no more than 10 residues, more typically no more than 5 residues, at the end of the antigen-binding domain in the CAR can be deleted. It may also be desirable to introduce a small number of amino acids, typically no more than 10 residues, more typically no more than 5 residues, at the border. Amino acid deletions or insertions may occur as a result of construction needs, providing convenient restriction sites, ease of manipulation, improved expression levels, etc. Additionally, substitution of one or more amino acids with different amino acids may occur for similar reasons, although typically no more than about 5 amino acids are substituted in a single domain.

[0189] In another embodiment, the nucleic acid construct further comprises a promoter and the correct translation initiation sequence, eg, a ribosome binding site and start codon, a stop codon, and a transcription termination sequence.

[0190] Nucleic acid constructs according to the present disclosure may also include other regulatory regions such as enhancers, silencers, and boundary elements / insulators to direct the level of transcription of a given gene.

[0191] Nucleic acid constructs encoding chimeric receptors according to the present disclosure can be prepared by conventional methods. In most cases, naturally occurring sequences may be employed, and the naturally occurring gene may be isolated and manipulated as necessary to allow for the appropriate combination of various components. Thus, nucleic acid sequences encoding the N- and C-terminal proteins of the chimeric receptor can be isolated by employing polymerase chain reaction (PCR) using appropriate primers that result in the deletion of undesired portions of the gene. Alternatively, restriction digestion of cloned genes can be used to generate chimeric constructs. In either case, sequences can be selected to provide restriction sites that are blunt-ended or have complementary overlapping restriction sites.

[0192] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of well-known techniques known and available in the art.

[0193] vector To express the CAR described herein, the expression cassette encoding the CAR can be inserted into a suitable vector.The term "vector" is used herein to refer to a nucleic acid molecule that can transport or carry another nucleic acid molecule.The nucleic acid to be transported is generally linked to, for example, inserted into, the vector nucleic acid molecule.Vector may have a sequence that directs autonomous replication in cells, or may contain a sufficient sequence that allows it to be integrated into host cell DNA.

[0194] As used herein, the term "expression cassette" refers to a gene sequence within a vector capable of expressing RNA and subsequently a protein. The nucleic acid cassette contains a gene of interest, such as a CAR. The nucleic acid cassette is positionally and sequentially oriented within the vector so that the nucleic acid within the cassette can be transcribed into RNA, translated into a protein or polypeptide as needed, and then transferred to the appropriate compartment for biological activity by targeting secretion into the appropriate intracellular or extracellular compartment, allowing it to undergo appropriate post-translational modifications necessary for the activity of the transformed cell. Preferably, the cassette has its 3' and 5' ends ready for insertion into a vector, e.g., it has restriction endonuclease sites at each end. In some embodiments, the nucleic acid cassette contains the sequence of a CAR used to enhance the cytotoxicity of HLA-G-expressing cancer cells. The cassette is removable and can be inserted into a plasmid or viral vector as a single unit.

[0195] Exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In some embodiments, the coding sequence of a CAR disclosed herein can be ligated into such an expression vector for expression of the CAR in mammalian cells. In some embodiments, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to T cells.

[0196] In some embodiments, the vector is a non-integrating vector, including, but not limited to, an episomal vector or a vector maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without integrating into the host's chromosomal DNA and without being gradually lost from dividing host cells, meaning that the vector replicates extrachromosomally or episomally. The vector is engineered to harbor an origin of DNA replication, or "ori," from lymphotropic herpesvirus or gammaherpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically a sequence encoding the lymphotropic herpesvirus or gammaherpesvirus replication origin corresponding to oriP of EBV. In certain embodiments, the lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), herpesvirus saimiri (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gammaherpesviruses. Typically, host cells contain viral replication transactivator proteins that activate replication.

[0197] In some embodiments, the polynucleotide is introduced into a target or host cell using a transposon-based vector system. In certain embodiments, the transposon-based vector system comprises a vector containing a transposable element and a polynucleotide as contemplated herein, and a transposase. In one embodiment, the transposon-based vector system is a single-transposase vector system. See, e.g., WO 2008 / 027384. Exemplary transposases include, but are not limited to, piggyBac, Sleeping Beauty, Mos1, Tc1 / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and derivatives thereof. The piggyBac transposon and transposase are described, for example, in U.S. Pat. No. 6,962,810, which is incorporated herein by reference in its entirety. Sleeping Beauty transposons and transposases are described, for example, in Izsvak et al., J. Mol. Biol. 302:93-102 (2000), which is incorporated herein by reference in its entirety. The Tol2 transposon, first isolated from the medaka fish Oryzias latipes and belonging to the hAT family of transposons, is described in Kawakami et al. (2000). Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al. (2006). Tol2 and Mini-Tol2 transposons, when acting in cooperation with the Tol2 transposase, promote the integration of transgenes into the genome of an organism. Frog Prince transposons and transposases are described, for example, in Miskey et al., Nucleic Acids Res. 31:6873-6881 (2003).

[0198] Preferably, the vectors disclosed herein are lentiviral vectors. In particular, vectors are derived from primate and non-primate lentiviruses. Examples of primate lentiviruses include human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Non-primate lentiviruses include the prototype "slow-release virus" Visna / Maeji virus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), and the more recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV). Prior art recombinant lentiviral vectors are known in the art. See, for example, U.S. Patent Nos. 6,924,123, 7,056,699, 7,07,993, 7,419,829, and 7,442,551, which are incorporated herein by reference.

[0199] Commercially available retroviral vectors for use in this disclosure include, but are not limited to, the pFB-neo vector (STRATAGENE®), Invitrogen's pLenti series versions 4, 6, and 6.2 "ViraPower" system produced by Lentigen Corp.; pHIV-7-GFP, which was produced and used in the lab at City of Hope Research Institute; the "Lenti-X" lentiviral vector, pLVX, produced by Clontech; pLKO.1-puro, produced by Sigma-Aldrich; pLmiR, produced by Open Biosystems; and pLV, which was produced and used in the lab at Charite Medical School, Institute of Virology (CBF), Berlin, Germany.

[0200] It will be apparent that viral vectors according to the present disclosure need not be limited to components of a particular virus. Viral vectors may contain components derived from two or more different viruses, or may contain synthetic components. Vector components may be engineered to obtain desired properties, such as target cell specificity.

[0201] US 6,924,123 discloses that certain retroviral sequences promote integration into the target cell genome. This patent teaches that each retroviral genome contains genes called gag, pol, and env, which encode virion proteins and enzymes. These genes are flanked on both sides by regions called long terminal repeats (LTRs). LTRs are involved in proviral integration and transcription. They also serve as enhancer-promoter sequences. Thus, vectors according to the present disclosure can include one or more of the integration features.

[0202] In some embodiments, particle components not encoded by the vector genome are provided in trans by additional nucleic acid sequences ("packaging systems," typically including gag / pol and / or env genes) expressed in the host cell, e.g., using a helper virus strategy. The set of sequences required for viral vector particle production may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixed manner, such as helper sequences. The techniques involved are known to those skilled in the art. For example, a retroviral construct may be a packaging plasmid containing at least one retroviral helper DNA sequence derived from the genome of a replication-incompetent retrovirus that encodes in trans all virion proteins required to package a replication-incompetent retroviral vector, for producing virion proteins capable of packaging a replication-incompetent retroviral vector at high titers without producing a replication-competent helper virus.

[0203] In the packaging step, the packaging plasmid expressing a CAR of the present disclosure and the retroviral vector are transiently co-transfected into a first population of mammalian cells capable of producing virus, such as human embryonic kidney cells, e.g., 293 cells (ATCC No. CRL1573, ATCC, Rockville, Md.), to produce a high-titer recombinant retrovirus-containing supernatant. In another method of the present disclosure, this transiently transfected first population of cells is then co-cultured with mammalian target cells, e.g., human lymphocytes, to transduce the target cells with the foreign gene at high efficiency. In yet another method of the present disclosure, the supernatant from the transiently transfected first population of cells described above is incubated with mammalian target cells, e.g., human lymphocytes or hematopoietic stem cells, to transduce the target cells with the foreign gene at high efficiency.

[0204] The nucleic acid construct of the present disclosure is inserted into a vector and packaged into retroviral or lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. In certain embodiments, the packaging plasmid is stably expressed in a first group of mammalian cells capable of producing virus, such as human embryonic kidney cells, e.g., 293 cells. The retroviral or lentiviral vector is introduced into cells by either co-transfection with a selectable marker or infection with a pseudotyped virus. In either case, the vector is integrated. Alternatively, the vector can be introduced into an episomally maintained plasmid.

[0205] "Control elements" or "regulatory sequences" present in expression vectors are non-translated regions of the vector (e.g., origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' untranslated regions) that interact with host cellular proteins to effect transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

[0206] In some embodiments, vectors include, but are not limited to, expression vectors and viral vectors, and may contain exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous" regulatory sequence is one that is naturally linked to a given gene in the genome. An "exogenous" regulatory sequence is one that is juxtaposed to a gene by genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" regulatory sequence is an exogenous sequence that originates from a species different from the cell being genetically engineered.

[0207] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In some embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins, and / or another sequence, a CNCAAT region (where N can be any nucleotide), found approximately 70-80 bases upstream from the start of transcription.

[0208] The term "enhancer" refers to a segment of DNA that contains sequences that can provide enhanced transcription, and in some cases can function independently of their orientation relative to another control sequence. Enhancers can function cooperatively or additively with a promoter and / or other enhancer elements. A "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions.

[0209] The term "operably linked" refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, such that the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0210] Exemplary ubiquitous promoters suitable for use in some embodiments include the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) (e.g., early or late) promoter, the spleen focus forming virus (SFFV) promoter, the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters of vaccinia virus, and the elongation factor 1 alpha (EF1α) promoter. Motor, early growth response 1 (EGR1) promoter, ferritin H (FerH) promoter, ferritin L (FerL) promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, heat shock 70 kDa protein 5 (HSPA5) promoter, heat shock protein 90 kDa β, member 1 (HSP90B1) promoter, heat shock protein 70 kDa (HSP70) promoter, β-kinesin (β-KIN) promoter, human ROSA Examples of such promoters include, but are not limited to, 26 loci (Irions et al., Nature Biotechnology, 25, 1477-1482 (2007)), ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deletion, and dl587rev primer binding site substitution (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)).

[0211] According to the present disclosure, the nucleic acid construct is transformed or introduced into a cell and transcribed and translated to produce a product (i.e., a chimeric receptor). Thus, the nucleic acid construct can further comprise at least one promoter to direct transcription of the CAR.

[0212] In one embodiment, promoters are operably linked to the nucleic acid sequence encoding the chimeric receptor of the present disclosure, i.e., they are positioned to promote transcription of messenger RNA from the DNA encoding the chimeric receptor. Promoters can be of genomic origin or synthetically produced. Various promoters for use in immune cells, particularly T cells, are well known in the art (e.g., the CD4 promoter disclosed by Marodon et al. (2003)). Promoters can be constitutive or inducible, with induction associated, for example, with a particular cell type or a particular maturation level, or with a drug (e.g., tetracycline or doxorubicin). Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Alternatively, several well-known viral promoters are also suitable. Promoters of interest include, but are not limited to, the β-actin promoter, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen focus-forming virus promoters, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukemia virus promoters, cytomegalovirus immediate early promoters, and Rous sarcoma virus promoters, as well as human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Promoters may or may not be associated with enhancers, and enhancers may be naturally associated with a particular promoter or may be associated with a different promoter.

[0213] In another embodiment, the nucleic acid construct further comprises a promoter and the correct translation initiation sequence, eg, a ribosome binding site and start codon, a stop codon, and a transcription termination sequence.

[0214] As used herein, "conditional expression" may refer to any type of conditional expression, including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state; and the like. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide or that causes increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest. Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionine promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-controllable system (Sirin et al., 2003, Gene, 323:67), cumic acid-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0215] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the vector contains at least one (usually two) site(s) for site-specific recombinase-mediated recombination. As used herein, the term "recombinase" or "site-specific recombinase" includes exclusive or integral proteins, enzymes, cofactors, or associated proteins involved in a recombination reaction that include one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequence or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0216] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct internal ribosome entry to an initiation codon, such as ATG, of a cistron (protein-coding region), thereby leading to cap-independent translation of the gene. See, e.g., Jackson et al., 1990. Trends Biochem. Sci. 15(12):477-83 and Jackson and Kaminski, 1995. RNA, 1(10):985-1000. In some embodiments, a vector comprises one or more polynucleotides of interest encoding one or more polypeptides. In some embodiments, to achieve efficient translation of each of multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences, or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the IRES of EMCV.

[0217] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts enhance heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In some embodiments, vectors contain a polyadenylation sequence 3' of the polynucleotide encoding the expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. Polyadenylation sequences promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to increased translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNAs contains two recognition elements flanking the cleavage-polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is accompanied by the addition of up to 250 adenosines to the 5' cleavage product. In some embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In some embodiments, the poly(A) sequence is an SV40 poly(A) sequence, bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0218] Exemplary methods of non-viral delivery of polynucleotides contemplated in some embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0219] Illustrative examples of polynucleotide delivery systems suitable for use in some contemplated embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, e.g., Liu et al. (2003), Gene Therapy. 10:180-187; and Balazs et al. (2011), Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterially derived, non-biological nanocell-based delivery is also contemplated in some embodiments.

[0220] Viral vectors containing contemplated polynucleotides in some embodiments can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or local application, as described below. Alternatively, the vectors can be delivered ex vivo to cells explanted from an individual patient (e.g., immobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or cells such as universal donor hematopoietic stem cells, which are then reimplanted into the patient.

[0221] In one embodiment, a viral vector containing a polynucleotide encoding an anti-HLA-G CAR is administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA can be administered. Administration can be by any of the routes typically used to introduce molecules into blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art. A particular composition can be administered using more than one route, although in many cases, certain routes can provide a more rapid and effective response than others.

[0222] Illustrative examples of viral vector systems suitable for use in some embodiments contemplated herein include, but are not limited to, adeno-associated viral (AAV), retroviral, herpes simplex viral, adenoviral, and vaccinia viral vectors.

[0223] genetically engineered cells In various embodiments, the present disclosure provides cells that have been genetically engineered to express the anti-HLA-G CAR described herein. As used herein, the term "genetically engineered" or "genetically engineered" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. As used herein, the term "gene therapy" refers to the introduction of additional genetic material in the form of DNA or RNA into the total genetic material in a cell for the purpose of restoring, correcting, or modifying the expression of a gene or expressing a therapeutic polypeptide, such as a CAR.

[0224] In some embodiments, the present disclosure provides engineered cells and populations thereof comprising an anti-HLA-G CAR. In some embodiments, the engineered cells comprise an anti-HLA-G CAR and one or more additional exogenous transgenes.

[0225] In some embodiments, the specificity of primary immune effector cells is redirected to cells expressing HLA-G, e.g., cancer cells, by genetically engineering the primary immune effector cells with a CAR contemplated herein. In various embodiments, a viral vector is used to genetically engineer the immune effector cells with a specific polynucleotide encoding a CAR that includes an anti-HLA-G antigen-binding domain that binds to an HLA-G polypeptide; a hinge domain; a transmembrane (TM) domain; a short oligo- or polypeptide linker that connects the TM domain to the intracellular signaling domain of the CAR; one or more intracellular costimulatory domains; and a signaling domain.

[0226] The cells can comprise a CAR that specifically binds to HLA-G isoforms that bind to β2M, preferably both HLA-G1 and HLA-G5 isoforms.

[0227] In one embodiment, the cells express at least two different CARs. For example, the cells may include a CAR that specifically binds to HLA-G isoforms that bind β2, preferably both HLA-G1 and HLA-G5, and another CAR that specifically binds to a different antigen.

[0228] Preferably, the cells comprise a CAR comprising an antigen-binding fragment derived from the LFTT-1 antibody as described in WO2020043899, which is incorporated herein by reference in its entirety.

[0229] In some embodiments, the engineered cells comprise a CAR comprising the sequence of SEQ ID NO: 68, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0230] The cells of the present disclosure can be prokaryotic or eukaryotic. Preferably, the cells are eukaryotic, such as mammalian, typically human, feline, or canine cells, more typically human cells, preferably primary human cells.

[0231] Immune effector cells In some embodiments, the present disclosure provides engineered cells (e.g., immune effector cells) and populations thereof comprising an anti-HLA-G CAR. In such embodiments, an anti-HLA-G CAR contemplated herein is introduced into and expressed on immune effector cells to redirect the specificity of the immune cells to a target antigen of interest, e.g., an HLA-G polypeptide. In some embodiments, the engineered cells express the anti-HLA-G CAR on the cell surface. In some embodiments, the engineered immune effector cells comprise an anti-HLA-G CAR and one or more additional exogenous transgenes.

[0232] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). Exemplary immune effector cells include T lymphocytes, particularly cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells), natural killer (NK) cells, and natural killer T (NKT) cells. Immune effector cells also include effector cell precursors, which can be induced to differentiate into immune effector cells in vivo or in vitro. In some embodiments, immune effector cells are natural killer (NK)-like cells, hematopoietic precursor cells, peripheral blood (PB)-derived T cells, or umbilical cord blood (UCB)-derived T cells.

[0233] In some embodiments, the immune effector cells engineered with anti-HLA-G CARs comprise T cells. The terms "T cells" or "T lymphocytes" are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells can be T helper cells (HTL; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells can be T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in some embodiments include naive T cells and memory T cells.

[0234] In some embodiments, the T cells are derived from a mammalian subject. In some embodiments, the T cells are derived from a primate subject, e.g., a human subject. T cells can be obtained from multiple sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as sedimentation, e.g., FICOLL™ separation. In one embodiment, cells derived from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing. The cells can be washed with PBS or other suitable solution that does not contain calcium, magnesium, or most (if not all) other divalent cations. As will be appreciated by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, such as using a semi-automated flow-through centrifuge, such as a Cobe 2991 cell processor or a Baxter CytoMate. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions, with or without buffer. In certain embodiments, undesirable components of the apheresis sample may be removed directly in the culture medium in which the cells are resuspended.

[0235] In certain embodiments, the immune cells are T cells, e.g., animal T cells, mammalian T cells, feline T cells, canine T cells, or human T cells. Subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells, and their subtypes, e.g., stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MANT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells. Non-limiting examples of commercially available T cell lines include BCL2(AAA) Jurkat (ATCC® CRL-2902™), BCL2(S70A) Jurkat (ATCC® CRL-2900™), BCL2(S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), Neo Jurkat (ATCC® CRL-2898™), and TALL-104 cytotoxic human T cell line (ATCC#CRL-11386).Further examples include mature T cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1~T14, TALL-1, TALL-101, T ALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4;11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162), etc. Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA), and the German Collection of Microorganisms and Cell Cultures.

[0236] In some embodiments, a population of cells comprising T cells, e.g., peripheral blood mononuclear cells (PBMCs), is genetically engineered according to the present disclosure. In some embodiments, the population of PBMCs is not subjected to positive or negative selection prior to activation, expansion, and / or genetic engineering. In other embodiments, T cells are isolated or purified from PBMCs prior to activation, expansion, and / or genetic engineering. In such embodiments, the population of PBMCs may be processed to lyse red blood cells and deplete monocytes, for example, by centrifugation through a PERCOLL™ gradient. In some embodiments, cytotoxic T lymphocytes and / or helper T lymphocytes are isolated from PBMCs. In some embodiments, the isolated T cells may be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and / or genetic engineering. In certain embodiments, specific subpopulations of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection.

[0237] In some embodiments, the immune effector cells are CD34 IgG1-positive cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood. + The population includes precursor cells of immune effector cells, such as hematopoietic stem cells (HSCs), that, upon administration to a subject, differentiate into mature immune effector cells or can be induced in vitro to differentiate into mature immune effector cells.

[0238] In some embodiments, the cells are natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), lymphokine-activated killer (LAK) cells, etc. NK cells may be either isolated or obtained from commercially available sources. Non-limiting examples of commercially available NK cell lines include lines NK-92 (ATCC® CRL-2407™), NK-92MI (ATCC® CRL-2408™). Further examples include, but are not limited to, NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA) and the German Collection of Microorganisms and Cell Cultures.

[0239] In some embodiments, the host cell presenting a CAR of the present disclosure is selected from a cytotoxic T cell (TC, also known as a cytotoxic T lymphocyte, CTL, T killer cell, lytic T cell, CD8+ T cell, or killer T cell) and a NK cell.

[0240] In some embodiments, the cells are B cells, monocytes or granulocytes, eg, myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0241] In some embodiments, the cells express an anti-HLA-G CAR disclosed herein and further express an inhibitor of one, two, or three immune checkpoint molecules. In some cases, the immune checkpoint molecule is PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM-1, CEACAM-3, CEACAM-5, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GALS, adenosine, or TGFR.

[0242] Genetic manipulation In some embodiments, the genetically engineered cells described herein comprise an anti-HLA-G CAR and one or more additional exogenous transgenes, hi some embodiments, the one or more additional exogenous transgenes encode a detectable tag, a safety switch system, or a chimeric switch receptor.

[0243] Detectable Tags In some embodiments, the genetically engineered cells described herein comprise an anti-HLA-G CAR and further comprise an exogenous transgene encoding a detectable tag. Examples of detectable markers include, but are not limited to, a FLAG tag, a polyhistidine tag (e.g., 6xHis), a SNAP tag, a Halo tag, a cMyc tag, a glutathione-S-transferase tag, avidin, an enzyme, a fluorescent protein, a luminescent protein, a chemiluminescent protein, a bioluminescent protein, and a phosphorescent protein.

[0244] In some embodiments, the fluorescent protein is a blue / UV protein (e.g., BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire), a cyan protein (e.g., CFP, eCFP, Cerulean, SCFP3A, mTurcoise, mTurcooise2, monomeric Midorishi-Cyan, TagCFP, and mTFP1), a green protein (e.g., GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeon). Green), yellow proteins (e.g., YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP), orange proteins (e.g., monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2), red proteins (e.g., RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2), far-red proteins (e.g., mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-infrared proteins (e.g., TagRFP657, IFP1.4, and iRFP), long Stokes shift proteins (e.g., mKeima Red, LSS-mKate1, LSS-mKate2, and mBeRFP), light-activated proteins (e.g., PA-GFP, PAmCherry1, and PATagRFP), photoconverting proteins (e.g., Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange, and PSmOrange), and photoswitching proteins (e.g., Dronpa).In some embodiments, the detectable marker can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed monomer, and / or AcGFP, all commercially available from Clontech.

[0245] In some embodiments, the detectable tag and the anti-HLA-G CAR are expressed from the same expression cassette. For example, in some embodiments, the engineered cells described herein comprise an expression cassette comprising a first polynucleotide sequence encoding an anti-HLA-G CAR and a second polynucleotide sequence encoding a detectable tag.

[0246] Manufacturing method Methods for introducing genes into cells and expressing genes in cells are known in the art.

[0247] In particular, methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In the context of expression vectors, the vectors can be readily introduced into host cells, e.g., mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological means, as described in more detail herein below.

[0248] In some embodiments, the methods include isolating cells from a subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient, before or after cryopreservation.

[0249] Specifically provided herein is a method of generating cells expressing an anti-HLA-G CAR, comprising, alternatively consisting essentially of, or alternatively consisting of, (i) transducing a population of isolated cells with a nucleic acid sequence encoding a CAR as described herein; and (ii) selecting a subpopulation of said isolated cells that are successfully transduced with said nucleic acid sequence of step (i), thereby generating anti-HLA-G CAR-expressing cells. In one embodiment, the isolated cells are selected from the group consisting of T cells and NK cells.

[0250] More particularly, there is provided herein a method for generating cells that express an anti-HLA-G CAR, comprising, alternatively consisting essentially of, or additionally consisting of the following steps: (i) obtaining a population of immune cells (e.g., blood cells), (ii) isolating a specific population of cells (e.g., T cells and / or NK cells), (iii) transducing the isolated population of cells with a nucleic acid sequence encoding a CAR as described herein, and (iv) selecting a subpopulation of said isolated cells that have been successfully transduced with said nucleic acid sequence of step (iii), thereby generating cells that express an anti-HLA-G CAR. These different steps are described in more detail below.

[0251] Cell acquisition Prior to expansion and genetic manipulation of cells as disclosed herein, the cells may be obtained, for example, from a subject in embodiments including autologous therapy, or from commercially available cultures.

[0252] Cells can be obtained from samples including tissues, bodily fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, e.g., separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation.

[0253] Cells can be obtained from numerous sources, including, but not limited to, whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus tissue, lymph node tissue, umbilical cord blood, tissue from a site of infection, ascites, pleural effusion, tissue biopsy, tumor, leukemia, lymphoma, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0254] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0255] In some embodiments, any number of T cell or NK cell lines available and known to those skilled in the art, such as those described above, may be used. In some embodiments, the cells may be derived from a healthy donor, a patient diagnosed with cancer, a patient diagnosed with an autoimmune or inflammatory disorder, or a patient diagnosed with an infectious disease. In some embodiments, the cells may be part of a mixed population of cells displaying different phenotypic characteristics.

[0256] Cell isolation As known to those skilled in the art, various methods for isolating immune cells from a subject are readily available or can be adapted for this application using, for example, the Life Technologies Dynabeads® system; STEMcell Technologies EasySep™, RboSep™, RosetteSep™, SepMate™, Miltenyi Biotec MACS™ cell separation kits, cell surface marker expression, and other commercially available cell separation and isolation kits (e.g., ISOCELL from Pierce, Rockford, IL). Specific subpopulations of immune cells may be isolated using beads or other binders available in such kits specific for unique cell surface markers. For example, MACS™ CD4+ and CD8+ microbeads may be used to isolate CD4+ and CD8+ T cells. Strategies to isolate and expand antigen-specific T cells as therapeutic interventions for human disease are also being tested in clinical trials (Riddell et al., 1992; Walter et al., 1995; Heslop et al., 1996).

[0257] In some embodiments, cell isolation involves one or more preparative and / or affinity-based cell separation steps. In some instances, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a particular reagent. In some instances, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to a particular component.

[0258] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium, and / or many or all divalent cations. In some aspects, the wash step is accomplished by a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended after washing in various biocompatible buffers, such as, for example, Ca2+ / Mg2+-free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0259] In some embodiments, isolation methods involve separating different cell types based on the cellular expression or presence of one or more specific molecules, such as, for example, surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation may be used. In some embodiments, separation is affinity- or immunoaffinity-based separation. For example, isolation in some aspects involves separating cells and cell populations based on the cellular expression or expression level of one or more markers, usually cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such markers, typically followed by a washing step and separation of cells bound to the antibody or binding partner from cells that do not. Such separation steps can be based on positive selection, in which cells that bind to the reagent are retained for further use, and / or negative selection, in which cells that do not bind to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when there are no antibodies available that specifically identify cell types in a heterogeneous population; as a result, separation is best performed based on markers expressed by cells other than the desired population.

[0260] In some embodiments, multiple separation steps are performed, and the positively or negatively selected fraction from one step is subjected to another separation step, for example, subsequent positive or negative selection. In some examples, a single separation step can simultaneously deplete cells expressing multiple markers, for example, by incubating cells with multiple antibodies or binding partners specific to markers that are each targeted by negative selection. Similarly, multiple cell types can simultaneously be positively selected by incubating cells with multiple antibodies or binding partners expressed in different cell types.

[0261] For example, in some embodiments, specific subpopulations of T cells, e.g., cells expressing positive or high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+, are isolated by positive or negative selection techniques. For example, CD3+ T cells can be expanded using CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T cell growth factor).

[0262] In some embodiments, isolation is achieved by enriching for a particular cell population by positive selection or by depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed or expressed at relatively high levels (marker+) on the positively or negatively selected cells, respectively.

[0263] In some embodiments, T cells are isolated from a sample by negative selection for markers expressed on B cells, monocytes, or other leukocytes, e.g., non-T cells, such as CD14. In some aspects, a CD4 or CD8 selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more subpopulations of naive T cells, memory T cells, and / or effector T cells.

[0264] In some embodiments, CD8+ cells are further enriched or depleted for naive cells, central memory cells, effector memory cells, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, e.g., improve long-term survival, proliferation, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012), Blood. 1:72-82; Wang et al. (2012), J. Immunother. 35(9):689-701. In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells with CD4+ T cells.

[0265] In embodiments, memory T cells reside in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8 fractions using, for example, anti-CD8 and anti-CD62L antibodies.

[0266] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection of cells expressing or overexpressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched in TCM cells is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which are subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0267] In some embodiments, enrichment for NK cells is based on positive or high surface expression of CD56 and CD16, and negative expression of CD3, and / or optionally the presence of NKp46 or NKp30 receptors.

[0268] In some aspects, a sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads or MACS beads). The magnetically responsive material, e.g., particles, is generally coupled, directly or indirectly, to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cell(s), cells, or cell population desired to be separated, e.g., negatively or positively selected. In some embodiments, the magnetic particle or bead comprises a magnetically responsive material coupled to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials for use in magnetic separation methods. Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342B, both of which are incorporated herein by reference. Colloidal-sized particles, such as those described in Oven, US Pat. No. 4,795,698, and Liberti et al., US Pat. No. 5,200,084, are other examples.

[0269] Incubation is generally carried out under conditions in which the antibody or binding partner or molecule, such as a secondary antibody or other reagent that specifically binds to the antibody or binding partner attached to the magnetic particle or bead, specifically binds to the cell surface molecule, if present on cells in the sample.

[0270] In some embodiments, the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles bound thereto are attracted to the magnet and separated from unlabeled cells. In the case of positive selection, cells attracted to the magnet are retained; in the case of negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed in the same selection step, and the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0271] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are bound to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, followed by the addition of magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (e.g., streptavidin). In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0272] In some embodiments, the magnetically responsive particles remain attached to the cells, which are then incubated, cultured, and / or manipulated; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies bound to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0273] In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, a system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize error, user interaction, and / or contamination. In one example, the system is a system described in International Patent Application Publication No. WO2009 / 072003 or US20110003380.

[0274] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) through the use of a microelectromechanical systems (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010), Lab. Chip, 10, 1567-1573; and Godin et al. See, e.g., (2008), J. Biophoton. l(5):355-376. In either case, cells can be labeled with multiple markers, allowing for the isolation of highly pure and well-defined T cell subsets. In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some instances, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers is performed in a fluid stream, such as by fluorescence-activated cell sorting (FACS), including preparative-scale (FACS) and / or microelectromechanical systems (MEMS) chips, coupled with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0275] In some embodiments, the method involves density-based cell separation, for example, preparing white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0276] In any of the above-mentioned separation steps, separation does not result in 100% enrichment or removal of specific cell populations or cells that express specific markers.For example, positive selection or enrichment for specific types of cells, such as cells that express markers, refers to increasing the number or proportion of such cells, but does not completely eliminate cells that do not express markers.Similarly, negative selection, removal or depletion of specific types of cells, such as cells that express markers, refers to reducing the number or proportion of such cells, but does not result in the complete removal of all such cells.

[0277] Alternatively, cells may be obtained via commercially available cell cultures, including, but not limited to, for T cells, the cell lines BCL2(AAA) Jurkat (ATCC® CRL-2902™), BCL2(S70A) Jurkat (ATCC® CRL-2900™), BCL2(S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), Neo Jurkat (ATCC® CRL-2898™), and TALL-104 (ATCC® CRL-11386); and for NK cells, the cell lines NK-92 (ATCC® CRL-2407™), and NK-92MI (ATCC® CRL-2408™).

[0278] Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA) and the German Collection of Microorganisms and Cell Cultures.

[0279] Cell preparation and growth The immune cells may be engineered to express the desired CAR, either before or after the immune cells have been engineered to express the desired CAR, using commonly known methods or other methods described in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,46 ... Cells can be activated and expanded using methods readily adapted for this application, such as those described in U.S. Patent Nos. 81, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005; the Life Technologies Dynabeads® System Activation and Expansion Kit, the BD Biosciences Phosflow™ Activation Kit, the Miltenyi Biotec MACS™ Activation and Expansion Kit, and other commercially available cell kits specific for the relevant cell activation moiety. Ex vivo stimulation with HLA-G antigen can activate and expand selected CAR-expressing cell subpopulations. Alternatively, the cells may be activated in vivo by interaction with the HLA-G antigen.

[0280] Incubation and / or manipulation may be performed in a culture vessel such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or culturing cells.

[0281] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culturing, culturing, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, growth, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to stimulate cells for genetic manipulation, such as the introduction of a recombinant antigen receptor. The conditions can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and other agents designed to activate cells.

[0282] In some embodiments, the immune cells of the present disclosure can be expanded in vitro by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0283] Generally, T cells of the present disclosure can be expanded by contacting them with, for example, an agent that stimulates the CD3TCR complex and a costimulatory molecule on the surface of the T cell to generate an activation signal for the T cell. For example, a chemical such as the ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or a mitogenic lectin such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cell.

[0284] In some embodiments, T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody, or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. In some embodiments, T cell populations may be stimulated in vitro by contact with muromonab-CD3 (OKT3). For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a T cell population can be incubated with an anti-CD3 antibody and an anti-CD28 antibody under conditions that stimulate T cell proliferation.

[0285] In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing PBMCs (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) to the culture start composition; and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rad to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0286] In some embodiments, costimulatory molecules are employed to enhance T cell activation, proliferation, and cytotoxicity generated by the CAR after antigen binding. Costimulatory ligands include B7-1 (CD80), B7-2 (CD86), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8a, CD8β, CD1a, LFA-1 (CD11a / CD18), CD1b, CD1c, CD1d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD30L, CD40, CD40MICA, CD49a, CD49D, CD49f, CD69, CD70, CD83, CD84, CD96(Tactile), CD100(SEMA4D), CD103, OX40(CD134), 4-1BB(CD137), SLAM(SLAMF1, CD150, IPO-3), CD160(BY55), SELPLG(CD162), DNAM1(CD226), Ly9(CD229), SLAMF4(CD244, 2B4), ICOS(CD278), CEACA M1, CDS, CRTAM, DAP10, GADS, GITR, HVEM(LIGHTR), HLA-G, IA4, ICAM-1, IL2Rβ, IL2Rγ, IL7Ra, ITGA4, ITGA6, ITGAD, IT GAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​MICB, NKG2C, NKG2D, NKp30, NKp44, N Examples of such proteins include, but are not limited to, Kp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PD-L1, PD-L2, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (rCAM), lymphotoxin beta receptor, 3TR6, ILT3, and ILT4.Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others.

[0287] In some embodiments, NK cell populations can be expanded in vitro using interleukin-2 (IL-2), IL-15, IL-15 / IL-15RA complex, IL-18, and IL-12.

[0288] Suitable conditions for culturing T cells and NK cells include an appropriate medium (e.g., Minimal Essential Media or RPMI Media 1640, or X-Vivo 10, X-Vivo 15, and X-Vivo 20 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-2, IL-15, IL-18, IL-21, TGF, and TNF, or other additives for cell growth known to those of skill in the art. In a preferred embodiment, T cells are stimulated in vitro by exposure to OKT3 and IL-2. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media can include RPMI1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 10, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free, or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokine(s) in an amount sufficient for T cell growth and proliferation.

[0289] Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures, not in the cultures of cells infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to various stimulation times may exhibit different characteristics.

[0290] In some embodiments, the preparation method includes a step of freezing the cells, e.g., for cryopreservation, either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing step removes granulocytes and some monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium, which is then diluted 1:1 with medium to a final concentration of 10% DMSO and 4% HSA, respectively. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0291] In some embodiments, the NK cells or T cells are expanded ex vivo for at least about 5 days, eg, about 10 days or more, about 15 days or more, or about 20 days or more, prior to administration to the patient.

[0292] In some embodiments, the NK cells or T cells are expanded at least about 100-fold, preferably at least about 200-fold, more preferably at least about 400-fold, preferably at least about 600-fold, more preferably at least about 1000-fold, and even more preferably at least about 1500-fold compared to day 0 of expansion prior to administration to the patient.

[0293] Cell transduction and expansion Nucleic acid constructs according to the present disclosure can be transduced into immune cells to generate immune cells that express anti-HLA-G CARs according to the present disclosure. In certain embodiments, cells are transduced to contain at least one CAR of the present disclosure.

[0294] It is contemplated that the chimeric nucleic acid construct can be introduced into the subject's own immune cells as naked DNA or in a suitable vector. Methods for stably transfecting immune cells, particularly T cells, by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding the chimeric receptor of the present disclosure contained in a plasmid expression vector in the appropriate orientation for expression. Advantageously, the use of naked DNA shortens the time required to generate T cells expressing the chimeric receptor of the present disclosure. Physical methods for introducing nucleic acid constructs into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc.

[0295] Alternatively, biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Various viral vectors, such as those described above, can be used to introduce the nucleic acid constructs of the present disclosure into immune cells. Vectors suitable for use in accordance with the methods of the present disclosure do not replicate in the subject's immune cells.

[0296] In one embodiment, a nucleic acid construct encoding a CAR according to the present disclosure is introduced into immune cells by a viral vector, in particular a lentiviral vector as described above.

[0297] Alternatively, chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0298] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the present disclosure, various assays may be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot), or by assays described herein to identify agents within the scope of the present disclosure. Methods for examining CARs for their ability to recognize target cells and antigen specificity are known in the art. For example, Clay et al., J. Immunol., 163:507-513 (1999) teaches methods for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin 2 (IL-2)). Furthermore, CAR function can be assessed by measuring cytotoxicity, as described in Zhao et al., J. Immunol., 174:4415-4423 (2005).

[0299] Once it has been confirmed that the transfected or transduced immune cells can express the chimeric receptor as a surface membrane protein at the desired regulation and desired level, it can be determined whether the chimeric receptor functions within the host cells and induces the desired signal. The transduced immune cells can then be reintroduced or administered to a subject to activate an anti-tumor response in the subject. To facilitate administration, the transduced T cells of the present disclosure can be made into a pharmaceutical composition together with a pharmaceutically acceptable carrier or diluent, or into a deposit suitable for in vivo administration.

[0300] Once cells expressing a CAR according to the present disclosure are administered to a subject, in some aspects, the biological activity of the engineered cell population and / or antibody is measured by any of several known methods. Parameters assessed include specific binding of engineered or natural T cells or other immune cells to an antigen in vivo, e.g., by imaging, or in vitro, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochderfer et al., J. Immunotherapy, 32(7)689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells can also be measured by assaying the expression and / or secretion of certain cytokines, such as GM-CSF, IL-3, MIP-1α, TNF-α, IL-10, IL-13, IFN-γ, or IL-2.

[0301] In some embodiments, biological activity is measured by assessing a clinical outcome such as reduction in tumor burden or burden, tumor stabilization, progression-free survival, or overall survival.

[0302] The manufacturing methods contemplated herein may further include cryopreservation of engineered immune cells for storage and / or preparation for use in human subjects. As used herein, "cryopreservation" refers to the preservation of cells by cooling to subzero temperatures, such as (usually) 77K or -196°C (the boiling point of liquid nitrogen).

[0303] In some embodiments, a method for preserving genetically engineered mouse, human, or humanized CAR protein-expressing immune effector cells that target HLA-G-expressing cells includes cryopreserving the immune effector cells so that the cells remain viable upon thawing. A fraction of the immune effector cells expressing the CAR protein can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with HLA-G-expressing cancer cells. If necessary, the cryopreserved transformed immune effector cells can be thawed, expanded, and propagated into more such cells.

[0304] Cryoprotectants are often used below freezing to prevent damage to stored cells due to freezing at low temperatures or warming to room temperature. Cryopreservatives and optimal cooling rates can protect against cell damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bilist, Nature, 1959; 183: 1394-1395; Ash Wood-Smith, Nature, 1961; 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfrat, Ann. NY. Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48).

[0305] Compositions and Formulations The present disclosure also relates to pharmaceutical or veterinary compositions comprising the anti-HLA-G antibodies or antibody fragments, CARs, nucleic acid constructs, vectors and / or cells described above.

[0306] In certain aspects, the present disclosure relates to pharmaceutical or veterinary compositions comprising cells, preferably immune cells, and comprising a CAR as described herein above and / or a nucleic acid construct encoding a CAR as described herein above. In one aspect, the pharmaceutical or veterinary composition may comprise a population of cells comprising a CAR that specifically binds to HLA-G that binds to β2M, preferably to both HLA-G1 and HLA-G5.

[0307] In a further embodiment, a pharmaceutical or veterinary composition may comprise a first population of cells expressing a CAR described above that targets an HLA-G isoform that binds β2M, and a second population of cells expressing a CAR that does not recognize HLA-G but recognizes an antigen known to be a target of interest in CAR therapy, such as anti-tumor and / or anti-viral therapy. It will be understood that such second population of CAR-expressing cells does not target HLA-G.

[0308] The present disclosure also relates to pharmaceutical or veterinary compositions containing a plurality of CAR-expressing cells of the present disclosure, such as T cells and / or NK cells. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds.

[0309] In one embodiment, the concentration of CAR-expressing cells according to the disclosure in the pharmaceutical or veterinary composition is at least 0.001 mg / ml, at least 0.1 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 5 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 20 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 35 mg / ml, at least 40 mg / ml, at least 45 mg / ml, at least 50 mg / ml, at least 55 mg / ml, at least 60 mg / ml, at least 65 mg / ml, at least 70 mg / ml 1, at least 75 mg / ml, at least 80 mg / ml, at least 85 mg / ml, at least 90 mg / ml, at least 95 mg / ml, at least 100 mg / ml, at least 105 mg / ml, at least 110 mg / ml, at least 115 mg / ml, at least 120 mg / ml, at least 125 mg / ml, at least 130 mg / ml, at least 135 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, at least 250 mg / ml, at least 275 mg / ml, or at least 300 mg / ml.

[0310] In another embodiment, the concentration of CAR-expressing cells of the present disclosure in the pharmaceutical or veterinary composition is between 0.001 and 0.01 mg / ml, between 0.01 and 0.1 mg / ml, between 0.1 and 1 mg / ml, between 1 and 10 mg / ml, between 10 and 50 mg / ml, between 50 and 100 mg / ml, between 50 and 150 mg / ml, between 50 and 200 mg / ml, between 50 and 250 mg / ml, between 50 and 300 mg / ml, between 100 and 200 mg / ml, between 100 and 300 mg / ml, or between 200 and 300 mg / ml.

[0311] In another embodiment, the pharmaceutical or veterinary composition comprises a CAR-expressing cell according to the disclosure, particularly at least 100 cells, at least 200 cells, at least 400 cells, at least 500 cells, at least 700 cells, at least 1000 cells, at least 1500 cells, at least 2000 cells, at least 3000 cells, at least 5000 cells, at least 10,000 cells, at least 100,000 cells, at least 1 million cells, or at least 10 million cells expressing a CAR according to the disclosure.

[0312] In some embodiments, the pharmaceutical compositions provided herein comprise a population of cells that express an anti-HLA-G CAR, wherein the population of cells comprises 70% or more viable cells.

[0313] In some embodiments, the pharmaceutical compositions provided herein comprise a population of cells expressing an anti-HLA-G CAR, wherein the composition comprises no more than 5 EU / kg of endotoxin. The amount of endotoxin is determined according to European Pharmacopoeia 2.6.14 United States Pharmacopoeia (USP) No. <85> Chapter Measured by bacterial endotoxin test.

[0314] In some embodiments, the pharmaceutical compositions provided herein comprise a population of cells expressing an anti-HLA-G CAR, wherein the composition comprises less than 50 copies / μg of replication-competent lentivirus, as measured by PCR.

[0315] In some embodiments, the pharmaceutical compositions provided herein comprise a population of cells expressing an anti-HLA-G CAR, wherein the cells contain an average of 5 or fewer vector copies per transduced cell, as measured by ddPCR.

[0316] In some embodiments, the pharmaceutical compositions provided herein comprise a population of cells expressing an anti-HLA-G CAR, wherein (a) the population of cells comprises 70% or more viable cells; (b) the composition comprises 5 EU / kg or less endotoxin; (c) the composition comprises less than 50 copies / μg of a replication-competent lentivirus; and (d) the cells comprise an average of 5 or less vector copies per transduced cell.

[0317] Pharmaceutical or veterinary compositions according to the present disclosure may be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration.

[0318] Preferably, pharmaceutical or veterinary compositions according to the present disclosure may be administered by enteral or parenteral routes of administration. When administered parenterally, pharmaceutical or veterinary compositions according to the present disclosure are preferably administered by the intravenous route. When administered enterally, pharmaceutical or veterinary compositions according to the present disclosure are preferably administered by the oral route of administration.

[0319] It will be understood by those skilled in the art that the formulations of the present disclosure may be isotonic with human blood, i.e., have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure of about 250 mOsm to 350 mOsm. Isotonicity can be measured, for example, using a vapor pressure osmometer or a freezing osmometer. The osmotic pressure of the formulation is adjusted using an osmotic agent. An "osmotic agent" is a pharmaceutically acceptable inert substance that can be added to a formulation to provide isotonicity to the formulation. Osmotic agents suitable for the present disclosure include, but are not limited to, sugars, salts, and amino acids.

[0320] Compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, Carder compounds, and other pharmaceutically acceptable carriers or excipients.

[0321] Pharmaceutical or veterinary compositions according to the present disclosure may further comprise a pharmaceutically acceptable vehicle. Accordingly, a further aspect of the present disclosure relates to compositions comprising a carrier and one or more of the products described in the embodiments disclosed herein, such as an anti-HLA-G CAR, a nucleic acid, a vector, an anti-HLA-G antibody, or a cell comprising an antibody fragment. The formulation can be sterilized and, if desired, can be mixed with auxiliary agents such as carriers and excipients that do not significantly interact with the product of the formulation, such as an anti-HLA-G CAR, a nucleic acid, a vector, an anti-HLA-G antibody, or an antibody fragment.

[0322] Preferably, pharmaceutical or veterinary compositions of the present disclosure, including but not limited to any one of the claimed compositions, may comprise CAR-expressing cells as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, as described below, preferably in a pharmaceutically acceptable form that does not adversely affect the desired immune enhancing effect of the recombinant cells of the present disclosure.

[0323] To facilitate administration, immune cells, preferably T cells and / or NK cells, transduced with a nucleic acid construct encoding a CAR of the present disclosure can be formulated into pharmaceutical compositions for in vivo administration together with a suitable pharmaceutically acceptable carrier or diluent. Means for preparing such compositions are described in the art (see, e.g., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)).

[0324] In particular, formulations comprising a population of CAR-expressing cells may include pharmaceutically acceptable excipient(s). The excipients included in the formulation will have different purposes depending, for example, on the CAR construct, the subpopulation of cells used, and the mode of administration. Formulations comprising a population of CAR-expressing cells are typically prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).

[0325] The formulations or compositions may also contain multiple active ingredients useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with complementary activities to the binding molecules or cells, where each activity does not adversely affect the other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0326] Pharmaceutical or veterinary compositions, in some embodiments, can employ time-release, delayed-release, and sustained-release delivery systems so that delivery of the composition occurs before and within sufficient time to cause sensitization of the area to be treated. Means known in the art can be used to prevent or minimize release and absorption of the composition or to ensure timed release of the composition until it reaches the target tissue or organ. Such systems can avoid repeated administration of the composition, thereby increasing convenience for the subject and the physician.

[0327] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. As used herein, a "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and any other compatible substance employed in pharmaceutical formulations.

[0328] In some embodiments, the composition comprises an effective amount of anti-HLA-G CAR-expressing immune effector cells. As used herein, an "effective amount" of engineered cells, e.g., T cells, is the amount of cells necessary to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result. An effective amount of engineered therapeutic cells may vary depending on factors such as the individual's condition, age, sex, and weight, and includes an amount effective to "treat" the subject. An effective amount of the compositions described herein suitable for administration to a subject can be determined by a physician, taking into account differences in the subject's age, weight, extent of disease, and condition.

[0329] In some embodiments, the pharmaceutical compositions comprising the T cells described herein are administered in a dose of 10 2 ~10 10 cells / kg body weight, preferably 10 5 ~10 6 The composition may be administered in doses of 10 cells / kg body weight, inclusive of all integer values ​​within these ranges. The number of cells will depend on the intended end use of the composition and the type of cells included in the composition. A clinically relevant number of immune cells can be administered cumulatively in doses of 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The CAR-expressing cell composition may be distributed over multiple infusions equal to or exceeding 100 cells. The CAR-expressing cell composition may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing treatment. If desired, the treatment may also enhance the induction of an immune response by administering mitogens (e.g., PHA), cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.).

[0330] Subjects, Regimen, and Administration The present disclosure relates to a pharmaceutical composition of the present disclosure or a CAR-expressing cell of the present disclosure for use as a drug or for treating a disease or disorder in a subject.The present disclosure also relates to the use of a pharmaceutical composition of the present disclosure or a CAR-expressing cell of the present disclosure in the manufacture of a drug for treating a disease or disorder in a subject.Finally, the present disclosure relates to a method for treating a disease or disorder in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present disclosure or a CAR-expressing cell of the present disclosure to the subject.

[0331] A human subject according to the present disclosure may be a human in the prenatal stage, a newborn, a child, an infant, an adolescent or an adult, in particular an adult at least 40 years of age, an adult at least 50 years of age, an adult at least 60 years of age, or an adult at least 70 years of age.

[0332] In some embodiments, the subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing adverse outcomes such as cytokine release syndrome (CRS).

[0333] In some embodiments, the subject has persistent or recurrent disease after treatment with other therapies, including other immunotherapy and / or chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject's resistance to other treatments. In some embodiments, the subject has not relapsed but is determined to be at risk of relapse, e.g., at high risk of relapse, and therefore the compound or composition is administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse.

[0334] In some embodiments, the present disclosure includes administering a CAR-expressing cell of the present disclosure, or a composition containing a CAR-expressing cell, to a subject, e.g., a subject at risk of or suspected of having a disease, condition, or disorder. In some embodiments, the cells and compositions are administered to a subject having the particular disease or condition being treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to a subject, e.g., a subject having or at risk for a disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate, one or more symptoms of the disease or condition. In some embodiments, the subject has been diagnosed with an immune disease, preferably cancer. Methods for diagnosing autoimmune diseases or cancer are well known to those of skill in the art.

[0335] In some embodiments, the subject has already undergone at least one line of treatment, and preferably several lines of treatment, prior to administration of the immune cells according to the present disclosure or the pharmaceutical or veterinary composition according to the present disclosure.

[0336] Preferably, the therapeutic agent is administered periodically, preferably daily to monthly, more preferably daily to every two weeks, even more preferably daily to weekly, and even more preferably daily. In certain embodiments, the therapeutic agent is administered several times daily, preferably two or three times daily, and even more preferably three times daily.

[0337] The duration of treatment with a vector according to the present disclosure, an immune cell according to the present disclosure, or a pharmaceutical or veterinary composition according to the present disclosure is preferably between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, even more preferably between 1 day and 4 weeks, and even more preferably between 1 day and 2 weeks. In some embodiments, the duration of treatment is about 1 week. Alternatively, treatment may continue for as long as the disease persists.

[0338] The form, route of administration and dosage of the pharmaceutical or veterinary composition of the immune cells of the present disclosure or the pharmaceutical composition of the present disclosure can be adjusted by one skilled in the art depending on the type and severity of the infection, and on the patient, particularly their age, weight, sex and general physical condition. The compositions of the present disclosure may be administered in a number of ways, depending on whether local or systemic treatment is desired.

[0339] In the case of adoptive cell therapy, methods for administering cells for adoptive cell therapy are known and may be used in conjunction with the provided methods and compositions. For example, adoptive T cell therapy is described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg, (2011), Nat. Rev. Clin. Oncol. 8(10):577-85. See, for example, Themeli et al. (2013), Nat. Biotechnol. 31(10):928-933; Tsukahara et al. (2013), Biochem. Biophys. Res. Commun. 438(1):84-9; Davila et al. (2013), PLoS ONE, 8(4):e61338.

[0340] Those skilled in the art will recognize that multiple routes of administration can be used, and that certain routes may provide more immediate and effective responses than other routes.For example, intradermal delivery may be used more advantageously than inhalation for the treatment of melanoma.Local or systemic delivery can be achieved by administration including application of formulations to body cavities or instillation, aerosol inhalation or insufflation, or by parenteral introduction, including intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous or intradermal administration.

[0341] Although systemic (intravenous, IV) injection is preferred for clinical use due to ease of administration, several preclinical studies (Carpenito, et al. (2009), Proc. Natl. Acad. Sci. USA, 106:3360-3365; Song, et al. (2011), Cancer Res. 71:4617-4627; Parente-Pereira, et al. (2011), J. Clin. Immunol. 31:710-718) suggest that local (intratumoral, IT or intraperitoneal, IP) administration of T cells may result in optimal therapeutic efficacy, which may be due in part to increased T cell trafficking to the tumor. For example, CAR T cells have been shown to remain at the site of inoculation with minimal systemic absorption when delivered via IP or IT routes (Parente-Pereira, et al. (2011), J. Clin. Immunol. 31:710-718). In contrast, after intravenous administration, CAR T cells first reach the lungs and then redistribute to the spleen, liver, and lymph nodes. Furthermore, T cells electroporated with RNA CARs may be particularly suitable for local administration due to the transient nature of CAR expression in T cells (Zhao, et al. (2010), Cancer Res. 70:9053-9061). Furthermore, clinical trials have demonstrated the feasibility and safety of both intratumoral and intraperitoneal injection of T cells (Canevari, et al. (1995), J. Natl. Cancer Inst. 87:1463-1469; Duval, et al. (2006), Clin. Cancer Res. 12:1229-1236). Overall, a local route of administration of recombinant T cells may provide optimal therapeutic efficacy and reduce the potential for "on-target, extra-organ" toxicity. Thus, in one embodiment, the CAR-expressing cells of the present disclosure are administered locally, preferably by intratumoral and intraperitoneal injection.

[0342] In some embodiments, the pharmaceutical composition contains CAR cells in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. In the case of repeated administration over several days or longer, depending on the condition, treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition. The amount of immune cells according to the present disclosure or the amount of pharmaceutical composition according to the present disclosure to be administered can be determined by standard procedures well known to those skilled in the art. The appropriate dosage should be determined taking into account the patient's physiological data (e.g., the recipient's age, size, weight, and health status and weight, type of concomitant treatment, if any, frequency of treatment, and the nature of the desired effect) and the route of administration, so that a therapeutically effective amount is administered to the patient. In particular, appropriate dosages and dosing schedules can be based on clinical trials or well-established cell-based therapies (see, e.g., Topalian & Rosenberg (1987), Acta. Haematol. 78, Suppl. 1:75-6; U.S. Pat. No. 4,690,915), or alternative continuous infusion strategies can be employed.

[0343] In some embodiments, an effective amount or number of cells or pharmaceutical compositions comprising the cells are administered parenterally. In some embodiments, administration can be intravenous. In some embodiments, administration can be by injection directly into the tumor.

[0344] In certain embodiments, in the context of engineered cells expressing a CAR, a subject has a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, or about 90 million cells). cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and optionally, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), etc., or any value between these ranges, and / or such number of cells per kg of the subject's body weight. For example, in some embodiments, administration of a cell or population of cells can include administration of about 10 to about 10 cells per kg of body weight, including all integer values ​​of cell number within those ranges; for example, the cell compositions of the present disclosure can be administered in doses or dosages, each dose including at least 10 cells / kg of body weight, at least 100 cells / kg of body weight, at least 1000 cells / kg of body weight, at least 10,000 cells; at least 100,000 cells; at least 1 million cells; at least 10 million cells; at least 100 million cells; at least 100 million cells; or at least 10 billion cells / kg of body weight.

[0345] In particular, a sufficient number of transduced immune cells are introduced to achieve the desired therapeutic response. Desirably, an effective amount or sufficient number of isolated transduced cells are present in a composition and introduced into a subject so as to establish a long-term, specific, anti-tumor or anti-infectious agent response to reduce tumor size or regrowth or the proliferation of an infectious agent compared to the absence of such treatment. Desirably, the amount of transduced immune cells, preferably T cells, reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% reduction in tumor size when compared to the same conditions except for the absence of the transduced immune cells.

[0346] The compositions of the present disclosure can be provided in unit dosage forms, with each dosage unit, e.g., injection, containing a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary administration for human and animal subjects, each unit containing a predetermined amount of the composition of the present disclosure, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, optionally in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of the novel unit dosage forms of the present disclosure depend on the specific pharmacodynamics associated with the pharmaceutical composition in a particular subject.

[0347] The cells or population of cells may be administered in one or more doses. In some embodiments, the effective amount or number of cells may be administered as a single dose. In some embodiments, the effective amount or number of cells may be administered as multiple doses over a period of time. The timing of administration is within the discretion of the supervising physician and depends on the clinical condition of the patient. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of one of ordinary skill in the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit.

[0348] For purposes of this disclosure, the amount or dose of a CAR substance administered should be sufficient to produce a therapeutic or prophylactic response in a subject over a reasonable time frame. For example, the dose of a CAR substance should be sufficient to bind to an antigen, e.g., an HLA-G isoform(s), or detect, treat, or prevent a disease for a period of about 2 hours or more, e.g., about 12 hours to about 24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose will be determined by the efficacy of the particular CAR substance and the condition of the subject, as well as the weight of the subject being treated.

[0349] For purposes of this disclosure, for example, an assay involving comparing the extent to which target cells are lysed or IFN-γ is secreted by CAR-expressing T cells upon administration of a given dose of such T cells to a mammal among a set of mammals each administered different doses of T cells can be used to determine the starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a particular dose can be measured by methods known in the art.

[0350] use Use in the treatment of diseases or disorders and in combination therapy In some aspects, the disease or disorder to be treated is a condition selected from a proliferative disease or disorder, preferably cancer; an infectious disease or disorder, preferably viral infection; an inflammatory disease or disorder; and an immune disease or disorder, preferably autoimmune or autoimmune disease, allergy, and graft-versus-host rejection. In some embodiments, the condition may be cancer.

[0351] The present disclosure relates to the use of antibodies, cells, nucleic acid constructs, vectors, and / or pharmaceutical compositions according to the present disclosure to interfere with or neutralize immune downregulation caused by HLA-G protein in a host in need thereof.

[0352] In particular, the cells, nucleic acid constructs, vectors and / or pharmaceutical compositions of the present disclosure are particularly suitable for the treatment of viral infections, such as, for example, HIV-1, Hepatitis B virus and Hepatitis C virus infections.

[0353] In particular, the cells, nucleic acid constructs, vectors and / or pharmaceutical compositions according to the present disclosure are even more preferably particularly suitable for the treatment of cancer, in particular solid tumors or hematopoietic cancers, where the availability of good selective single targets is limited.

[0354] The immune system can specifically identify and eliminate tumor cells based on the expression of tumor-specific antigens or molecules induced during malignant cell transformation. This process is called tumor immunosurveillance. Despite tumor immunosurveillance, tumors can still develop in the presence of a functional immune system. This occurs through a process known as tumor immunoediting, which involves three major stages: 1) the elimination phase, in which most immunogenic tumor cells are eliminated by cytotoxic T cells and NK cells; 2) the equilibrium phase, in which less immunogenic tumor cells are selected; and 3) the escape phase, in which variants that no longer respond to the host immune system are maintained (Urosevic and Dummer, 2008). HLA-G is involved in all stages of tumor immunoediting by reducing tumor cell elimination, inhibiting the cytotoxic function of T cells and NK cells, and by trogocytosis (the cell-to-cell transfer of viable HLA-G molecules), which renders competent cytotoxic cells unresponsive to tumor antigens (LeMaoult et al., 2007; CauMartin et al., 2007). Thus, the chimeric constructs of the present disclosure are applied to subjects having or suspected of having a disease, disorder, or particular condition, particularly subjects having or suspected of having cancer, to thereby reduce tumor size, prevent tumor growth or regrowth, or prevent the induction of an immunosuppressive microenvironment in these subjects.

[0355] Accordingly, the present disclosure also relates to methods for inhibiting tumor growth in a subject in need thereof and / or methods for treating a cancer patient in need thereof. The tumor may be a solid tumor or a liquid tumor. In some embodiments, the tumor or cancer expresses or overexpresses HLA-G. In certain embodiments, these methods comprise, alternatively consist essentially of, or even consist of administering to the subject or patient an effective amount of isolated cells. In still further embodiments, the cells expressing a CAR according to the present disclosure are T cells or NK cells. The isolated cells may be allogeneic or autologous to the subject or patient being treated. In a further aspect, the tumor expresses or overexpresses HLA-G antigen, and the subject has been selected for treatment by diagnosis.

[0356] In one embodiment, the present disclosure relates to a method for reducing tumor growth or preventing tumor formation in a subject by introducing a chimeric construct of the present disclosure into a subject's immune cells, preferably T cells or NK cells, and reintroducing the transformed immune cells into the subject to express a CAR according to the present disclosure, resulting in an anti-tumor response that reduces or eliminates the tumor in the subject. The step of delivering a nucleic acid construct to a subject generally involves introducing a nucleic acid construct of the present disclosure into isolated immune cells (e.g., autologous immune cells isolated from PBMCs or immune cells from an allogeneic third-party donor) and introducing the transformed immune cells into the subject, thereby resulting in an anti-tumor response that reduces or eliminates the tumor in the subject, similar to adoptive T cell therapy. For example, the immune cells may include T cells, and the subject is suffering from, or suspected to be suffering from, a tumor or cancer, e.g., an HLA-G-expressing cancer, or has been diagnosed with a tumor or cancer. For example, an anti-HLA-G CAR molecule encoded by an exemplary nucleic acid construct of the present disclosure may be administered to the subject in the form of recombinant immune cells engineered to express the anti-HLA-G CAR molecule.

[0357] CAR-expressing cells according to the present disclosure and obtained by the methods described above, or cell lines derived from such cells, can be used as drugs in the treatment of a disease, disorder, or condition in a subject. In some embodiments, such drugs can be used to treat cancer.

[0358] In some embodiments, administering a therapeutic agent to a subject may include adoptive cell therapy (ACT) using immune cells harvested from the subject or from one or more donors. Thus, the cells can be xenogeneic, allogeneic, or autologous to the subject. Generally, the cells are autologous to the subject.

[0359] In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, in which cells are isolated and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment, and following isolation and processing, the cells are administered to the same subject.

[0360] In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed by allogeneic transplantation, in which cells are isolated and / or otherwise prepared from a subject other than the subject receiving or ultimately receiving cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. The cells of the present disclosure may be capable of killing target cells, such as cancer cells. The target cells may be recognizable by a defined pattern of antigen expression, e.g., expression of antigen A or antigen B.

[0361] In some embodiments, ACT may include isolating primary immune cells from a subject or one or more donors, transducing the primary immune cells with the nucleic acid construct or constructs of any of the preceding embodiments, expressing the CAR in the transduced primary immune cells, and delivering the transduced immune cells to the subject. ACT may further include stimulating and / or expanding the transduced immune cells prior to delivering them to the subject.

[0362] For example, in some embodiments, ACT may involve harvesting autologous or allogeneic T cells, transducing these T cells with one or more nucleic acid constructs such that the T cells express CAR-mediated inflammatory cytokine expression, and then infusing the cells into a subject in need thereof.

[0363] The present disclosure also provides methods of treating cancer, comprising delivering to a subject in need thereof an effective amount of a nucleic acid construct, vector or vectors, or transduced immune cells or pharmaceutical composition according to any of the preceding embodiments, thereby treating the cancer. In some embodiments, the treatment of cancer may be measured by a reduction in tumor cell burden or by an increase in survival rate.

[0364] The present disclosure further provides methods of immunotherapy comprising administering to a subject a nucleic acid construct or constructs, vector or vectors, recombinant cell, or pharmaceutical composition according to any of the foregoing embodiments. Treatment with the cells of the present disclosure may help prevent tumor cell escape or release, which often occurs with standard approaches.

[0365] In certain embodiments, CAR-expressing cells are modified in various ways to enhance their therapeutic or prophylactic efficacy. For example, CARs may be conjugated to a targeting moiety either directly or indirectly via a linker. The practice of conjugating compounds, e.g., CARs, to targeting moieties is known in the art. See, e.g., Wadhwa et al., J. Drug Targeting, 1995;3(2):111-127, and U.S. Pat. No. 5,087,616. In particular, the present disclosure includes a type of cell therapy in which isolated cells are genetically engineered to express a CAR and the CAR cells are infused into a subject in need thereof. Such administration can promote cell activation (e.g., T cell activation) in a target-specific manner, thereby targeting disease or disorder cells for destruction. When the cells are T cells, CAR T cells, unlike antibody therapies, can replicate in vivo, resulting in long-term persistence that can lead to sustained control of the targeted disease, disorder, or condition.

[0366] The CAR-expressing cells disclosed herein may be administered alone or in combination with diluents, known anti-cancer therapeutics, and / or other components such as immunostimulatory cytokines or other cell populations, which may be administered as a first-line therapy, a second-line therapy, a third-line therapy, or an additional therapy.

[0367] In some embodiments, the CAR-expressing cells are administered as part of a combination therapy, either simultaneously with another therapeutic intervention, e.g., an antibody or engineered cell or receptor, or agent, e.g., a cytotoxic or therapeutic agent, or sequentially, in any order. The cells or antibodies, in some embodiments, are administered with or in conjunction with one or more additional therapeutic agents, either simultaneously or sequentially, in any order. In some situations, the cells are co-administered with another therapy sufficiently close in time so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered before one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after one or more additional therapeutic agents, e.g., anti-cancer agents. An "anti-cancer agent" can adversely affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, slowing the rate of cancer cell proliferation, reducing the incidence or number of metastases, decreasing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the longevity of a subject with cancer. More generally, these other compositions can be provided in a combined amount effective to kill the cells or inhibit the proliferation of the cells. This process can include simultaneously contacting the cancer cells with cells expressing a CAR of the present disclosure and the agent(s) or multiple factor(s). This can be accomplished by contacting the cells with a single composition or pharmacological formulation containing both agents, or by simultaneously contacting the cells with two different compositions or formulations, where one composition contains the expression construct and the other composition contains the second agent(s).

[0368] In some embodiments, cells expressing a CAR against an HLA-G isoform according to the present disclosure are administered as part of a combination therapy, in any order, simultaneously or sequentially, with other CAR-expressing cells that do not recognize HLA-G but are known to be useful in other CAR therapies, such as anti-tumor and / or anti-viral CAR therapies. Such CAR-expressing cells preferably target an antigen involved in a disease such as cancer or viral infection, preferably an antigen that is a target of cancer therapy or viral therapy. It will be understood that such antigen is not HLA-G.

[0369] In the context of the present disclosure, it is contemplated that cell therapy may also be used in combination with chemotherapy, radiation therapy, or immunotherapy intervention, as well as with pro-apoptotic or cell cycle modulating agents such as immune checkpoint inhibitors.

[0370] Alternatively, the present treatment can precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments in which the other agent and the present disclosure are administered to an individual separately, this generally ensures that no significant time period elapses between the times of delivery, thereby still allowing the agent and treatment to exert their beneficial combined effect on the cells. In such cases, it is contemplated that both modalities may be administered to the cells within about 12-24 hours of each other, and more preferably within about 6-12 hours of each other. However, in some circumstances, it may be desirable to extend the treatment period significantly, with days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsing between each administration.

[0371] It is expected that this treatment cycle would be repeated as necessary. It is also contemplated that various standard treatments, as well as surgical intervention, may be applied in combination with the cell therapy.

[0372] Targeted cancers HLA-G is aberrantly expressed in many human in situ solid malignant tumors and hematopoietic malignancies, including breast cancer, ovarian cancer, clear cell renal cell carcinoma, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, and hepatocellular carcinoma, as well as acute myeloid leukemia and chronic lymphocytic leukemia (B-CLL). Abnormal expression of HLA-G in malignant neoplasms is significantly correlated with poor clinical outcomes in patients with colorectal cancer (CRC), gastric cancer (GC), non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), breast cancer, hepatocellular carcinoma, and B-CLL. Furthermore, serum soluble HLA-G is elevated in patients with various types of cancer (including melanoma, acute leukemia, multiple myeloma, neuroblastoma, lymphoproliferative disorders, breast or ovarian cancer, non-small cell lung cancer, esophageal cancer, colorectal cancer, gastric cancer, and hepatocellular carcinoma) compared with normal healthy controls or benign disease cases.

[0373] Cancers that may be treated by a CAR-expressing cell, nucleic acid construct, vector, or pharmaceutical composition according to the present disclosure include vascularized tumors as well as tumors that are not vascularized or not yet substantially vascularized.

[0374] The CAR-expressing cells, nucleic acid constructs, vectors, or pharmaceutical compositions of the present disclosure may be used to treat cancers of the oral cavity and pharynx (including tongue cancer, oral cavity cancer, and pharyngeal cancer), cancers of the digestive system (including esophageal cancer, stomach cancer, and colorectal cancer), cancers of the liver and biliary system (including hepatocellular carcinoma and cholangiocarcinoma), cancers of the respiratory system (including bronchial carcinoma, lung cancer, and laryngeal cancer); cancers of the bone and joints, including osteosarcoma, cancers of the skin, including melanoma, breast cancer, cancers of the reproductive organs, including uterine cancer, endometrial cancer, ovarian cancer, and cervical cancer in women, prostate cancer and testicular cancer in men, cancers of the kidney, including renal cell carcinoma and transitional cell carcinoma of the urinary tract or bladder; gastrointestinal stromal tumors, pancreatic cancer, kidney cancer, colon cancer, cervical cancer, and glioma. The compounds may be used to treat cancers of the endocrine system, including brain tumors, including glioblastoma multiforme and medulloblastoma, thyroid cancer, adrenal cancer, and cancers associated with multiple endocrine neoplasia syndrome; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, B-cell lymphoma, monocytic lymphoma, marginal zone lymphoma, Burkitt's lymphoma, T- and B-lymphoma, multiple myeloma, plasmacytoma; acute and chronic leukemia, prohemocytic leukemia, acute nonlymphoblastic leukemia (ANLL), acute lymphoblastic leukemia (ALL), erythroblastic leukemia, myeloid leukemia, or lymphocytic leukemia; and cancers of other and unspecified sites, including neuroblastoma. In some embodiments, the cancer is selected from the group consisting of renal cell carcinoma (RCC), melanoma, kidney cancer, and bladder cancer.

[0375] Cancer may include non-solid tumors (such as blood tumors, e.g., leukemia and lymphoma) or solid tumors. As used herein, a "solid tumor" is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).

[0376] Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, malignant lymphoma, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary tract carcinoma, gallbladder ... These include ductal carcinoma, chorioma, Werms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (gliomas (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0377] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological cancers (or hematopoietic cancers) include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroid leukemias), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0378] In some embodiments, cancer cells express or overexpress HLA-G. Preferably, cancer cells express or overexpress HLA-G1 and / or HLA-G5. Preferably, when such cancer cells express these particular HLA-G isoforms, the CAR of the present disclosure specifically binds to HLA-G1 and HLA-G5, respectively.

[0379] Diagnosis and Prognosis The anti-HLA-G monoclonal antibodies or scFvs disclosed herein are also useful in diagnostic and prognostic methods. Thus, the present disclosure relates to the use of the antibodies disclosed herein in diagnosing an HLA-G-associated condition in a subject.

[0380] The monoclonal antibodies or scFvs disclosed herein are useful in art-known methods related to localization and / or quantification of HLA-G polypeptides (e.g., for measuring the level of HLA-G polypeptides in appropriate physiological samples, for use in diagnostic methods, for use in imaging polypeptides, etc.). The monoclonal antibodies or scFvs disclosed herein are useful for isolating HLA-G polypeptides by standard techniques such as Western blotting, affinity chromatography methods for isolating cells, or flow cytometry-based cell analysis or cell sorting, or immunoprecipitation. The HLA-G antibodies disclosed herein can facilitate the purification of native HLA-G polypeptides from biological samples, such as mammalian serum or cells, as well as recombinantly produced HLA-G polypeptides expressed in host systems. Furthermore, HLA-G monoclonal antibodies or scFvs can be used to detect HLA-G polypeptides (e.g., in plasma, cell lysates, or cell supernatants) to assess the abundance and expression pattern of the polypeptides. The HLA-G antibodies disclosed herein can be used diagnostically to monitor HLA-G levels in tissues as part of a clinical testing procedure, e.g., to determine the effectiveness of a given therapeutic regimen. Detection can be facilitated by coupling (i.e., physically linking) the HLA-G antibodies disclosed herein to a detectable substance such that the HLA-G antibody or fragment thereof is detectably labeled. The term "labeled" with respect to an antibody is intended to encompass both direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, and indirect labeling of the antibody by reactivity with another compound that is directly labeled. Non-limiting examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin for detection with fluorescently labeled streptavidin.

[0381] The detection methods of the present disclosure can be used to detect the expression level of HLA-G polypeptides in biological samples both in vitro and in vivo. In vitro techniques for detecting HLA-G polypeptides include enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry, immunoprecipitation, radioimmunoassay, and immunofluorescence (e.g., IHC). Additionally, in vivo techniques for detecting HLA-G polypeptides include introducing a labeled anti-HLA-G antibody into a subject. For example, an antibody can be labeled with a radioactive marker, and its presence and location can be detected in a subject using standard imaging techniques.

[0382] In some embodiments, HLA-G antibodies containing structural modifications that facilitate rapid binding and cellular uptake and / or sustained release are useful in in vivo imaging detection methods. In some embodiments, the HLA-G antibodies contain deletions in the CH2 constant heavy chain region of the antibody to facilitate rapid binding and cellular uptake and / or sustained release. In some embodiments, Fab fragments are used to facilitate rapid binding and cellular uptake and / or sustained release. In some embodiments, F(ab)'2 fragments are used to facilitate rapid binding and cellular uptake and / or sustained release.

[0383] Thus, the present disclosure also provides prognostic (or predictive) assays for determining whether a subject is at risk for developing a medical disease or condition associated with increased HLA-G polypeptide expression or activity (e.g., detecting precancerous cells). Such assays can be used for prognostic or predictive purposes to prophylactically treat individuals prior to the onset of a medical disease or condition characterized by or associated with HLA-G polypeptide expression.

[0384] Another aspect of the present disclosure provides a method for determining HLA-G expression in a subject and thereby selecting an appropriate therapeutic or prophylactic compound for that subject.

[0385] Alternatively, prognostic assays can be used to identify subjects who have or are at risk of developing cancer and / or solid tumors. Accordingly, the present disclosure provides methods for identifying diseases or conditions associated with elevated expression levels of HLA-G isoform(s), in which a test sample is obtained from a subject and HLA-G isoform(s) is detected, wherein the presence of elevated levels of HLA-G polypeptide compared to a control sample predicts the subject having or at risk of developing a disease or condition associated with elevated expression levels of HLA-G isoform(s). In some embodiments, the disease or condition associated with elevated expression levels of HLA-G isoform(s) is selected from the group consisting of those related to the development of cancer and / or solid tumors.

[0386] In another embodiment, the present disclosure provides a method for determining whether a subject can be effectively treated with a compound for a disorder or condition associated with elevated HLA-G expression, wherein a biological sample is obtained from the subject, and HLA-G isoform(s) are detected using an HLA-G antibody or scFv as described above. The expression level of HLA-G polypeptide in the biological sample obtained from the subject is determined and compared to the HLA-G expression level found in a biological sample obtained from a disease-free subject. An elevated level of HLA-G in a sample obtained from a subject suspected of having a disease or condition compared to a sample obtained from a healthy subject indicates an HLA-G-related disease or condition in the subject being investigated.

[0387] There are many disease states in which elevated expression levels of HLA-G isoform(s) are known to indicate whether a subject with the disease is likely to respond to a particular type of therapy or treatment. Therefore, the method of detecting HLA-G isoform(s) in a biological sample can be used as a prognostic method to, for example, assess the likelihood that a subject will respond to a therapy or treatment.

[0388]

[0010] A further aspect of the present disclosure relates to a method for determining whether a patient is likely or unlikely to respond to HLA-G CAR therapy. In a specific embodiment, the method comprises contacting a tumor sample isolated from the patient with an effective amount of HLA-G antibody and detecting the presence of any antibody bound to the tumor sample. In a further embodiment, the presence of antibody bound to the tumor sample indicates that the patient is likely to respond to HLA-G CAR therapy, and the absence of antibody bound to the tumor sample indicates that the patient is unlikely to respond to HLA-G therapy. In some embodiments, the method comprises the additional step of administering an effective amount of HLA-G CAR therapy to the patient determined to be likely to respond to HLA-G CAR therapy.

[0389] A further aspect of the present disclosure relates to a method for determining whether a patient is likely to respond or not to HLA-G CAR therapy depending on the isoform(s) expressed by the tumor, in particular the isoform(s) selected from HLA-G1 and HLA-G5. By identifying the HLA-G expressed isoform(s) prior to treatment, the most suitable CARs that specifically bind to the HLA-G1 and HLA-G5 isoforms can be selected and used for efficient treatments such as cell therapy.

[0390] kit Any of the compositions described herein may be included in a kit provided by the present disclosure. Thus, the kit comprises recombinant / engineered cells of the present disclosure, and / or vectors encoding nucleic acid constructs of the present disclosure, and / or nucleic acid constructs or associated reagents in suitable container means. In some embodiments, the kit further comprises an additional agent for treating cancer or an infectious disease, which may be combined with the nucleic acid construct(s) or cells or other components of the kit of the present disclosure, or may be provided separately in the kit. In some embodiments, means for obtaining a sample from an individual and / or means for assaying the sample may be provided in the kit. In certain embodiments, the kit includes, for example, cells, buffers, cell culture media, vectors, primers, restriction enzymes, salts, etc. The kit may also include a means for containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0391] The components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably suitably aliquoted. Where there are multiple components in the kit, the kit will also generally contain second, third, or other additional containers into which the additional components may be placed separately. However, various combinations of components may be contained within vials. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained. The kits of the present disclosure will also typically include means for containing the components in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained.

[0392] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The composition may also be formulated into a syringe-compatible composition. In this case, the container means may itself be a syringe, pipette, and / or other such device from which the formulation may be applied to an infected site on the body, injected into an animal, and / or further applied to other components of the kit and / or mixed with other components. However, the components of the kit may also be provided as a dry powder(s). When reagents and / or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate container means.

[0393] In some embodiments of the present disclosure, the cells used for cell therapy are provided in a kit, and in some cases, the cells are essentially the only component of the kit. The kit may include reagents and materials for producing the desired cells. In specific embodiments, the reagents and materials include primers for amplifying the desired sequence, nucleotides, appropriate buffers or buffering reagents, salts, etc., and in some cases, the reagents include a vector and / or DNA encoding a CAR and / or its regulatory elements as described herein.

[0394] In some embodiments, the kit includes one or more devices suitable for extracting one or more samples from an individual. The device may be a syringe, a scalpel, or the like.

[0395] In some cases of the present disclosure, the kits, in addition to the cell therapy embodiments, also include a second cancer therapy, such as, for example, chemotherapy and / or immunotherapy. The kit(s) may be tailored to an individual's particular cancer and may include a respective second cancer therapy for the individual, as described above.

[0396] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be construed as, an admission or in any manner suggestion that they constitute available prior art or form part of the common general knowledge in any country in the world. [Example]

[0397] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are offered by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize that there are a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

[0398] Example 1 - V of humanized and chimeric LFTT-1 H and V L Mutant generation A series of humanized V LFTT-1 antibodies H and V L The amino acid sequences of LFTT-1 were created by selectively backmutating specific humanized amino acid residues to murine amino acid residues. Residues in the Vernier zone, adjacent CDRs, and residues important for the stability of the variable region were backmutated in all designs. The degree of humanization was defined either as the number of backmutations (lower numbers indicate a higher degree of humanization) or the degree of homology that the IGV shares with the human germline sequence. Exemplary humanized and chimeric LFTT-1 Vs H and V L The mutation types of V are shown in Table 5 below. H and V L The parent array of is V H 0 and VL It is called 0. V H 1 and V L 1 is most similar to the mouse parent sequence, while V H 5 and V L The one designated 5 is most similar to the optimal human template. [Table 5] For the variable domains, the sequences of CDR1, CDR2, CDR3 (from left to right) are underlined; humanized residues are in bold; and backmutated residues are underlined / bold.

[0399] Humanized V for LFTT-1 mutants H or V L A DNA construct for the expression of V H and V L ) were used to co-transfect HEK293EBNA cells for expression of humanized LFTT-1 variants. Construct pairs for expression of humanized and chimeric full antibodies are shown below in Table 6. [Table 6-1] [Table 6-2]

[0400] The HLA-G binding efficiency of humanized LFTT-1 variants was assessed using a flow cytometry-based competitive assay. K562-HLAG1 cells expressing human HLA-G1 antigen and control K562 wild-type cells were seeded at 1E+6 cells / well in a 96-well U-bottom plate (Corning, Amsterdam, The Netherlands). After washing with DPBS + 10% FBS, the cells were titrated with each test antibody (8-point 3-fold dilution series ranging from 83.33 nM to 0.04 nM), resuspended, and incubated for 1 h in a static incubator at 37°C. After repeated centrifugation and washing with DPBS + 10% FBS to remove unbound antibody from the plate, the cells were resuspended and incubated with a fixed molar concentration (1.6 nM) of biotinylated mouse LFTT-1 for 1 h in a static incubator at 37°C. Cells were washed and resuspended three more times in DPBS + 10% FBS before being resuspended in eBioscience™ Streptavidin PE Conjugate (Thermofisher, Loughborough, UK) and then incubated for a further 30 minutes at 4°C. After further washing, plates were read on an Attune flow cytometer, and mean fluorescence intensity was recorded using the Attuny YFP laser. To determine IC50 values, the mean MFI of technical replicates was plotted against the molar concentration of the test antibody using Prism (GraphPad Software, La Jolla, USA). Background subtraction was performed using MFI values ​​determined using wild-type K562 cells, followed by normalization of MFI readings so that the highest MFI in each series was defined as 100% in all cases and specific MFI was plotted against the molar concentration of antibody. IC50 values ​​were calculated by fitting a nonlinear one-site IC50 curve to the data points. To correct for plate-to-plate variability, the IC50 of each LFTT-1 variant was calculated for every plate. The relative IC50 for a given antibody variant is determined by dividing the IC50 value of the variant by that of chimeric LFTT-1 run on the same plate.Variants with relative IC50 values ​​greater than 1 are predicted to bind less efficiently to K562-HLA-G1 cells, while variants with values ​​less than 1 are predicted to have improved binding efficiency. The relative IC50 values ​​of the LFTT-1 variants are presented in Table 7. [Table 7]

[0401] The relative degree of humanization of the LFTT-1 variants was determined by determining the homology of the heavy and light chain IGHV / IGKV and IGKJ genes of each variant to the human germline sequences of the optimal human template. This was achieved by concatenating the amino acid sequences of IGHV to IGHJ and IGKV to IGKJ for all variants and the optimal human template. The concatenated sequences of each variant were compared to the concatenated sequences of the template in a pairwise alignment to determine the percentage of homology. The overall homology of the variants to the mouse parent was calculated, and reversions were counted. Figure 2 shows the degree of humanization of the LFTT-1 humanized variants by homology (to the parent and human germline) and by counting reversions.

[0402] Because there is often a trade-off between binding efficiency and similarity to human germline, both the degree of humanization and the relative IC50 score of each antibody must be considered. Generally, a more human-like design results in lower binding efficiency because fewer mouse residues are conserved in the design, increasing the likelihood that the antibody will lose affinity for its target. Generally, a more conservative design results in better binding efficiency. The IC50 scores of humanized variants are plotted against the degree of humanization in Figure 3. Antibody variants with lower relative IC50 scores and a higher degree of humanization represent the best lead candidates for retaining antibody affinity for the target and ensuring tolerance by the human immune system. H 4 / V L 5 is a strong contender on both counts.

[0403] MHC class II HLA-DR epitopes were detected by analyzing overlapping 9-amino acid peptides within the sequences of the humanized VH and VL regions of LFTT-1 variants for their potential to bind to 51 HLA-DR alleles. The relative IC50 values ​​calculated for each variant and the number of strong and moderate 9-amino acid MHC class II binding peptides predicted to bind to HLA-DR from both chains in the entire V region are presented in Table 8. [Table 8]

[0404] Table 8 shows that infliximab (chimeric antibody) contains approximately twice as many MHC class II-binding 9-amino acid sequences as trastuzumab (humanized antibody), consistent with its relative clinical immunogenicity. Furthermore, while the murine LFTT-1 variable region contains a similar number of MHC class II-binding 9-amino acid sequences to infliximab, its humanized counterparts contain progressively fewer epitopes, with the most "human" variant containing 16 epitopes. Humanized LFTT-1 antibodies VH4 / VL5 and VH5 / VL4 exhibit low relative IC50 values ​​and a relatively high degree of humanization. The number of strong and moderate MHC class II-binding 9-amino acid sequences for VH4 / VL5 and VH5 / VL4 (16 each) is slightly higher than that of the humanized antibody trastuzumab (11).

[0405] Based on relative IC50 values, degree of humanization, and MHC class II evaluation, three lead candidates were selected: LFTT-1, VH4 / VL5, VH5 / VL4, and VH5 / VL3. The properties of the three lead candidates are presented in Table 9. [Table 9]

[0406] The rate constants for the three LFTT-1 lead candidates were determined by LigandTracer analysis according to a two-to-one bivalent binding model, using a weighted average (according to Bmax) of the KD measurements for both binding events determined for each candidate. The rate constants for each LFTT-1 lead candidate are presented in Table 10. Ka (1 / Ms, on-rate) is the association constant and is a measure of the rate of complex formation between HLA-G1-expressing cells and the antibody under investigation. Kd (1 / sec, dissociation rate) is the dissociation constant and is a measure of the rate at which the complex dissociates. KD (M) is the dissociation constant of the antibody-antigen complex and is determined by dividing Kd by Ka. LigandTracer revealed that all three antibodies bind to cells expressing human HLA-G1 with KD values ​​in the low nanomolar range. [Table 10]

[0407] Example 2 - Humanized LFTT-1 V using a human plasma membrane protein cell array H 4 / V L 5. Evaluation of Binding Profiles Using Retrogenix cell microarray technology, V H 4 / V L A humanized murine IgG1 antibody known as 5 was screened for specific off-target binding interactions.

[0408] 2, 5, or 20 μg / mL of hLFTT-1 VH4 / VL5, 1 μg / mL of rituximab follow-on biologic, or PBS alone were added to slides of untransfected HEK293 cells (non-spotted areas) and HEK293 cells overexpressing HLA-G, HLA-G+B2M, CD20, or EGFR (spotted areas) either post- or pre-fixation. Slides were then incubated with AF647 anti-hIgG Fc detection antibody followed by fluorescent imaging. hLFTT-1 V H 4 / V L5 showed specific interaction with its primary target, HLA-G+β2M, in both fixed and live cell microarrays. Significant interactions with HLA-G and EPHB6 were observed only on live cell microarrays.

[0409] hLFTT-1 V H 4 / V L The interaction of 5 with HLA-G, HLA-G+β2M, and EPHB6 was confirmed by single-dose flow cytometry follow-up. Human HEK293 cells were transfected with expression vectors encoding ZsGreen1 alone, or ZsGreen1 and HLA-G, HLA-G+β2M, EPHB6, or CD20 (assay control). Live transfectants were incubated with 5 μg / mL hLFTT-1 VH4 / VL5, 1 μg / mL rituximab biosimilar (assay control), or assay buffer alone. Cells were washed and incubated with the same AF647 anti-human IgG Fc detection antibody used in the cell microarray screening. Cells were washed again and analyzed by flow cytometry using an Accuri flow cytometer (BD). Dead cells were excluded using 7AAD live / dead dye, and ZsGreen+ (transfected) cells were selected and analyzed. hLFTT-1 VH4 / VL5 showed moderate (average 8.2-fold) and strong (average 69.5-fold) binding to its primary targets HLA-G and HLA-G+β2M, respectively. Strong (average 13.9-fold) binding was also observed with EPHB6. H 4 / V L The binding results of 5 to the target antigen are shown in Table 11. [Table 11]

[0410] Binding to HLA-G and HLA-G+β2M showed a biphasic profile, and binding to EPHB6 was not maximal at the highest dose tested, 300 μg / mL, so EC50 values ​​could not be determined in transiently transfected cells in further dose-response follow-up studies. An EC50 greater than 300 μg / mL makes it unlikely that the interaction with EPHB6 is physiologically relevant. Binding results are presented in Table 12. [Table 12]

[0411] In further dose-response experiments, HLA-G and HLA-G+β2M transiently transfected cells, as well as stably transfected HEK-HLA-G1 cells, were treated with hLFTT-1 V H 4 / V L hLFTT-1 V was incubated with a range of doses of either 5 or a commercially available anti-HLA-G antibody to determine the EC50 values ​​for the primary targets, HLA-G and HLA-G+β2M, to determine whether the commercially available anti-human HLA-G antibody also exhibited a biphasic binding profile with its primary target. H 4 / V L 5 showed a biphasic binding profile with the primary targets HLA-G, HLA-G + β2M, and stably transfected HEK-HLA-G1 cells. At the top two doses (167 μg / mL and 500 μg / mL), high background binding was observed with cells transfected with ZsGreen alone, suggesting that at these very high concentrations, the test antibody interacts with endogenously produced proteins on HEK293 cells. Thus, in target-transfected cells, the first inflection point is the interaction with the transfectant, and the second inflection point is the interaction with endogenous HEK cell proteins (either HEK293 or HEK-HLA-G1 cells). For cells stably transfected with HLA-G, HLA-G+β2M, and HEK-HLA-G1, the EC50 values ​​were 0.35, 0.18, and 0.05 μg / mL, respectively, except for the doses of 167 μg / mL and 500 μg / mL. H 4 / VL The binding results of 5 to the target antigen are presented in Table 13. [Table 13]

[0412] A commercially available anti-human HLA-G antibody (clone 87G) did not show a biphasic profile, and EC50 values ​​of 1.6, 3.1, and 1.9 μg / mL were calculated for its interaction with HLA-G, HLA-G+β2M, and stable HEK-HLA-G1 cells, respectively. H 4 / V L EC50 values ​​for clone 5 and clone 87G are presented in Table 14. [Table 14]

[0413] Example 3 - Preclinical evaluation of CAR-T efficacy in solid tumors Cytolytic function of humanized anti-HLA-G LFTT-1 CAR-T in vitro To evaluate specific cytotoxicity against solid tumors in vitro, humanized LFTT-1 CAR-T cells were incubated with multiple chemotherapy-resistant SKOV-3-HLA-G+ cell lines at ratios of 12:1, 6:1, and 3:1. Figure 4 shows the percentage of tumor cell lysis after CAR-T cell incubation. We observed that anti-HLA-G CAR-T cells exhibited dose-dependent specific cytotoxicity against multiple chemotherapy-resistant SKOV-3-HLA-G+ cell lines, while showing minimal cytotoxicity against HLA-G-negative cells.

[0414] In vitro infiltration and activation of humanized anti-HLA-G LFTT-1 CAR-T To assess the extent of infiltration of humanized anti-HLA-G LFTT-1 CAR-T cells into solid tumor tissues, immunohistochemistry (IHC) was performed on renal cell carcinoma tissues after incubation with anti-HLA-G CAR-T cells. Potent and specific in vitro activation of anti-HLA-G CAR-T cells was observed upon infiltration into human clear cell renal cell carcinoma (ccRCC) biopsies and RCC-HLA-G+ tumor models, respectively. Figure 5A shows representative IHC images of anti-HLA-G CAR-T cells on human ccRCC tissue samples. Significant numbers of anti-HLA-G CAR-T cells were observed on human ccRCC tissue samples. Figure 5B shows the activation ratio of CAR-T cells compared to untransduced control cells. There is a significant increase in CAR-T cell activity compared to untransduced control cells. Figure 5C summarizes the activation ratio of CAR-T cells on PDX RCC and human ccRCC, respectively. Significant activation of anti-HLA-G CAR-T cells was observed in HLA-G-expressing PDX RCC and human ccRCC tissue samples. Results of in vitro assays of CAR-T cells may provide predictive value for in vivo efficacy.

[0415] In vivo function of humanized anti-HLA-G LFTT-1 CAR-T cells To investigate the in vivo function of humanized anti-HLA-G LFTT-1 CAR-T cells, NGS mice were implanted with renal cell carcinoma patient-derived xenografts (RCC PDXs) and then inoculated with humanized anti-HLA-G LFTT-1 CAR-T cells on day 22 (Figure 6A). The cytotoxicity of anti-HLA-G CAR-T cells against HLA-G tumor cells was monitored. A significant therapeutic effect of anti-HLA-G LFTT-1 CAR-T cells was observed. Figure 6B shows tumor volume measurements in NGS mice after tumor implantation over a 79-day period. NGS mice inoculated with humanized anti-HLA-G LFTT-1 CAR-T cells demonstrated significantly more effective control and elimination of the PDX primary tumor than control T cells. These results demonstrate the significant therapeutic effect of anti-HLA-G LFTT-1 CAR-T cells in an in vivo renal carcinoma system.

[0416] Example 4 - GMP manufacturing of anti-HLA-G CAR-T cells This disclosure provides a closed, continuous, and automated process for GMP (Good Manufacturing Process) manufacturing of anti-HLA-G CAR-T cell finished drug product (FDP). This process is adapted from the Miltenyi Prodigy TCT process. The formulated cryopreserved product is referred to as the FDP.

[0417] All raw materials are GMP grade and sourced from qualified vendors. The manufacturing of anti-HLA-G CAR-T cells is supported by a qualified contract development and manufacturing organization (CDMO) and operates under a quality agreement. All materials are tested / examined according to standard operating procedures before use. Critical raw materials are listed in Table 15. [Table 15]

[0418] Anti-HLA-G CAR-T cell products are manufactured using autologous peripheral blood leukocytes. Leukapheresis is performed at one or more clinical sites by experienced and licensed staff using specific leukapheresis equipment, following local standard clinical procedures. Donors are tested for applicable infectious diseases, and donor suitability must be assessed prior to the apheresis procedure, according to clinical protocols. A Certificate of Analysis is provided to verify the testing and quality of the leukapheresis. These procedures are also compliant with European Directive 2006 / 17 / EC and Directive 2004 / 23 / EC.

[0419] Anti-HLA-G CAR expression and CAR-T cell proliferation were evaluated in peripheral blood mononuclear cells (PBMCs) isolated from four different healthy donors using the CliniMACS® Prodigy-TCT manufacturing process. Anti-HLA-G CAR-T cells were identified by flow cytometry as a CD3+CD19+ double-positive population. Anti-HLA-G CAR-T cells were successfully expressed at large scale (4-5x10), with 69% to 85% transduction efficacy and >90% viability of T cells expressing the CAR construct (CD3+CD19+ double-positive). 9 The table shows the CD3 expression levels as a function of the lentivirus MOI used. + 1 shows the characterization of anti-HLA-G CAR CD4 / CD8 T cell composition from cells. [Table 16]

[0420] Based on these primary nonclinical study results, anti-HLA-G CAR-T cells can be produced in large quantities with a transduction efficiency of 69–85% of T cells expressing the CAR construct at a low MOI.

[0421] Anti-HLA-G CAR-T cells are composed of autologous CD3+ T cells, which typically comprise more than 90% of the total cell population. Product-related impurities are cellular impurities derived from leukocyte-depleted materials such as red blood cells, granulocytes, dead cells, and B cells / B-lineage lymphoblasts. Typical cellular impurity content is less than 2% of the total anti-HLA-G CAR-T cell final product. Cell viability is controlled during the manufacturing and formulation process.

[0422] Ancillary materials and reagents not intended to be present in the final product are evaluated as part of the final drug product characterization testing.

[0423] The quality of the anti-HLA-G CAR-T cell product is assessed using a product release quality control test plan (Table 17). Table 17 summarizes the analytical test methods used to evaluate the critical quality attributes of the anti-HLA-G CAR-T cell product in support of process development / optimization, raw material selection, intermediates, drug substance (day 12 harvest), and final product characterization (FDP formulation and cryopreservation). [Table 17-1] [Table 17-2]

[0424] The objective of the product release QC testing plan is to develop a strategy that can shorten the release testing period (approximately 2 weeks) for anti-HLA-G CAR-T cell FDPs while maintaining all safety and testing requirements necessary for FDP release prior to administration to patients.

[0425] Stability studies will be performed on GMP lots of anti-HLA-G CAR-T cell FDP. The purpose of these studies is to obtain information on long-term stability. The stability evaluation and study design are described in the table below. [Table 18]

[0426] The potency of GMP-grade anti-HLA-G CAR-T cells derived from three donors was evaluated in an in vitro cytotoxicity assay. Anti-HLA-G CAR-T cells were incubated on LCL cells expressing GFP and HLA-G (LCL-GFP-HLA-G). Figures 7A–7C show that GMP-grade anti-HLA-G CAR-T cells derived from three different donors lysed HLA-G-expressing cells in a dose-dependent manner, causing significantly greater cell death than non-transformed (NTD) CAR-T cells. The levels of IFN-γ secreted by anti-HLA-G CAR-T cells derived from three donors were measured using Mesoscale Discovery (MSD) multiplex cytokine analysis. Figures 8A–8C show that anti-HLA-G CAR-T cells secreted significantly more IFN-γ than NTD CAR-T cells. These results demonstrate that GMP-grade anti-HLA-G CAR-T cells are highly potent in vitro.

[0427] The effect of increasing concentrations of soluble HLA-G on the efficacy of GMP-grade anti-HLA-G CAR-T cells was evaluated in vitro. Elevated plasma levels of soluble HLA-G have been reported in patients with various tumors. Therefore, the effect of increasing soluble HLA-G concentrations on the efficacy of GMP-grade anti-HLA-G CAR-T cells derived from three donors was evaluated by in vitro cytotoxicity assays. Anti-HLA-G CAR-T cells were incubated with increasing concentrations of soluble HLA-G on HLA-G-expressing LCL cells (LCL-HLA-G). Figures 9A–9C show that soluble HLA-G at concentrations up to 10 μg / ml did not affect the cytotoxicity of anti-HLA-G CAR-T cells against HLA-G-expressing LCL cells in vitro.

[0428] Example 5 - Verification of HLA-G expression in tumor cells Validation studies were performed using an HLA-G IHC assay, which specifically detects the HLA class I histocompatibility antigen α-chain G in human tissue, to identify HLA-G-expressing tumor cells in tissue sections of formalin-fixed, paraffin-embedded (FFPE) samples.

[0429] FFPE human tissue blocks included normal placenta, clear cell renal cell carcinoma (ccRCC), epithelial ovarian cancer (EOC), pancreatic cancer (PCA), colorectal cancer (CRC), esophageal cancer (ESC), breast cancer, and BRCA tissue blocks. Anti-HLA-G (4H84) mouse monoclonal antibody was used for HLA-G IHC assays. Interpretation of HLA-G expression in ccRCC, EOC, PCA, CRC, ESC, BRCA, normal tissues, and controls was performed by a board-certified anatomic pathologist using conventional light microscopy.

[0430] Table 19 shows the HLA-G positivity rate by indication. A high incidence of HLA-G expression was found in ccRCC and EOC samples. The IHC data support ccRCC and EOC as primary indications for anti-HLA-G CAR T-cell therapy. [Table 19]

[0431] In another study, HLA-G isoforms were measured in patient RCC biopsies using an RT-PCR assay. Results showed that HLA-G1 and HLA-G5 were the predominant isoforms expressed in RCC tumors. Low levels of HLA-G6 were detected in RCC tumors, and expression of HLA-G2 and HLA-G4 was not detected. These data indicate that humanized LFTT-1 CAR T cells, which specifically bind to HLA-G1 and HLA-G5, are effective in treating RCC.

Claims

1. A chimeric antigen receptor (CAR), (A) an extracellular domain comprising an antigen-binding domain that specifically binds to human leukocyte antigen G (HLA-G); wherein the antigen-binding domain is (a) (i) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 25, and (ii) a light chain variable region (VL) comprising the sequence of SEQ ID NO: 35; (b) (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 50; or (c) the extracellular domain comprising (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 60; (B) a transmembrane domain; (C) an intracellular signaling domain.

2. A CAR, (A) an extracellular domain comprising an antigen-binding domain that specifically binds to HLA-G; wherein the antigen-binding domain is (a) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:25; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:35; (b) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 50; or (c) an extracellular domain comprising: (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:9; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:60; (B) a transmembrane domain; (C) an intracellular signaling domain.

3. The CAR of claim 1 or 2, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

4. The CAR of claim 3, wherein the antigen-binding domain comprises a sequence set forth in one of SEQ ID NOs: 63, 65, and 67.

5. The CAR according to claim 1 or 2, wherein the transmembrane domain is a CD28 transmembrane domain, and the intracellular signaling domain comprises a 4-1BB costimulatory signaling region and a CD3ζ endodomain.

6. the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 72; the 4-1BB costimulatory signaling region comprises the sequence of SEQ ID NO: 73; The CAR of claim 5, wherein the CD3ζ endodomain comprises the sequence of SEQ ID NO:

74.

7. The CAR of claim 1 or 2, wherein the intracellular signaling domain further comprises a CD28 costimulatory domain.

8. The CAR according to any one of claims 1 to 7, further comprising a signal peptide.

9. The CAR of claim 8, wherein the signal peptide comprises the sequence of SEQ ID NO:

69.

10. The CAR according to any one of claims 1 to 9, further comprising a hinge domain connecting the antigen-binding domain to the transmembrane domain.

11. The CAR of claim 10, wherein the hinge domain comprises the sequence of SEQ ID NO:

71.

12. The CAR according to any one of claims 1 to 11, further comprising a cleavable linker.

13. The CAR of claim 12, wherein the cleavable linker comprises the sequence of SEQ ID NO:

75.

14. The CAR according to any one of claims 1 to 13, further comprising a truncated human CD19.

15. The CAR of claim 14, wherein the truncated human CD19 comprises the sequence of SEQ ID NO:

76.

16. The CAR of claim 1 or 2, wherein the CAR comprises the sequence of SEQ ID NO:

68.

17. A nucleic acid molecule encoding the CAR of claim 1 or 2.

18. 18. An expression vector comprising the nucleic acid molecule of claim 17.

19. A cell comprising the nucleic acid molecule of claim 17.

20. A cell comprising the expression vector of claim 18.

21. A cell comprising the CAR of claim 1 or 2.

22. The cell of claim 21 , wherein the cell is a T cell, a B cell, a NK cell, a NKT cell, a monocyte cell, or a dendritic cell.

23. A pharmaceutical composition comprising the cells according to any one of claims 19 to 22 and a pharmaceutically acceptable carrier.

24. 24. The pharmaceutical composition of claim 23, wherein the cells contain no more than 5 copies of the expression vector in each transduced cell.

25. 24. The pharmaceutical composition of claim 23, further comprising 70 percent or more viable cells.

26. 24. The pharmaceutical composition of claim 23, further comprising 5 EU / kg or less endotoxin.

27. 24. The pharmaceutical composition of claim 23, further comprising a replication-competent lentivirus with less than 50 copies / μg.

28. The cell according to any one of claims 19 to 22 for use in treating cancer.

29. The pharmaceutical composition according to any one of claims 23 to 27, for use in the treatment of cancer.

30. 30. A method of treating cancer in a subject in need of treatment, comprising administering to said subject the pharmaceutical composition of claim 29.

31. 31. The method of claim 30, wherein the cancer is a solid tumor.

32. 31. The method of claim 30, wherein the cancer is a hematological cancer.

33. The cancer may be clear cell renal cell carcinoma, epithelial ovarian cancer, melanoma, kidney cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, renal cell carcinoma, colon cancer, gastric cancer, esophageal cancer, lung cancer, hepatocellular carcinoma, bile duct cancer, neuroblastoma, tongue cancer, oral and pharyngeal cancer, bronchogenic carcinoma, laryngeal cancer, osteosarcoma, prostate cancer, testicular cancer, gastrointestinal stromal tumor, pancreatic cancer, kidney cancer, colon cancer, glioma, glioblastoma multiforme, medulloblastoma, thyroid cancer, adrenal cancer, acute bone marrow cancer, or the like.

31. The method of claim 30, wherein the leukemia is myeloid leukemia, chronic lymphocytic leukemia, non-small cell lung cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, monocytic lymphoma, marginal zone lymphoma, Burkitt's lymphoma, T-cell lymphoma, B-cell lymphoma, plasmacytoma, prohemocytic leukemia, acute non-lymphoblastic leukemia, acute lymphoblastic leukemia, erythroleukemia, myeloid leukemia, or lymphocytic leukemia.

34. 31. The method of claim 30, wherein the cells are autologous.

35. 31. The method of claim 30, wherein the cells are allogeneic.

36. 31. The method of claim 30, wherein the pharmaceutical composition is administered to the subject by intravenous, intratumoral, intraperitoneal, intramuscular, intraportal, intrahepatic, subcutaneous or intradermal administration.

37. 31. The method of claim 30, wherein the subject is a human.

38. An anti-HLA-G antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment (a) (i) a VH comprising the sequence of SEQ ID NO: 25, and (ii) a VL comprising the sequence of SEQ ID NO: 35; (b) (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO: 50; or (c) The anti-HLA-G antibody or antigen-binding fragment thereof, comprising (i) a VH comprising the sequence of SEQ ID NO: 9, and (ii) a VL comprising the sequence of SEQ ID NO:

60.

39. An anti-HLA-G antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment (a) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:25; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:35; (b) (i) a VH comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 50; or (c) The anti-HLA-G antibody or antigen-binding fragment thereof, comprising: (i) a VH comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 9; and (ii) a VL comprising a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:

60.

40. The antibody or antigen-binding fragment of claim 38 or 39, wherein the antibody or antigen-binding fragment is chimeric.

41. The antibody or antigen-binding fragment of any one of claims 38 to 40, wherein the antibody or antigen-binding fragment is conjugated to a toxin.

42. The antigen-binding fragment of any one of claims 38 to 41, wherein the antigen-binding fragment is an scFv, Fv, Fab, Fab', or F(ab')2.

43. The antibody or antigen-binding fragment of any one of claims 38 to 42, wherein the antibody is monoclonal.

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