Rituximab-resistant chimeric antigen receptors and uses thereof

Abstract

To provide polynucleotides encoding chimeric antigen receptors (CARs) comprising a CD19 antigen binding domain that specifically binds to CD19 and is resistant to rituximab binding, and immune cells comprising these CD19-specific CARs, e.g., CAR-T cells.SOLUTION: An isolated polynucleotide encoding a polypeptide including an anti-CD19 chimeric antigen receptor (CAR) that is at least 70% identical to SEQ ID NO: 9, wherein the polypeptide does not include a rituximab binding site, and the polynucleotide includes a short EF1a promoter that is capable of expressing the anti-CD19 chimeric antigen receptor (CAR) in a mammalian T cell.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 839,455, filed Apr. 26, 2019, and U.S. Provisional Patent Application No. 63 / 005,041, filed Apr. 3, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to chimeric antigen receptors (CARs) including antigen - binding molecules that bind to CD19, polynucleotides encoding the same, and methods of using the same to treat cancer in a patient.

[0003] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created on Apr. 21, 2020, is named AT - 028_03WO_SL.txt and is 81,460 bytes in size.

Background Art

[0004] The adoptive transfer of genetically modified immune cells that recognize malignant tumor-related antigens is regarded as a promising new approach for treating cancer (see, for example, Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). Immune cells can be genetically modified to express a chimeric antigen receptor (CAR), a fusion protein consisting of a CD19 antigen recognition portion and a T cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993), and Sadelain et al., Curr. Opin. Immunol, 21(2):215-223 (2009)). Immune cells containing a CAR, such as CAR-T cells (CAR-T), are engineered to have antigen specificity while retaining or enhancing the ability to recognize and kill target cells.

[0005] There is a need for treatments for cancers, particularly malignancies, associated with abnormal expression of CD19. Provided herein are methods and compositions for addressing this need. SUMMARY OF THE INVENTION

[0006] Provided herein are chimeric antigen receptors (CARs) that include a CD19 antigen-binding domain that specifically binds to CD19, polynucleotides encoding these CARs, and immune cells, such as CAR-T cells, that express these CD19-specific CARs. Also provided are methods of making and using these CD19-specific CARs, and immune cells that include these CD19-specific CARs.

[0007] In one aspect, the present disclosure provides an isolated polynucleotide encoding a polypeptide comprising an anti-CD19 chimeric antigen receptor (CAR) that is at least 70% identical to SEQ ID NO: 9, the polypeptide does not contain a rituximab binding site, and the polynucleotide contains a short EF1a promoter capable of expressing the anti-CD19 chimeric antigen receptor (CAR) in mammalian T cells.

[0008] In some embodiments, the short EF1a promoter does not contain an intron sequence within the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the intron contains the nucleic acid sequence of SEQ ID NO: 39.

[0009] In some embodiments, the promoter contains the nucleic acid sequence of SEQ ID NO: 16.

[0010] In some embodiments, the promoter is a full-length EF1a promoter containing the nucleic acid sequence of SEQ ID NO: 15.

[0011] In some embodiments, the promoter contains the nucleic acid sequence of SEQ ID NO: 15, and the polynucleotide encodes a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 8-14.

[0012] In some embodiments, the polypeptide further comprises a safety switch.

[0013] In some embodiments, the safety switch is linked to the CD19 CAR using a linker peptide.

[0014] In some embodiments, the safety switch is linked to the anti-CD19 CAR using a T2A linker.

[0015] In some embodiments, the safety switch contains an antibody binding site.

[0016] In some embodiments, the safety switch contains a mutant CD20 mimotope.

[0017] In some embodiments, the polypeptide further comprises a CD8 hinge / transmembrane domain.

[0018] In some embodiments, the polypeptide comprises a CD34 epitope.

[0019] In some embodiments, the CD34 epitope is the QBEND-10 epitope.

[0020] In some embodiments, the isolated polynucleotide comprises a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1-7.

[0021] In some embodiments, the isolated polynucleotide encodes a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 8-14.

[0022] In another aspect, the disclosure provides a vector comprising the isolated polynucleotide described herein.

[0023] In some embodiments, the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof.

[0024] In one aspect, the disclosure provides an engineered immune cell comprising the isolated polynucleotide described herein.

[0025] In some embodiments, the disclosure provides an engineered immune cell comprising a polynucleotide comprising a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 3.

[0026] In some embodiments, the present disclosure provides engineered immune cells comprising a polynucleotide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an anti-CD19 CAR v1.2, v1.3, v1.4, v1.5, or v1.6 lentiviral construct, as shown in Table 1.

[0027] In some embodiments, the present disclosure provides engineered immune cells comprising a polynucleotide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an anti-CD19 CAR v1.2 lentiviral construct, as shown in Table 1.

[0028] In some embodiments, the present disclosure provides engineered immune cells comprising a polynucleotide encoding a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10, regardless of the presence or absence of a signal sequence.

[0029] In some embodiments, the present disclosure provides engineered immune cells comprising a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 3, which is driven by a short promoter of EF1a comprising the nucleic acid of SEQ ID NO: 16.

[0030] In some embodiments, the present disclosure provides engineered immune cells comprising a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 9 or SEQ ID NO: 10, regardless of the presence or absence of a signal sequence, which is driven by a short promoter of EF1a comprising the nucleic acid of SEQ ID NO: 16. In some embodiments, the promoter does not include the first intron of the EF1a gene.

[0031] In some embodiments, the engineered immune cells do not include a mimotope of rituximab.

[0032] In some embodiments, the engineered immune cells comprise a polynucleotide comprising the CD19 CAR v1.2 lentiviral construct (which can also be referred to as ALLO-501A) shown in FIG. 1.

[0033] In some embodiments, the engineered immune cells comprise a vector described herein.

[0034] In some embodiments, the immune cells are T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.

[0035] In some embodiments, the cells are autologous T cells.

[0036] In some embodiments, the cells are allogeneic T cells.

[0037] In one aspect, the disclosure provides an engineered immune cell described herein, wherein the cell is resistant to rituximab.

[0038] In another aspect, the disclosure provides a pharmaceutical composition comprising an engineered immune cell described herein.

[0039] In one aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an engineered immune cell described herein, or a pharmaceutical composition described herein.

[0040] In some embodiments, the disease or disorder is non-Hodgkin lymphoma (NHL).

[0041] In some embodiments, the subject has been treated with rituximab or is currently being treated with rituximab.

[0042] In one aspect, the disclosure provides an article of manufacture comprising an engineered immune cell that expresses a chimeric antigen receptor described herein or a pharmaceutical composition comprising an engineered immune cell described herein.

Brief Description of the Drawings

[0043]

Figure 1

[0044]

Figures 2A - 2B

[0045]

Figure 3

[0046]

Figures 4A - 4D

[0047]

Figures 5A - 5D

[0048]

Figures 6A - 6D

[0049]

Figure 7

[0050]

Figures 8A - 8D

[0051]

Figure 9

[0052]

Figure 10

[0053]

Figures 11A - 11D

[0054]

Figure 12

[0055]

Figures 13A - 13B

DETAILED DESCRIPTION OF THE INVENTION

[0056] Chimeric antigen receptor (CAR) therapy is a promising approach for cancer treatment. The CAR construct described herein as v1.0 is an exemplary anti-CD19 CAR that expresses the synthetic peptide RQR8 that serves as a safety switch. RQR8 contains two rituximab-binding mimotopes. In the case of adverse events, patients may be treated with rituximab to deplete the levels of anti-CD19 v1.0 in the circulating blood. Also, rituximab is used as a standard treatment in several non-Hodgkin lymphoma (NHL) indications and is administered at high doses. Due to the long half-life of rituximab, anti-CD19 v1.0 cannot be administered to patients until the levels of circulating rituximab reach low concentrations. With rituximab-resistant CD19 CAR therapy, patients previously treated with rituximab can receive CAR-T therapy immediately without waiting for the levels of rituximab to decrease and without the patient undergoing apheresis. Provided herein is an anti-CD19 chimeric antigen receptor (CAR) that is resistant to the CD20-binding antibody rituximab. The novel CAR construct is designed to remove rituximab binding while retaining CAR expression and activity.

[0057] I. Chimeric Antigen Receptor As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to the target antigen, the CAR can activate immune cells to attack and destroy cells having that antigen (e.g., cancer cells). The CAR can also incorporate a co-stimulatory domain or a signaling domain to increase their potency. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797, Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465, and 6,319,494.

[0058] The chimeric antigen receptors described herein include an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain includes a CD19 antigen-binding domain that specifically binds to CD19. In some embodiments, the CD19-specific CAR includes the following elements from 5' to 3': a signal sequence, a CD19 antigen-binding domain (e.g., an scFv derived from 4G7), a hinge and transmembrane region, and one or more consecutive signaling domains. In some embodiments, the antibody-binding domain binds to CD19 and treats a blood cancer associated with the expression of CD19.

[0059] The scFv portion of the chimeric antigen receptor (CAR) used in the allogeneic anti-CD19 CAR v1.0 is derived from the mouse anti-human CD19 antibody clone 4G7. 4G7 is a CD19 monoclonal antibody that recognizes CD19. The single chain variable fragment (scFv) formed from 4G7 contains several chimeric antigen receptor (CAR) targeting components (see WO2014184143A1). In some embodiments, the scFv derived from the CD19 monoclonal antibody 4G7 contains a portion of the CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain (GenBank: CAD88275.1, SEQ ID NO: 17) and a portion of the CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain (GenBank: CAD88204.1, SEQ ID NO: 35) linked together by a flexible linker. (Peipp M., D. Saul, et al., 2004. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications. J. Immunol. Methods 285:265-280). In some embodiments, the scFv comprises a variable fragment of the CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain and a variable fragment of the anti-CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain linked together by a flexible linker.

[0060] CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain (signal sequence underlined)

[0061] MEWSWIFLFLLSGTAGVHSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 17)

[0062] CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain (signal sequence is underlined)

[0063] MRCLAEFLGLLVLWIPGAIG DIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO: 18)

[0064] In some embodiments, the scFv comprises a portion of the amino acid sequence of SEQ ID NO: 17 and / or SEQ ID NO: 18. In some embodiments, the scFv has at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the variable region of the amino acid sequence of SEQ ID NO: 34 and / or SEQ ID NO: 35. Disclosed herein are antigen-binding molecules that specifically bind to an anti-CD19 scFv derived from 4G7, as well as molecules comprising these sequences, and cells presenting such molecules. Humanized forms of the antigen-binding molecules are also formed as aspects of the present disclosure. The applications and uses of these antigen-binding molecules are also disclosed.

[0065] a. Antigen-binding domain As described above, the CD19 CAR described herein comprises an antigen-binding domain. As used herein, "antigen-binding domain" means any polypeptide that binds to a specific target antigen, for example, the specific target antigen may be a CD19 protein or a fragment thereof. In some embodiments, the antigen-binding domain binds to the CD19 antigen on tumor cells. In some embodiments, the antigen-binding domain binds to the CD19 antigen on cells involved in proliferative diseases.

[0066] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) and comprises light chain CDRs CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3. Variants of the antigen-binding domain (e.g., variants of the CDRs, VH, and / or VL) are also within the scope of the present disclosure and include, for example, variable light chains and / or variable heavy chains that each have at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or more than 99% identity to the amino acid sequence of the antigen-binding domain sequences described herein. In some instances, such molecules comprise at least one heavy chain and one light chain, while in other instances, the variant forms comprise two variable light chains and two variable heavy chains (or sub-portions thereof). One of ordinary skill in the art can determine suitable variants of the antigen-binding domains described herein using well-known techniques. In certain embodiments, one of ordinary skill in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not considered critical for activity.

[0067] In certain embodiments, the polypeptide structure of the antigen-binding domain is antibody-based and includes, but is not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the antigen-binding domain comprises or consists of an affimer.

[0068] A CD19 antigen-binding domain is said to be "selective" if it binds more strongly to one target than to a second target. In some embodiments, the CD19 antigen-binding domain is an scFv.

[0069] In some embodiments, the present disclosure relates to an isolated polynucleotide encoding any one of the CD19 chimeric antigen receptors (CARs) described herein. In some embodiments, the present disclosure relates to an isolated polynucleotide encoding the CD19 CARs described in Table 1. Also provided herein are vectors containing the polynucleotide, and methods of making the polynucleotide. [Table 1] TIFF2025109712000001.tif249167 TIFF2025109712000002.tif250165 TIFF2025109712000003.tif250162 TIFF2025109712000004.tif249165 TIFF2025109712000005.tif249164 TIFF2025109712000006.tif100170

[0070] b. Safety switches and monoclonal antibody-specific epitopes Safety switches It can be seen that adverse events can be minimized by transducing immune cells (containing one or more CARs) with a suicide gene other than the rituximab-binding epitope. Also, it may be desirable to incorporate an inducible "on" or "promoter" switch into the immune cells. Suitable techniques include the use of inducible caspase-9 (U.S. Appl. 2011 / 0286980) or thymidine kinase, either before, after, or simultaneously with the cells being transduced with the CAR constructs of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

[0071] According to the present disclosure, additional on-off or other types of control switch technologies can be incorporated herein. These technologies can employ the use of such dimerization domains of domain dimerization and any activators. These technologies include, for example, those described by Wu et al., Science 2014 350(6258), which utilize the FKBP / rapalog dimerization system in certain cells, the content of which is hereby incorporated by reference in its entirety. Additional dimerization technologies are described, for example, in Fegan et al. Chem.Rev.2010, 110, 3315-3336, and U.S. Pat. Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the content of which is also hereby incorporated by reference in its entirety. Additional dimerization pairs can include cyclosporin-A / cyclophilin, receptors, estrogen / estrogen receptor (optionally using tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, vitamin D / vitamin D receptor. Further examples of dimerization technologies are described, for example, in WO2014 / 127261, WO2015 / 090229, US2014 / 0286987, US2015 / 0266973, US2016 / 0046700, U.S. Pat. No. 8,486,693, US2014 / 0171649, and US2012 / 0130076, the content of which is hereby incorporated by reference in its entirety.

[0072] In some embodiments, the CAR immune cells (e.g., CAR-T cells) of the present disclosure comprise a polynucleotide encoding a suicide polypeptide lacking rituximab binding. In some embodiments, the suicide peptide comprises a mutant RQR8 sequence. See, for example, WO2013153391A, the content of which is hereby incorporated by reference in its entirety. In CAR immune cells (e.g., CAR-T cells) comprising the polynucleotide, the suicide polypeptide is expressed on the surface of the CAR immune cells (e.g., CAR-T cells). In some embodiments, the suicide polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19 CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 19).

[0073] The suicide polypeptide may also include a signal peptide at the amino terminus, for example, MGTSLLCWMALCLLGADHADA (SEQ ID NO: 20). In some embodiments, the suicide polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21 and includes the signal sequence of SEQ ID NO: 20. MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 21).

[0074] In certain embodiments, the suicide peptide comprises an amino acid sequence that includes one or more mutant residues, inserted residues, or deleted residues that reduce or eliminate binding of rituximab.

[0075] When the suicide polypeptide is expressed on the surface of CAR immune cells (e.g., CAR-T cells), an antibody that binds to the suicide gene epitope of the polypeptide causes lysis of the cells. Depletion of CD19-specific CAR immune cells (e.g., CAR-T cells) can occur in vivo, for example, by administering a suicide agent to the patient. The decision to eliminate the introduced cells can result from undesirable effects detected in the patient that are attributable to the introduced cells, such as when an unacceptable level of toxicity is detected. As used herein, a "suicide agent" refers to a molecule that binds to CAR immune cells and causes lysis of CAR-expressing immune cells.

[0076] In some embodiments, the suicide polypeptide is expressed on the surface of the cell. In some embodiments, the suicide polypeptide is included in the CAR construct. In some embodiments, the suicide polypeptide is not part of the CD19 CAR construct.

[0077] In some embodiments, the extracellular domain of any one of the CD19-specific CARs disclosed herein may include one or more epitopes specific to a monoclonal antibody (i.e., specifically recognized by a monoclonal antibody). These epitopes are also referred to herein as mAb-specific epitopes. Examples of mAb-specific epitopes are disclosed in International Patent Publication No. WO2016 / 120216, which is incorporated herein by reference in its entirety. In these embodiments, the extracellular domain of the CAR includes an antigen-binding domain that specifically binds to CD19 and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). The CARs containing mAb-specific epitopes can be single-chain or multi-chain.

[0078] The inclusion of epitopes specific to monoclonal antibodies in the extracellular domain of the CARs described herein enables the sorting and depletion of engineered immune cells expressing the CAR. In some embodiments, this function also promotes the recovery of endogenous CD19-expressing cells depleted by the administration of engineered immune cells expressing the CAR. In some embodiments, enabling depletion provides, for example, a safety switch in the event of adverse effects upon administration to a subject.

[0079] Accordingly, in some embodiments, the present disclosure relates to methods of sorting and / or depleting engineered immune cells bearing a CAR containing an mAb-specific epitope, as well as methods of promoting the recovery of endogenous CD19-expressing cells.

[0080] Using some epitope - monoclonal antibody bindings, CARs containing monoclonal - antibody - specific epitopes can be generated, in particular, CARs that have already been approved for medical use or use in GMP manufacturing, such as, by way of non - limiting example, the CD34 epitope / QBEND - 10, can be generated.

[0081] The present disclosure also encompasses a method of sorting engineered immune cells endowed with a CD19 - specific CAR that expresses an mAb - specific epitope, and a therapeutic method in which the activation of engineered immune cells endowed with these CARs is regulated by depleting cells using an antibody that targets the external ligand - binding domain of the aforementioned CAR. Table 2 provides exemplary mimotope sequences that can be inserted into the extracellular domain of the CARs of the present disclosure. [Table 2] Table 2: Exemplary mimotope sequences TIFF2025109712000007.tif104154

[0082] In certain embodiments, the CAR comprises an epitope or mimotope amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the epitope or mimotope amino acid sequences described herein in Table 2. In certain embodiments, the CAR comprises an epitope or mimotope amino acid sequence that is not, or does not contain, the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the CAR comprises an epitope or mimotope that comprises the amino acid sequence of SEQ ID NO: 30.

[0083] c. Hinge domain The extracellular domain of the CAR of the present disclosure may include a "hinge" domain (or hinge region). The term generally includes any polypeptide that functions to link the transmembrane domain in the CAR to the extracellular antigen-binding domain in the CAR. In particular, the hinge domain can be used to provide greater flexibility and accessibility to the extracellular antigen-binding domain.

[0084] The hinge domain can contain up to 300 amino acids, and in some embodiments contains 10-100 amino acids, or in some embodiments contains 25-50 amino acids. The hinge domain can arise from all or part of a natural molecule derived from the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (in particular, all or part of the hinge region of IgG, i.e., can include some or all or fragments of members of immunoglobulins such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, etc.) or all or part of the constant region of the antibody heavy chain. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence or a fully synthetic hinge sequence. In some embodiments, the aforementioned hinge domain is part of the human CD8α chain (e.g., NP_001139345.1). In another specific embodiment, the aforementioned hinge domain and transmembrane domain include part of the human CD8α chain. In some embodiments, the hinge domain of the CAR described herein includes a sub-sequence of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα, particularly the hinge region of any of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain includes the human CD8α hinge, the human IgG1 hinge, human IgG4, human PD-1, or the human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein includes an scFv, a human CD8α hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 3 provides exemplary amino acid sequences of hinges provided herein. [Table 3] Table 3: Exemplary Hinges TIFF2025109712000008.tif62125

[0085] In certain embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the hinge domain amino acid sequences described herein in Table 3.

[0086] d. Transmembrane domain The CARs of the present disclosure are designed using a transmembrane domain fused to the extracellular domain of the CAR. This can similarly be fused to the intracellular domain of the CAR. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins and to minimize interaction with other members of the receptor complex. In some embodiments, a short linker can form a bond between any or part of the extracellular domain, transmembrane domain, and intracellular domain of the CAR. In some embodiments, the linker comprises a glycine repeat sequence. In some embodiments, the linker comprises (GGGGS)n, where n is 1, 2, 3, 4, or 5 (SEQ ID NO: 41).

[0087] Suitable transmembrane domains for the CARs disclosed herein have the ability to (a) be expressed, for example, on the surface of immune cells such as lymphocyte cells such as T helper (T h ) cells, cytotoxic T (T c ) cells, T regulatory (T reg ) cells, or natural killer (NK) cells, and / or (b) interact with the extracellular antigen-binding domain and intracellular signaling domain to induce a cellular response in order to direct a cellular response of the immune cell against a target cell.

[0088] The transmembrane domain can be derived from either a natural or a synthetic source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein.

[0089] The transmembrane regions for specific uses in the present disclosure include ligands that specifically bind to CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, toll-like receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or are derived from (including or corresponding to) any combination thereof.

[0090] As a non-limiting example, the transmembrane region may be derived from, for example, in particular Fcγ receptor III or CD proteins, such as the α, β, γ or δ polypeptides that make up the CD3 complex, the IL-2 receptor p55 (α chain), p75 (β chain), or γ chain, a subunit chain of the Fc receptor, or may be part of the T cell receptor. Alternatively, the transmembrane domain may be synthetic and may mainly contain hydrophobic residues such as leucine and valine. In some embodiments, the aforementioned transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[0091] In some embodiments, the transmembrane domain in the CAR of the present disclosure is the CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 35). In some embodiments, the CD8α transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence of SEQ ID NO: 35. In some embodiments, the hinge and transmembrane domain in the CAR of the present disclosure is a CD8α hinge and transmembrane domain comprising the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 36).

[0092] e. Intracellular domain The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide activation of at least one of the normal effector functions of immune cells comprising the CAR. The effector functions of T cells can refer to, for example, cytolytic activity or helper activity including cytokine secretion. In some embodiments, the activating intracellular signaling domain for use in the CAR is, for example, but not limited to, the cytoplasmic sequences of the T cell receptor and co-receptor that act in concert to initiate signaling after engagement of the antigen receptor, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same function.

[0093] Suitable (e.g., activating) intracellular domains include, but are not limited to, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, toll-like receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, and signal transduction domains derived from (or corresponding to) ligands that specifically bind to them, or any combination thereof.

[0094] In addition to the activation domains described above, the intracellular domain of the CARs of the present disclosure can incorporate co-stimulatory signaling domains (alternatively shown herein as co-stimulatory molecules) to increase their potency. The co-stimulatory domains can provide signals in addition to the primary signal provided by the activating molecules described herein.

[0095] Suitable co-stimulatory domains within the scope of the present disclosure include, for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1(CD11a / CD18)), CD247, CD276(B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signal transduction lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof.It is understood that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0096] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the 41BB / CD137 domain by itself or may be combined with any other desired intracellular domain useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of 41BB / CD137 is described in NCBI reference sequence: NP_001552.2. The complete native 41BB / CD137 nucleic acid sequence is described in NCBI reference sequence: NM_001561.5.

[0097] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the CD28 domain by itself or may be combined with any other desired intracellular domain useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI reference sequence: NM_006139.1.

[0098] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the CD3 zeta domain by itself or may be combined with any other desired intracellular domain useful in the context of the CARs of the present disclosure. In some embodiments, the intracellular signaling domain of the CAR may include a CD3ζ signaling domain having an amino acid sequence with at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 38. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a portion of a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CARs of the present disclosure can be linked to each other in a random or specific order. In some embodiments, the intracellular domain is designed to include the activation domain of CD3 zeta and the signaling domain of CD28.

[0099] In some embodiments, the intracellular domain is designed to include the activation domain of CD3 zeta and the signaling domain of 4-1BB. In some embodiments, 4-1BB (intracellular domain) has the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 37).

[0100] The CD3 zeta amino acid sequence may include SEQ ID NO: 38. LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 38).

[0101] In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes the domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes a portion of a costimulatory molecule selected from the group consisting of fragments of 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of the CAR includes an amino acid sequence having at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequences shown in SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes an amino acid sequence having at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 37 and / or at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 38.

[0102] In an exemplary embodiment, the CAR of the present disclosure comprises, from the N-terminus to the C-terminus: a CD8α signal sequence, a CD19 scFv, a CD8α hinge and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain.

[0103] III. Immune Cells Comprising CAR a. Immune Cells Engineered immune cells that express the CAR of the present disclosure (e.g., CAR-T cells) are provided herein.

[0104] In some embodiments, the engineered immune cells comprise a population of CARs, each CAR comprising a different extracellular antigen-binding domain. In some embodiments, the immune cells comprise a population of CARs, each CAR comprising an extracellular antigen-binding domain.

[0105] The engineered immune cells can be allogeneic or autologous.

[0106] In some embodiments, the engineered immune cells are T cells (e.g., inflammatory T lymphocyte cells, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor-infiltrating lymphocytes (TIL)), NK cells, NK-T cells, TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the cells can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are gamma delta T cells. In some exemplary embodiments, the engineered immune cells are macrophages.

[0107] In some embodiments, the engineered immune cells can be derived from, for example but not limited to, stem cells. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0108] In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells. In some embodiments, the cells are transfected or transduced by a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistic (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplex.

[0109] In some embodiments, the engineered immune cells expressing the CD19-specific CAR of the present disclosure comprise a proportion of stem cell memory and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the engineered immune cells expressing the CD19-specific CAR of the present disclosure comprise a proportion of stem cell memory and central memory cells of about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, about 90% to about 100%, about 25% to about 50%, about 75% to about 100%, or about 50% to about 75%.

[0110] In some embodiments, the immune cells are inflammatory T lymphocytes that express any one of the CARs described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes that express any one of the CARs described herein. In some embodiments, the immune cells are regulatory T lymphocytes that express any one of the CARs described herein. In some embodiments, the immune cells are helper T lymphocytes that express any one of the CARs described herein.

[0111] Prior to expansion and genetic modification, the cell source can be obtained from a subject via a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, stem cells or iPSC-derived T cells or NK cells, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those of skill in the art can be used. In some embodiments, the cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In some embodiments, the cells may be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0112] Also provided herein are cell lines obtained from transformed immune cells (e.g., T cells) according to any of the methods described above. Also provided herein are modified cells resistant to immunosuppressive therapy. In some embodiments, the isolated cells according to the present disclosure comprise a polynucleotide encoding a CAR.

[0113] The immune cells of the present disclosure can be activated and proliferated using generally known methods, either before or after genetic modification of the immune cells. Generally, the engineered immune cells of the present disclosure can be proliferated, for example, by contacting T cells with an agent that stimulates the CD3 TCR complex and co-stimulatory molecules on the surface of the T cells to create an activation signal for the T cells. For example, chemical substances such as mitogenic lectins such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or phytohemagglutinin (PHA) can be used to create an activation signal for T cells.

[0114] In some embodiments, the T cell population can be stimulated in vitro by contacting, for example, with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD28 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For co-stimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. The anti-CD3 antibody and the anti-CD28 antibody can be disposed on beads or plates or other substrates. Conditions appropriate for T cell culture include an appropriate medium (e.g., minimum essential medium or RPMI medium 1640, or X-vivo 15, (Lonza)), which may contain factors necessary for growth and viability, and may include serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF beta, and TNF or any other additives for cell growth well-known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer with additional amino acids, sodium pyruvate, and vitamins, and can be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or a sufficient amount of cytokines for the growth of T cells (e.g., IL-7 and / or IL-15). Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in cultures of cells to be injected into a subject. 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 different stimulation times may exhibit different characteristics. In some embodiments, the cells of the present disclosure can be grown by co-culturing with a tissue or cells. The cells can also be grown in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0115] In some embodiments, the engineered immune cells according to the present disclosure may include one or more damaged or inactivated genes. In some embodiments, the engineered immune cells according to the present disclosure include one damaged or inactivated gene selected from the group consisting of CD52, CD19, GR, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and / or express a CAR, a multi-chain CAR, and / or a pTα transgene. In some embodiments, the isolated cells include a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to the present disclosure include two damaged or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, CD19 and CD52, CD19 and TCRα, CD19 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ and / or express a CAR, a multi-chain CAR, and a pTα transgene. In some embodiments, the method includes disrupting or inactivating one or more genes by introducing an endonuclease into the cell, which can selectively inactivate genes by selective DNA cleavage.In some embodiments, the endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE nuclease), or a CRIPR (e.g., Cas9) endonuclease.

[0116] In some embodiments, the TCR does not function in the cells according to the present disclosure by disrupting or inactivating the TCRα gene and / or the TCRβ gene. In some embodiments, a method for obtaining modified cells derived from an individual is provided, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway are readily obtained by the present method and are therefore encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used for the treatment of patients in need thereof against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Thus, within the scope of the present disclosure, a method for treating a patient in need thereof against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) comprises treating the aforementioned patient by administering to the aforementioned patient an effective amount of modified cells comprising a disrupted or inactivated TCRα gene and / or a TCRβ gene.

[0117] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agent may be, for example, a purine nucleotide analog (PNA), and thus, generate immune cells suitable for cancer treatment that combines adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, cladribine, fludarabine, cyclophosphamide, and cytarabine, either alone or in combination. The PNA is metabolized by deoxycytidine kinase (dCK) into monophosphate PNA, diphosphate PNA, and triphosphate PNA. Their triphosphate forms compete with ATP for DNA synthesis, act as apoptosis promoters, and are potent inhibitors of ribonucleotide reductase (RNR) involved in trinucleotide production. CD19-specific CAR-T cells containing a disrupted or inactivated dCK gene are provided herein. In some embodiments, the dCK knockout cells are generated, for example, by transfection of T cells using a polynucleotide encoding a TAL nuclease specific for the dCK gene by electroporation of mRNA. The dCK knockout CD19-specific CAR-T cells are resistant to PNA containing, for example, cladribine and / or fludarabine, and maintain T cell cytotoxic activity against CD19-expressing cells.

[0118] In some embodiments, the isolated cells or cell lines of the present disclosure may contain pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRα gene.

[0119] The present disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein.

[0120] c. Preparation method Methods for making the CARs and CAR-containing immune cells of the present disclosure are provided herein. Various known techniques can be utilized for the preparation of polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, etc. according to the present disclosure.

[0121] Polynucleotides and Vectors In some embodiments, the CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector may also contain a selectable marker, for example, that provides for the identification and / or selection of cells that have received the vector.

[0122] The CAR polypeptide can be synthesized in situ within the cell after introduction of the polynucleotide encoding the CAR polypeptide into the cell. Alternatively, the CAR polypeptide can be produced outside of the cell and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, a stable transformation method (for example, using a lentiviral vector) can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used to transiently express the polynucleotide construct, and polynucleotide constructs that are not integrated into the genome of the cell. In other embodiments, a virus-mediated method can be used. The polynucleotide can be introduced into the cell by any suitable means, for example, a recombinant viral vector (for example, a retrovirus, an adenovirus), liposomes, or the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide may be contained in a vector such as a plasmid vector or a viral vector.

[0123] In some embodiments, an isolated nucleic acid is provided that comprises a promoter operably linked to a first polynucleotide encoding a CD19 antigen-binding domain, at least one co-stimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a murine leukemia virus vector, an SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid.

[0124] In one aspect, the present disclosure provides a polynucleotide sequence comprising a promoter capable of expressing a CAR transgene in mammalian T cells. In some embodiments, the promoter is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex involved in the enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into lentiviral vectors. See, for example, Milone et al., Mol. Ther. 17(8):1453-1464 (2009). In some embodiments, the EF1a promoter comprises the sequence provided as SEQ ID NO: 15. TIFF2025109712000009.tif143170

[0125] The EF1a promoter sequence shown above includes the first exon (bold) and the first intron (underlined, SEQ ID NO: 39) of the EF1a gene, followed by the N-terminal portion of the second exon. In some embodiments, the polynucleotides provided herein include a short EF1a promoter. In some embodiments, the polynucleotides provided herein include an EF1a promoter that is shorter than the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the polynucleotides provided herein include an EF1a promoter that does not include the first intron of the EF1a gene. In some embodiments, the polynucleotides provided herein include an EF1a promoter that does not include the nucleic acid sequence of SEQ ID NO: 39.

[0126] In some embodiments, the promoter includes the sequence provided as SEQ ID NO: 16. GCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG (SEQ ID NO: 16)

[0127] Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. Cells expressing the CD19 CAR can be derived from an allogeneic or autologous process.

[0128] Raw materials In some embodiments, the immune cells include T cells. T cells can be obtained from a number of sources including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, the T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ separation.

[0129] Cells can be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing.

[0130] In certain embodiments, T cells are isolated from PBMCs, for example, using centrifugation through a PERCOLL™ gradient, by lysing red blood cells and depleting monocytes. Specific subpopulations of T cells (e.g., CD28+, CD4+, CD8+, CD45RA−, and CD45RO+ T cells or CD28+, CD4+, CD8+, CD45RA−, CD45RO+, and CD62L+ T cells) can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers specific to the negatively selected cells. One method for use herein is negative magnetic immunoadherence or cell sorting and / or selection via flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the cell population of interest for use in the present disclosure.

[0131] PBMCs can be used directly for genetic modification with immune cells (such as CAR or TCR) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be classified into naive, memory, and effector T cell subsets, either before or after genetic modification and / or expansion.

[0132] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO−, CD62L+, CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In certain embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be classified into naive, central memory, and effector cells by identifying cell populations having cell surface antigens.

[0133] Stem cell-derived immune cells In some embodiments, the immune cells can be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Suitable HSCs, ES cells, iPS cells, and other stem cells can be cultured from immortalized cell lines or isolated directly from a patient. Various methods for isolating, expressing, and / or culturing stem cells are known in the art and can be used in the practice of the present invention.

[0134] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the starting material may be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. The starting material can be embryonic stem cells. The starting material can be B cells, or any other cells of any other somatic cell type of a peripheral blood mononuclear cell isolate, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells.

[0135] Genetic Modification of Isolated Cells Immune cells such as T cells can be genetically modified after isolation using known methods, or immune cells can be activated and proliferated (or differentiated in the case of progenitor cells) in vitro before being genetically modified. In some embodiments, isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRα and / or CD52. In some embodiments, cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, zinc finger nucleases (ZFNs), TALENs, MegaTALs, meganucleases) to reduce or eliminate the expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells such as T cells are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro. Methods for activating and proliferating T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, and PCT WO2012 / 079000, the contents of which are incorporated herein by reference in their entirety. Generally, such methods further comprise contacting PBMCs or isolated T cells with a stimulating molecule and a costimulatory molecule, such as an anti-CD3 antibody and an anti-CD28 antibody, generally attached to beads or other surfaces, in a culture medium having an appropriate cytokine such as IL-2. The anti-CD3 antibody and anti-CD28 antibody bound to the beads serve as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator for the physiological activation of human T cells. In other embodiments, methods described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and WO2012129514, the contents of which are incorporated herein by reference in their entirety, are used to activate and stimulate T cells to proliferate with feeder cells and appropriate antibodies and cytokines.

[0136] Certain methods for constructing and engineering the immune cells disclosed herein are described in PCT application PCT / US15 / 14520, the content of which is incorporated herein by reference in its entirety.

[0137] It will be appreciated that PBMCs may further comprise other cytotoxic lymphocytes such as NK cells or NKT cells. Expression vectors carrying the coding sequences of the chimeric receptors disclosed herein can be introduced into populations of human donor T cells, NK cells, or NKT cells. T cells successfully transduced with the expression vector are sorted using flow cytometry to isolate CD3-positive T cells, which are then further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibody and IL-2 or other methods known in the art described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing CARs for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of products derived from non-human animals such as fetal calf serum and fetal bovine serum.

[0138] For cloning of polynucleotides, a vector can be introduced into a host cell (an isolated host cell) to enable replication of the vector itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors can generally contain sequence components, including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, the origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0139] In certain embodiments, the present disclosure provides an isolated host cell containing the vectors provided herein. A host cell containing a vector can be useful for the expression or cloning of a polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, for example, eubacteria such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such as the genus Escherichia, for example, Escherichia coli, the genus Enterobacter, the genus Erwinia, the genus Klebsiella, the genus Proteus, the genus Salmonella, for example, Salmonella typhimurium, the genus Serratia, for example, Serratia marcescens, and the genus Shigella, as well as bacilli such as Bacillus subtilis and B. licheniformis, Pseudomonas such as Pseudomonas aeruginosa, and the genus Streptomyces, but are not limited thereto.

[0140] The vector can be introduced into the host cell using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, particle bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of the vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used when genetically modifying a donor population of immune effector cells, and each vector encodes a different CAR disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, and the proportion of engineered cells expresses one or more different CARs.

[0141]

[0142] In some embodiments, the vector comprises a lentiviral vector. A lentiviral vector containing the CAR coding sequence can be introduced into a lentiviral packaging cell line, and the lentivirus produced by the packaging cell line can be used for transducing T cells to generate CAR-T cells. To produce a lentivirus encoding a CAR, HEK-293T cells can be seeded at 400,000 cells / mL on day 0 in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific) per well of a 6-well plate. On day 1, lentivirus can be prepared by mixing 1.5 μg of psPAX2, 0.5 μg of pMD2G, and 0.5 μg of the appropriate transfer CAR vector in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 μL of Lipofectamine 2000 (Invitrogen) in 250 μL of Opti-MEM can be incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture can be incubated at room temperature for 20 minutes and added slowly to the side of the well containing HEK-293T with a total volume of 500 μL. General methods for CAR-containing lentivirus production and transduction are generally known in the art, see, for example, Milone et al., Leukemia, 2018, 32:1529-1541; Sanber et al., Construction of stable packaging cell lines for clinical lentiviral vector production, Nature 2015, DOI:10.1038, Roddie et al., Cytotherapy 2019, 21:327-340, all of which are incorporated herein by reference in their entirety. In one embodiment, the present disclosure provides a method of preserving genetically engineered cells that express a TCR targeting a CAR or CD19 protein. This involves cryopreserving immune cells such that the cells continue to survive upon thawing.The fraction of immune cells expressing CAR is cryopreserved by methods known in the art, providing a permanent source of such cells for future treatment of patients suffering from malignant tumors. If necessary, the cryopreserved transformed immune cells can be thawed, expanded, and propagated to obtain more of such cells.

[0143] In some embodiments, the cells are formulated by first recovering the cells from their culture medium and then washing and concentrating the cells in a medium and container system suitable for administration at a therapeutically effective amount (in a pharmaceutically acceptable carrier). Suitable infusion media are any isotonic media formulations, typically saline, Normosol® R (Abbott), or Plasma-Lyte® A (Baxter), but 5% dextrose in water or Ringer's lactate can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0144] Allogeneic CAR T cells The process for manufacturing allogeneic CAR T therapies, or AlloCARs®, involves obtaining healthy, selected, screened, and tested T cells from healthy donors. The T cells are then engineered to express a CAR that recognizes a specific cell surface protein (e.g., CD19) expressed on hematological tumors or solid tumors. The allogeneic T cells are gene-edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene can be knocked out to render the CAR T product resistant to anti-CD52 antibody therapy. Thus, anti-CD52 antibody therapy can be used to suppress the host immune system so that the CAR T can engraft and achieve a complete therapeutic effect. The engineered T cells then undergo a purification process and are ultimately cryopreserved in vials for delivery to patients.

[0145] Autologous CAR T cells Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting a patient's own cells (e.g., white blood cells including T cells), genetically engineering the T cells to express a CAR that recognizes a target expressed on the cell surface of one or more specific cancer cells and kills the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient.

[0146] IV. Treatment Methods The present disclosure includes methods of treating or preventing a condition associated with undesired and / or increased CD19 levels in a patient, comprising administering to a patient in need thereof an effective amount of at least one CAR, or an immune cell comprising a CAR disclosed herein.

[0147] Methods for treating a disease or disorder including cancer are provided. In some embodiments, the present disclosure relates to generating a T cell-mediated immune response in a subject and comprises administering to the subject an effective amount of the engineered immune cells of the present application. In some embodiments, the T cell-mediated immune response is directed against target cells. In some embodiments, the engineered immune cells comprise a chimeric antigen receptor (CAR). In some embodiments, the target cells are tumor cells. In some aspects, the present disclosure includes methods for treating or preventing a malignant tumor, the foregoing methods comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding domain described herein. In some aspects, the present disclosure includes methods for treating or preventing a malignant tumor, the foregoing methods comprising administering to a subject in need thereof an effective amount of at least one immune cell, the immune cell comprising at least one chimeric antigen receptor, a T cell receptor, and / or an isolated antigen-binding domain described herein.

[0148] The CAR containing immune cells of the present disclosure can be used to treat malignant tumors involved in the abnormal expression of CD19. In some embodiments, the CAR containing immune cells of the present disclosure can be used to treat cancer. As used herein, the term "cancer" includes, but is not limited to, solid tumors and hematopoietic tumors. The term "cancer" refers to diseases of skin tissue, organs, blood, and blood vessels, including, but not limited to, cancers of the bladder, bone or blood, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lymph node, lung, mouth, neck, ovary, pancreas, prostate, rectum, stomach, testis, larynx, and uterus. Specific cancers include, but are not limited to: advanced malignant tumors, amyloidosis, neuroblastoma, meningioma, perivascular cell tumor, multiple brain transfer enzymes, glioblastoma multiforme, glioblastoma, brainstem glioma, malignant brain tumor with poor prognosis, malignant glioma, recurrent glioma, undifferentiated astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C&D colorectal cancer, unresectable colorectal cancer, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karyotype acute myeloblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), cutaneous T cell lymphoma, cutaneous B cell lymphoma, diffuse large B cell lymphoma, low-grade follicular lymphoma, malignant melanoma, malignant mesothelioma, malignant pleural mesothelioma syndrome, peritoneal cancer, papillary serous carcinoma, gynecological sarcoma, soft tissue sarcoma, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, progressive ossifying fibrodysplasia, hormone-resistant prostate cancer, resected high-risk soft tissue sarcoma, unresectable hepatocellular carcinoma, Waldenström's hypergammaglobulinemia, indolent myeloma, slowly progressive myeloma, fallopian tube cancer, androgen-independent prostate cancer, androgen-dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, and leiomyoma. In certain embodiments, the cancer is metastatic. In another embodiment, the cancer is refractory or resistant to chemotherapy or radiation.

[0149] In exemplary embodiments, for example, CAR-containing immune cells, such as the CAR-T cells of the present disclosure, are used to treat NHL.

[0150] Also, a method for reducing the size of a tumor in a subject, comprising administering to the subject the engineered cells of the present disclosure, wherein the cells comprise a chimeric antigen receptor comprising a CD19 antigen-binding domain and bind to the CD19 antigen on the tumor.

[0151] In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the engineered cells are delivered to the tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are xenogeneic immune cells, such as xenogeneic T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells of the present application are transfected or transduced ex vivo. As used herein, the term "in vitro cell" refers to any cell cultured ex vivo.

[0152] A "therapeutically effective amount", "effective dose", "effective amount" or "therapeutically effective dose" of a therapeutic agent, such as engineered CART cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease, or increases the frequency and duration of disease-free periods, as indicated by a decrease in the severity of symptoms of the disease or by promoting disease regression, or prevents functional impairment or disability due to the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0153] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as those with a formally diagnosed disorder, those without a formally recognized disorder, those that draw medical attention, those at risk of developing a disorder, and the like.

[0154] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and uses that reduce the risk that a subject will develop a disorder or other risk factor. Treatment does not require complete cure of the disorder and encompasses embodiments that reduce symptoms or underlying risk factors. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of the event occurring is reduced in the presence of a compound or method.

[0155] A desired therapeutic amount of cells in a composition is generally at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset), or more typically 10 2 cells or more, up to 10 6 cells, up to 10 8 cells or 10 9 cells, and can be 10 10 cells or more. The number of cells depends on the desired use for which the composition is intended and the type of cells contained therein. The desired density of cells is typically greater than 10 6 cells / ml, generally greater than 10 7 cells / ml, generally greater than 10 8 cells / ml. A clinically significant number of immune cells can be distributed over multiple injections that cumulatively equal or exceed 10 5 10 6 10 7 10 8 10 9 10 10 10 11 or 10 12 cells. In some aspects of the disclosure, particularly where all injected cells are redirected to a specific target antigen (CD19), 10 6 / kilogram (10 per patient6 ~10 11 It is possible to administer a smaller number of cells within the range of ( ). CAR therapy can be administered multiple times at doses within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient receiving the therapy.

[0156] In some embodiments, the therapeutically effective amount of CAR T cells is about 1X10 5 cells / kg, about 2X10 5 cells / kg, about 3X10 5 cells / kg, about 4X10 5 cells / kg, about 5X10 5 cells / kg, about 6X10 5 cells / kg, about 7X10 5 cells / kg, about 8X10 5 cells / kg, about 9X10 5 cells / kg, 2X10 6 cells / kg, about 3X10 6 cells / kg, about 4X10 6 cells / kg, about 5X10 6 cells / kg, about 6X10 6 cells / kg, about 7X10 6 cells / kg, about 8X10 6 cells / kg, about 9X10 6 cells / kg, about 1X10 7 cells / kg, about 2X10 7 cells / kg, about 3X10 7 cells / kg, about 4X10 7 cells / kg, about 5X10 7 cells / kg, about 6X10 7 cells / kg, about 7X10 7 cells / kg, about 8X10 7 cells / kg, or about 9X10 7 cells / kg.

[0157] In some embodiments, the target dose of CAR+ / CAR-T+ / TCR+ cells is 1×10 6 ~2×10 8 cells / kg, for example, 2×10 6It is in the range of cells / kg. Doses above and below this range may be appropriate for a particular subject, and the appropriate dose level can be determined by medical personnel as needed. Additionally, multiple doses of cells can be provided in accordance with the present disclosure.

[0158] In some aspects, the present disclosure includes a pharmaceutical composition comprising at least one antigen-binding domain described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0159] The CAR-expressing cell population of the present disclosure can be administered as a pharmaceutical composition alone, or in combination with a diluent, and / or in combination with other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present disclosure can comprise a CAR- or TCR-expressing cell population, such as the T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffered solutions such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, amino acids such as polypeptides or glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0160] The pharmaceutical composition (solution, suspension, etc.) may contain one or more of the following: sterile diluents such as water for injection, physiological saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides that can serve as solvents or suspension media, polyethylene glycol, glycerin, propylene glycol or other solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

[0161] In some embodiments, when administered to a patient, engineered immune cells that express any one of the CD19-specific CARs described herein on their cell surface can reduce, kill, or lyse the patient's endogenous CD19-expressing cells. In one embodiment, the rate of reduction or lysis of endogenous CD19-expressing cells or cells of a cell line expressing CD19 by engineered immune cells expressing any one of the CD19-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 95%. In one embodiment, the rate of reduction or lysis of endogenous CD19-expressing cells or cells of a cell line expressing CD19 by engineered immune cells expressing any one of the CD19-specific CARs described herein is from about 5% to about 95%, from about 10% to about 95%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 25% to about 75%, or from about 25% to about 60%. In one embodiment, the endogenous CD19-expressing cells are endogenous CD19-expressing myeloid cells.

[0162] One embodiment can measure the rate of reduction or lysis of target cells, such as a cell line expressing CD19, by engineered immune cells expressing the CD19-specific CAR of the present disclosure on their cell surface membrane, using the assays disclosed herein.

[0163] The method can further comprise administering one or more chemotherapeutic agents. In certain embodiments, the chemotherapeutic agent is lymphodepleting (pre-conditioning) chemotherapy. For example, a particular beneficial dose of cyclophosphamide (200 mg / m 2 / day to 2000 mg / m 2 / day, about 100 mg / m 2 / day to about 2000 mg / m 2 / day, for example, about 100 mg / m2 / day, about 200 mg / m 2 / day, about 300 mg / m 2 / day, about 400 mg / m 2 / day, about 500 mg / m 2 / day, about 600 mg / m 2 / day, about 700 mg / m 2 / day, about 800 mg / m 2 / day, about 900 mg / m 2 / day, about 1000 mg / m 2 / day, about 1500 mg / m 2 / day or about 2000 mg / m 2 / day) and a specific dose of fludarabine (20 mg / m 2 / day to 900 mg / m 2 / day, about 10 mg / m 2 / day to about 900 mg / m 2 / day; for example, about 10 mg / m 2 / day, about 20 mg / m 2 / day, about 30 mg / m 2 / day, about 40 mg / m 2 / day, about 40 mg / m 2 / day, about 50 mg / m 2 / day, about 60 mg / m 2 / day, about 70 mg / m 2 / day, about 80 mg / m 2 / day, about 90 mg / m 2 / day, about 100 mg / m 2 / day, about 500 mg / m 2 / day or about 900 mg / m 2 / day) and conditioning a patient in need of T cell therapy that includes administering to the patient. A preferred dosing regimen is about 300 mg / m 2 / day of cyclophosphamide and about 30 mg / m 2 / day of fludarabine for 3 days, including treating the patient.

[0164] In some embodiments, lymphodepletion further includes administration of a CD52 antibody. In some embodiments, the CD52 antibody is administered at an intravenous dosing of about 13 mg / day.

[0165] In other embodiments, the antigen-binding domain, the transduced (or otherwise engineered) cells, and the chemotherapeutic agent are each administered in an amount effective to treat the subject's disease or condition.

[0166] In certain embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimine and methylamelamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novobiocin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.; nitrosoureas; antibiotics such as aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, carzinophilin, carzinomycin, carminomycin, cardifilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodomycin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioguanine, thiamiprine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens, such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testosterone; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid, aceglutamide; aldophosphamide glycoside; aminolevulinic acid, amsacrine; bestrabucil; bisantrene; edatraxate, defofamine; demeclocycline; diaziquone; elformithine; elliptinium acetate, etoglucid; gallium nitrate, hydroxyurea; lentinan; lonidamine; mitoguazone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid, 2-ethylhydrazide; procarbazine; PSK (registered trademark), razoxane; sizofiran; spirogermanium; tenuazonic acid, triaziquone; 2,2’,2’-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C), cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL (trademark), Bristol-Myers Squibb) and docetaxel (TAXOTERE (registered trademark), Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs, such as mercaptopurine, methotrexate cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16), ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinoid acid derivatives, such as targretin (trademark) (bexarotene), panretin (trademark), (alitretinoin); ONTAK (trademark) (denileukin diftitox); esperamicin, capecitabine;Also included are any of the aforementioned pharmaceutically acceptable salts, acids or derivatives. This definition includes, for example, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin, which are anti-hormonal agents that modulate or inhibit the hormonal action on tumors; and also any of the aforementioned pharmaceutically acceptable salts, acids or derivatives. Combinations of chemotherapeutic agents are also administered, where appropriate, and include, but are not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.;

[0167] In some embodiments, the chemotherapeutic agent is administered after, simultaneously with, or within one week of administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month prior to administering the cell, polypeptide, or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0168] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0169] Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (Kadcyla®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tibosutinib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, luxolutinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as CDK inhibitor (palbociclib).

[0170] In some embodiments, the composition comprising CAR-containing immune cells can be administered in a treatment regimen to prevent cytokine release syndrome (CRS) or neurotoxicity. The treatment regimen for preventing cytokine release syndrome (CRS) or neurotoxicity can include, but is not limited to, ranizumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, the composition comprising CAR-containing immune cells can be administered with anti-inflammatory agents. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and non-steroidal anti-inflammatory drugs (NSAIDs) including mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialic acid. Exemplary analgesics include acetaminophen, oxycodone, tramadol of propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, e.g., TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0171] In certain embodiments, the compositions described herein are administered in combination with a cytokine. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibiting substance, mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet-derived growth factor; transforming growth factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and -II; erythropoietin (EPO); osteogenic factor; interferons-alpha, beta, and -gamma; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture, as well as biologically active equivalents of native sequence cytokines.

[0172] V. Methods of Sorting and Depletion In some embodiments, a method of in vitro sorting of an immune cell population is provided, wherein a subset of the immune cell population comprises engineered immune cells expressing any one of CD19-specific CARs comprising an epitope specific for a monoclonal antibody (e.g., an exemplary mimotope sequence). The method comprises contacting the immune cell population with a monoclonal antibody specific for the epitope and selecting immune cells that bind to the monoclonal antibody to obtain a cell population enriched in engineered immune cells expressing the CD19-specific CAR.

[0173] In some embodiments, the aforementioned monoclonal antibody specific for the aforementioned epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0174] In some embodiments, the aforementioned monoclonal antibody specific for the aforementioned epitope is optionally conjugated to magnetic particles. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by magnetic-activated cell sorting (MACS).

[0175] In some embodiments, the mAb used in the method of sorting immune cells expressing a CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and / or ustekinumab. In some embodiments, the aforementioned mAb is rituximab. In another embodiment, the aforementioned mAb is QBEND-10.

[0176] In some embodiments, the population of CAR-expressing immune cells obtained when using the method of sorting the above-described CAR-expressing immune cells in vitro comprises at least 70%, 75%, 80%, 85%, 90%, 95% CAR-expressing immune cells. In some embodiments, the population of CAR-expressing immune cells obtained when using the in vitro sorting method of CAR-expressing immune cells comprises at least 85% CAR-expressing immune cells.

[0177] In some embodiments, the population of CAR-expressing immune cells obtained when using the above-described in vitro selection method of CAR-expressing immune cells shows an increase in cytotoxic activity in vitro as compared to the initial (non-sorted) cell population. In some embodiments, the aforementioned cytotoxic activity increases by 10%, 20%, 30%, or 50% in vitro. In some embodiments, the immune cells are T cells.

[0178] In some embodiments, the mAb is previously bound on a support or surface. Non-limiting examples of solid supports can include beads, agarose beads, magnetic beads, plastic well plates, glass well plates, ceramic well plates, columns, or cell culture bags.

[0179] The CAR-expressing immune cells administered to the recipient can be concentrated in vitro from the source population. Methods of expanding the source population can include using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting to select cells expressing antigens such as the CD34 antigen.

[0180] Flow cytometry can be used to quantify specific cell types within a cell population. Generally, flow cytometry is a method for quantifying the components or structural features of cells mainly by optical means. Since different cell types can be distinguished by quantifying the structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0181] Flow cytometry analysis involves two main steps: 1) labeling the selected cell type with one or more labeled markers, and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling the cell type includes binding an antibody labeled with a marker expressed by a specific cell type. The antibody can be directly labeled with a fluorescent compound or indirectly labeled, for example, with a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0182] In some embodiments, the method used to sort T cells expressing a CAR is magnetic-activated cell sorting (MACS). Magnetic-activated cell sorting (MACS) is a method for separating various cell populations according to their surface antigens (CD molecules) using superparamagnetic nanoparticles and columns. Using MACS, a pure cell population can be obtained. Cells in a single-cell suspension can be magnetically labeled with microbeads. The sample is applied to a column consisting of ferromagnetic spheres, covered with a cell-friendly coating, allowing for rapid and gentle separation of the cells. Unlabeled cells pass through, while magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0183] Detailed protocols for the purification of specific cell populations, such as T cells, can be found in Basu S et al. (2010). (See Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. (41):1546).

[0184] In some embodiments, the present disclosure provides a method of depleting CD19-specific CAR-expressing immune cells by in vivo depletion. In vivo depletion can include administering a therapeutic agent (e.g., a molecule that binds to an epitope on the CAR) to a mammalian organism, with the aim of inhibiting or eliminating the proliferation of CAR-expressing immune cells.

[0185] One aspect of the invention relates to a method of in vivo depleting engineered immune cells expressing a CD19 CAR comprising an mAb-specific epitope, the method comprising contacting the engineered immune cells or the CAR-expressing immune cells as described above with at least one epitope-specific mAb. Another aspect of the invention relates to a method of in vivo depleting CAR-expressing immune cells comprising a chimeric scFv (e.g., formed by insertion of an mAb-specific epitope) by contacting the engineered immune cells as described above with an epitope-specific antibody. In some embodiments, the immune cells are T cells and / or the antibody is a monoclonal antibody.

[0186] According to one embodiment, in vivo depletion of the engineered cells is performed on the engineered immune cells previously sorted using the in vitro methods of the present disclosure. In this case, the injected mAb can be used. In some embodiments, the mAb-specific antigen is the CD20 antigen, and the epitope-specific mAb is rituximab. In some embodiments, the present invention relates to a method of in vivo depleting engineered immune cells expressing a CAR that contain an mAb-specific epitope (CAR-expressing immune cells) in a patient, the method comprising contacting the aforementioned CAR-expressing immune cells with at least one epitope-specific mAb.

[0187] In some embodiments, the step of contacting the aforementioned engineered immune cells or the aforementioned CAR-expressing immune cells with at least one epitope-specific mAb comprises injecting the epitope-specific mAb (e.g., rituximab) into the patient. In some embodiments, the amount of the epitope-specific mAb administered to the patient is sufficient to deplete at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the CAR-expressing immune cells in the patient.

[0188] In some embodiments, the step of contacting the aforementioned engineered immune cells or the aforementioned CAR-expressing immune cells with at least one epitope-specific mAb comprises injecting 375 mg / m 2 of rituximab into the patient one or more times. In some embodiments, the mAb (e.g., rituximab) is administered once a week.

[0189] In some embodiments, when immune cells expressing a CAR that includes an mAb-specific epitope (CAR-expressing immune cells) are depleted in a complement-dependent cytotoxicity (CDC) assay using an epitope-specific mAb, the amount of surviving CAR-expressing immune cells decreases. In some embodiments, the amount of surviving CAR-expressing immune cells decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the aforementioned mAb-specific epitope is a CD20 epitope or a mimotope, and / or the epitope-specific mAb is rituximab.

[0190] In certain embodiments, depletion of CAR-engineered immune cells in vivo is effected by injecting a bispecific antibody. By definition, a bispecific monoclonal antibody (BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigens. The use of these BsAbs and their use in immunotherapy are outlined in Muller D and Kontermann R.E. (2010) Bispecific Antibodies for Cancer Immunotherapy, BioDrugs 24(2):89-98.

[0191] According to another specific embodiment, the injected bispecific mAb can bind to both the mAb-specific epitope carried by the engineered immune cells expressing a chimeric scFv and the surface antigens of effector and cytotoxic cells (e.g., immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTLs), etc.). By doing so, depletion of the engineered immune cells induced by the BsAb can occur via antibody-dependent cell-mediated cytotoxicity (ADCC). (Deo Y M, Sundarapandiyan K, Keler T, Wallace PK, and Graziano RF, (2000), Journal of Immunology, 165(10):5954-5961]).

[0192] In some embodiments, the cytotoxic agent is conjugated to an epitope-specific mAb that can be used to deplete CAR-expressing immune cells. By combining the targeting ability of the monoclonal antibody with the cancer-killing ability of the cytotoxic drug, an antibody-drug conjugate (ADC) enables discrimination of sensitivity between healthy and diseased tissues as compared to the use of the drug alone. Market approvals have been granted for some ADCs, and in particular the technology for making them, especially on the linker, is described in (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al. (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epeneto (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172, U.S. Patent No. 4,975,278).

[0193] In some embodiments, the injected epitope-specific mAb is pre-conjugated to a molecule that can promote complement-dependent cytotoxicity (CDC). Thus, the complement system helps or complements the ability of the antibody to remove pathogens from the organism. When stimulated, the activation cascade drives as a large-scale amplification of the response and activation of the cell-killing membrane attack complex. Different molecules can be used to bind the mAb, such as glycans [Courtois, A, Gac-Breton, S., Berthou, C, Guezennec, J., Bordron, A. and Boisset, C. (2012), Complement dependent cytotoxicity activity of therapeutic antibody fragments can be acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN:0717-3458; http: / / www.ejbiotechnology.info DOI:10.2225 / voll5-issue5).

[0194] VI. Kits and Manufactured Articles This application provides a kit comprising any one of CD19 containing immune cells containing the CAR or CD19 CAR described herein, and a pharmaceutical composition thereof. In some exemplary embodiments, the kits of the disclosure include allogeneic CD19 CAR-containing T cells and CD52 antibodies for administering a lymphodepletion regiment and a CAR-T regiment to a subject.

[0195] This application also provides a manufactured article comprising any one of the therapeutic compositions or kits described herein. Examples of manufactured articles include vials (e.g., sealed vials).

Examples

[0196] Example 1: Generation of rituximab - resistant CD19 CAR immune cells As shown in Figure 1 and Table 4, a rituximab-resistant anti-CD19 chimeric antigen receptor construct that does not express the rituximab binding site was generated. A lentiviral vector construct was introduced into a viral packaging cell line, and an allogeneic anti-CD19 CAR containing lentivirus was produced.

[0197] Pan T cells (541, 604, 410, and 2593) from 4 human donors were thawed and activated with large-scale T cell TransAct™ (1:15 ratio) at 1.5×10 6 cells / ml in the presence of IL-2 (100 IU / ml). Two days later, 1.5×10 6 cells (3 ml) were transduced with 2 ml of fresh lentivirus containing the vector described in Table 4. A schematic diagram of the modified vector is shown in Figure 1. IL-2 (100 IU / ml) was added on days 0, 2, 5, 7, 9, and 12. 6×10 6 total cells were transferred to a 6-well G-Rex plate on day 5, and the medium was changed on days 9 and 12. The cells were frozen on day 13. [Table 4] Table 4: Rituximab-resistant CD19 CAR vector TIFF2025109712000010.tif52166 co = codon optimization

[0198] Flow cytometry experiments were performed on lymphocytes, viable CD3+, CAR+, CD4 / CD8, and transduced cells gated on downstream markers. Human transduction check and CD34 panels were performed on days 5 and 13 using antibodies against CD3, CD4, CD8, viability, CD34, and anti-idiotype against anti-CD19 CAR (4G7 anti-Id). Figures 2A and 2B show flow cytometry plots demonstrating CAR expression on day 5 from pan T cells transduced with the CAR expression vectors shown in Table 4 using the anti-CD19 CAR anti-Id antibody.

[0199] Flow cytometry using the human phenotype and activation panel was performed on days 9 and 13. The panel included CD3, CD4, CD8, viability, CD45RO, CD62L, CD25, 4-1BB, PD-1, anti-idiotype against the antibody against anti-CD19 CAR, and TIM3. Cells were normalized on day 13 for cell proliferation and final CAR expression from all 4 donors (Figure 3). The data in Figure 3 show that v1.2 had a higher transduction rate (%CAR+), but v1.2 transduced cells showed lower levels of CAR expression (CAR MFI) compared to, for example, v1.0 and v1.1. Time-course cell proliferation and CAR expression of pan T cells from donors 541 (Figure 4A), 604 (Figure 4B), 410 (Figure 4C), 2593 (Figure 4D) transduced with the rituximab-resistant CAR expression vector are shown.

[0200] The CD4 / CD8 ratios of pan T cells on days 5, 9, and 13 from donors 541 (Figure 5A), 604 (Figure 5B), 410 (Figure 5C), and 2593 (Figure 5D) transfected with the rituximab-resistant CAR expression vector were measured. Figures 6A - 6D show the phenotypes and activities of pan T cells on day 9 from donors 541 (Figure 6A), 604 (Figure 6B), 410 (Figure 6C), and 2593 (Figure 6D) transfected with the rituximab-resistant CAR expression vector. Figure 7 shows the phenotypes, activation %CD8+, and T cell anergy measured using TIM3 and PD1 staining averaged from all four donors on day 9. The phenotypes and activities of pan T cells on day 13 from donors 541 (Figure 8A), 604 (Figure 8B), 410 (Figure 8C), and 2593 (Figure 8D) transfected with the rituximab-resistant CAR expression vector were measured. Figure 9 shows the phenotypes, activation %CD8+, and T cell anergy measured using TIM3 and PD1 staining from all four donors on day 13.

[0201] Example 2: Short - term and long - term in vitro killing assays The transfected CAR cells from Example 1 were tested for short-term and long-term killing capabilities. Co-cultures of CAR T cells with Raji cells (2:1 E-to-T) were prepared for later Luminex assays. An average short-term (24-hour) killing assay using Raji cells as target cells was determined for each CAR construct (Figure 10). Figures 11A - 11D show the average long-term killing assays using A549-CD19+ cells as target cells, with an E:T of 8:1 (Figure 11A), 4:1 (Figure 11B), 2:1 (Figure 11C), and 1:1 (Figure 11D) for each CAR construct.

[0202] The killing assay results were analyzed for correlation with phenotypic characteristics. The killing rate on day 7 at an E-to-T of 1:1 was CAR+CD4+41BB+, CAR+CD4+Tim3+ (p = 0.0352), CAR+CD4+TE M+(p = 0.0328), CAR + CD8 + PD - 1+(p = 0.0269), and negatively correlated with %CAR expression (p = 0.0245). The killing rate on day 7 at an E-to-T ratio of 1:1 was for CAR + CD8 + T SCM + and was positively correlated. The killing rate on day 9 at an E-to-T ratio of 1:1 was for CAR + CD4 + Tim3 +, CAR + CD4 + T EM +(p = 0.0031), CAR + CD8 + T cm +(p = 0.0182), and negatively correlated with %CAR expression (p = 0.0469). The killing rate on day 9 at an E-to-T ratio of 1:1 was for CAR + CD8 + T SCM +, CAR + CD8 + Tim3 - PD - 1-(p = 0.0318), and CAR + CD4 + T SCM +(p = 0.0289) and was positively correlated.

[0203] Example 3: Analysis of the titers of lentiviruses containing different lentiviral constructs In this experiment, a lentiviral vector construct was introduced into a viral packaging cell line to generate an anti-CD19 CAR-containing lentivirus, and the titer was determined with Lentigen (Gaithersberg, MD) under the same protocol as in Example 1.

[0204] The lentiviral titer was evaluated either by measuring the physical titer of the viral protein p24 level or by measuring the transduction titer. Unexpectedly, it was found that removing the safety switch RQR8 from the lentiviral construct v1.0 significantly reduced the viral titer (comparing v1.0 to v1.1 in Table 5). When the EF1a promoter of v1.1 was replaced with a short or truncated EF1a promoter such as v1.2 (EF1a (short) promoter), the titer was improved. [Table 5] Table 5 Viral titers of lentiviruses with rituximab-sensitive and -resistant CD19 CAR constructs TIFF2025109712000011.tif36143 1 TU = Transduction Unit

[0205] To analyze the robustness of lentiviral preparations of anti-CD19 CAR v1.0, v1.2, and v1.3, viral titration assays were performed. Serial volume dilutions of the lentiviral preparations of v1.0, v1.2, and v1.3 were analyzed for CAR+ T cell % on day 5 after transduction of pan T cells. The results show that at low dilutions (e.g., 10% v / v), all three constructs showed similar acceptable transduction efficiencies. However, at increased dilutions (e.g., 3.3%, 1.1% v / v), the transduction efficiency of the rituximab-resistant anti-CD19 CAR construct v1.3 decreased more significantly compared to the other rituximab-resistant anti-CD19 CAR construct v1.2. See Figure 12. Construct v1.2 was selected for in vivo analysis.

[0206] Example 4: In vivo titer assay In this experiment, the in vivo antitumor titer of ALLO-501v1.2 was analyzed compared to ALLO-501v1.0 in a mouse tumor model. CD19-positive Raji cells carrying the luciferase reporter gene were injected into NSG mice. Lentivirus containing the lentiviral construct of v1.0 or v1.2 was transduced into pan T cells from two donors 541 and 604. NSG mice were inoculated with 100,000 luciferase Raji cells by tail vein injection. On day 4 after inoculation, the CAR construct was administered to Raji-bearing NSG mice at the indicated doses. The engraftment and progression of Raji were evaluated by intraperitoneal injection of luciferase substrate, followed by measurement of the cumulative luciferase signal. The results are shown in Figure 13A (donor 541) and Figure 13B (donor 604).

[0207] The teachings of the present disclosure have been described with reference to various uses, methods, kits, and compositions, but it will be understood that various changes and modifications can be made without departing from the teachings of this specification and the inventions of the following claims. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the present teachings have been described in terms of these exemplary embodiments, those skilled in the art will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the teachings of the present invention. [Table 6] TIFF2025109712000012.tif250168 TIFF2025109712000013.tif250163 TIFF2025109712000014.tif250165 TIFF2025109712000015.tif251167 TIFF2025109712000016.tif251164 TIFF2025109712000017.tif248161 TIFF2025109712000018.tif250164 TIFF2025109712000019.tif250165 TIFF2025109712000020.tif250167 TIFF2025109712000021.tif147170

Claims

1. An isolated polynucleotide encoding a polypeptide comprising an anti-CD19 chimeric antigen receptor (CAR) that is at least 70% identical to SEQ ID NO: 9, wherein the polypeptide does not contain a rituximab binding site, and the polynucleotide comprises a short EF1a promoter capable of expressing the anti-CD19 chimeric antigen receptor (CAR) in mammalian T cells.

2. The isolated polynucleotide according to claim 1, wherein the short EF1a promoter does not contain an intron containing SEQ ID NO:

15.

3. The isolated polynucleotide according to claim 2, wherein the intron contains the nucleic acid sequence of SEQ ID NO:

39.

4. The isolated polynucleotide according to any one of claims 1 to 3, wherein the promoter contains the nucleic acid sequence of SEQ ID NO:

16.

5. The isolated polynucleotide according to any one of claims 1 to 4, wherein the polypeptide further comprises a safety switch.

6. The isolated polynucleotide according to claim 5, wherein the safety switch is linked to the CD19 CAR using a linker peptide.

7. The isolated polynucleotide according to claim 5, wherein the safety switch is linked to the anti-CD19 CAR using a T2A linker.

8. The isolated polynucleotide according to any one of claims 5 to 7, wherein the safety switch contains an antibody binding site.

9. The isolated polynucleotide according to any one of claims 5 to 7, wherein the safety switch contains a mutant CD20 mimotope.

10. The isolated polynucleotide according to any one of claims 5 to 9, wherein the polypeptide contains a CD34 epitope.

11. The isolated polynucleotide according to claim 10, wherein the CD34 epitope is the Qbend-10 epitope.

12. The isolated polynucleotide according to any one of claims 5 to 11, wherein the polypeptide further comprises a CD8 hinge / membrane-spanning domain. The isolated polynucleotide according to any one of claims 1 to 11, comprising a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1 to 7.

13. An isolated polynucleotide according to any one of claims 1 to 12, encoding a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 8 to 14.

14. A vector comprising the isolated polynucleotide according to any one of claims 1 to 13.

15. The vector according to claim 15, wherein the vector is a retroviral vector, DNA vector, plasmid, RNA vector, adenoviral vector, adeno-associated vector, lentiviral vector, or any combination thereof.

16. An engineered immune cell comprising the isolated polynucleotide according to any one of claims 1 to 14, which does not express a rituximab binding site.

17. An engineered immune cell comprising the vector according to claim 14 or 16, which does not express a rituximab binding site.

18. The engineered immune cell according to any one of claims 17 or 18, wherein the immune cell is a T cell, tumor-infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell, or NK-T cell.

19. The engineered immune cell according to claim 19, wherein the cell is an autologous T cell.

20. The engineered immune cell according to claim 19, wherein the cell is an allogeneic T cell.

21. The engineered immune cell according to any one of claims 17 to 21, wherein the cell comprises a polynucleotide that is at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:

3.

22. The engineered immune cell according to any one of claims 17 to 22, wherein the cell is resistant to rituximab.

23. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 17 to 23.

24. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject the engineered immune cell according to any one of claims 17 to 23, or the pharmaceutical composition according to claim 24.

25. The method according to claim 25, wherein the disease or disorder is non-Hodgkin lymphoma (NHL).

26. The method according to claim 25 or 26, wherein the subject has been treated with or is currently being treated with rituximab.

27. An article of manufacture comprising the engineered immune cell according to any one of claims 17 to 23, or the pharmaceutical composition according to claim 24.

Images