Anti-rituximab chimeric antigen receptor and its use

Abstract

Provided herein are polynucleotides encoding chimeric antigen receptors (CARs) comprising CD19 antigen-binding domains that specifically bind to CD19 and are resistant to rituximab binding; and immune cells, such as CAR-T cells, comprising these CD19-specific CARs. The present invention also provides methods for manufacturing and using these CD19-specific CARs, and immune cells comprising these CD19-specific CARs.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 839,455, filed April 26, 2019; and U.S. Provisional Application No. 63 / 005,041, filed April 3, 2020, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to chimeric antigen receptors (CARs) comprising antigen binding molecules that bind to CD19, polynucleotides encoding the same, and methods of using the same to treat cancer in patients.

[0004] Sequence Listing

[0005] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 21, 2020, is named AT-028_03WO_SL.txt and is 81,460 bytes in size. Background Art

[0006] Adoptive transfer of immune cells genetically modified to recognize malignancy-associated antigens has shown promise as a novel approach to treating cancer (see, e.g., 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), which is a fusion protein consisting of a CD19 antigen recognition portion and a T cell activation domain (see, e.g., 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)). CAR-containing immune cells, such as CAR-T cells (CAR-T), are engineered to confer antigen specificity while maintaining or enhancing their ability to recognize and kill target cells.

[0007] There is a need for treatments for cancer, and more specifically, for malignancies involving aberrant expression of CD 19. Provided herein are methods and compositions that address this need. Summary of the Invention

[0008] Provided herein is a chimeric antigen receptor (CAR), comprising a CD19 antigen binding domain specifically bound to CD19; polynucleotides encoding these CARs; and immune cells expressing these CD19 specific CARs, such as CAR-T cells. The present invention also provides methods for manufacturing and using these CD19 specific CARs, and immune cells comprising these CD19 specific CARs.

[0009] 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, wherein the polypeptide does not comprise a rituximab binding site, and wherein the polynucleotide comprises a short EF1a promoter capable of expressing the anti-CD19 chimeric antigen receptor (CAR) in mammalian T cells.

[0010] In some embodiments, the short EF1a promoter does not comprise an intron sequence within the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 39.

[0011] In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 16,

[0012] In some embodiments, the promoter is the full-length EF1a promoter comprising the nucleic acid sequence of SEQ ID NO:15.

[0013] In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 15, and the polynucleotide encodes a polypeptide at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 8-14.

[0014] In some embodiments, the polypeptide further comprises a safety switch.

[0015] In some embodiments, a linker peptide is used to link the safety switch to the CD19 CAR.

[0016] In some embodiments, the safety switch is linked to the anti-CD19 CAR using a T2A linker.

[0017] In some embodiments, the safety switch comprises an antibody binding site.

[0018] In some embodiments, the safety switch comprises a mutated CD20 mimotope.

[0019] In some embodiments, the polypeptide further comprises a CD8 hinge / transmembrane domain.

[0020] In some embodiments, the polypeptide comprises a CD34 epitope.

[0021] In some embodiments, the CD34 epitope is a QBEND-10 epitope.

[0022] In some embodiments, the isolated polynucleotide comprises a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-7.

[0023] In some embodiments, the isolated polynucleotide encodes a polypeptide at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 8-14.

[0024] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide described herein.

[0025] In some embodiments, the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0026] In one aspect, the present disclosure provides an engineered immune cell comprising the isolated polynucleotide described herein.

[0027] In some embodiments, the present 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.

[0028] In some embodiments, the present disclosure provides an engineered immune cell 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.

[0029] In some embodiments, the present disclosure provides an engineered immune cell 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.

[0030] In some embodiments, the present disclosure provides an engineered immune cell comprising a polynucleotide encoding a polypeptide at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10, with or without a signal sequence.

[0031] In some embodiments, the present disclosure provides an engineered immune cell comprising a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 3, driven by an EF1a short promoter comprising the nucleic acid of SEQ ID NO: 16.

[0032] In some embodiments, the present disclosure provides an engineered immune cell comprising a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 9 or 10, with or without a signal sequence, driven by the EF1a short promoter comprising the nucleic acid of SEQ ID NO: 16. In some embodiments, the promoter does not comprise the first intron of the EF1a gene.

[0033] In some embodiments, the engineered immune cells do not comprise a rituximab mimotope.

[0034] In some embodiments, the engineered immune cell comprises a polynucleotide comprising Figure 1 The CD19 CAR v1.2 lentiviral construct (also referred to as ALLO-501A) is shown.

[0035] In some embodiments, the engineered immune cells comprise a vector described herein.

[0036] In some embodiments, the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), a NK cell, a cell expressing a TCR, a dendritic cell, or a NK-T cell.

[0037] In some embodiments, the cells are autologous T cells.

[0038] In some embodiments, the cells are allogeneic T cells.

[0039] In one aspect, the present disclosure provides the engineered immune cell described herein, wherein the cell is resistant to rituximab.

[0040] In another aspect, the present disclosure provides a pharmaceutical composition comprising the engineered immune cells as described above.

[0041] In one aspect, the present 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.

[0042] In some embodiments, the disease or disorder is non-Hodgkin lymphoma (NHL).

[0043] In some embodiments, the individual has been or is currently being treated with rituximab.

[0044] In one aspect, the present disclosure provides an article of manufacture comprising an engineered immune cell or a pharmaceutical composition comprising an engineered immune cell expressing a chimeric antigen receptor described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 A schematic diagram of the anti-rituximab CD19 chimeric antigen receptor is shown.

[0046] Figure 2A and 2B Shown are flow cytometry graphs showing CAR expression on day 5 of Pan T cells transduced with an anti-rituximab CAR expression vector. Figure 2A CAR expression on cells from donors 541 and 604 is shown. Figure 2B Shown are CAR and CD34 expression on cells from donor 410. A similar profile was observed for donor 2593 (data not shown).

[0047] Figure 3 Normalized cell expansion and final CAR expression from all four donors at day 13 are shown.

[0048] Figures 4A to 4D Figure 2 shows the transduction of anti-rituximab CAR expression vector from donor 541 ( Figure 4A )、604( Figure 4B )、410( Figure 4C )、2593( Figure 4D ) of Pan T cells over time and CAR expression.

[0049] Figures 5A to 5D Figure 2 shows the transduction of anti-rituximab CAR expression vector from donor 541 ( Figure 5A )、604( Figure 5B )、410( Figure 5C )、2593( Figure 5D ) of Pan T cells on days 5, 9, and 13.

[0050] Figures 6A to 6D Figure 2 shows the transduction of anti-rituximab CAR expression vector from donor 541 ( Figure 6A )、604( Figure 6B )、410( Figure 6C)、2593( Figure 6D ) Pan T cells phenotype and activation on day 9.

[0051] Figure 7 Shown are the phenotype, activated CD8+ % and anergy averaged from all four donors measured at day 9 using TIM3 and PD1 staining.

[0052] Figures 8A to 8D Figure 2 shows the transduction of anti-rituximab CAR expression vector from donor 541 ( Figure 8A )、604( Figure 8B )、410( Figure 8C )、2593( Figure 8D ) Pan T cells on day 13 phenotype and activation.

[0053] Figure 9 Shown are the phenotypes, activated CD8+ % and anergy measured from all four donors at day 13 using TIM3 and PD1 staining.

[0054] Figure 10 Shown are the average short-term (24 h) killing assays for each CAR construct using Raji cells as target cells.

[0055] Figures 11A to 11D The results show that A549-CD19+ cells were used as target cells in each CAR construct with an E:T ratio of 8:1. Figure 11A )、4:1( Figure 11B )、2:1( Figure 11C ) and 1:1( Figure 11D ) in the presence of an average long-term killing assay.

[0056] Figure 12 Shown are the % levels of CAR+ T cells on day 5 after transduction of Pan T cells with serial dilutions of lentiviral preparations of rituximab-resistant lentiviral constructs (ALLO-501v1.2 and v1.3) or rituximab-sensitive lentiviral constructs (ALLO-501v1.0).

[0057] Figures 13A to 13B Figure 3 shows the expression of CARs from donor 541 (ALLO-501v1.2) transduced with either a rituximab-resistant CAR expression vector (ALLO-501v1.2) or a rituximab-sensitive CAR expression vector (ALLO-501v1.0) when tested in the NSG mouse tumor model bearing Raji cells. Figure 13A ) and 604( Figure 13B ) in vivo efficacy of pan T cells. DETAILED DESCRIPTION

[0058] Chimeric antigen receptor (CAR) therapy is a promising method for cancer treatment. The CAR construct described herein as v1.0 is an exemplary anti-CD19 CAR expressing a synthetic peptide RQR8, which acts as a safety switch. RQR8 contains two mimetic epitopes that bind rituximab. In the case of adverse events, patients can be treated with rituximab to deplete the level of anti-CD19 v1.0 in the circulation. Rituximab is also used as the standard treatment for some non-Hodgkin's lymphoma (NHL) indications and is administered in high doses. Due to the long half-life of rituximab, it is impossible to administer anti-CD19 v1.0 to patients until the level of circulating rituximab has reached a low concentration. Anti-rituximab CD19 CAR therapy will allow patients previously treated with rituximab to receive CAR-T therapy immediately without having to wait for rituximab levels to decrease and patients do not need to undergo apheresis. Provided herein are anti-CD19 chimeric antigen receptors (CARs) that are resistant to the CD20-binding antibody rituximab. The novel CAR constructs are designed to eliminate rituximab binding while retaining CAR expression and activity.

[0059] I. Chimeric Antigen Receptors

[0060] 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 a target antigen, CAR can activate immune cells to attack and destroy cells carrying the antigen (e.g., cancer cells). CAR can also have co-stimulatory or signaling domains to increase its effectiveness. See Krause et al., Journal of Experimental Medicine, Vol. 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 2003 (619-626). 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.

[0061] The chimeric antigen receptor described herein comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a CD19 antigen binding domain that specifically binds to CD19. In some embodiments, the CD19-specific CAR comprises the following elements from 5' to 3': a signal sequence, a CD19 antigen binding domain (e.g., a scFv derived from 4G7), a hinge and a transmembrane region, and one or more continuous signaling domains. In some embodiments, the antibody binding domain binds to CD19 to treat blood cancers associated with the expression of CD19.

[0062] 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 by 4G7 contains some targeting components of the chimeric antigen receptor (CAR) (see WO2014184143A1). In some embodiments, the scFv derived from the CD19 monoclonal antibody 4G7 comprises a portion of the CD19 monoclonal antibody 4G7 immunoglobulin γ1 heavy chain (GenBank: CAD88275.1; SEQ ID NO: 17) and a portion of the CD19 monoclonal antibody 4G7 immunoglobulin κ light chain (GenBank: CAD88204.1; SEQ ID NO: 35) connected 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 CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain, linked by a flexible linker.

[0063] CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain (signal sequence underlined)

[0064] MEWSWIFLFLLSGTAGVHSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:17)

[0065] CD19 monoclonal antibody 4G7 immunoglobulin κ light chain (signal sequence underlined)

[0066] MRCLAEFLGLLVLWIPGAIG DIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:18)

[0067] 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 comprises at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the variable region of the amino acid sequence of SEQ ID NO: 34 and / or SEQ ID NO: 35. Antigen binding molecules are disclosed herein, including antibodies that specifically bind to 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. Applications and uses of these antigen binding molecules are also disclosed.

[0068] a. Antigen binding domain

[0069] As discussed above, the CD19 CAR described herein comprises an antigen binding domain. As used herein, "antigen binding domain" means any polypeptide that binds to a specified target antigen, which may be, for example, a CD19 protein or a fragment thereof. In some embodiments, the antigen binding domain binds to a CD19 antigen on a tumor cell. In some embodiments, the antigen binding domain binds to a CD19 antigen on a cell involved in a hyperproliferative disease.

[0070] In some embodiments, the antigen-binding domains include variable heavy chains, variable light chains and / or one or more CDRs. In some embodiments, the antigen-binding domains are single-chain variable fragments (scFv), including light chain CDRs CDR1, CDR2 and CDR3, and heavy chain CDRs CDR1, CDR2 and CDR3. Variants of antigen-binding domains (e.g., variants of CDRs VH and / or VL) are also within the scope of the present disclosure, such as each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99% or higher than 99% consistency of the amino acid sequence of the antigen-binding domain sequences described herein. Variable light and / or variable heavy chains. In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, variant forms contain two variable light chains and two variable heavy chains (or sub-parts thereof). Those skilled in the art will be able to use well-known technology to determine suitable variants of antigen-binding domains as shown herein. In certain embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions not thought to be important for activity.

[0071] In certain embodiments, the polypeptide structure of the antigen binding domain is based on an antibody, including but not limited to monoclonal antibodies, bispecific antibodies, miniantibodies, 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 avimer.

[0072] A CD19 antigen binding domain is said to be "selective" when it binds to one target more tightly than it binds to a second target. In some embodiments, the CD19 antigen binding domain is a scFv.

[0073] In some embodiments, the present disclosure relates to isolated polynucleotides encoding any one of the CD19 chimeric antigen receptors (CARs) described herein. In some embodiments, the present disclosure relates to isolated polynucleotides encoding the CD19 CARs described in Table 1. Also provided herein are vectors comprising polynucleotides and methods for preparing the same.

[0074] Table 1. Exemplary CD19-targeting polynucleotide sequences of CARs

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] b. Safety Switch and Monoclonal Antibody Specificity-Epitope

[0083] Safety switch

[0084] It should be understood that adverse events can be minimized by transducing immune cells (containing one or more CARs) with suicide genes other than the epitope binding rituximab. It may also be necessary to incorporate an inducible "on" or "accelerator" switch into immune cells. Suitable techniques include using inducible caspase-9 (U.S. application 2011 / 0286980) or thymidine kinase before, after, or simultaneously with the CAR construct transduction cells disclosed herein. Other methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other technologies known in the art.

[0085] According to the present disclosure, other on-off types or other types of control switch technologies can be incorporated herein. These technologies can use dimerization domains and optional activators of such domain dimerization. These technologies include, for example, those technologies utilizing FKBP / Rapalog dimerization systems in certain cells described by Wu et al., Science 2014 350 (6258), the contents of which are incorporated herein by reference in their entirety. Other dimerization technologies are described in, for example, Fegan et al., Chem. Rev. 2010, 110, 3315-3336 and U.S. Patent No. 5,830,462; No. 5,834,266; No. 5,869,337; and No. 6,165,787, the contents of which are also incorporated herein by reference in their entirety. Other dimerization pairs can include cyclosporine-A / cyclophilin, receptor, estrogen / estrogen receptor (optionally with tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, vitamin D / vitamin D receptor. Other examples of dimerization technology can be found in, for example, WO 2014 / 127261, WO 2015 / 090229, US 2014 / 0286987, US 2015 / 0266973, US 2016 / 0046700, U.S. Patent No. 8,486,693, US 2014 / 0171649, and US 2012 / 0130076, the contents of which are further incorporated herein by reference in their entireties.

[0086] In some embodiments, the CAR immune cells (e.g., CAR-T cells) of the present disclosure include polynucleotides encoding suicide polypeptides that are defective in terms of rituximab binding. In some embodiments, the suicide peptide includes a mutated RQR8 sequence. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In CAR immune cells (e.g., CAR-T cells) comprising polynucleotides, the suicide polypeptide is expressed at the surface of the CAR immune cells (e.g., CAR-T cells). In some embodiments, the suicide polypeptide includes the amino acid sequence shown in SEQ ID NO: 19.

[0087] CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 19).

[0088] The suicide polypeptide may further comprise a signal peptide at the amino terminus, such as MGTSLLCWMALCLLGADHADA (SEQ ID NO: 20). In some embodiments, the suicide polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21, which includes the signal sequence of SEQ ID NO: 20.

[0089] MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 21).

[0090] In certain embodiments, the suicide peptide comprises an amino acid sequence comprising one or more mutated residues, inserted residues, or deleted residues that reduce or eliminate rituximab binding.

[0091] When suicide polypeptide is expressed at the surface of CAR immune cells (such as CAR-T cells), the combination of the suicide gene epitope of the antibody and the polypeptide causes cell lysis. The loss of CD19-specific CAR immune cells (such as CAR-T cells) can occur in vivo, for example, by administering a suicide agent to the patient. The decision to delete the transferred cells may result from undesirable effects attributable to transferred cells detected in the patient, for example, when unacceptable toxicity levels are detected. As used herein, "suicide agent" refers to a molecule that is bound to a CAR immune cell and causes the cracking of the immune cell expressing CAR.

[0092] In some embodiments, the suicide polypeptide is expressed on the cell surface. In some embodiments, the CAR construct includes a suicide polypeptide. In some embodiments, the suicide polypeptide is not a part of the CD19CAR construct.

[0093] In some embodiments, the extracellular domain of any one of the CD19 specific CARs disclosed herein may include one or more epitopes that are specific to monoclonal antibodies (i.e., specifically recognized by monoclonal antibodies). These epitopes are also referred to herein as mAb specific epitopes. Exemplary 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 CAR includes an antigen binding domain that specifically binds to CD19 and one or more epitopes that are bound to one or more monoclonal antibodies (mAb). The CAR comprising mAb specific epitopes can be single-chain or multi-chain.

[0094] Including an epitope specific for a monoclonal antibody in the extracellular domain of the CAR described herein allows for sorting and exhaustion of engineered immune cells expressing CAR. In some embodiments, this feature also promotes the recovery of cells that endogenously express CD19, which are exhausted by administering engineered immune cells expressing CAR. In some embodiments, in the event of a deleterious effect, such as when administered to an individual, exhaustion provides a safety switch.

[0095] Thus, in some embodiments, the present disclosure relates to a method for sorting and / or depleting engineered immune cells having a CAR comprising a mAb-specific epitope and a method for promoting recovery of cells that endogenously express CD19.

[0096] Several epitope-monoclonal antibody conjugates are available for generating CARs comprising monoclonal antibody-specific epitopes; specifically, those that have been approved for medical use or for GMP production, such as the CD34 epitope / QBEND-10 as a non-limiting example.

[0097] The present disclosure also encompasses methods for sorting engineered immune cells having CD19-specific CARs expressing mAb-specific epitopes, and methods of treatment, wherein the activation of engineered immune cells having these CARs is regulated by depleting cells with antibodies targeting the ligand binding domain of the CAR. Table 2 provides exemplary mimotope sequences that can be inserted into the extracellular domain of the CAR of the present disclosure.

[0098] Table 2: Exemplary mimotope sequences

[0099]

[0100]

[0101] 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 an epitope or mimotope amino acid sequence shown in Table 2. In certain embodiments, the CAR comprises an epitope or mimotope amino acid sequence that is not or does not comprise SEQ ID NO: 22. In certain embodiments, the CAR comprises an epitope or mimotope amino acid sequence that comprises the amino acid sequence of SEQ ID NO: 30.

[0102] c. Hinge domain

[0103] The extracellular domain of the CAR of the present disclosure may include a "hinge" domain (or hinge region). The term is generally directed to any polypeptide used to connect the transmembrane domain in CAR to the extracellular antigen-binding domain in CAR. Specifically, the hinge domain can be used to provide more flexibility and accessibility of the extracellular antigen-binding domain.

[0104] The hinge domain may comprise up to 300 amino acids - in some embodiments 10 to 100 amino acids or in some embodiments, 25 to 50 amino acids. The hinge domain may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, CD28, 4-1BB or IgG (specifically, the hinge region of IgG; it should be understood that the hinge region may contain some or all of the members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or a fragment thereof), or all or part of the constant region of the heavy chain of an antibody. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence or may be an entire synthetic hinge sequence. In some embodiments, the hinge domain is a part of a human CD8 α chain (e.g., NP_001139345.1). In another specific embodiment, the hinge and transmembrane domain comprise a part of a human CD8 α chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα, specifically, a hinge region of any one of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein comprises a scFv, a CD8α human hinge and a transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 3 provides the amino acid sequences of exemplary hinges provided herein.

[0105] Table 3: Exemplary hinges

[0106]

[0107] 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 sequence shown in Table 3.

[0108] d. Transmembrane domain

[0109] The CAR of the present disclosure is designed to have a membrane-spanning domain fused with the extracellular domain of CAR. It can similarly be fused with the intracellular domain of CAR. In some cases, the membrane-spanning domain can be selected or modified by amino acid substitution to avoid such domains from being combined with the membrane-spanning domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex. In certain embodiments, a short linker can form a bond between any one or some of the extracellular, transmembrane and intracellular domains of CAR. In certain embodiments, the linker includes a glycine repeat sequence. In certain embodiments, the linker includes (GGGGS) n, wherein n is 1, 2, 3, 4 or 5 (SEQ ID NO: 41).

[0110] Suitable transmembrane domains of the CARs disclosed herein have the ability to: (a) express on the surface of immune cells, such as, but not limited to, lymphocytes, such as T helper cells (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 the intracellular signaling domain to direct the cellular response of the immune cell against the target cell.

[0111] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0112] The transmembrane region of specific use in the present disclosure may be derived from (contain or correspond to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, D3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, 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, a ligand that specifically binds to CD83, or any combination thereof.

[0113] As non-limiting examples, the transmembrane region may be derived from or be a portion of a T cell receptor (such as α, β, γ or δ), a polypeptide constituting the CD3 complex, IL-2 receptor p55 (α chain), p75 (β chain) or γ chain, a subunit chain of an Fc receptor (specifically, Fcγ receptor III) or a CD protein. Alternatively, the transmembrane domain may be synthetic and may comprise primarily hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from a human CD8 α chain (e.g., NP_001139345.1).

[0114] In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8 α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8 α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 35). In some embodiments, the CD8 α 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 are CD8 α hinge and transmembrane domains comprising the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 36).

[0115] e. Intracellular domain

[0116] The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide at least one activation of the normal effector function of the immune cell comprising CAR.For example, the effector function of T cells can refer to cytolytic activity or auxiliary activity, including the secretion of cytokines.

[0117] In some embodiments, the activating intracellular signaling domain for use in a CAR may be, for example, but not limited to, cytoplasmic sequences of T cell receptors and co-receptors that act coordinately to initiate signal transduction after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0118] It will be appreciated that suitable (e.g., activation) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, D3ε, CD247, CD276 (B7-H3), LIGH, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptors, MHC Class 1 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, 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, a ligand that specifically binds to CD83, or any combination thereof.

[0119] The intracellular domain of the CAR of the present disclosure may also be incorporated into a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule) in addition to the above-mentioned activation domain to increase its effectiveness. The costimulatory domain may provide a signal other than the primary signal provided by the activation molecule as described herein.

[0120] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure may be derived from (or correspond to) for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), 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 (CD1 1a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptors, MHC Class I molecules, TNFR, integrin, signaling lymphocyte activation molecules, 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, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD4 9D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITG B2, 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 ligand, or a fragment or combination thereof. It should be understood that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0121] In some embodiments, the intracellular / cytoplasmic domain of CAR can be designed to include the 41BB / CD137 domain itself or in combination with any other desired intracellular domain suitable for the context of the CAR 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.

[0122] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to contain the CD28 domain itself or in combination with any other desired intracellular domain suitable for the context of the CAR 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.

[0123] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to contain a CD3 zeta domain by itself or in combination with any other desired intracellular domain that is suitable for the context of the CAR of the present disclosure. In some embodiments, the intracellular signaling domain of the CAR may comprise a CD3 zeta signaling domain having an amino acid sequence having 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 comprise a portion of the CD3 zeta chain and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR of the present disclosure may be linked to each other randomly or in a specified order. In some embodiments, the intracellular domain is designed to comprise the activation domain of CD3 zeta and the signaling domain of CD28.

[0124] In some embodiments, the intracellular domain is designed to include the activation domain of CD3ζ and the signaling domain of 4-1BB. In some embodiments, 4-1BB (intracellular domain) comprises the amino acid sequence

[0125] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 37).

[0126] The CD3zeta amino acid sequence may comprise SEQ ID NO:38.

[0127] LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 38).

[0128] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of the CAR of the present disclosure comprises 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 of the present disclosure comprises an amino acid sequence comprising 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 SEQ ID NO: 38. In some embodiments, the intracellular signaling domain of the CAR of the present disclosure comprises an amino acid sequence comprising 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.

[0129] In an exemplary embodiment, the CAR of the present disclosure comprises, from N-terminus to C-terminus: a CD8α signal sequence, a CD19scFv, a CD8-hinge and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain.

[0130] III. CAR-containing immune cells

[0131] a. Immune cells

[0132] Provided herein are engineered immune cells (e.g., CAR-T cells) that express the CARs of the present disclosure.

[0133] In some embodiments, the engineered immune cell comprises a CAR population, each CAR comprising a different extracellular antigen binding domain. In some embodiments, the immune cell comprises a CAR population, each CAR comprising the same extracellular antigen binding domain.

[0134] Engineered immune cells can be allogeneic or autologous.

[0135] In some embodiments, the engineered immune cells are T cells (e.g., inflammatory T lymphocytes, 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 may be derived from a 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 γδ T cells. In some exemplary embodiments, the engineered immune cells are macrophages.

[0136] In some embodiments, 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 specifically, 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.

[0137] In some embodiments, the cells are obtained from or prepared from peripheral blood. In some embodiments, the cells are obtained from or prepared from peripheral blood mononuclear cells (PBMC). In some embodiments, the cells are obtained from or prepared from bone marrow. In some embodiments, the cells are obtained from or prepared from umbilical cord blood. In some embodiments, the cells are human cells. In some embodiments, the cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, a gene gun (e.g., a gene gun), lipofection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplexes.

[0138] In some embodiments, the engineered immune cells expressing the CD19-specific CAR of the present invention at their cell surface membranes comprise greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of stem cell memory and central memory cells. In some embodiments, the engineered immune cells expressing the CD19-specific CAR of the present invention at their cell surface membranes comprise 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 100%, about 15% to about 90%, about 10% to about 100%, about 15 ... 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% of the percentage of stem cell memory and central memory cells.

[0139] In some embodiments, the immune cell is an inflammatory T lymphocyte expressing any one of the CARs described herein. In some embodiments, the immune cell is a cytotoxic T lymphocyte expressing any one of the CARs described herein. In some embodiments, the immune cell is a regulatory T lymphocyte expressing any one of the CARs described herein. In some embodiments, the immune cell is a helper T lymphocyte expressing any one of the CARs described herein.

[0140] Before amplification and genetic modification, cell sources can be obtained from individuals by 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, umbilical cord blood, thymus tissue, stem cells or iPSC-derived T cells or NK cells, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumor. In certain embodiments, any number of available and known T cell lines of those skilled in the art can be used. In certain embodiments, cells can be derived from healthy donors, from patients diagnosed with cancer, or from patients diagnosed with infection. In certain embodiments, cells can be part of a mixed cell population exhibiting different phenotypic characteristics.

[0141] Also provided herein is a cell line obtained by transforming immune cells (e.g., T cells) according to any of the above methods. Also provided herein is a modified cell resistant to immunosuppressive therapy. In certain embodiments, the isolated cell according to the present disclosure includes a polynucleotide encoding CAR.

[0142] The immune cells of the present invention can be activated and amplified using methods such as those generally known before or after the genetic modification of the immune cells. Typically, the engineered immune cells of the present invention can be amplified, for example, by contacting with agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of the T cells to form activation signals for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectin-like phytohemagglutinin (PHA) can be used to generate activation signals for T cells.

[0143] In some embodiments, T cell populations can be stimulated in vitro by contacting, for example, an anti-CD3 antibody or its antigen-binding fragment or an anti-CD28 antibody fixed on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ion carrier. In order to costimulate the auxiliary molecules on the surface of the T cells, a ligand that binds to the auxiliary molecule is used. For example, under conditions suitable for stimulating T cell proliferation, the T cell population can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. Anti-CD3 antibodies and anti-CD28 antibodies can be placed on beads or plates or other substrates. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors required for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFβ, and TNF, or any other additives for cell growth known to the skilled artisan. Other additives for cell growth include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture medium may include RPMI1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, optimizer (Optimizer) and added amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined group of hormones, and / or one or more cytokines (e.g., IL-7 and / or IL-15) sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and are not included in cell cultures to be infused into individuals. Target cells are maintained under conditions required to support growth, such as an appropriate temperature (e.g., 37° C.) and an atmosphere (e.g., air plus 5% CO 2). T cells exposed to different stimulation times may exhibit different characteristics.

[0144] In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissues or cells. After the cells are administered to a subject, the cells can also be expanded in vivo, for example, in the blood of the subject.

[0145] In some embodiments, the engineered immune cells according to the present disclosure may include one or more destroyed or inactivated genes. In some embodiments, the engineered immune cells according to the present disclosure include a destroyed 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 CAR, multi-chain CAR and / or 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 comprise two disrupted 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. β, 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 expressing CAR, multi-chain CAR and pTα transgene. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cell an endonuclease that can selectively inactivate genes by selective DNA cleavage. In some embodiments, the endonuclease can be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease (TALE-nuclease) a transcription activator-like effector nuclease (TALE-nuclease), or a CRIPR (e.g., Cas9) endonuclease.

[0146] In some embodiments, the TCR is rendered non-functional in cells according to the present disclosure by destroying or inactivating the TCR alpha gene and / or the TCR beta 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 and are easily obtained by this method are encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used to treat patients in need of combating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD); therefore, within the scope of the present disclosure is a method for treating patients in need of combating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising treating the patient by administering to the patient an effective amount of modified cells comprising destroyed or inactivated TCR alpha and / or TCR beta genes.

[0147] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapeutic drugs. The chemotherapeutic drug may be, for example, a purine nucleotide analog (PNA), so that immune cells are suitable for cancer treatment in combination with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, either alone or in combination. PNA is metabolized into single, two, and three phosphate PNAs by deoxycytidine kinase (dCK). Its triphosphate form competes with ATP for DNA synthesis, acts as a pro-apoptotic agent, and is an effective inhibitor of ribonucleotide reductase (RNR) involved in trinucleotide production. Provided herein are CD19-specific CAR-T cells comprising a fractured or inactivated dCK gene. In some embodiments, dCK gene knockout cells are obtained by transfecting T cells using polynucleotides encoding specific TAL-nucleases for dCK genes, such as by electroporation of mRNA. dCK gene knockout CD19-specific CAR-T cells are resistant to PNA, including, for example, clofarabine and / or fludarabine, and maintain T cell cytotoxic activity against cells expressing CD19.

[0148] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise pTalpha or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRalpha gene.

[0149] The present disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein.

[0150] c. Manufacturing method

[0151] Provided herein are methods for preparing CARs and CAR-containing immune cells of the present disclosure. Various known techniques can be used to prepare polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, etc. according to the present disclosure.

[0152] Polynucleotides and vectors

[0153] In certain embodiments, CAR can be introduced into immune cells as transgene via a plasmid vector.In certain embodiments, the plasmid vector can also contain, for example, a selection marker, which provides identification and / or selection of cells that receive the vector.

[0154] After the polynucleotide encoding the CAR polypeptide is introduced into the cell, the CAR polypeptide can be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. The method for introducing a polynucleotide construct into a cell is known in the art. In some embodiments, a stable transformation method (e.g., 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 a polynucleotide construct and a polynucleotide construct that is not integrated into the genome of the cell. In other embodiments, a viral-mediated method can be used. Polynucleotides can be introduced into cells by any suitable means, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment. Polynucleotides may be included in a vector, such as a plasmid vector or a viral vector.

[0155] In some embodiments, an isolated nucleic acid is provided, comprising a promoter operably linked to a first polynucleotide encoding a CD19 antigen binding domain, at least one costimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained in a viral vector. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a murine leukemia virus vector, a SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained in a plasmid.

[0156] 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 an EF1a promoter. The natural EF1a promoter drives the expression of the α subunit of the elongation factor-1 complex, which is responsible for 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 effectively drive CAR expression from a transgenic cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). In some embodiments, the EF1a promoter comprises a sequence provided as SEQ ID NO: 15.

[0157]

[0158] The EF1a promoter sequence shown above comprises 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 comprise a short EF1a promoter. In some embodiments, the polynucleotides provided herein comprise an EF1a promoter that is shorter than the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the polynucleotides provided herein comprise an EF1a promoter that does not comprise the first intron of the EF1a gene. In some embodiments, the polynucleotides provided herein comprise an EF1a promoter that does not comprise the nucleic acid sequence of SEQ ID NO: 39.

[0159] In some embodiments, the promoter comprises the sequence provided as SEQ ID NO:16.

[0160] GCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG (SEQ ID NO:16)

[0161] Prior to in vitro manipulation or genetic modification of the immune cells described herein, cells can be obtained from an individual. Cells expressing CD19 CAR can be derived from allogeneic or autologous processes.

[0162] Source Material

[0163] In some embodiments, immune cells comprise T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to those skilled in the art, such as FICOLL TM Isolate and obtain T cells from one unit of blood collected from an individual.

[0164] Cells can be obtained from a person's circulating blood by apheresis. The apheresis product typically contains 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 plasma components and placed in an appropriate buffer or culture medium for subsequent processing.

[0165] In some embodiments, for example, using TM Gradient centrifugation, by lysing red blood cells and consuming monocytes, separates T cells from PBMC. Specific subsets of T cells, (such as CD28+, CD4+, CD5+, CD45RA- and CD45RO+ T cells or CD28+, CD4+, CD5+, CD45RA-, CD45RO+ and CD62L+ T cells) can be further separated by positive or negative selection techniques known in the art. For example, the enrichment of T cell colonies by negative selection can be achieved with a combination of antibodies for surface markers unique to negatively selected cells. One of the methods used herein is to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies for cell surface markers present on negatively selected cells. For example, in order to enrich for CD4+ cells by negative selection, a monoclonal antibody mixture typically includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0166] PBMC can be directly used for carrying out genetic modification with immune cells (such as CAR or TCR) using methods as described herein.In certain embodiments, after separation of PBMC, T lymphocytes can be further separated, and before or after genetic modification and / or amplification, cytotoxic and helper T lymphocytes can be sorted into primary, memory and effector T cell subsets.

[0167] In some embodiments, CD8+ cells are further sorted into primary, stem cell memory, central memory, and effector cells by identifying the cell surface antigens associated with each of those 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 are positive for granzyme B and perforin. In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into primary, central memory, and effector cells by identifying cell populations with cell surface antigens.

[0168] Stem cell-derived immune cells

[0169] In certain embodiments, immune cells can be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Suitable HSC, ES cells, iPS cells and other stem cells can be immortalized cell lines cultivated or directly isolated from patients. Various methods for isolating, producing and / or cultivating stem cells are known in the art and can be used to practice the present invention.

[0170] In certain embodiments, immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In certain embodiments, the source material can be an induced pluripotent stem cell (iPSC) derived from a T cell or a non-T cell. The source material can be an embryonic stem cell. The source material can be a B cell, or any other cell from a peripheral blood mononuclear cell isolate, a hematopoietic progenitor cell, a hematopoietic stem cell, a mesenchymal stem cell, an adipose stem cell, or any other somatic cell type.

[0171] Genetic modification of isolated cells

[0172] Immune cells, such as T cells, can be genetically modified using known methods after separation, or immune cells can be activated and amplified (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 TCR α and / or CD52. In some embodiments, cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, zinc finger nucleases (ZFNs), TALENs, MegaTALs, large-range nucleases) to reduce or eliminate the expression of endogenous proteins (e.g., TCR α and / or CD52). In another embodiment, immune cells (such as T cells) are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding CAR), and subsequently activated and / or amplified in vitro. Methods for activating and expanding 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 hereby incorporated herein by reference in their entirety. Typically, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory molecules, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same beads act as "surrogate" antigen presenting cells (APCs). One example is for physiological activation of human T cells. System, CD3 / CD28 activator / stimulator system. In other embodiments, feeder cells and appropriate antibodies and cytokines can be used to activate and stimulate T cells to proliferate using methods such as those described in U.S. Patent No. 6,040,177; U.S. Patent No. 5,827,642; and WO2012129514, the contents of which are hereby incorporated by reference in their entirety.

[0173] Certain methods for making the constructs and engineering immune cells of the present disclosure are described in PCT application PCT / US15 / 14520, the contents of which are incorporated herein by reference in their entirety.

[0174] It should be understood that PBMC may also include other cytotoxic lymphocytes, such as NK cells or NKT cells. The expression vector carrying the coding sequence of the chimeric receptor as disclosed herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Flow cytometry can be used to sort the T cells carrying the successful transduction of the expression vector to separate CD3 positive T cells, and then further propagate to increase the number of these CAR expression T cells except for the cell activation of other methods as described elsewhere herein known in the art using anti-CD3 antibodies and IL-2 or other methods. Standard procedures are used to cryopreserve the T cells expressing CAR, to store and / or prepare for human individuals. In one embodiment, in the absence of non-human animal derived products, such as fetal bovine serum (fetal calf serum / fetal bovine serum), T cells are transduced in vitro, cultured and / or expanded.

[0175] In order to clone a polynucleotide, a vector can be introduced into a host cell (isolated host cell) to allow replication of the vector itself, and thus amplify copies of the polynucleotide contained therein. The cloning vector can contain sequence components, generally including but not limited to a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be selected as appropriate by those of ordinary skill in the art. For example, a replication origin can be selected to promote autonomous replication of the vector in the host cell.

[0176] In certain embodiments, the present disclosure provides an isolated host cell containing a vector provided herein. The host cell containing the vector can be used for expression or cloning of the polynucleotide contained in the vector. Suitable host cells may 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, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens. marcescens); and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces.

[0177] 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 method, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, gene gun method, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan and peptide. Standard methods for transfection and transformation of cells for expressing the vector of interest are well known in the art. In another embodiment, a mixture of different expression vectors can be used for a donor population of genetically modified immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a certain proportion of engineered cells express more than one different CAR.

[0178] In some embodiments, the vector comprises a lentiviral vector. The lentiviral vector comprising a 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 the transduction of T cells to produce CAR-T cells. In order to prepare the lentivirus encoding CAR, at day 0, HEK-293T cells can be plated with 400,000 cells / mL in 2mL DMEM (Gibco) per well of a 6-well plate supplemented with 10% FBS (Hyclone or JR Scientific). On day 1, lentivirus can be prepared by mixing together 0.5ug of the appropriate transfer CAR vector ("DNA mixture") in 250uLOpti-MEM (Gibco) per well of a 6-well plate with lentiviral packaging vector 1.5ug psPAX2, 0.5ug pMD2G. 10uL lipofectamine 2000 (Invitrogen) in 250uLOpti-MEM can be incubated at room temperature for 5 minutes and then added to the DNA mixture. The mixture can be incubated at room temperature for 20 minutes, and a total volume of 500uL is slowly added to the side of the hole containing HEK-293T. The general method of lentivirus production and transduction containing CAR is generally known in the art, for example, see Milone et al., "Leukemia", 2018, 32: 1529-1541; Sanber et al., Construction of stable packing celllines for clinical lendiviol 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 for storing genetically engineered cells expressing CAR or TCR, wherein the CAR or TCR targets CD19 protein. This involves cryopreserving immune cells so that the cells remain alive after thawing. A portion of the CAR-expressing immune cells can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignant tumors. When needed, the cryopreserved transformed immune cells can be thawed, grown, and expanded to obtain more such cells.

[0179] In some embodiments, cells are formulated by first collecting the cells from their culture medium and then washing and concentrating the cells in a culture medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline (Normosol). TM R (Abbott) or Plasma-Lyte TM A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0180] Allogeneic CAR T cells

[0181] For the manufacture of allogeneic CAR T therapies or AlloCARs TM The method involves collecting healthy, selected, screened and tested T cells from healthy donors. Then, T cells are engineered to express CAR, which recognizes certain cell surface proteins (such as CD19) expressed in blood or solid tumors. 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 make the CAR T product resistant to anti-CD52 antibody therapy. Therefore, anti-CD52 antibody therapy can be used to suppress the host immune system and allow CAR T to remain implanted to achieve a complete therapeutic effect. The engineered T cells are then subjected to a purification step and ultimately cryopreserved in vials for delivery to the patient.

[0182] Autologous CAR T cells

[0183] Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting a patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express CARs that recognize targets expressed on the cell surface of one or more specific cancer cells and kill the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient.

[0184] IV. Treatment Methods

[0185] The present disclosure includes methods for treating or preventing a condition associated with undesirable and / or elevated 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.

[0186] Provided is a method for treating a disease or condition including cancer. In some embodiments, the present disclosure relates to generating a T cell-mediated immune response in an individual, comprising administering an effective amount of an engineered immune cell of the present application to the individual. In some embodiments, the T cell-mediated immune response is directed to a target cell or cell. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the present disclosure includes a method for treating or preventing a malignant tumor, comprising administering an effective amount of at least one separated antigen-binding domain as described herein to an individual in need. In some aspects, the present disclosure includes a method for treating or preventing a malignant tumor, comprising administering an effective amount of at least one immune cell to an individual in need, wherein the immune cell comprises at least one chimeric antigen receptor, T cell receptor, and / or separated antigen-binding domain as described herein.

[0187] The CAR-containing immune cells of the present disclosure can be used to treat malignant tumors involving 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 hematogenous tumors. The term "cancer" refers to diseases of skin tissue, organs, blood and blood vessels, including but not limited to bladder cancer, bone cancer or blood cancer, brain cancer, breast cancer, cervical cancer, chest cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer, kidney cancer, liver cancer, lymph node cancer, lung cancer, oral cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, stomach cancer, testicular cancer, laryngeal cancer and uterine cancer. Specific cancers include, but are not limited to, advanced malignancies, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastases, glioblastoma multiforms, glioblastoma, brainstem glioma, poor prognosis malignant brain tumors, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumors, rectal adenocarcinoma, Dukes C&D colorectal cancer, unresectable colorectal cancer, metastatic hepatocellular carcinoma, Kaposi's sarcoma, and sarcoma), Carroll's acute myeloid 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 effusion-mesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressiva, hormone-refractory prostate cancer, resectable high-risk soft tissue sarcoma, unresectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia In one embodiment, the cancer is metastatic. In another embodiment, the cancer is refractory or resistant to chemotherapy or radiation.

[0188] In an exemplary embodiment, CAR-containing immune cells (e.g., CAR-T cells of the present disclosure) are used to treat NHL.

[0189] Also provided are methods for reducing tumor size in a subject, comprising administering to the subject an engineered cell of the disclosure, wherein the cell comprises a chimeric antigen receptor comprising a CD19 antigen binding domain and binds to a CD19 antigen on the tumor.

[0190] In some embodiments, the individual suffers from a solid tumor or a hematological malignancy, such as a 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 individual. 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 heterologous immune cells, such as heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term "in vitro cell" refers to any cell cultured ex vivo.

[0191] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent, e.g., an engineered CART cell, is any amount that, when used alone or in combination with another therapeutic agent, protects an individual from the onset of disease or promotes disease regression, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of disorders or disabilities caused by disease ailments. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by analyzing the activity of the agent in in vitro assays.

[0192] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as those with a formally diagnosed condition, those without a formally recognized condition, those under medical observation, those at risk of developing the condition, etc.

[0193] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and any application that reduces the risk that a subject will develop a condition or other risk factor. Treatment does not require a complete cure of the condition and encompasses embodiments in which symptoms or underlying risk factors are reduced. The term "prevent" does not require 100% elimination of the likelihood of an event occurring. Rather, it means that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0194] The desired therapeutic amount of cells in the composition is typically 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 greater than 10 2 cells, and up to 10 6 , up to and including 10 8 or 10 9 cells, and can exceed 10 10 The number of cells will depend on the intended use of the composition and the type of cells included therein. The desired cell density is generally greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 108 cells / ml or greater. Clinically relevant numbers of immune cells can be allocated to cumulative amounts equal to or greater than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In some aspects of the present disclosure, specifically, since all infused cells will be redirected to a specific target antigen (CD19), 10 6 / kg range (10 per patient 6 -10 11 ) of cells. CAR therapy can be administered multiple times at doses within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient being treated.

[0195] In some embodiments, the therapeutically effective amount of CAR T cells is about 1×10 5 cells / kg, about 2×10 5 cells / kg, about 3×10 5 cells / kg, about 4×10 5 cells / kg, about 5×10 5 cells / kg, about 6×10 5 cells / kg, about 7×10 5 cells / kg, about 8×10 5 cells / kg, about 9×10 5 cells / kg, 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×106 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg, or approximately 9×10 7 cells / kg.

[0196] 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 6 In some embodiments, the dosage of the cell is in the range of 100 cells / kg. It will be appreciated that dosages above and below this range may be suitable for certain individuals, and appropriate dosage levels may be determined by a healthcare provider as needed. Additionally, multiple doses of cells may be provided according to the present disclosure.

[0197] In some aspects, the present disclosure comprises a pharmaceutical composition comprising at least one antigen binding domain as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0198] The cell colonies expressing CAR of the present disclosure can be administered alone or in the form of a pharmaceutical composition in combination with a diluent and / or with other components (such as IL-2 or other cytokines or cell colonies). The pharmaceutical composition of the present disclosure may include CAR expression or TCR expression cell colonies, such as T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. This composition may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid, such as glycine; antioxidant; chelating agent, such as EDTA or glutathione; adjuvant (e.g., aluminum hydroxide); and preservative. The present disclosure composition is preferably formulated for intravenous administration.

[0199] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides that can serve as solvents or suspension media; polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0200] In some embodiments, after administration to the patient, the engineered immune cells expressing any one of the CD19 specific CARs described herein at their cell surface can reduce, kill or lyse the patient's endogenous expression CD19 cells. In one embodiment, the reduction or lysis percentage of the cell of the endogenous cell or cell line expressing CD19 of the engineered immune cells expressing any one of the CD19 specific CARs described herein is at least about or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In one embodiment, the reduction or lysis percentage of endogenous cells expressing CD19 or cell lines expressing CD19 by an engineered immune cell expressing any one of the CD19-specific CARs described herein is about 5% to about 95%, about 10% to about 95%, 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 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 25% to about 75%, or about 25% to about 60%. In one embodiment, the cells endogenously expressing CD19 are bone marrow cells that endogenously express CD19.

[0201] In one embodiment, the assays disclosed herein can be used to measure the percentage of reduction or lysis of a target cell, such as a cell line expressing CD19, by an engineered immune cell expressing a CD19-specific CAR of the present disclosure at its cell surface membrane.

[0202] The method may further comprise administering one or more chemotherapeutic agents. In certain embodiments, the chemotherapeutic agent is a lymphocyte depletion (pretreatment) chemotherapeutic agent. For example, a method of conditioning a patient in need of T cell therapy comprises administering to the patient a prescribed beneficial dose of cyclophosphamide (at 200 mg / m 2 / day and 2000mg / m 2 / day, about 100mg / m 2 / day and about 2000 mg / m 2 / day; for example, about 100 mg / m 2 / 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 the indicated dose of fludarabine (at 20 mg / m 2 / day and 900 mg / m 2 / day, about 10mg / m 2 / day and about 900 mg / m 2 / day; for example, about 10 mg / m 2 / day, about 20 mg / m 2 / day, about 30mg / m 2 / day, about 40mg / m 2 / day, about 40mg / m 2 / day, about 50mg / m 2 / day, about 60mg / m 2 / day, about 70mg / m 2 / day, about 80mg / m 2 / day, about 90mg / m 2 / day, about 100 mg / m 2 / day, about 500 mg / m 2 / day or about 900 mg / m 2 / day). A preferred dosing regimen involves treating a patient comprising administering to the patient about 300 mg / m2 daily prior to administering to the patient a therapeutically effective amount of engineered T cells. 2 / day of cyclophosphamide and approximately 30 mg / m 2 / day of fludarabine for three days.

[0203] In some embodiments, lymphocyte depletion further comprises administering a CD52 antibody. In some embodiments, the CD52 antibody is administered IV at a dose of about 13 mg / day.

[0204] In other embodiments, the antigen binding domain, transduced (or otherwise engineered) cells, and chemotherapeutic agent are each administered in an amount effective to treat the disease or condition in the subject.

[0205] In certain embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide. TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trishydroxymethylmelamine; nitrogen mustards, such as chlorambucil, naphthyl mustard, chlorphosphamide, estramustine, ifosfamide, dichloromethane, dichloromethane oxide hydrochloride, melphalan, novemb ichin), cholesterol-lowering phenylacetic acid nitrogen mustard, prednisone nitrogen mustard, trolofosamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as aclarubicin, actinomycin, oslamycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carmomycin, chromomycin, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L-leucine alanine, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, peplomycin, sphingomycin, puromycin, quinamycin, rodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zinostatin, levorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dinotetin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, Labine, 6-mercaptopurine, thiabendine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as captestosterone, drotosterone propionate, cyclothiocarbamate, melastane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestab; bisantrene; idatroq; defuvalamide; colchicine; diazocone; efamicin; elliptonium acetate; etoglucose; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; niterac;Pentostatin; phenamet; pirarubicin; podophyllic acid; 2-acetylhydrazine; procarbazine; Razoxane; sizolan; spirogermanamine; tricosporic acid; triazoline; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; metoclopramide; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel ( Rhne-Poulenc Rorer, Antony, France; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; mitoxantrone; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as targretin TM (Besselotine), Panretin TM (Yali Cuitonin); ONTAK TM (denileukin); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents used to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibition 4 (5) -imidazole, 4-hydroxytamoxifen, troxifene, raloxifene hydrochloride, LY117018, onapristone and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered when appropriate, including but not limited to CHOP, i.e., cyclophosphamide Doxorubicin (hydroxydoxorubicin), vincristine and prednisone.

[0206] In some embodiments, the chemotherapeutic agent is administered simultaneously or within a week after the engineered cells, polypeptides, or nucleic acids are administered. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks, 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 the engineered cells, polypeptides, or nucleic acids are administered. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to the administration of the cells, polypeptides, or nucleic acids. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0207] 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. pembrolizumab Pembrolizumab, pidilizumab, and atezolizumab.

[0208] Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to: ibrutinib Ofatumumab Rituximab bevacizumab trastuzumab trastuzumab emtansine Imatinib Cetuximab panitumumab Catumaxomab, ibritumomab, osetumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib ntedanib), pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, tocianib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurinib, ruxolitinib inib), 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; CDK inhibitors such as the CDK inhibitor palbociclib.

[0209] In some embodiments, the composition comprising the immune cells containing CAR can be administered together with a treatment regimen for preventing cytokine release syndrome (CRS) or neurotoxicity. Treatment regimens for preventing cytokine release syndrome (CRS) or neurotoxicity may include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (eg, L-NIL or 1400W). In additional embodiments, the composition comprising the immune cells containing CAR can be administered together with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); nonsteroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Exemplary analgesics include acetaminophen, oxycodone, tramadol with propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors (e.g., TNF antagonists (e.g., etanercept), Adalimumab and infliximab )), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.

[0210] In certain embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are 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); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, β and γ; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, 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-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

[0211] V. Sorting and Consumption Methods

[0212] In some embodiments, a method for sorting an immune cell colony in vitro is provided, wherein a subset of the immune cell colony includes an engineered immune cell expressing any one of CD19 specific CARs, and the CD19 specific CARs include an epitope (e.g., an exemplary mimicking epitope sequence) specific for a monoclonal antibody. The method includes contacting an immune cell colony with a monoclonal antibody specific for an epitope, and selecting the immune cell bound to the monoclonal antibody to obtain a cell colony enriched for an engineered immune cell expressing CD19 specific CARs.

[0213] In some embodiments, the monoclonal antibody specific for the 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).

[0214] In some embodiments, the monoclonal antibody specific for the epitope is optionally bound 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).

[0215] In some embodiments, the mAb used in the method of sorting CAR-expressing immune cells is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10.

[0216] In some embodiments, the CAR-expressing immune cell population obtained when the method for in vitro sorting of CAR-expressing immune cells described above is used comprises at least 70%, 75%, 80%, 85%, 90%, 95% of CAR-expressing immune cells. In some embodiments, the CAR-expressing immune cell population obtained when the method for in vitro sorting of CAR-expressing immune cells is used comprises at least 85% of CAR-expressing immune cells.

[0217] In some embodiments, the colony of the immune cells expressing CAR obtained when the method for in vitro sorting of immune cells expressing CAR is used as described above shows an increase in in vitro cytotoxic activity compared to the initial (unsorted) cell population. In some embodiments, the cytotoxic activity is increased by 10%, 20%, 30% or 50% in vitro. In some embodiments, the immune cells are T cells.

[0218] In some embodiments, the mAb is previously bound to 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.

[0219] CAR-expressing immune cells to be administered to a recipient can be enriched in vitro from a source population. Methods for expanding a source population can include selecting cells expressing an antigen (such as the CD34 antigen) using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[0220] Flow cytometry can be used to quantify specific cell types within a cell population. Generally speaking, flow cytometry is a method that quantifies cellular components or structural features primarily through optical means. Because different cell types can be distinguished by quantifying structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes within a mixture.

[0221] Flow cytometry analysis involves two basic steps: 1) labeling selected cell types 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, methods for labeling cell types include binding a labeled antibody to a marker expressed by a specific cell type. The antibody can be directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0222] In some embodiments, the method for sorting CAR-expressing T cells is magnetic activated cell sorting (MACS). Magnetic activated cell sorting (MACS) is a method for separating a variety of cell populations by using superparamagnetic nanoparticles and columns, depending on cell population surface antigens (CD molecules). MACS can be used to obtain pure cell populations. Cells in single cell suspensions can be magnetically labeled with microbeads. The sample is applied to a column consisting of ferromagnetic spheres, which is covered with a cell-friendly coating that allows rapid and gentle separation of cells. Unlabeled cells pass through and magnetically labeled cells remain in the column. The flow-through can be collected as an unlabeled cell portion. After the washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0223] Detailed protocols for purifying specific cell populations (e.g., T cells) can be found in Basu S et al. (2010). (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. (41): 1546).

[0224] In some aspects, the present disclosure provides a method for consuming immune cells expressing CD19-specific CARs by in vivo consumption. In vivo consumption can include administering treatment (e.g., a molecule combining an epitope on CAR) to a mammalian organism, the treatment being intended to prevent the proliferation of immune cells expressing CARs by suppressing or eliminating them.

[0225] One aspect of the present invention relates to a method for consuming in vivo the engineered immune cells of CD19 CARs expressing mAb specific epitopes, comprising contacting the engineered immune cells or the immune cells expressing CAR with at least one epitope-specific mAb. Another aspect of the present invention relates to a method for consuming in vivo the immune cells expressing CAR, the immune cells comprising chimeric scFv (e.g., formed by inserting mAb specific epitopes), the method being achieved by contacting the engineered immune cells with epitope-specific antibodies. In certain embodiments, the immune cells are T cells and / or antibodies are monoclonal.

[0226] According to one embodiment, the engineered immune cells previously sorted using the in vitro method of the present invention are subjected to in vivo consumption of immune engineered cells. In this case, the mAb of the same infusion can be used. In certain embodiments, mAb specific antigen is CD20 antigen, and epitope specific mAb is rituximab. In certain embodiments, the present invention relates to a method for consuming in vivo in a patient an engineered immune cell (immune cell expressing CAR) expressing a CAR comprising a mAb specific epitope, the method comprising contacting the immune cell expressing CAR with at least one epitope specific mAb.

[0227] In some embodiments, the step of contacting the engineered immune cell or the immune cell expressing CAR with at least one epitope-specific mAb comprises infusing an epitope-specific mAb (e.g., rituximab) to the patient. In some embodiments, the amount of the epitope-specific mAb administered to the patient is sufficient to eliminate at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the immune cells expressing CAR in the patient.

[0228] In some embodiments, the step of contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb comprises infusing 375 mg / m 2 In some embodiments, the mAb (e.g., rituximab) is administered once per week.

[0229] In some embodiments, when the immune cells (immune cells expressing CAR) of the CAR comprising mAb specific epitopes are consumed using epitope-specific mAb in complement dependent cytotoxicity (CDC) analysis, the amount of immune cells expressing survival CAR is reduced. In some embodiments, the amount of immune cells expressing survival CAR is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, the mAb specific epitope is a CD20 epitope or a mimotope, and / or the epitope-specific mAb is rituximab.

[0230] In certain embodiments, in vivo depletion of CAR-engineered immune cells is performed by infusion of bispecific antibodies. By definition, bispecific monoclonal antibodies (BsAbs) are artificial proteins composed of fragments of two different monoclonal antibodies and thus bind to two different types of antigens. These BsAbs and their use in immunotherapy have been described in Muller D and Kontermann RE (2010) "Bispecific Antibodies for Cancer Immunotherapy", BioDrugs 24(2):89-98.

[0231] According to another specific embodiment, the infused bispecific mAb is capable of binding to mAb-specific epitopes carried on engineered immune cells expressing chimeric scFvs as well as surface antigens bound to effector and cytotoxic cells (e.g., immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTLs)). By doing so, the depletion of engineered immune cells triggered by the BsAb can occur through antibody-dependent cellular cytotoxicity (ADCC). (Deo YM, Sundarapandiyan K, Keler T, Wallace PK, and Graziano RF, (2000), Journal of Immunology, 165(10):5954-5961]).

[0232] In some embodiments, cytotoxic drugs are coupled to epitope-specific mAbs that can be used to deplete CAR-expressing immune cells. By combining the targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, antibody-drug conjugates (ADCs) provide sensitive discrimination between healthy and diseased tissues when compared to using individual drugs. Several ADCs have received market approval; their manufacturing techniques, particularly utilizing linkers, are described in (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al. (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278).

[0233] In some embodiments, the epitope-specific mAb to be infused is pre-bound to a molecule capable of promoting complement-dependent cytotoxicity (CDC). Thus, the complement system assists or supplements the ability of antibodies to clear pathogens from an organism. When stimulated, an activation cascade is triggered due to the large-scale amplification of responses and the activation of the membrane attack complex of killer cells. Different molecules can be used to bind mAbs, 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 acquired by immunogenic glycan coupling", Electronic Journal of Biotechnology ISSN: 0717-3458; http: / / www.ejbiotechnology.info DOI: 10.2225 / Volume 15-Issue 5).

[0234] VI. Kits and Articles

[0235] The present application provides a kit comprising any of the CD19-containing CAR or CD19 CAR-containing immune cells described herein, and pharmaceutical compositions thereof. In some exemplary embodiments, the kit of the present disclosure comprises allogeneic CD19 CAR-containing T cells and CD52 antibodies for administering a lymphocyte depletion regimen and a CAR-T regimen to an individual.

[0236] The present application also provides an article of manufacture comprising any of the therapeutic compositions or kits described herein.Examples of articles of manufacture include vials (eg, sealed vials).

[0237] Examples

[0238] Example 1: Anti-rituximab CD19 Generation of CAR immune cells

[0239] like Figure 1 Anti-rituximab anti-CD19 chimeric antigen receptor constructs that do not express the rituximab binding site were generated as shown in Table 4. The lentiviral vector constructs were introduced into a viral packaging cell line, and lentivirus containing the anti-CD19 CAR was produced at Allogene.

[0240] Pan T cells from four human donors (541, 604, 410, and 2593) were thawed and cultured at 1.5 × 10 6 1.5×10 cells / ml were activated with large-scale T cell TransAct™ (1:15 ratio). After 2 days, 1.5×10 cells / ml were transduced with 2 ml of fresh lentivirus containing the vectors described in Table 4. 6 cells (3 ml). The schematic diagram of the modified vector is as follows Figure 1 IL-2 (100 IU / ml) was added on day 0, day 2, day 5, day 7, day 9, and day 12. On day 5, a total of 6 × 10 6 Cells were transferred to 6-well G-Rex plates and medium was exchanged on days 9 and 12. Cells were frozen on day 13.

[0241] Table 4: Anti-rituximab CD19 CAR vectors

[0242] Carrier: name: Anti-CD19 CAR v1.0 pCLS-m4G7 CAR (with RQR8 safety switch) Anti-CD19 CAR v1.1 pCLS-4G7_CAR (same EF1a promoter sequence as in v1.0) Anti-CD19 CAR v1.2 pCLS-EF1a(short)-4G7(co) Anti-CD19 CAR v1.3 pCLS-(δRQR)8-4G7(co) (same EF1a promoter sequence as in v1.0) Anti-CD19 CAR v1.4 pCLS-1Q8-4G7(co) (same EF1a promoter sequence as in v1.0) Anti-CD19 CAR v1.5 pCLS-LQL8-4G7(co) (same EF1a promoter sequence as in v1.0) Anti-CD19 CAR v1.6 pCLS-Q38-4G7(co) (same EF1a promoter sequence as in v1.0)

[0243] co = codon optimization

[0244] Flow cytometry experiments were performed on transduced cells gated on lymphocytes, live CD3+, CAR+, CD4 / CD8, and downstream markers. On days 5 and 13, human transduction and CD34 panels were examined using a panel of CD3, CD4, CD8, viability, CD34, and anti-idiotype antibodies against anti-CD19 CAR (4G7 anti-Id). Figure 2A and 2B Shown are flow cytometry graphs demonstrating CAR expression of Pan T cells transduced with the CAR expression vectors shown in Table 4 on day 5 using anti-CD19 CAR anti-Id antibody.

[0245] Flow cytometry of human phenotype and activation panels was performed on days 9 and 13. The panel included CD3, CD4, CD8, viability, CD45RO, CD62L, CD25, 4-1BB, PD-1, anti-idiotype and TIM3 for antibodies against CD19 CAR. At day 13, cells were normalized for cell expansion and final CAR expression from all four donors ( Figure 3 ). Figure 3 The data in Figure 2 show that although v1.2 showed a higher transduction rate (CAR+%), v1.2 transduced cells showed lower CAR expression levels (CAR MFI) compared to, for example, v1.0 and v1.1. Figure 4A )、604( Figure 4B)、410( Figure 4C )、2593( Figure 4D ) of Pan T cells over time and CAR expression.

[0246] Measurement of the anti-rituximab CAR expression vector transduced from donor 541 ( Figure 5A )、604( Figure 5B )、410( Figure 5C )、2593( Figure 5D ) of Pan T cells on days 5, 9, and 13. Figures 6A to 6D Figure 2 shows the transduction of anti-rituximab CAR expression vector from donor 541 ( Figure 6A )、604( Figure 6B )、410( Figure 6C )、2593( Figure 6D ) Pan T cells phenotype and activation on day 9. Figure 7 Figure 9 shows the phenotype, activated CD8+% and T cell anergy averaged from all four donors measured using TIM3 and PD1 staining at day 9. Figure 8A )、604( Figure 8B )、410( Figure 8C )、2593( Figure 8D ) Pan T cells on day 13 phenotype and activation. Figure 9 Shown are the phenotypes, activated CD8+ % and T cell anergy measured from all four donors at day 13 using TIM3 and PD1 staining.

[0247] Example 2: Short-term and long-term in vitro killing assays

[0248] The transduced CAR cells from Example 1 were tested for short-term and long-term killing ability. CAR T cell co-cultures with Raji cells (2:1 E to T) were prepared for subsequent flow fluorometry (Luminex) assays. The average short-term (24 h) killing assay ( Figure 10 ). Figures 11A to 11D The results show that A549-CD19+ cells were used as target cells in each CAR construct with an E:T ratio of 8:1. Figure 11A )、4:1( Figure 11B )、2:1( Figure 11C ) and 1:1( Figure 11D ) in the presence of an average long-term killing assay.

[0249] The correlation between the killing assay results and phenotypic characteristics was analyzed. The killing percentage at day 7 under 1:1 E ratio T was significantly associated with CAR+CD4+41BB+, CAR+CD4+Tim3+ (p=0.0352), CAR+CD4+T EM + (p = 0.0328), CAR + CD8 + PD-1 + (p = 0.0269) and CAR expression % on day 13 (p = 0.0245). The killing percentage at 1:1 E ratio T on day 7 was negatively correlated with CAR + CD8 + T SCM + positively correlated. On day 9, the killing percentage at 1:1 E ratio T was significantly higher than that of CAR+CD4+Tim3+, CAR+CD4+T EM + (p=0.0031), CAR+CD8+T cm + (p = 0.0182) and negatively correlated with CAR expression% on day 13 (p = 0.0469). SCM +, CAR+CD8+Tim3-PD-1- (p=0.0318) and CAR+CD4+T SCM +(p=0.0289) positive correlation.

[0250] Example 3: Titer analysis of lentivirus containing different lentiviral constructs

[0251] In this experiment, the lentiviral vector construct was introduced into a viral packaging cell line, and the anti-CD19 CAR containing lentivirus was produced and titered at Lentigen (Gaithersberg, MD) according to a similar protocol as in Example 1.

[0252] Lentiviral titers were assessed by measuring physical titers of viral protein p24 levels or by measuring transduction titers. Surprisingly, it was found that when the safety switch RQR8 was removed from the lentiviral construct v1.0, viral titers were significantly reduced (compare v1.0 with v1.1 in Table 5). Titers were improved when the EF1a promoter in v1.1 was replaced with a short or truncated EF1a promoter as in v1.2 ("EF1a (short) promoter").

[0253] Table 5 Viral titers of lentiviruses carrying rituximab-sensitive and -resistant CD19 CAR constructs

[0254] Construct Physical titer p24 Transduction titer Anti-CD19 CAR v1.0 9293 ng / mL <![CDATA[1.9x10 9 TU / mL 1 ]]> Anti-CD19 CAR v1.1 2464 ng / mL <![CDATA[5.4x10 8 TU / mL]]> Anti-CD19 CAR v1.2 8208 ng / mL <![CDATA[7x10 9 TU / mL]]> Anti-CD19 CAR v1.3 4494 ng / mL <![CDATA[7x10 8 TU / mL]]> Anti-CD19 CAR v1.4 9686 ng / mL <![CDATA[2.4x10 9 TU / mL]]>

[0255] 1 TU = transduction unit

[0256] To analyze the robustness of the lentiviral formulations of anti-CD19 CAR v1.0, v1.2, and v1.3, a viral titration assay was performed. On day 5, after transduction of pan T cells, serial volume dilutions of the lentiviral formulations of v1.0, v1.2, and v1.3 were analyzed for % CAR+T cells. The results showed that at low dilutions (e.g., 10% v / v), all three constructs exhibited similar acceptable transduction efficiencies. However, at increasing dilutions (e.g., 3.3%, 1.1% v / v), the transduction efficiency of the anti-rituximab anti-CD19 CAR construct v1.3 decreased more significantly compared to the other anti-rituximab anti-CD19 CAR construct v1.2. See Figure 12 Construct v1.2 was selected for in vivo analysis.

[0257] Example 4: In vivo potency assay

[0258] In this experiment, the in vivo anti-tumor potency of ALLO-501v1.2 was analyzed compared to ALLO-501v1.0 in a mouse tumor model. CD19-positive Raji cells carrying a luciferase reporter gene were injected into NSG mice. Lentivirus containing v1.0 or v1.2 lentiviral constructs were transduced into pan T cells of two donors, 541 and 604. NSG mice were inoculated with 100,000 luciferase Raji cells via tail vein injection. On the 4th day after inoculation, NSG mice carrying Raji were administered the CAR construct at a specified dose. Raji engraftment and progression 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).

[0259] Although the disclosed teachings about various applications, methods, kits and compositions have been described, it will be appreciated that various changes and modifications may be made without departing from the teachings herein and the inventions claimed below. The above 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, it will be readily understood by those skilled in the art that a large number of changes and modifications to these exemplary embodiments are possible without excessive experimentation. All such changes and modifications are within the scope of existing teachings.

[0260] SEQ ID NO. chart

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

Claims

1. An isolated polynucleotide encoding a polypeptide comprising an anti-CD19 chimeric antigen receptor (CAR) consisting of SEQ ID NO: 9, wherein the polypeptide does not comprise a rituximab binding site, and wherein the polynucleotide comprises a short EF1a promoter capable of expressing an anti-CD19 chimeric antigen receptor (CAR) in mammalian T cells, The short EF1a promoter consists of the nucleic acid sequence of SEQ ID NO:

16.

2. The isolated polynucleotide of claim 1, wherein the polypeptide further comprises a safety switch.

3. The isolated polynucleotide of claim 2, wherein the safety switch is linked to the anti-CD19 CAR using a linker peptide.

4. The isolated polynucleotide of claim 2, wherein the safety switch is linked to the anti-CD19 CAR using a T2A linker.

5. The isolated polynucleotide of any one of claims 2 to 4, wherein the safety switch comprises an antibody binding site.

6. The isolated polynucleotide of any one of claims 2 to 4, wherein the safety switch comprises a mutated CD20 mimotope.

7. The isolated polynucleotide of any one of claims 2 to 4, wherein the polypeptide comprises a CD34 epitope.

8. The isolated polynucleotide of claim 7, wherein the CD34 epitope is a QBEND-10 epitope.

9. The isolated polynucleotide of any one of claims 2 to 4, wherein the polypeptide further comprises a CD8 hinge / transmembrane domain.

10. The isolated polynucleotide according to any one of claims 1 to 4, comprising a nucleic acid sequence identical to any one of SEQ ID NOs: 2-7.

11. The isolated polynucleotide of any one of claims 1 to 4, encoding a polypeptide identical to any one of SEQ ID NOs: 11-14.

12. A vector comprising the isolated polynucleotide according to any one of the preceding claims.

13. The vector of claim 12, wherein the vector is a retroviral vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof. The vector according to claim 12 , wherein the vector is a DNA vector.

15. An engineered immune cell comprising the isolated polynucleotide of any one of claims 1 to 11, wherein the engineered immune cell does not express a rituximab binding site.

16. An engineered immune cell comprising the vector of any one of claims 12 to 14, wherein the engineered immune cell does not express a rituximab binding site.

17. The engineered immune cell of claim 15 or 16, wherein the immune cell is a T cell.

18. The engineered immune cell of claim 15 or 16, wherein the immune cell is a tumor infiltrating lymphocyte (TIL).

19. The engineered immune cell of claim 15 or 16, wherein the immune cell is a NK cell.

20. The engineered immune cell of claim 15 or 16, wherein the immune cell is a TCR-expressing cell.

21. The engineered immune cell according to claim 15 or 16, wherein the immune cell is a T cell that is further genetically modified by destroying or inactivating the TCRα gene.

22. The engineered immune cell of claim 15 or 16, wherein the immune cell is a dendritic cell.

23. The engineered immune cell of claim 15 or 16, wherein the immune cell is a NK-T cell.

24. The engineered immune cell of any one of claims 17 to 23, wherein the cell is an autologous T cell.

25. The engineered immune cell of any one of claims 17 to 23, wherein the cell is an allogeneic T cell.

26. The engineered immune cell of claim 15 or 16, wherein the cell comprises a polypeptide consistent with SEQ ID NO:

3.

27. The engineered immune cell of claim 15 or 16, wherein the cell is resistant to rituximab.

28. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 15 to 27.

29. Use of the engineered immune cell according to any one of claims 15 to 27 or the pharmaceutical composition according to claim 28 in the preparation of a medicament for treating non-Hodgkin's lymphoma (NHL).

30. The use according to claim 29, wherein the subject has been or is currently being treated with rituximab.

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