Genetically modified cells containing heterologous nucleic acid molecules inserted at the CD5 gene locus.

Abstract

This embodiment provides cells containing heterologous nucleic acid molecules inserted at the CD5 gene locus. A composition containing such cells, as well as methods for producing and using such cells and compositions, are also provided herein.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 63 / 503,809, filed on 23 May 2023, which is incorporated by reference in its entirety herein.

[0002] Reference to sequence listings filed electronically This application includes a sequence listing submitted electronically in XML format, the entire sequence listing being incorporated herein by reference. The XML copy created on 16 May 2024 is named VTB-005WO_SL and has a size of 96,478 bytes.

[0003] This embodiment generally relates to cells, and examples include immune cells (e.g., T cells, NK cells) that contain heterologous nucleic acid molecules at the CD5 locus, which can be used, for example, in the treatment of cancer. [Background technology]

[0004] Gene editing and gene integration technologies have expanded the ability to modify cells to contain heterologous nucleic acid molecules that can be used to express heterologous molecules (such as chimeric antigen receptors, target nucleic acid molecules, or target proteins). However, when inserting heterologous nucleic acid molecules into cells, there is always a risk that such insertions may create cells with undesirable properties. Therefore, it is necessary to identify safe harbor gene loci that allow for the insertion of heterologous nucleic acid molecules without harmful or undesirable effects on cells. This embodiment satisfies these needs, as well as other needs. [Overview of the Initiative]

[0005] In some embodiments, cells containing a heterologous nucleic acid molecule are provided, which is inserted at the CD5 locus. In some embodiments, the cells have reduced or absent CD5 expression and / or function. In some embodiments, the heterologous nucleic acid molecule replaces the CD5 locus.

[0006] In some embodiments, the cells are immune cells such as immune effector cells. In some embodiments, the immune cells are T cells, NK cells, or B cells. In some embodiments, the T cells are αβ T cells. In some embodiments, the B cells are B1 B cells.

[0007] In some embodiments, pharmaceutical compositions comprising multiple cells are provided herein.

[0008] In some embodiments, methods for producing cells containing heterologous nucleic acid molecules inserted at the CD5 locus are described herein. In some embodiments, the method involves contacting cells with a gene editing system under conditions sufficient to produce cells containing heterologous nucleic acid molecules inserted at the CD5 locus. In some embodiments, the cells are contacted with the gene editing system in vivo. In some embodiments, the cells are contacted with the gene editing system ex vivo.

[0009] In some embodiments, the gene editing system is a CRISPR system (e.g., CRISPR / CAS9) or a zinc finger nuclease.

[0010] In some embodiments, methods are provided for treating subjects having diseases such as cancer. In some embodiments, the method involves administering cells, or compositions containing cells, to the subject as shown herein. In some embodiments, cells containing heterologous nucleic acid molecules inserted at the CD5 locus are more effective in treating cancer or disease than cells containing heterologous nucleic acid molecules not inserted at the CD5 locus. [Brief explanation of the drawing]

[0011] [Figure 1] This demonstrates how a nonviral CRISPR / Cas9 knock-in platform, along with the addition of ssDNA-tCTS (cleavage-type Cas9 target sequence) oligos, precisely edits the human CD5 locus (RAB11A). [Figure 2A] Panels A, B, and C show the timeline (Figure 2, Panel A) for activating and preparing CD4 / CD8+ T cells using TransAct CD3 / CD28 beads and electroporation (EP), as well as flow cytometry results from two donors tested, 7 days (Figure 2, Panel B) and 9 days (Figure 2, Panel C) after activation, demonstrating that up to 20% GFP knock-in was achieved by the CRISPR / Cas9-mediated ssDNA-tCTS approach. [Figure 2B] Panels A, B, and C show the timeline (Figure 2, Panel A) for activating and preparing CD4 / CD8+ T cells using TransAct CD3 / CD28 beads and electroporation (EP), as well as flow cytometry results from two donors tested, 7 days (Figure 2, Panel B) and 9 days (Figure 2, Panel C) after activation, demonstrating that up to 20% GFP knock-in was achieved by the CRISPR / Cas9-mediated ssDNA-tCTS approach. [Figure 2C] Panels A, B, and C show the timeline (Figure 2, Panel A) for activating and preparing CD4 / CD8+ T cells using TransAct CD3 / CD28 beads and electroporation (EP), as well as flow cytometry results from two donors tested, 7 days (Figure 2, Panel B) and 9 days (Figure 2, Panel C) after activation, demonstrating that up to 20% GFP knock-in was achieved by the CRISPR / Cas9-mediated ssDNA-tCTS approach. [Figure 3]Figure 3A shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 48 hours after "knock-in". CD4 His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. Results for untreated cells are shown. Figure 3B shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 48 hours after "knock-in". CD4 His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. Results for CD5 KO CAR-negative cells are shown. Figure 3C shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 48 hours after "knock-in". CD4 His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. Results for CD5 KO-CART5 MSCV knock-in cells in 4 pmol ssCTS are shown. Figure 3D shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 48 hours after "knock-in". CD4HisPE represents the labeled CD5 antigen, which is detected on the surface expression of CAR. The results for CD5 KO-CART5 MSCV knock-in cells in 8 pmol ssCTS are shown. [Figure 4]Figure 4A shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". CD4His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. Results for untreated cells are shown. Figure 4B shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". CD4His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. Results for CD5 KO CAR-negative cells are shown. Figure 4C shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". CD4His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. Results for CD5 KO-CART5 MSCV knock-in cells in 4pmol ssCTS are shown. Figure 4D shows the expression of CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". CD4His PE represents the labeled CD5 antigen detecting the surface expression of the CAR. The results for CD5 KO - CART5 MSCV knock-in cells in 8 pmol ssCTS are shown. [Figure 5]Figure 5A shows the expression of the CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". G4S FITC represents the detection of the linker region within the CD5 CAR. CD5 APC represents the anti-CD5 antibody detecting the surface expression of CD5. Results for untreated cells are shown. Figure 5B shows the expression of the CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". G4S FITC represents the detection of the linker region within the CD5 CAR. CD5 APC represents the anti-CD5 antibody detecting the surface expression of CD5. Results for CD5 KO CAR-negative cells are shown. Figure 5C shows the expression of the CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". G4S FITC represents the detection of the linker region within the CD5 CAR. CD5 APC represents the anti-CD5 antibody detecting the surface expression of CD5. The results for CD5 KO-CART5 MSCV knock-in cells in 4 pmol ssCTS are shown. Figure 5D shows the expression of the CD5 CAR (CART5) inserted into the CD5 locus 5 days after "knock-in". G4S FITC represents the detection of the linker portion within the CD5 CAR. CD5 APC represents the anti-CD5 antibody detecting the surface expression of CD5. The results for CD5 KO-CART5 MSCV knock-in cells in 8 pmol ssCTS are shown. [Figure 6A] The results of PCR analysis of Jurkat cells in the following conditions are shown: untreated, RNP only, RNP + 4pmol MSCV CART5 ssCTS, and RNP + 8pmol MSCV CART5 ssCTS. Primer designs are also shown. [Figure 6B] The results of PCR analysis of Jurkat cells in the following states are shown: untreated, RNP only, RNP + 4pmol MSCV CART5 ssCTS, and RNP + 8pmol MSCV CART5 ssCTS. Representative agarose gels of the amplified products are shown. [Figure 7]The results of IFNγ efficacy assays in Jurkat cells that were untreated, RNP-only, RNP + 4pmol MSCV CART5 ssCTS, or RNP + 8pmol MSCV CART5 ssCTS are shown. Jurkat cells were either CD5 KO-CAR negative, WT, or target cell-only. [Modes for carrying out the invention]

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the embodiments relate.

[0013] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of articles. For example, "an element" means one or more elements.

[0014] When referring to measurable values ​​(such as quantity or duration), the term "approximately" means that it encompasses a variation of ±20%, or possibly ±10%, or possibly ±5%, or possibly ±1%, or possibly ±0.1%, from the specified value, provided that such variation is appropriate for carrying out the disclosed method.

[0015] The term “chimeric antigen receptor,” or alternatively “CAR,” refers to a polypeptide that, when present in an immune effector cell, provides the cell with specificity to target cells (such as cancer cells) and intracellular signaling. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”), the cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulating molecule and / or a co-stimulating molecule as defined below. In some embodiments, the domains are contiguous within a single polypeptide or multiple polypeptides. In some embodiments, the polypeptide may include a dimerization switch, the dimerization switch which can couple polypeptides to each other in the presence of a dimerizing molecule, for example, by coupling an antigen-binding domain to an intracellular signaling domain. In some embodiments, the stimulating molecule is a zeta chain associated with a T cell receptor complex. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulating molecule as defined below. In some embodiments, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from the stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, a functional signaling domain derived from the co-stimulatory molecule, and an intracellular signaling domain comprising a functional signaling domain derived from the stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules, and a functional signaling domain derived from the stimulatory molecule.In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules, and a functional signaling domain derived from the stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino terminus (N terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0016] A CAR containing an antigen-binding domain (e.g., scFv or TCR) that targets a specific tumor maker X, as described herein, is also called an XCAR. For example, a CAR containing an antigen-binding domain that targets CD19 is called a CD19CAR.

[0017] The term "signaling domain" refers to the functional portion of a protein that functions by transmitting information within a cell and regulating cellular activity through defined signaling pathways, either by generating second messengers or by acting as an effector in response to such messengers.

[0018] As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from a natural recombinant source or a recombinant source. Antibodies may be tetramers of immunoglobulin molecules.

[0019] The term "antibody fragment" refers to at least one portion of an antibody that possesses the ability to specifically interact with an antigen epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, scFv antibody fragments, disulfide-linked Fv(sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb(either VL or VH), multispecific antibodies formed from antibody fragments such as the VHH domain of a camelid, a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, CD5 labodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be transplanted into polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0020] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked, for example, via a synthetic linker (e.g., a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, the scFv may have the VL and VH variable regions in either order, and for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may contain a VL-linker-VH or a VH-linker-VL.

[0021] The portion of the CAR containing an antibody or antibody fragment may exist in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR contains an antibody fragment. In some embodiments, the CAR contains an antibody fragment containing an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using one of several well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), A1-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or a combination thereof.

[0022] As used herein, the terms “binding domain” or “antibody molecule” refer to a protein (e.g., an immunoglobulin chain or fragment thereof) containing at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” encompass antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, for example, comprising multiple immunoglobulin variable domain sequences, where multiple first immunoglobulin variable domain sequences have binding specificity to a first epitope, and multiple second immunoglobulin variable domain sequences have binding specificity to a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity to two or fewer antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope, and a second immunoglobulin variable domain sequence having binding specificity to a second epitope.

[0023] The portion of the CAR containing an antibody or antibody fragment may exist in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR contains an antibody fragment. In some embodiments, the CAR contains an antibody fragment containing an scFv.

[0024] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule within its natural conformation. This usually determines the class to which the antibody belongs.

[0025] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules within their natural conformation. Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.

[0026] The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, examples of which include antibodies expressed by bacteriophage or yeast expression systems. This term should also be interpreted to mean antibodies produced by the synthesis of DNA molecules, which encode antibodies and express antibody proteins or amino acid sequences that specify the antibody, and the DNA or amino acid sequences are obtained using recombinant DNA or amino acid sequence technologies that are available and well known in the art.

[0027] The terms “antigen” or “Ag” refer to molecules that trigger an immune response. This immune response may involve antibody production, activation of specific immune-qualified cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may originate from recombinant DNA or genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that triggers an immune response encodes the term “antigen” as used herein. Furthermore, those skilled in the art will understand that an antigen does not need to be encoded by a full-length nucleotide sequence of a gene alone. This embodiment includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to encode polypeptides that trigger a desired immune response. Moreover, a skilled craftsman will understand that an antigen does not need to be encoded by a “gene” at all. It is readily apparent that antigens may be synthesized, produced, derived from biological samples, or even macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids containing other biological components.

[0028] The term "anti-cancer effect" refers to biological effects that can be manifested by various means, including, but are not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an extension of life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival rate, or an improvement in various physiological symptoms associated with the pathogenesis of cancer. "Anti-cancer effects" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the development of cancer in the first place. The term "anti-tumor effect" refers to biological effects that can be manifested by various means, including, but are not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival rate.

[0029] The term "self" refers to any material originating from the same individual that is later reintroduced into that individual.

[0030] The term "homogeneous" refers to any material originating from different animals of the same species as the individual into which it is introduced. Two or more individuals are said to be homogeneous if their genes at one or more loci are not identical. In some embodiments, homogeneous material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0031] The term "heterogeneous" refers to grafts derived from animals of different species.

[0032] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein. These include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In this specification, the terms "tumor" and "cancer" are used interchangeably, for example, both terms encompass solid and liquid tumors, such as diffusive or circulating tumors. As used herein, the terms "cancer" or "tumor" include precancerous cancers and malignant cancers as well as tumors.

[0033] As used herein, the term "derived from" indicates a relationship between a first molecule and a second molecule. This generally refers to a structural similarity between the first and second molecules and does not imply or include any limitations on processing or sources for the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the necessary function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include any limitations on specific processing to produce the intracellular signaling domain. For example, it does not mean that to provide an intracellular signaling domain, one must start from a CD3 zeta sequence and delete or mutate unwanted sequences to reach the intracellular signaling domain.

[0034] The phrase "diseases associated with the expression of tumor antigens as described herein" includes, but is not limited to, diseases associated with the expression of tumor antigens as described herein, or conditions associated with cells expressing tumor antigens as described herein (including, for example, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndromes, or preleukemia), or non-cancer-related indications associated with cells expressing tumor antigens as described herein. In some embodiments, cancers associated with the expression of tumor antigens as described herein are hematological cancers. In some embodiments, cancers associated with the expression of tumor antigens as described herein are solid tumors. Further diseases associated with the expression of tumor antigens as described herein include, but are not limited to, atypical and / or non-typical cancers, malignant tumors, precancerous conditions, or proliferative disorders associated with the expression of tumor antigens as described herein. Non-cancer-related indications associated with the expression of tumor antigens as described herein include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express mRNA encoding the tumor antigen, or express it at any given time. In some embodiments, tumor antigen-expressing cells produce tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In some embodiments, tumor antigen-expressing cells produce a detectable level of tumor antigen protein at some point in time, and then substantially no longer produce detectable levels of tumor antigen protein.

[0035] The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in a CAR can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0036] The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex or CAR) to its homologous ligand (or, in the case of a CAR, a tumor antigen), thereby mediating a signaling event. Examples of signaling events include, but are not limited to, signaling via the TCR / CD3 complex or signaling via the appropriate NK receptor or signaling domain of a CAR. Stimulation can mediate changes in the expression of specific molecules.

[0037] The term “stimulating molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides cytoplasmic signaling sequences (may be multiple) that control the activation of immune cells in a stimulating manner to at least some aspects of an immune cell signaling pathway. In some embodiments, the signal is a primary signal initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, and leading to the mediation of a T cell response (including, but not limited to, proliferation, activation, and differentiation). The primary cytoplasmic signaling sequence that acts in a stimulating manner (also called the “primary signaling domain”) may contain a signaling motif known as an immune receptor tyrosine activation motif or ITAM. Examples of ITAM-containing cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, any one or more intracellular signaling domains within a CAR include an intracellular signaling sequence (e.g., the primary signaling sequence of CD3 zeta). In some embodiments, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 18, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 20, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0038] The term "antigen-presenting cell" or "APC" refers to immune system cells, including accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens on their surface, often complexed with major histocompatibility complexes (MHC). T cells may recognize these complexes using T cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0039] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains generate signals that promote immune effector functions in CAR-containing cells (e.g., CART cells). Examples of immune effector functions, in CART cells, include cytolytic activity and helper activity, including cytokine secretion.

[0040] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. An exemplary primary intracellular signaling domain may be derived from a molecule responsible for primary stimulation or antigen-dependent simulation. In some embodiments, the intracellular signaling domain may include a co-stimulatory intracellular domain. An exemplary co-stimulatory intracellular signaling domain may be derived from a molecule responsible for co-stimulatory signaling or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule.

[0041] The primary intracellular signaling domain may contain signaling motifs known as immune receptor tyrosine activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0042] The terms “Zeta” or alternatively “Zeta chain,” “CD3-Zeta,” or “TCR-Zeta” are defined as the protein provided as GenBan accession number BAG36664.1, or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.), and the “Zeta-stimulating domain” or alternatively “CD3-zeta-stimulating domain” or “TCR-zeta-stimulating domain” are defined as amino acid residues from the cytoplasmic domain of the Zeta chain or its functional derivatives sufficient to functionally transmit the initial signals necessary for T cell activation. In some embodiments, the cytoplasmic domain of Zeta includes residues 52-164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.) that are functional orthologues thereof. In some embodiments, the “Zeta-stimulating domain” or “CD3-zeta-stimulating domain” is the sequence provided as Sequence ID No. 18. In some embodiments, the "zeta-stimulating domain" or "CD3 zeta-stimulating domain" is the sequence provided as Sequence ID No. 20.

[0043] The term "costimulatory molecule" refers to a congenital binding partner on a T cell, which specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell (including, but not limited to, proliferation). Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, and DNAM1. The product contains ligands that specifically bind to CD83, including CD226, SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0044] The co-stimulatory intracellular signaling domain may be the intracellular portion of a co-stimulatory molecule. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), and activated NK cell receptors. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and CD83.

[0045] The intracellular signaling domain may include the entire intracellular portion of the derived molecule, the entire intrinsic intracellular signaling domain, or a functional fragment or derivative thereof.

[0046] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In some embodiments, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 14, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0047] As used herein, the term “immune effector cell” refers to a cell involved in an immune response (e.g., promoting an immune effector response). Examples of immune effector cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.

[0048] As used herein, the term “immune effector function or immune effector response” refers to a function or response (e.g., of an immune effector cell) that enhances or promotes an immune attack against a target cell. For example, immune effector function or response refers to the properties of a T cell or NK cell that promote the killing of a target cell or the inhibition of its growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0049] The term "encode" refers to the inherent properties of a specific nucleotide sequence within a polynucleotide (such as a gene, cDNA, or mRNA) that serve as a template for the synthesis of other polymers and macromolecules in biological processes, possessing a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein when a protein is produced in a cell or other biological system by the transcription and translation of the mRNA corresponding to that gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually shown in the sequence listing) and the non-coding strand (used as a template for the transcription of the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0050] Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” includes all nucleotide sequences that encode the same amino acid sequence, including degenerate versions of each other. The phrase “nucleotide sequences encoding a protein or RNA” may also include introns, insofar as protein-encoding nucleotide sequences may contain introns in some variant form.

[0051] The terms “effective dose” or “therapeutic dose” are used interchangeably herein and refer to the amount of any compound, formulation, material or composition described herein that is effective in achieving a particular biological outcome.

[0052] The term "endogenous" refers to any material obtained from or produced within an organism, cell, tissue, or system.

[0053] The term "exogenous" refers to any material that is introduced from outside an organism, cell, tissue, or system, or produced outside of them.

[0054] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0055] The term “transfer vector” refers to a composition of substance which comprises isolated nucleic acids and can be used to deliver isolated nucleic acids into cells. Numerous vectors are known in the art. These include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transport of nucleic acids into cells, examples of which include, for example, polylysine compounds and liposomes. Examples of viral transport vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0056] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operatively ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by host cells or in an in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (e.g., naked or liposome-containing), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0057] The term "lentivirus" refers to a genus of retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and are one of the most efficient methods of gene delivery vectors because they can deliver large amounts of genetic information to the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses.

[0058] The term “lentiviral vector” refers to a vector derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors (as shown in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009)). Other examples of lentiviral vectors available for clinical use include, but are not limited to, Oxford BioMedica’s LENTIVECTOR® gene delivery technology and Lentigen’s LENTIMAX® vector system. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0059] The terms "homologous" or "identical" refer to the subunit sequence identity between two polymer molecules (e.g., two nucleic acid molecules, such as two DNA molecules or two RNA molecules) or two polypeptide molecules. If both subunit positions in two molecules are occupied by the same monomeric subunit, for example, if each position in two DNA molecules is occupied by adenine, then those molecules are homologous or identical at that position. Homologousness between two sequences is a direct function of the number of matching or homologous positions. For example, two sequences are 50% homologous if half of the positions in them (e.g., five positions in a polymer of length 10 subunits) are homologous. Two sequences are 90% homologous if 90% of the positions (e.g., nine out of ten) are matching or homologous.

[0060] The "humanized" form of non-human (e.g., murine) antibodies is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of the antibody), which contains minimal sequences derived from the non-human immunoglobulin. In most cases, the humanized antibody and its antibody fragment are human immunoglobulin (recipient antibody or antibody fragment), where residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody) with desired specificity, affinity, and capability, such as mouse, rat, or rabbit. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody / antibody fragment may contain residues not found in either the recipient antibody or the imported CDR or framework sequence. These modifications can further improve and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or antibody fragments contain substantially all of at least one, typically two, variable domains, with all or substantially all of their CDR region corresponding to the CDR region of a non-human immunoglobulin, and all or most of their FR region being the FR region of a human immunoglobulin sequence. Humanized antibodies or antibody fragments may also contain at least a portion of the immunoglobulin constant region (Fc) (typically of human immunoglobulin). For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0061] "Completely human" refers to immunoglobulins, examples of which include antibodies or antibody fragments. Here, the entire molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody or immunoglobulin.

[0062] The term "isolation" means being altered or removed from its natural state. For example, nucleic acids or peptides that naturally exist in a living animal are not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from the coexisting material in its natural state. Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment (e.g., a host cell).

[0063] The following abbreviations are commonly used for the nucleic acid bases that are produced: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0064] The terms "operably linked" or "transcriptional control" refer to a functional linkage between a control sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, when a first nucleic acid sequence is positioned in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, when a promoter affects the transcription or expression of a coding sequence, that promoter is operably linked to the coding sequence. Operatively linked DNA sequences can be contiguous with each other, for example, if two protein-coding regions need to be joined, they are within the same reading frame.

[0065] The term "parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0066] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded forms, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes not only the explicitly stated sequence, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitution may be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0067] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include peptides or proteins containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short-chain (e.g., commonly called peptides, oligopeptides, and oligomers in the art) and long-chain (generally called proteins in the art), of which there are many types. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0068] The term "promoter" refers to a DNA sequence recognized by the cell's synthetic machinery, or introduced synthetic machinery, which is necessary to initiate the specific transcription of a polynucleotide sequence.

[0069] The term "promoter / control sequence" refers to a nucleic acid sequence necessary for the expression of a gene product, operably ligated to a promoter / control sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, it may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. The promoter / control sequence may, for example, express the gene product in a tissue-specific manner.

[0070] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or designating a gene product, causes the gene product to be produced within the cell under most or all physiological conditions.

[0071] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell only if an inducer substantially corresponding to the promoter is present within the cell.

[0072] The term "tissue-specific" promoter refers to a nucleotide sequence that, when encoded by a gene or operably linked to a polynucleotide specified by the gene, ensures that gene products are produced within the cell only if the cell is of the tissue type corresponding to the promoter.

[0073] Interchangeably, the terms “cancer-associated antigen” or “tumor antigen” refer to molecules (usually proteins, carbohydrates, or lipids) expressed either as whole or as fragments (e.g., MHC / peptides) on the surface of cancer cells, which are useful for pharmacological agents to preferentially target cancer cells. In some embodiments, tumor antigens are markers expressed by both normal and cancer cells, examples of which include lineage markers, such as CD19 on B cells. In some embodiments, tumor antigens are cell surface molecules that are overexpressed in cancer cells compared to normal cells, e.g., 1x overexpression, 2x overexpression, 3x overexpression, or more compared to normal cells. In some embodiments, tumor antigens are cell surface molecules that are improperly synthesized within cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, tumor antigens are expressed exclusively on the cell surface of cancer cells, either whole or as fragments (e.g., MHC / peptides), and are not synthesized or expressed on the surface of normal cells. In some embodiments, the CAR comprises an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presenting peptide. Typically, peptides derived from endogenous proteins fill the pocket of a major histocompatibility complex (MHC) class I molecule and are recognized by the T cell receptor (TCR) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets in immunotherapy.TCR-like antibodies targeting viral or tumor antigen-derived peptides in relation to human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0074] Interchangeably, the terms “tumor-supporting antigen” or “cancer-supporting antigen” refer to molecules (usually proteins, carbohydrates, or lipids) expressed on the surface of cells that are not cancerous themselves but support cancer cells (for example, by promoting their growth or survival, or by providing resistance to immune cells). Exemplary cells of this type include stromal cells and bone marrow-derived suppressor cells (MDSCs). Tumor-supporting antigens themselves do not need to play a supporting role for tumor cells as long as they are present on cells that support cancer cells.

[0075] The terms “flexible polypeptide linker” or “linker” as used in relation to scFv refer to peptide linkers consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together. In some embodiments, the flexible polypeptide linker is a Gly / Ser linker comprising the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer of 1 or greater. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10 (SEQ ID NO: 28). In some embodiments, the flexible polypeptide linker comprises, but is not limited to, (Gly4 Ser)4 (SEQ ID NO: 29) or (Gly4 Ser)3 (SEQ ID NO: 30). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 31). Other linkers include those described in WO2012 / 138475, which are incorporated herein by reference.

[0076] When used herein, a 5' cap (RNA cap, RNA 7-methylguanosine cap or RNA m) is used. 7 A 5' cap (also known as a G-cap) is a modified guanine nucleotide added to the "front" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of terminal groups linked to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. The addition of the cap is coupled to transcription and occurs cotranscribely, meaning each affects the other. Immediately after transcription initiation, the 5' end of the synthesizing mRNA is ligated by a cap synthesis complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping region can be modified to regulate the functionality of mRNA, such as stability or translation efficiency.

[0077] As used herein, “in vitro transcribed RNA” refers to RNA (preferably mRNA) synthesized in vitro. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used to produce in vitro transcribed RNA.

[0078] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In preferred embodiments of transient expression constructs, poly(A) is 50 to 5000 (SEQ ID NO: 34), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate the functionality of the mRNA, such as localization, stability, or translation efficiency.

[0079] As used herein, “polyadenylation” refers to the covalent binding of a polyadenylyl portion or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules have a polyadenylated 3' end. The 3' poly(A) tail is a long sequence (often hundreds) of adenine nucleotides added to pre-mRNA by the action of an enzyme called polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is attached to a transcript containing a specific sequence, i.e., a polyadenylation signal. The poly(A) tail and the protein bound to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription from DNA to RNA, but can also occur later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0080] As used herein, “transient” refers to the expression of an unintegrated transgene over a period of several hours, days, or weeks, the duration of which is shorter than the duration of expression of a gene when it is integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0081] As used herein, the terms “to treat,” “treatment,” and “to treat” refer to the reduction or improvement of the progression, severity, and / or duration of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as CARs), or the improvement of one or more symptoms (preferably one or more identifiable symptoms) of the proliferative disorder. In specific embodiments, the terms “to treat,” “treatment,” and “to treat” refer to the improvement of at least one measurable physical parameter of a proliferative disorder (e.g., tumor growth) that is not necessarily identifiable by the patient. In other embodiments, the terms “to treat,” “treatment,” and “to treat” refer to inhibiting the progression of a proliferative disorder, either physically, for example by stabilizing an identifiable symptom, or physiologically, for example by stabilizing a physical parameter, or both. In other embodiments, the terms “to treat,” “treatment,” and “to treat” refer to a reduction or stabilization of tumor size or cancer cell count.

[0082] The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting signals from one part of a cell to another. The term "cell surface receptor" includes molecules and complexes of molecules that can receive and transmit signals across the cell membrane.

[0083] The term "target" includes organisms that can trigger an immune response (e.g., mammals, humans).

[0084] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells isolated from other cell types that are normally associated with them in their naturally occurring state. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells isolated from naturally associated cells in their natural state. In some embodiments, the cells are cultured in vitro. In some embodiments, the cells are not cultured in vitro.

[0085] As used herein, the term “treatment” means a procedure. Therapeutic effects are obtained by reducing, suppressing, relieving, or eradicating the symptoms of a disease.

[0086] As used herein, the term “prevention” means preventive or protective measures against disease or medical condition.

[0087] "Tumor antigen," "hyperproliferative disorder antigen," or "antigen associated with hyperproliferative disorder" refers to an antigen common to a specific hyperproliferative disorder. In some embodiments, the hyperproliferative disorder antigen is derived from cancer, including, but is not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma (such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer).

[0088] The terms “transfected,” “transformed,” or “transduced” refer to the process of introducing or transferring exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Such cells include primary target cells and their progeny.

[0089] The term "specifically binding" refers to an antibody or ligand that recognizes and binds to a binding partner protein (e.g., a tumor antigen) present in the sample, but does not substantially recognize or bind to other molecules in the sample.

[0090] As used herein, the term “controllable chimeric antigen receptor (RCAR)” refers to a set of polypeptides (typically two in the simplest embodiment). When present in RCARX cells, these polypeptides provide RCARX cells with specificity to target cells (typically cancer cells) and controllable intracellular signaling or proliferation that can optimize the immunoeffector properties of RCARX cells. RCARX cells provide specificity to target cells containing the antigen bound by the antigen-binding domain, at least in part, depending on the antigen-binding domain. In some embodiments, the RCAR includes a dimerization switch that allows the intracellular signaling domain to be coupled to the antigen-binding domain in the presence of a dimerizing molecule.

[0091] As used herein, the terms “membrane anchor” or “membrane CD5 hering domain” refer to a polypeptide or moiety (e.g., a myristoyl group) sufficient to anchor an extracellular or intracellular domain to the cell membrane.

[0092] As used herein, the term “switch domain” refers to an entity (usually a polypeptide-based entity) associated with another switch domain in the presence of a dimerizing molecule, for example, when referring to RCAR. This association results in a functional binding between a first entity linked (e.g., fused) to the first switch domain and a second entity linked (e.g., fused) to the second switch domain. The first and second switch domains are collectively referred to as a dimerizing switch. In some embodiments, the first and second switch domains are identical (e.g., polypeptides having the same primary amino acid sequence) and are collectively called a homodimerizing switch. In some embodiments, the first and second switch domains are different (e.g., polypeptides having different primary amino acid sequences) and are collectively called a heterodimerizing switch. In some embodiments, the switch is intracellular. In some embodiments, the switch is extracellular. In some embodiments, the switch domain is a polypeptide-based entity (e.g., FKBP or FRB-based) and the dimerizing molecule is a small molecule (e.g., a rapalog). In some embodiments, the switch domain is a polypeptide-based entity (e.g., an scFv that binds a myc peptide), and the dimerizing molecule is a polypeptide, a fragment thereof, or a polypeptide polymer (e.g., a myc ligand that binds to one or more myc scFvs, or a polymer of myc ligands). In some embodiments, the switch domain is a polypeptide-based entity (e.g., a myc receptor), and the dimerizing molecule is an antibody or a fragment thereof (e.g., a myc antibody).

[0093] As used herein, the term “dimerizing molecule” refers to a molecule that facilitates the association between the first and second switch domains, for example, when referring to RCAR. In some embodiments, the dimerizing molecule does not occur spontaneously in the subject, nor does it occur at concentrations that result in significant dimerization. In some embodiments, the dimerizing molecule is a small molecule (e.g., rapamycin) or a rapalog (e.g., RAD001).

[0094] The term "biologically equivalent" refers to the amount of an agent other than the reference compound (e.g., RAD001) required to produce an effect equivalent to the effect produced by the reference dose or amount of the reference compound (e.g., RAD001). In one embodiment, this effect is the level of mTOR inhibition (e.g., as measured by P70 S6 kinase inhibition, as evaluated, for example, by an in vivo or in vitro assay, as measured, for example, by the assay described herein, for example, as measured by a Bouley assay). In some embodiments, the effect is a change in the ratio of PD-1-positive T cells to PD-1-negative T cells, which is measured by cell sorting. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as the reference dose or amount of the reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the ratio of PD-1-positive T cells to PD-1-negative T cells as the reference dose or amount of the reference compound.

[0095] The term “immunoenhancing low dose” refers, when used in conjunction with an mTOR inhibitor (e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor), to a dose of an mTOR inhibitor that partially but not completely inhibits mTOR activity (e.g., as measured by inhibition of P70 S6 kinase activity). This specification discusses, for example, a method for evaluating mTOR activity by inhibition of P70 S6 kinase. This dose is insufficient to produce complete immunosuppression but sufficient to enhance the immune response. In some embodiments, an immunoenhancing low dose mTOR inhibitor results in a decrease in the number of PD-1-positive T cells and / or an increase in the number of PD-1-negative T cells, or an increase in the PD-1-negative T cell / PD-1-positive T cell ratio. In some embodiments, an immunoenhancing low dose mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, an immunoenhancing low dose mTOR inhibitor results in one or more of the following:

[0096] Marker CD62L high CD127 high CD27 + and increased expression of one or more of the BCL2 (for example, in memory T cells, an example being memory T cell precursors);

[0097] Decreased expression of KLRG1 (for example, in memory T cells, an example being memory T cell precursors); and

[0098] Memory T cell precursor (e.g., CD62L high Increase, CD127 high Increase, CD27 + An increase in the number of cells having one or a combination of the following characteristics: an increase in KLRG1, a decrease in KLRG1, and an increase in BCL2;

[0099] Any of the above changes occur, for example, compared to the untreated subjects, at least transiently.

[0100] As used herein, “refractory” refers to a disease (e.g., cancer) that does not respond to treatment. In some embodiments, refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, refractory cancer may become resistant during treatment. Refractory cancer is also called resistant cancer.

[0101] As used herein, “relapse” refers to a return of a disease such as cancer (e.g., cancer) or the signs and symptoms of a disease such as cancer, after a period of improvement (e.g., after pretreatment, e.g., cancer treatment).

[0102] Scope: Throughout this disclosure, various embodiments may be presented in scope form. It should be understood that scope form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the embodiments. Therefore, scope descriptions should be considered to specifically disclose all possible sub-ranges, and even the individual numerical values ​​within those ranges. For example, a scope description such as 1-6 should be considered to have specifically disclosed sub-ranges (1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc.), and even the individual numerical values ​​within those ranges (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6). As another example, a scope such as 95-99% identity includes those having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-ranges (96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity, etc.). This applies regardless of the scope.

[0103] As used herein, the term "CD5" refers to the gene for differentiation antigen group 5 and the protein encoded by that gene. CD5 may also be known as T1 or LEU1. In the human genome, CD5 is located on chromosome 11, and its GenBank number is NC_00011.10.

[0104] In this specification, the term “system” as used in relation to gene editing or CD5 inhibition refers to a group of molecules (e.g., one or more molecules) that act together to achieve a desired function.

[0105] As used herein, the term “gene editing system” refers to a system (e.g., one or more molecules) that induces and achieves a modification (e.g., deletion) of one or more nucleic acids at or near a site of genomic DNA targeted by the system. Gene editing systems are known in the art and are described more comprehensively below.

[0106] In this specification, the term "binding partner" as used in relation to CD5 binding partners refers to a molecule (e.g., a protein) that interacts with (e.g., binds to) the CD5 protein.

[0107] A "dominant-negative" gene product or protein interferes with the function of another gene product or protein. The other affected gene product may be the same as or different from the dominant-negative protein. Dominant-negative gene products can take many forms, including full-length proteins containing cleavage, point mutations or fragments thereof, or fusions of full-length wild-type or mutant proteins or fragments thereof with other proteins. The observed inhibition levels may be very low. For example, a large excess of the dominant-negative protein may be required compared to the functional protein(s) involved in the treatment to confirm the effect. It may be difficult to confirm the effect under normal biological assay conditions. In some embodiments, the dominant-negative CD5 is catalytically inactive CD5.

[0108] While we do not wish to be bound by theory, cells possessing the "central memory T cell (Tcm) phenotype" express CCR7 and CD45RO. In some embodiments, cells possessing the central memory T cell phenotype express CCR7 and CD45RO and / or do not express CD45RA or express it at a lower level compared to naive T cells. In some embodiments, cells possessing the central memory T cell phenotype express CD45RO and CD62L and / or do not express CD45RA or express it at a lower level compared to naive T cells. In some embodiments, cells possessing the central memory T cell phenotype express CCR7, CD45RO, and CD62L and / or do not express CD45RA or express it at a lower level compared to naive T cells.

[0109] While I don't want to be constrained by theory, cells with the "effector memory T cell (Tem) phenotype" either do not express CCR7 or express it at lower levels, and express CD45RO at higher levels compared to naive T cells.

[0110] The pathways described herein are explained, for example, by gene ontology consortia (e.g., biological processing ontology) and / or gene set enrichment analysis (GSEA) (e.g., Hallmark pathway gene set or Canonical pathway gene set).

[0111] For information on biological processing ontologeries, see, for example, Ashburner et al. Gene ontology: tool for the unification of biology (2000) Nat Genet 25(1):25-9; The Gene Ontology Consortium. Gene Ontology Consortium: going forward. (2015) Nucl Acids Res 43 Database issue D1049-D1056. For information on the Hallmark gene set and the Canonical pathway gene set, see, for example, Tamayo, et al. (2005) PNAS 102, 15545-15550; Mootha, Lindgren, et al. (2003) Nat Genet 34, 267-273.

[0112] As used herein, “leukocyte differentiation pathway” refers to the process by which relatively undifferentiated hematopoietic progenitor cells acquire the specific characteristics of leukocytes, including one or more processes classified in GO:0002521 of the Biological Processes Ontology.

[0113] As used herein, “a pathway of positive control of immune system processing” means a process that activates or increases the frequency, rate, or extent of immune system processing, including one or more processes classified in GO:0002684 of the Biological Processing Ontology.

[0114] As used herein, “transmembrane receptor protein tyrosine kinase signaling pathway” refers to a signaling pathway initiated by the binding of an extracellular ligand to a cell surface receptor when the cell surface receptor has tyrosine kinase activity, and examples of such pathways include one or more pathways classified in GO:0007169 of the Biological Processing Ontology.

[0115] As used herein, “controlling pathways for morphogenesis of anatomical structures” refers to processes that regulate the frequency, rate, or degree of morphogenesis of anatomical structures, including one or more processes classified in GO:0022603 of the Biological Processes Ontology.

[0116] As used herein, the “NFKB-mediated TNFA signaling pathway” refers to a process regulated by NFκB in response to TNF, such as a process involving one or more genes classified as M5890 in the Hallmark gene set (GSEA).

[0117] As used herein, “positive regulatory pathways of hydrolase activity” refers to a process that activates or increases the frequency, rate, and / or degree of hydrolase activity, examples of which include one or more processes classified in GO:0051345 of the Biological Treatment Ontology.

[0118] As used herein, “wound healing pathway” refers to a process that restores the integrity (e.g., partial or complete integrity) of tissue damaged after trauma, and examples of such processes include one or more processes classified under GO:0042060 of the Biological Treatment Ontology.

[0119] As used herein, “α-β T cell activation pathway” refers to a treatment that results in changes in the morphology and / or behavior of αβ T cells, for example, as a result of exposure to a mitogen, cytokine, chemokine, cell ligand, or antigen to which it is specific, including one or more changes classified in the Biological Treatment Ontology GO:0046631.

[0120] As used herein, “control pathways for the migration of cellular components” refers to processes that regulate the frequency, rate, and / or degree of migration of cellular components, including one or more processes classified under GO:0051270 of the Biological Processes Ontology.

[0121] As used herein, “inflammatory response pathway” refers to a defensive response by, for example, vertebrate tissue to infection or injury caused by chemical or physical factors (e.g., an immediate defensive response), examples of which include one or more responses classified in GO:0006954 of the Biological Treatment Ontology. In some embodiments, this treatment is characterized by local vasodilation, extravasation of plasma into the intercellular space, and / or accumulation of leukocytes and macrophages.

[0122] As used herein, “myelocyte differentiation pathway” refers to a process by which relatively undifferentiated myelocyte progenitor cells acquire specific characteristics of any cell in the myelocyte, megakaryocyte, platelet, or erythrocyte lineage, including one or more processes classified under GO:0030099 in the Biological Processes Ontology.

[0123] As used herein, “cytokine production pathway” refers to a process in which cytokines are synthesized or secreted following cell stimulation, resulting in an increase in their intracellular or extracellular levels, examples of which include one or more processes classified in GO:0001816 of the Biological Treatment Ontology.

[0124] As used herein, “downregulation pathway in UV response” refers to a process involving genes that are downregulated in response to ultraviolet (UV) radiation, such as one or more genes classified as M5942 in the Hallmark gene set.

[0125] As used herein, “negative control pathways of multicellular biological processing” means processing that stops, prevents or reduces the frequency, rate and / or range of biological processing and relates to biological functions above the cellular level (e.g., incorporation processing of tissues and organs), examples of which include one or more processing classified in GO:0051241 of the Biological Processing Ontology.

[0126] As used herein, “vascular morphogenetic pathway” refers to a process in which the anatomical structure of blood vessels is generated and organized, and examples include one or more processes classified in GO:0048514 of the Biological Processes Ontology.

[0127] As used herein, “NFAT-dependent transcription pathway” refers to processing related to genes involved in calcineurin-regulated NFAT-dependent transcription in lymphocytes, examples of which include one or more genes classified as M60 in the Canonical pathway gene set.

[0128] As used herein, “a positive regulatory pathway of the apoptotic process” means a treatment that activates or increases the frequency, rate, and / or degree of apoptosis, including one or more treatments classified in GO:0043065 of the Biological Treatment Ontology.

[0129] As used herein, “hypoxia pathway” refers to a process involving genes that are upregulated in response to hypoxic conditions, examples of which include one or more genes classified as M5891 in the Hallmark gene set.

[0130] As used herein, “KRAS signaling-mediated upregulation pathway” refers to a process involving a gene that is upregulated by KRAS activation, such as one or more genes classified as M5953 in the Hallmark gene set.

[0131] As used herein, “stress-activated protein kinase signaling cascade pathway” means a signaling pathway in which a stress-activated protein kinase (SAPK) cascade relays one or more signals, including one or more signaling pathways classified in GO:0031098 of the Biological Processing Ontology.

[0132] explanation This embodiment provides cells containing a heterogeneous nucleic acid molecule inserted at the CD5 locus. The heterogeneous nucleic acid molecule can encode a protein of interest (such as a chimeric antigen receptor) or another nucleic acid molecule (such as an antisense receptor or siRNA). In some embodiments, the protein of interest is an antibody, cytokine, growth hormone, etc. In some embodiments, the cytokine is IL-2 or IL-12. The heterogeneous nucleic acid molecule may also be referred to as the “target heterogeneous molecule” intentionally inserted at the CD5 locus. As used herein, the phrase “intentionally inserted at the CD5 locus” in relation to the target heterogeneous molecule refers to the target heterogeneous molecule that has been intentionally edited into the CD5 locus, as opposed to random insertions that occur at the CD5 locus. Thus, in some embodiments, cells containing a heterogeneous nucleic acid molecule intentionally inserted at the CD5 locus are provided. In some embodiments, the heterogeneous nucleic acid molecule does not encode CD5. In some embodiments, the advantage of inserting a heterologous nucleic acid molecule at the CD5 locus is that, surprisingly, the locus was identified as one that can support the expression of molecules encoded by heterologous nucleic acid molecules other than CD5. Thus, the CD5 locus can be used as a location in the cellular genome for the expression of other molecules encoded by heterologous nucleic acid molecules, and the cell can still survive because disruption of the CD5 gene does not prevent the cell from expressing the encoded molecules.

[0133] As used herein, the term “heterogeneous” as used in relation to the term “nucleic acid molecule” means a nucleic acid molecule (such as DNA or RNA) that is introduced exogenously into a cell and edited, for example, at the CD5 locus in the embodiments shown herein. In some embodiments, the nucleic acid molecule is DNA inserted into the CD5 locus.

[0134] Therefore, in some embodiments, cells containing heterologous nucleic acid molecules are provided. In some embodiments, the heterologous nucleic acid molecules are inserted at the CD5 locus. In some embodiments, the cells have reduced or absent CD5 expression and / or function.

[0135] As used herein, a heterologous nucleic acid molecule inserted at the CD5 locus can either replace a portion of the CD5 gene or be inserted into the genomic sequence of CD5. In some embodiments, the insertion causes a frameshift in the CD5 gene, resulting in the cell being unable to express the CD5 transcript. In some embodiments, the heterologous nucleic acid molecule replaces the CD5 locus. This can replace either an exon or an intron of CD5. In some embodiments, this sequence replaces a CD5 exon (such as exons 1, 2, 3, 4, or 5 of CD5). In some embodiments, all exons are replaced. In some embodiments, exons 1, 2, 3, or 4 are replaced.

[0136] In some embodiments, heterologous nucleic acid molecules are inserted into the CD5 locus. However, such insertions result in reduced or lost CD5 expression and / or function. In some embodiments, heterologous nucleic acid molecules inserted into the CD5 locus produce cells having a CD5 locus containing a frameshift CD5 locus. The frameshift causes either cleavage of the CD5 transcript and protein, or the production of a nonsense transcript or protein.

[0137] In some embodiments, the cells are immune cells (such as those shown herein), and these immune cells include, but are not limited to, T cells, NK cells, or B cells. In some embodiments, the T cells are αβ T cells. In some embodiments, the B cells are B1 B cells.

[0138] In some embodiments, the cells containing the heterologous nucleic acid molecule do not express CD5 or do not express functional CD5 from the native CD5 locus.

[0139] In some embodiments, the heterologous nucleic acid molecule encodes a protein of interest. In some embodiments, the heterologous nucleic acid molecule encodes a chimeric antigen receptor. Non-limiting examples of chimeric antigen receptors are provided herein. In some embodiments, the heterologous nucleic acid molecule encodes an antibody, chemokine, hormone, cytokine, etc. In some embodiments, the heterologous nucleic acid molecule encodes an RNA molecule, examples of which include miRNA, siRNA, mRNA, antisense molecules, etc.

[0140] Also provided herein are pharmaceutical compositions comprising the cells provided herein. As indicated herein, the composition can contain more than one million cells. In some embodiments, the composition contains from about 1×10 6 to about 1×10 9 ; from about 1×10 7 to about 1×10 9 ; from about 1×10 8 to about 1×10 9 ; from about 2×10 8 to about 1×10 9 ; from about 3×10 8 to about 1×10 9 ; from about 3×10 8 to about 1×10 9 ; from about 3×10 8 to about 1×10 9 ; from about 4×10 8 to about 1×10 9 ; from about 5×10 8 to about 1×10 9 ; from about 6×10 8 ; from about 7×10 8 to about 1×10 9 ; from about 8×10 8 ; from about 9×10 8 to about 1×10 9 cells.

[0141] Furthermore, a method for producing cells containing heterologous nucleic acid molecules inserted at the CD5 locus is also described herein. This method involves contacting cells with a gene editing system under conditions sufficient to produce cells containing heterologous nucleic acid molecules inserted at the CD5 locus. In some embodiments, the produced cells have reduced or absent CD5 expression and / or function.

[0142] In some embodiments, a cell-producing process produces cells in which a heterologous nucleic acid molecule inserted into the CD5 locus replaces the CD5 locus. In some embodiments, the heterologous nucleic acid molecule inserted into the CD5 locus reduces or eliminates the expression and / or function of CD5. In some embodiments, the heterologous nucleic acid molecule inserted into the CD5 locus produces cells having a CD5 locus that includes a frameshift CD5 locus. In some embodiments, the produced cells do not express CD5 or do not express functional CD5 from the native CD5 locus.

[0143] In some embodiments, the cells are immune cells, examples of which are shown herein.

[0144] In some embodiments, the gene editing system includes a targeting molecule that binds to a target sequence in an early exon or intron of the CD5 gene. In some embodiments, the gene editing system includes a targeting molecule that binds to a target sequence in a late exon or intron of the CD5 locus. In some embodiments, the gene editing system includes a targeting molecule that binds to a target sequence of the CD5 locus, where the target sequence is downstream of the fourth to last exon, for example, in the third to last exon of the CD5 locus, the second to last exon, or the last exon.

[0145] In some embodiments, the gene editing system is selected from the group consisting of CRISPR or CRISPR / Cas9 systems, zinc finger nuclease systems, TALEN systems, and meganuclease systems.

[0146] In some embodiments, the gene editing system is a CRISPR system. In some embodiments, the CRISPR system includes a gRNA molecule that hybridizes to a target sequence at the CD5 locus. In some embodiments, the CRISPR system is a CRISPR / Cas system.

[0147] Cells can be produced either ex vivo or in vivo. Methods for extracting, growing, and activating T cells are known in the art. Cells can be modified to express heterologous sequences either ex vivo or in vivo. Cells can be contacted with plasmids or viral vectors containing components to be transfected or introduced into the cells. For example, viruses (or multiple viruses), such as lentiviruses, can be used to transfect immune cells either in vivo to edit the cells so that heterologous nucleic acid molecules are inserted into the CD5 locus.

[0148] This composition and cells can be used to treat subjects suffering from cancer, and this method includes administering cells or compositions as shown herein to the subject. In some embodiments, cells containing heterologous nucleic acid molecules inserted at the CD5 locus are more effective in treating cancer than cells containing heterologous nucleic acid molecules not inserted at the CD5 locus.

[0149] Gene editing systems As described herein, gene editing systems can be used to insert heterologous nucleic acid molecules into the CD5 gene locus. Examples of such gene editing systems include, but are not limited to, those described herein and above, as well as the following:

[0150] CRISPR / Cas9 gene editing system

[0151] The naturally occurring CRISPR / Cas system is found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements (such as plasmids and phages) and provides a form of adaptive immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

[0152] The CRISPR / Cas system has been modified for use in gene editing (silencing, enhancing, or modifying specific genes) in eukaryotes (such as mice or primates). Wiedenheft et al. (2012) Nature 482: 331-8. This is achieved, for example, by introducing a plasmid containing a specifically designed CRISPR and one or more suitable Cass into eukaryotic cells.

[0153] A CRISPR sequence (sometimes called a CRISPR locus) contains alternating repeat sequences and spacers. In naturally occurring CRISPRs, spacers typically contain sequences that are exogenous to the bacterium (such as plasmid or phage sequences). In the exemplary CD5 CRISPR / Cas system, the spacers originate from the CD5 gene sequence or the sequences of its regulatory elements.

[0154] RNA from the CRISPR locus is constitutively expressed and processed into small RNAs. These contain spacers adjacent to the repetitive sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. Thus, the spacers function as templates for RNA molecules, similar to siRNA. Pennisi (2013) Science 341: 833-836.

[0155] Because these genes are naturally present in many different types of bacteria, the structure, function, and number of CRISPR and Cas genes, as well as the precise arrangement of their products, vary somewhat between species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype, E. coli) protein (e.g., CasA) forms a functional complex called a cascade. This cascade processes CRISPR RNA transcripts into spacer-repeat units held by the cascade. Brouns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes CRISPR transcripts. Inactivation of CRISPR-based phages in E. coli requires the cascade and Cas3, but not Cas1 or Cas2. In Purococcus phryusus and other prokaryotes, the Cmr (Cas RAMP module) protein forms a functional complex with small CRISPR RNAs that recognize and cleave complementary target RNAs. A simpler CRISPR system relies on the protein Cas9 (one on each strand of the double helix), which is a nuclease with two active cleavage sites. The combination of Cas9 and modified CRISPR locus RNA can be used in gene editing systems. Pennisi (2013) Science 341: 833-836.

[0156] Therefore, the CRISPR / Cas system can be used to modify (e.g., delete) one or more nucleic acids, the CD5 gene, or CD5 gene regulatory elements, or to introduce early arrest that reduces functional CD5 expression, and this can be done to insert heterologous nucleic acid molecules into CD loci containing CD5 gene regulatory elements (e.g., promoters, enhancers, etc.). Alternatively, the CRISPR / Cas system can be used like RNA interference to reversibly turn off the CD5 gene and / or insert heterologous nucleic acid molecules into the CD5 locus if the insertion does not inhibit CD5 gene expression.

[0157] CRISPR / Cas systems for gene editing in eukaryotic cells typically involve (1) a guide RNA molecule (gRNA) containing a targeting sequence (which can be hybridized to a genomic DNA target sequence) and a sequence that can bind to an enzyme (e.g., Cas9 enzyme), and (2) a Cas (e.g., Cas9) protein. The targeting sequence and the Cas (e.g., Cas9 enzyme)-binding sequence may be located on the same molecule or on different molecules. If located on different molecules, each includes a hybridization domain that allows the molecules to associate, for example, through hybridization.

[0158] Artificial CRISPR / Cas systems can be generated targeting insertions at the CD5 locus using techniques known in the art, such as those described in U.S. Publication No. 20140068797, WO2015 / 048577, and Cong (2013) Science 339: 819-823. Other artificial CRISPR / Cas systems known in the art may also be generated, such as those described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Patents No. 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359, the contents of which are incorporated herein by reference in their entirety. Such systems can be used to generate a target heterologous gene and manipulate the CRISPR / Cas system to include a gRNA molecule containing a targeting sequence that hybridizes to, for example, the sequence of the CD5 gene. In some embodiments, the gRNA contains a targeting sequence that is perfectly complementary to 15-25 nucleotides (e.g., 20 nucleotides) of the CD5 gene. In some embodiments, 15-25 nucleotides (e.g., 20 nucleotides) of the CD5 gene are located immediately 5' to the protospacer adjacent motif (PAM) sequence recognized by the Cas protein of the CRISPR / Cas system (for example, if the system contains the Streptococcus pyogenes Cas9 protein, the PAM sequence contains NGG, where N is A, T, G, or C).

[0159] Therefore, in some embodiments, heterologous nucleic acid molecules can be introduced into cells together with a CRISPR / Cas system (e.g., CAR-encoding DNA) as described herein, and this process can be used to incorporate CAR-encoding DNA at or near a site targeted by the CRISPR / Cas system, as described herein, depending on the sequence and chromosomal sequence of the foreign DNA. As shown herein, and not limited to theory, such incorporation may lead to CAR expression and even disruption of the CD5 gene. Such foreign DNA molecules are referred to herein as “template DNA”. In some embodiments, the template DNA further includes homology arms on the 5', 3', or both 5' and 3' ends of the nucleic acid of the template DNA encoding the target molecule (or more) (e.g., the CAR-encoding molecule described herein), wherein the homology arms are complementary to genomic DNA sequences adjacent to the target sequence.

[0160] In some embodiments, the CRISPR / Cas system comprises Cas9 (e.g., Streptococcus pyogenes Cas9) and a gRNA containing a targeting sequence that hybridizes to the CD5 gene sequence. In some embodiments, the CRISPR / Cas system comprises a nucleic acid encoding the CD5 gRNA and a nucleic acid encoding the Cas protein (e.g., Cas9, e.g., Streptococcus pyogenes Cas9). In some embodiments, the CRISPR / Cas system comprises the CD5 gRNA and a nucleic acid encoding the Cas protein (e.g., Cas9, e.g., Streptococcus pyogenes Cas9).

[0161] TALEN gene editing system

[0162] TALENs are artificially produced by fusing a TAL effector DNA-binding domain with a DNA-cleaving domain. The transcriptional activator-like effect (TALE) can be manipulated to bind to any desired DNA sequence, including HLA or TCR gene segments. By combining the manipulated TALE with a DNA-cleaving domain, restriction enzymes specific to any desired DNA sequence, including HLA or TCR sequences, can be produced. These can then be introduced into cells and used for genome editing. (Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.)

[0163] TALE is a protein secreted by Xanthomonas bacteria. Its DNA-binding domain contains a repeating and highly conserved sequence of 33-34 amino acids, excluding the 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. Therefore, they can be manipulated to bind to a desired DNA sequence.

[0164] To produce TALENs, the TALE protein is fused to a nuclease (N) (which is, for example, wild-type or mutant FokI endonuclease). Several mutations have been developed for FokI for use in TALENs. These improve, for example, the specificity or activity of cleavage. Cermak et al. (2011) Nucl. Acids Res. 39: e82;Miller et al. (2011) Nature Biotech. 29: 143-8;Hockemeyer et al. (2011) Nature Biotech. 29: 731-734;Wood et al. (2011) Science 333: 307;Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96.

[0165] The FokI domain functions as a dimer, requiring two constructs, each possessing a unique DNA-binding domain for a target site within the genome with appropriate orientation and spacing. Both the number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN-binding sites, appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8.

[0166] CD5 TALENs can be used inside cells to produce double-strand breaks (DSBs). If the repair mechanism improperly repairs the break via non-homologous end joining, mutations and insertions may be introduced at the break site. For example, improper repair may introduce a frameshift mutation. Alternatively, foreign DNA (such as a heterologous nucleic acid molecule) can be introduced into cells together with a TALEN (e.g., CAR-encoding DNA) as described herein, and this process can be used to incorporate CAR-encoding DNA at or near the site targeted by the TALEN, as described herein, depending on the sequence of the foreign DNA and the chromosomal sequence. As shown herein, in the examples, and not bound by theory, such incorporation may lead to CAR expression and even disruption of the CD5 gene. Such foreign DNA molecules are referred to herein as “template DNA”. In some embodiments, the template DNA further includes homology arms on the 5', 3', or both 5' and 3' ends of the nucleic acid of the template DNA encoding the target molecule(s) (e.g., the CAR encoding molecule described herein), wherein the homology arms are complementary to genomic DNA sequences adjacent to the target sequence.

[0167] TALENs specific to the CD5 sequence can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509; U.S. Patent No. 8,420,782; U.S. Patent No. 8,470,973 (these contents are incorporated herein by reference in their entirety).

[0168] Zinc finger nuclease

[0169] "ZFN" or "zinc finger nuclease" refers to zinc finger nucleases, which are artificial nucleases that can be used to modify a desired nucleic acid sequence (for example, to delete one or more nucleic acids).

[0170] Similar to TALENs, ZFNs contain a FokI nuclease domain (or a derivative thereof) fused to a DNA-binding domain. In the case of ZFNs, the DNA-binding domain contains one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0171] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can include, for example, Cys2His2 and can recognize sequences of approximately 3 bp. By combining various zinc fingers with known specificities, multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp can be produced. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage displays, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0172] Similar to TALENs, ZFNs need to be dimerized to cleave DNA. Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. The two individual ZFNs need to ligate the opposite strands of DNA, with their nucleases separated by an appropriate interval. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5.

[0173] Furthermore, similar to TALENs, ZFNs can create double-strand breaks in DNA, and if improperly repaired, can result in frameshift mutations, potentially leading to decreased CD5 expression and levels in cells. ZFNs can also be used in conjunction with homologous recombination to mutate the CD5 gene or to introduce heterologous nucleic acid molecules (such as those encoding CARs) into or near a targeted sequence. As discussed above, the nucleic acid encoding the CAR may be introduced as part of the template DNA. In some embodiments, the template DNA further includes homology arms on the 5', 3', or both the 5' and 3' ends of the nucleic acid of the template DNA encoding the target molecule(s) of interest (e.g., the CAR-encoding molecules described herein), wherein the homology arms are complementary to genomic DNA sequences adjacent to the target sequence.

[0174] A ZFN specific to the CD5 gene sequence can be constructed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; U.S. Patent Publication No. 2011 / 0158957; and U.S. Patent Publication No. 2012 / 0060230 (the contents of which are incorporated herein by reference as a whole). In some embodiments, the ZFN gene editing system may also include nucleic acids encoding one or more components of the ZFN gene editing system (e.g., a ZFN gene editing system targeting CD5).

[0175] While not theoretically bound, it is thought that using a gene editing system targeting CD5 (e.g., a CRISPR / Cas gene editing system) may inhibit one or more functions of CD5, for example, by inducing an editing event that results in the expression of cleaved CD5. Also, while not theoretically bound, such cleaved CD5 proteins may be desirable because they may inhibit one or more other functions of CD5 (e.g., catalytic function) while maintaining one or more functions of CD5 (e.g., scaffold function). In this regard, gene editing systems targeting late exons or introns of the CD5 gene may be particularly desirable. In some embodiments, gene editing system CD5 inhibitors target late exons or introns of the CD5 gene.

[0176] While not bound by theory, in other embodiments, it may be preferable to target the early exons or introns of the CD5 gene, for example, by introducing an immature stop codon into the targeted gene, resulting in the expression of a gene product that is not expressed or is not fully functional. In this regard, gene editing systems that target the early exons or introns of the CD5 gene may be particularly preferred. In some embodiments, the gene editing system CD5 inhibitor targets the early exons or introns of the CD5 gene.

[0177] Modulator If CD5 expression is not inhibited by the insertion of heterologous nucleic acid molecules, CD5 activity can be regulated by inhibiting either CD5 itself or CD5-related genes. While not bound by any particular theory, cells with inhibited CD5 activity, achieved either directly or via CD5-related genes, may have a greater effect on tumor killing than cells with uninhibited CD5 activity. Therefore, CD5 can also be inhibited by the use of modulators.

[0178] As used herein, “CD5-related gene” refers to a gene whose structure, expression, and / or function are related to (e.g., influenced by or regulated by) CD5, or a gene that encodes a gene product (e.g., mRNA or polypeptide). A CD5-related gene does not include the CD5 gene. In some embodiments, a CD5-related gene includes one or more of the genes described herein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, a CD5-related gene includes one or more of the genes selected from IFNG, NOTCH2, CD28, ICOS, IL2RA, or PRDM1 (e.g., 1, 2, 3, 4, 5, or all of them).

[0179] In one embodiment, the CD5-related gene includes IFNG. In one embodiment, the CD5-related gene includes NOTCH2. In one embodiment, the CD5-related gene includes CD28. In one embodiment, the CD5-related gene includes ICOS. In one embodiment, the CD5-related gene includes IL2RA. In one embodiment, the CD5-related gene includes PRDM1.

[0180] In one embodiment, the CD5-related genes include IFNG and NOTCH2. In one embodiment, the CD5-related genes include IFNG and CD28. In one embodiment, the CD5-related genes include IFNG and ICOS. In one embodiment, the CD5-related genes include IFNG and IL2RA. In one embodiment, the CD5-related genes include IFNG and PRDM1. In one embodiment, the CD5-related genes include NOTCH2 and CD28. In one embodiment, the CD5-related genes include NOTCH2 and ICOS. In one embodiment, the CD5-related genes include NOTCH2 and IL2RA. In one embodiment, the CD5-related genes include NOTCH2 and PRDM1. In one embodiment, the CD5-related genes include CD28 and ICOS. In one embodiment, the CD5-related genes include CD28 and IL2RA. In one embodiment, the CD5-related genes include CD28 and PRDM1. In one embodiment, the CD5-related genes include ICOS and IL2RA. In one embodiment, the CD5-related genes include ICOS and PRDM1. In one embodiment, the CD5-related genes include IL2RA and PRDM1.

[0181] In one embodiment, the CD5-related genes include IFNG, NOTCH2, and CD28. In one embodiment, the CD5-related genes include IFNG, NOTCH2, and ICOS. In one embodiment, the CD5-related genes include IFNG, NOTCH2, and IL2RA. In one embodiment, the CD5-related genes include IFNG, NOTCH2, and PRDM1. In one embodiment, the CD5-related genes include IFNG, CD28, and ICOS. In one embodiment, the CD5-related genes include IFNG, CD28, and IL2RA. In one embodiment, the CD5-related genes include IFNG, CD28, and PRDM1. In one embodiment, the CD5-related genes include IFNG, ICOS, and IL2RA. In one embodiment, the CD5-related genes include IFNG, ICOS, and PRDM1. In one embodiment, the CD5-related genes include IFNG, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, CD28, and ICOS. In one embodiment, the CD5-related genes include NOTCH2, CD28, and IL2RA. In one embodiment, the CD5-related genes include NOTCH2, CD28, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, ICOS, and IL2RA. In one embodiment, the CD5-related genes include NOTCH2, ICOS, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include CD28, ICOS, and IL2RA. In one embodiment, the CD5-related genes include CD28, ICOS, and PRDM1. In one embodiment, the CD5-related genes include CD28, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include ICOS, IL2RA, and PRDM1.

[0182] In one embodiment, the CD5-related genes include CD28, ICOS, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, ICOS, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, CD28, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, CD28, ICOS, and PRDM1. In one embodiment, the CD5-related genes include NOTCH2, CD28, ICOS, and IL2RA. In one embodiment, the CD5-related genes include IFNG, ICOS, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include IFNG, CD28, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include IFNG, CD28, ICOS, and PRDM1. In one embodiment, the CD5-related genes include IFNG, CD28, ICOS, and IL2RA. In one embodiment, the CD5-related genes include IFNG, NOTCH2, IL2RA, and PRDM1. In one embodiment, the CD5-related genes include IFNG, NOTCH2, ICOS, and PRDM1. In one embodiment, the CD5-related genes include IFNG, NOTCH2, ICOS, and IL2RA. In one embodiment, the CD5-related genes include IFNG, NOTCH2, CD28, and PRDM1. In one embodiment, the CD5-related genes include IFNG, NOTCH2, CD28, and IL2RA. In one embodiment, the CD5-related genes include IFNG, NOTCH2, CD28, and ICOS.

[0183] In some embodiments, the CD5-related genes include IFNG, NOTCH2, CD28, ICOS, and IL2RA. In some embodiments, the CD5-related genes include IFNG, NOTCH2, CD28, ICOS, and PRDM1. In some embodiments, the CD5-related genes include IFNG, NOTCH2, CD28, IL2RA, and PRDM1. In some embodiments, the CD5-related genes include IFNG, NOTCH2, ICOS, IL2RA, and PRDM1. In some embodiments, the CD5-related genes include IFNG, CD28, ICOS, IL2RA, and PRDM1. In some embodiments, the CD5-related genes include NOTCH2, CD28, ICOS, IL2RA, and PRDM1.

[0184] In some embodiments, the CD5-related genes include IFNG, NOTCH2, CD28, ICOS, IL2RA, and PRDM1.

[0185] Double-stranded RNA (e.g., siRNA or shRNA), modulator

[0186] For example, double-stranded RNA ("dsRNA"), such as siRNA or shRNA, can be used as a modulator (e.g., inhibitor) of CD5-related genes and / or CD5 genes. Furthermore, the use of nucleic acids encoding such dsRNA modulators (e.g., inhibitors) of CD5-related genes and / or CD5 genes is intended in this embodiment.

[0187] In some embodiments, the CD5-related gene and / or CD5 gene modulator (e.g., inhibitor) is a nucleic acid, such as dsRNA; such example is an siRNA or shRNA specific to the nucleic acid encoding the CD5-related gene or gene product and / or the CD5 gene or gene product; such example is a genomic DNA or mRNA encoding the CD5-related gene product and / or the CD5 gene product.

[0188] In some embodiments, the embodiments provide a composition comprising dsRNA, examples of which include siRNA or shRNA, comprising at least 15 consecutive nucleotides, examples of which include 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides, and examples of which include 21 consecutive nucleotides, which are complementary (e.g., 100% complementary) to the sequence of a CD5-related gene and / or CD5 gene nucleic acid sequence (e.g., genomic DNA or mRNA encoding a CD5-related gene product and / or CD5 gene product). While some target sequences and / or shRNA molecules are understood to be presented as DNA, dsRNA agents that target or contain these sequences may be RNA, or any nucleotide, modified nucleotide or substituent disclosed herein and / or known in the art (however, the molecule may still mediate RNA interference).

[0189] In some embodiments, nucleic acid molecules encoding dsRNA molecules that inhibit the expression of CD5-related genes and / or CD5 genes are operably ligated to a promoter (e.g., a promoter derived from H1 or U6), resulting in the expression of CD5-related genes and / or dsRNA molecules that inhibit the expression of CD5 genes within CAR-expressing cells. See, for example, Tiscornia G., “Development of Lentiviral Vectors Expressing siRNA,” Chapter 3, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007; Brummelkamp TR, et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol. 19: 497-500. In one embodiment, the nucleic acid molecule encoding the CD5-related gene and / or the dsRNA molecule that inhibits the expression of the CD5 gene resides on the same vector (e.g., a lentiviral vector) as the nucleic acid molecules encoding the components of the CAR (e.g., all components). In such embodiments, the nucleic acid molecule encoding the CD5-related gene and / or the dsRNA molecule that inhibits the expression of the CD5 gene is placed on the vector of the CAR, an example of which is a lentiviral vector on the 5' or 3' side of the nucleic acid encoding the components, an example of which is all of the components. The nucleic acid molecule encoding the CD5-related gene and / or the dsRNA molecule that inhibits the expression of the CD5 gene can be transcribed in the same direction as or different from the nucleic acid encoding the components of the CAR (e.g., all of the components).In one embodiment, the nucleic acid molecule encoding the CD5-related gene and / or the dsRNA molecule that inhibits the expression of the CD5 gene resides on a vector other than the vector containing the nucleic acid molecules encoding the components of CAR (e.g., all of the components). In some embodiments, the nucleic acid molecule encoding the CD5-related gene and / or the dsRNA molecule that inhibits the expression of the CD5 gene is transiently expressed in CAR-expressing cells. In some embodiments, the nucleic acid molecule encoding the CD5-related gene and / or the dsRNA molecule that inhibits the expression of the CD5 gene is stably integrated into the genome of CAR-expressing cells.

[0190] In some embodiments, double-stranded RNA ("dsRNA") (e.g., siRNA or shRNA) can be used as a modulator (e.g., inhibitor) of CD5-related genes and / or CD5 genes. Furthermore, the use of nucleic acids encoding such dsRNA modulators (e.g., inhibitors) of CD5-related genes and / or CD5 genes is also intended.

[0191] In some embodiments, the CD5-related gene and / or CD5 gene modulator (e.g., inhibitor) is a nucleic acid, such as dsRNA; such example is an siRNA or shRNA specific to the nucleic acid encoding the CD5-related gene or gene product and / or the CD5 gene or gene product; such example is a genomic DNA or mRNA encoding the CD5-related gene product and / or the CD5 gene product.

[0192] vector As described herein, the embodiments shown herein allow for the transduction or transfection of cells using, for example, vectors as described herein. The vector may be a non-viral vector or, in the case of a plasmid vector, may contain nucleic acid molecules or proteins for transporting molecules into the cell. The vector may be a viral vector, a plasmid, or a liposomal vesicle vector that encapsulates what is delivered to the cell. Such vectors can be targeted to transport molecules only to cells of interest (such as immune cells as described herein).

[0193] In some embodiments, the embodiments provide a method for producing the cells described herein (e.g., the method described above). In some embodiments, the method includes the step of introducing a gene editing system into cells, examples of which include a CD5-targeting CRISPR / Cas gene editing system, and examples of which include a CRISPR / Cas system comprising a gRNA having a targeting sequence complementary to the target sequence of the CD5 gene. In some embodiments, the CRISPR / Cas system is introduced into the cells as a ribonucleoprotein complex of gRNA and Cas enzyme, for example by electroporation. In some embodiments, the method includes introducing a nucleic acid encoding one or more components of the CRISPR / Cas system into the cells. In some embodiments, the nucleic acid is placed on a vector encoding a CAR as described herein.

[0194] In some embodiments, the embodiments provide a method (e.g., the method described above) that includes the step of introducing a CD5-targeting inhibitory dsRNA (e.g., shRNA or siRNA) into cells. In some embodiments, the method includes introducing a nucleic acid encoding a CD5-targeting inhibitory dsRNA (e.g., shRNA or siRNA) into the cells. In some embodiments, the nucleic acid is placed on a vector encoding a CAR (e.g., a CAR as described herein). This can be performed after or concurrently with the editing of the cells to insert a heterologous nucleic acid molecule into the CD5 locus.

[0195] Additional components of CAR and CAR T cells, as well as methods related to embodiments, are described herein, as above and below.

[0196] This specification provides compositions and methods of use for treating diseases such as cancer, using CAR-mediated immunoeffector cells (e.g., T cells, NK cells).

[0197] In some embodiments, the antigen-binding domain of the CAR described herein is an scFv antibody fragment. In some embodiments, such an antibody fragment is functional in that it retains equivalent binding affinity, for example, binding the same antigen with equivalent affinity to the IgG antibody from which it is derived. In other embodiments, the antibody fragment has low binding affinity, for example, binding the same antigen with lower binding affinity than the antibody from which it is derived, but is functional in that it provides the biological response described herein. In some embodiments, the CAR molecule has a binding affinity KD of 10 to the target antigen. -4 M~10 -8 It contains an antibody fragment that is M, and an example of this is 10 -5 M~10 -7 There is an M, and one example is 10 -6 M or 10 -7M is present. In some embodiments, the antibody fragment has a binding affinity of at least 5, 10, 20, 30, 50, 100, or less than 1,000 times that of the reference antibody (e.g., the antibody described herein).

[0198] In some embodiments, as will be understood by skilled craftsmen, such antibody fragments are functional in that they provide a biological response, which may include, but is not limited to, activation of an immune response, inhibition of signal initiation from its target antigen, or inhibition of kinase activity.

[0199] In some embodiments, the antigen-binding domain of the CAR is a humanized scFv antibody fragment compared to the murine sequence of the derived scFv.

[0200] In some embodiments, the antigen-binding domain of the CAR (e.g., scFv) is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In some embodiments, the entire CAR construct is encoded by a nucleic acid molecule whose entire sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons encoding the same amino acid) when encoding DNA is biased across different species. Such codon degeneracy makes it possible to encode the same polypeptide with various nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in at least U.S. Patents 5,786,464 and 6,114,148.

[0201] In some embodiments, the CAR combines the antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in some embodiments, the intracellular signaling molecule includes, but is not limited to, CD3 zeta chains, 4-1BB and CD28 signaling modules, and combinations thereof. In some embodiments, the antigen-binding domain binds to a tumor antigen as described herein.

[0202] Furthermore, this embodiment provides the use of CARs and CAR-expressing cells, as well as agents or methods for treating cancer or any malignant tumor or autoimmune disease involving cells or tissues expressing tumor antigens as described herein, among many other diseases.

[0203] In some embodiments, CAR can be used to eradicate normal cells expressing tumor antigens as described herein, thereby making it applicable as a cell conditioning therapy before cell transplantation. In some embodiments, the normal cells expressing tumor antigens as described herein are normal stem cells, and the cell transplantation is stem cell transplantation.

[0204] In some embodiments, the embodiments provide immune effector cells (e.g., T cells, NK cells) engineered to express a chimeric antigen receptor (CAR), and the engineered immune effector cells exhibit antitumor properties. In some embodiments, the antigen is a cancer-associated antigen (i.e., a tumor antigen) as described herein. In some embodiments, the antigen-binding domain of the CAR comprises a partially humanized antibody fragment. In some embodiments, the antigen-binding domain of the CAR comprises a partially humanized scFv. Thus, the embodiments provide CARs engineered into cells (e.g., T cells or NK cells) and containing a humanized antigen-binding domain, and methods for using them for adoptive therapy.

[0205] In some embodiments, the CAR comprises at least one intracellular domain selected from the group consisting of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD27 signaling domain, a CD3 zeta signaling domain, and any combination thereof. In some embodiments, the CAR comprises at least one intracellular signaling domain selected from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.

[0206] Table 1 lists some example sequences of various components of CAR, where aa represents an amino acid and na represents the nucleic acid encoding the corresponding polypeptide. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0207] Cancer-related antigens This embodiment provides immune effector cells (e.g., T cells, NK cells) that have been engineered to contain one or more CARs that induce cancer in immune effector cells. This is achieved through antigen-binding domains on the CARs that are specific to cancer-associated antigens. In some embodiments, there are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs: (1) cancer-associated antigens expressed on the surface of cancer cells, and (2) cancer-associated antigens that are intracellular themselves, but fragments (peptides) of such antigens are presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0208] Therefore, in some embodiments, the cancer-related antigens (tumor antigens) targeted by the provided CAR include CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, PRSS21, SSEA-4, CD20, Folate Receptor Alpha, and ERBB2. (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, regmine, HPV E6, E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutation, prostain, sulbibin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutation, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0209] Tumor-supporting antigen The CARs described herein can include an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein). In some embodiments, the tumor-supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSC). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit the proliferation and activation of T cells. Without wishing to be bound by theory, in some embodiments, CAR-expressing cells destroy tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.

[0210] In some embodiments, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin. In some embodiments, the FAP-specific antibody competes for binding with sibrotuzumab or has the same CDRs as sibrotuzumab. In some embodiments, the MDSC antigen is selected from one or more of CD33, CD11b, C14, CD15, and CD66b. Thus, in some embodiments, the tumor-supporting antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b.

[0211] Chimeric antigen receptor (CAR) This embodiment includes a recombinant DNA construct inserted into a CD5 locus containing a sequence encoding a CAR, the CAR comprising an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that specifically binds to a cancer-associated antigen as described herein, the sequence of the antigen-binding domain being contiguous with and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain may include a co-stimulatory signaling domain and / or a primary signaling domain (e.g., a zeta chain). The co-stimulatory signaling domain refers to a portion of the CAR that includes at least a portion of the intracellular domain of a co-stimulatory molecule.

[0212] In some embodiments, the CAR construct includes an scFv domain, which may be preceded by an optional leader sequence (an example of which is provided in SEQ ID NO: 2), followed by an optional hinge sequence (an example of which is provided in SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10), a transmembrane region (an example of which is provided in SEQ ID NO: 12), an intracellular signaling domain including SEQ ID NO: 14 or SEQ ID NO: 16, and a CD3 zeta sequence including SEQ ID NO: 18 or SEQ ID NO: 20, for example, these domains consecutively within the same reading frame to form a single fusion protein.

[0213] In some embodiments, the exemplary CAR construct comprises an optional leader sequence (e.g., the leader sequence described herein), an extracellular antigen-binding domain (e.g., the antigen-binding domain described herein), a hinge (e.g., the hinge region described herein), a transmembrane domain (e.g., the transmembrane domain described herein), and an intracellular stimulatory domain (e.g., the intracellular stimulatory domain described herein). In some embodiments, the exemplary CAR construct comprises an optional leader sequence (e.g., the leader sequence described herein), an extracellular antigen-binding domain (e.g., the antigen-binding domain described herein), a hinge (e.g., the hinge region described herein), a transmembrane domain (e.g., the transmembrane domain described herein), an intracellular co-stimulatory signaling domain (e.g., the co-stimulatory signaling domain described herein), and / or an intracellular primary signaling domain (e.g., the primary signaling domain described herein).

[0214] An exemplary leader sequence is provided as SEQ ID NO: 2. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 12. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 14. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 16. An exemplary CD3 zeta domain sequence is provided as SEQ ID NO: 18 or SEQ ID NO: 20.

[0215] In some embodiments, a recombinant nucleic acid construct is provided comprising a nucleic acid molecule encoding a CAR, the nucleic acid molecule comprising a nucleic acid sequence encoding an antigen-binding domain, for example, as described herein, which is contiguous with and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain.

[0216] In some embodiments, recombinant nucleic acid constructs are provided that include a nucleic acid molecule encoding a CAR. The nucleic acid molecule includes a nucleic acid sequence encoding an antigen-binding domain, which is contiguous with a nucleic acid sequence encoding an intracellular signaling domain and is within the same reading frame. Exemplary intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains such as CD3-zeta, CD28, CD27, and 4-1BB. In some cases, a CAR may include any combination of CD3-zeta, CD28, and 4-1BB.

[0217] Nucleic acid sequences encoding desired molecules can be obtained using recombinant methods known in the art, such as screening libraries from cells expressing nucleic acid molecules, inducing nucleic acid molecules from vectors known to contain nucleic acid molecules, or directly isolating nucleic acid molecules from cells and tissues using standard techniques. Alternatively, the nucleic acid of interest can be produced by synthesis rather than cloning.

[0218] In some embodiments, retroviral and lentiviral vector constructs expressing a CAR (which can be directly transduced into cells in cooperation with a gene editing system to insert the CAR into the CD5 gene locus) are provided.

[0219] This embodiment may also include RNA constructs that can be directly transfected into cells for the same gene editing purposes. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template using specially designed primers, followed by poly-A addition, to produce a construct containing 3' and 5' untranslated sequences ("UTRs") (e.g., the 3' and / or 5'UTRs described herein), a 5' cap (e.g., the 5' cap described herein), and / or an internal ribosome entry site (IRES) (e.g., the IRES described herein), the nucleic acid to be expressed, and a poly-A tail typically 50 to 2000 nucleotides long (SEQ ID NO: 32). The RNA thus produced can be efficiently transfected into different types of cells. In some embodiments, the template includes a CAR sequence. In some embodiments, the RNA CAR vector is transduced into cells (e.g., T cells or NK cells) by electroporation.

[0220] antigen-binding domain

[0221] In some embodiments, the CAR includes a target-specific binding element (otherwise also called an antigen-binding domain). The selection of this element depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that function as cell surface markers on target cells associated with a particular disease condition. Thus, examples of cell surface markers that may function as ligands for the antigen-binding domain of a CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0222] In some embodiments, a CAR-mediated T cell response can be induced to a target antigen by manipulating an antigen-binding domain that specifically binds to a desired antigen into the CAR.

[0223] In some embodiments, the portion of the CAR containing the antigen-binding domain includes an antigen-binding domain that targets a tumor antigen (e.g., the tumor antigens described herein).

[0224] The antigen-binding domain may be any domain that binds to an antigen, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but is not limited to, single-domain antibodies (such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of nanobodies derived from camelids), and alternative scaffolds known in the art to function as antigen-binding domains (such as recombinant fibronectin domains, T cell receptors (TCRs), or fragments thereof) (e.g., single-chain TCRs). In some cases, it may be beneficial for the antigen-binding domain to originate from the same species in which the CAR will ultimately be used. For example, when used in humans, it may be beneficial for the antigen-binding domain of the CAR to contain human or humanized residues for the antigen-binding domain of the antibody or antibody fragment.

[0225] In some embodiments, CD19 CAR is referred to in U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260 (2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122(25):4129-39 (2013); or 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst The CD19 CAR described in 10 (each of which is incorporated herein by reference as a whole). In some embodiments, the antigen-binding domain for CD19 is, for example, the CAR, antibody, or antigen-binding portion (e.g., CDR) of its antigen-binding fragment, as described in PCT Publication WO2012 / 079000 (which is incorporated herein by reference as a whole). In some embodiments, the antigen-binding domain for CD19 is, for example, a CAR, antibody, or the antigen-binding portion (e.g., CDR) of its antigen-binding fragment, as described in PCT Publication WO2014 / 153270; Kochenderfer, JN et al., J. Immunother. 32 (7), 689-702 (2009); Kochenderfer, JN, et al., Blood, 116 (20), 4099-4102 (2010); PCT Publication WO2014 / 031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or U.S. Patent No. 7,446,190 (each of which is incorporated herein by reference as a whole).

[0226] In some embodiments, the CAR comprises an antigen-binding domain against CD5. In some embodiments, the antigen-binding domain against CD5 comprises a CD5 antibody or a fragment thereof. In some embodiments, the antibody fragment is as shown herein, and examples thereof include, but are not limited to, scFv antibodies, antigen-binding domains, ankyrin repeats (e.g., DARPIN), VHH domain antibodies, nanobodies, single-domain antibodies, FN3 domains, or any combination thereof. In some embodiments, the anti-CD5 antibody comprises a peptide selected from the following table showing CDRs based on Kabat numbering. [Table 2]

[0227] In some embodiments, the CAR comprises an antigen-binding domain against CD5 comprising (i) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 sequences (the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 59, the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 60, and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 61); and (ii) a heavy chain variable region comprising heavy chain CDR1, CDR2 and CDR3 sequences (the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 56, the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 57, and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 58).

[0228] In the foregoing paragraphs, mention may be made of CDRs in the Kabat system, but equivalent CDR sequences can be used from the IMGT and Chothia notations.

[0229] In some embodiments, the CAR comprises an antigen-binding domain against CD5 comprising a heavy chain variable region peptide having the sequence shown in the following table. <00009​​​​​​​In some embodiments, the CAR includes an antigen-binding domain for CD5 comprising a light chain variable region peptide having the sequence shown in the table below. [Table 4]

[0231] In some embodiments, CAR is V of Sequence ID 62 H It contains an antigen-binding domain for CD5 containing a peptide. In some embodiments, the CAR is V of SEQ ID NO: 63 L It contains an antigen-binding domain for CD5 containing a peptide. In some embodiments, the CAR is V H Peptides and V L It contains an antigen-binding domain for CD5 containing a peptide, V H The peptide contains the amino acid sequence of SEQ ID NO: 62, V L The peptide contains the amino acid sequence of SEQ ID NO: 63. In some embodiments, CAR is V H Peptides and V L It contains an antigen-binding domain for CD5 containing a peptide, V H The peptide contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 62, V L The peptide contains an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 63. In some embodiments, CAR is V H Peptides and V L It contains an antigen-binding domain for CD5 containing a peptide, V H The peptide contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 62, however, V H It must include HCDR1 containing the amino acid sequence of SEQ ID NO: 56, HCDR2 containing the amino acid sequence of SEQ ID NO: 57, and HCDR3 containing the amino acid sequence of SEQ ID NO: 58. L The peptide contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 63, however, V LIt must include LCDR1 containing the amino acid sequence of SEQ ID NO: 59, LCDR2 containing the amino acid sequence of SEQ ID NO: 60, and LCDR3 containing the amino acid sequence of SEQ ID NO: 61.

[0232] V H Array and V L The array is V H Region and V L The regions may be in any form, including but not limited to the scFv format, in which they are linked by peptide linkers. Examples of peptide linkers that can be used to link various peptides are shown herein.

[0233] In some embodiments, the CAR includes an antigen-binding domain to CD5 comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 64. EVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK(Sequence ID 64)

[0234] In some embodiments, the CAR includes an antigen-binding domain for CD5 containing an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 64. In some embodiments, the CAR includes an antigen-binding domain for CD5 containing an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 64. In some embodiments, the CAR includes an antigen-binding domain for CD5 containing an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 64. In some embodiments, the CAR includes an antigen-binding domain for CD5 containing the amino acid sequence of SEQ ID NO: 64.

[0235] In some embodiments, the CAR includes an antigen-binding domain to CD5 comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 65. NIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSS (Sequence ID 65)

[0236] In some embodiments, the CAR includes an antigen-binding domain for CD5 containing an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 65. In some embodiments, the CAR includes an antigen-binding domain for CD5 containing an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 65. In some embodiments, the CAR includes an antigen-binding domain for CD5 containing an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 65. In some embodiments, the CAR includes an antigen-binding domain for CD5 containing the amino acid sequence of SEQ ID NO: 65.

[0237] In some embodiments, the CAR containing the antigen-binding domain for CD5 includes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 66. MALPVTALLLPLALLLHAARPGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDG YYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYD NLWTFGGGTKLEIKSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 66)

[0238] In some embodiments, the CAR containing the antigen-binding domain for CD5 includes an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 66. In some embodiments, the CAR containing the antigen-binding domain for CD5 includes an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 66. In some embodiments, the CAR containing the antigen-binding domain for CD5 includes an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 66. In some embodiments, the CAR containing the antigen-binding domain for CD5 includes the amino acid sequence of SEQ ID NO: 66.

[0239] The embodiments described herein for CARs containing an antigen-binding domain to CD5 are illustrative and not intended to limit the scope of use. Additional CAR constructs (CD5 or otherwise) suitable for use in this disclosure are described, for example, in International Patent Publication WO 2020 / 132327, U.S. Patent No. 11,673,964, and U.S. Patent Application Publication 2023 / 0331864, each incorporated herein by reference as a whole.

[0240] In some embodiments, the antigen-binding domain for mesothelin may be, for example, an antigen-binding domain of an antibody, antigen-binding fragment, or CAR described in PCT Publication WO2015 / 090230 (e.g., CDR, scFv, or VH and VL) or derived therefrom (in one embodiment, the CAR is a CAR described in WO2015 / 090230, and its contents are incorporated herein in whole). In some embodiments, the antigen-binding domain for mesothelin is, for example, an antibody, antigen-binding fragment, or antigen-binding portion of a CAR (e.g., CDR, scFv, or VH and VL) described in PCT Publications WO1997 / 025068, WO1999 / 028471, WO2005 / 014652, WO2006 / 099141, WO2009 / 045957, WO2009 / 068204, WO2013 / 142034, WO2013 / 040557, or WO2013 / 063419 (each of which is incorporated herein by reference as a whole).

[0241] In some embodiments, the antigen-binding domain for CD123 is, for example, an antibody, antigen-binding fragment, or antigen-binding portion of a CAR (e.g., CDR, scFv, or VH and VL) as described in PCT Publication WO2014 / 130635 (incorporated herein in whole by reference). In some embodiments, the antigen-binding domain for CD123 is, for example, an antibody, antigen-binding fragment, or antigen-binding portion of a CAR (e.g., CDR, scFv, or VH and VL) as described in PCT Publication WO2016 / 028896 (incorporated herein in whole by reference). In some embodiments, the CAR is a CAR as described in WO2016 / 028896. In some embodiments, the antigen-binding domain for CD123 is, for example, PCT publications WO1997 / 024373, WO2008 / 127735 (e.g., CD123-binding domains of 26292, 32701, 37716 or 32703), WO2014 / 138805 (For example, the CD123-binding domain of CSL362), WO2014 / 138819, WO2013 / 173820, WO2014 / 144622, WO2001 / 66139, WO2010 / 126066 (for example, the CD123-binding domain of any of Old4, Old5, Old17, Old19, New102 or Old6), WO2014 / 144622 or US2009 / 0252742 (each of which is incorporated herein by reference as a whole) are or derived from antibodies, antigen-binding fragments or antigen-binding portions of CARs (for example, CDR, scFv, or VL and VH).

[0242] In some embodiments, the antigen-binding domain for CD22 is the antigen-binding portion (e.g., CDR) of an antibody described, for example, Haso et al., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res 16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1): 83-88 (2013); Creative BioMart (creativebiomart.net): MOM-18047-S(P).

[0243] In some embodiments, the antigen-binding domain for CS-1 is the antigen-binding portion of elotuzumab (BMS) (e.g., CDR), see, for example, Tai et al., 2008, Blood 112(4):1329-37; Tai et al., 2007, Blood. 110(5):1656-63.

[0244] In some embodiments, the antigen-binding domain for CLL-1 is, for example, an antibody, antigen-binding fragment, or antigen-binding portion of a CAR (e.g., CDR or VH and VL) described in PCT Publication WO2016 / 014535, the contents of which are incorporated herein by reference as a whole. In some embodiments, the antigen-binding domain for CLL-1 is an antigen-binding portion of an antibody (e.g., CDR) available from R&D, ebiosciences, and Abcam, examples of which include PE-CLL1-hu catalog number 353604 (BioLegend); and PE-CLL1 (CLEC12A) catalog number 562566 (BD).

[0245] In some embodiments, the antigen-binding domain to CD33 is, for example, Bross et al., Clin Cancer Res 7(6):1490-1496 (2001) (Gemtuzumab Ozogamicin, hP67.6), Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab, HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012) (AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al., Adv hematol 2012:683065 (2012), and Pizzitola et al., Leukemia This refers to the antigen-binding portion (e.g., CDR) of the antibody described in doi:10.1038 / Lue.2014.62 (2014). Exemplary CAR molecules targeting CD33 are described herein and are listed in WO2016 / 014576 (e.g., Table 2 of WO2016 / 014576) (incorporated by reference as a whole).

[0246] In some embodiments, the antigen-binding domain for GD2 is the antigen-binding portion (e.g., CDR) of an antibody described, for example, Mujoo et al., Cancer Res. 47(4):1098-1104 (1987); Cheung et al., Cancer Res 45(6):2642-2649 (1985); Cheung et al., J Clin Oncol 5(9):1430-1440 (1987); Cheung et al., J Clin Oncol 16(9):3053-3060 (1998); Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). In some embodiments, the antigen-binding domain for GD2 is the antigen-binding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9 (see, for example, WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061, WO2013074916, and WO201385552). In some embodiments, the antigen-binding domain for GD2 is the antigen-binding portion of an antibody described in U.S. Publication No. 20100150910 or PCT Publication No. WO 2011160119.

[0247] In some embodiments, the antigen-binding domain for BCMA is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in WO2012163805, WO200112812, and WO2003062401. In some embodiments, additional exemplary BCMA CAR constructs are generated using antigen-binding domains, examples of which include CDR, scFv, or VH and VL sequences from PCT Publication WO2012 / 0163805 (whose contents are incorporated in whole by reference herein). In some embodiments, additional exemplary BCMA CAR constructs are generated using antigen-binding domains, examples of which include CDR, scFv, or VH and VL sequences from PCT Publication WO2016 / 014565 (whose contents are incorporated in whole by reference herein). In some embodiments, additional exemplary BCMA CAR constructs are generated using antigen-binding domains, examples of which include CDR, scFv, or VH and VL sequences from PCT Publication WO2014 / 122144 (whose contents are incorporated in whole by reference herein). In some embodiments, additional exemplary BCMA CAR constructs are generated using a CAR molecule and / or a BCMA-binding domain (e.g., CDR, scFv, or VH and VL sequences) from PCT Publication WO2016 / 014789 (whose contents are incorporated in whole by reference herein). In some embodiments, additional exemplary BCMA CAR constructs are generated using a CAR molecule and / or a BCMA-binding domain (e.g., CDR, scFv, or VH and VL sequences) from PCT Publication WO2014 / 089335 (whose contents are incorporated in whole by reference herein). In some embodiments, additional exemplary BCMA CAR constructs are generated using BCMA-binding domains (e.g., CDR, scFv, or VH and VL sequences) from the CAR molecule and / or PCT Publication WO2014 / 140248 (the contents of which are incorporated as a whole by reference herein).

[0248] In some embodiments, the antigen-binding domain for the Tn antigen is the antigen-binding portion (e.g., CDR) of an antibody described, for example, US2014 / 0178365, U.S. Patent No. 8,440,798, Brooks et al., PNAS 107(22):10056-10061 (2010), and Stone et al., OncoImmunology 1(6):863-873 (2012).

[0249] In some embodiments, the antigen-binding domain to PSMA is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Parker et al., Protein Expr Purif 89(2):136-145 (2013), US20110268656 (J591 ScFv); Frigerio et al., European J Cancer 49(9):2223-2232 (2013) (scFvD2B); WO 2006125481 (mAbs 3 / A12, 3 / E7 and 3 / F11), and single-chain antibody fragments (scFv A5 and D7).

[0250] In some embodiments, the antigen-binding domain for ROR1 is the antigen-binding portion (e.g., CDR) of an antibody described, for example, Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; and US20130101607.

[0251] In some embodiments, the antigen-binding domain for FLT3 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in WO2011076922, U.S. Patent No. 5,777,084, EP0754230, U.S.20090297529, and several commercial catalog antibodies (R&D, ebiosciences, Abcam).

[0252] In some embodiments, the antigen-binding domain for TAG72 is, for example, the antigen-binding portion of an antibody (e.g., CDR) as described in Hombach et al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691.

[0253] In some embodiments, the antigen-binding domain for FAP is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAPS), U.S. Patent Publication No. 2009 / 0304718; cibrotuzumab (see, for example, Hofheinz et al., Oncology Research and Treatment 26(1), 2003); and Tran et al., J Exp Med 210(6):1125-1135 (2013).

[0254] In some embodiments, the antigen-binding domain for CD38 is the antigen-binding portion (e.g., CDR) of daratumumab (e.g., Groen et al., Blood 116(21):1261-1262 (2010); see MOR202 (e.g., U.S. Patent No. 8,263,746)) or an antibody described in U.S. Patent No. 8,362,211.

[0255] In some embodiments, the antigen-binding domain for CD44v6 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Casucci et al., Blood 122(20):3461-3472 (2013).

[0256] In some embodiments, the antigen-binding domain for CEA is, for example, the antigen-binding portion of an antibody (e.g., CDR) as described in Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012).

[0257] In some embodiments, the antigen-binding domain for EPCAM is the antigen-binding portion (e.g., CDR)S of an antibody selected from MT110, EpCAM-CD3 bispecific Ab (see, e.g., clinicaltrials.gov / ct2 / show / NCT00635596), edrecolomab, 3622W94, ING-1, and adecatumumab (MT201).

[0258] In some embodiments, the antigen-binding domain for PRSS21 is the antigen-binding portion of an antibody described in U.S. Patent No. 8,080,650 (e.g., CDR).

[0259] In some embodiments, the antigen-binding domain for B7H3 is the antigen-binding portion (e.g., CDR) of the antibody MGA271 (Macrogenics).

[0260] In some embodiments, the antigen-binding domain for KIT is, for example, the antigen-binding portion of an antibody (e.g., CDR) as described in U.S. Patent No. 7,915,391, U.S. Patent No. 20120288506, and some commercial catalog antibodies.

[0261] In some embodiments, the antigen-binding domain for IL-13Ra2 is, for example, WO2008 / 146911, WO2004087758, some commercial catalog antibodies, and the antigen-binding portion (e.g., CDR) of the antibody described in WO2004087758.

[0262] In some embodiments, the antigen-binding domain for CD30 is the antigen-binding portion (e.g., CDR) of an antibody described, for example, U.S. Patent No. 7,090,843B1 and EP0805871.

[0263] In some embodiments, the antigen-binding domain for GD3 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in U.S. Patent Nos. 7,253,263; 8,207,308; US20120276046; EP1013761; WO2005035577; and U.S. Patent No. 6,437,098.

[0264] In some embodiments, the antigen-binding domain for CD171 is the antigen-binding portion of an antibody (e.g., CDR) as described, for example, in Hong et al., J Immunother 37(2):93-104 (2014).

[0265] In some embodiments, the antigen-binding domain for IL-11Ra is the antigen-binding portion (e.g., CDR) of an antibody available from Abcam (catalog no. ab55262) ​​or Novus Biologicals (catalog no. EPR5446). In another embodiment, the antigen-binding domain for IL-11Ra is a peptide. See, for example, Huang et al., Cancer Res 72(1):271-281 (2012).

[0266] In some embodiments, the antigen-binding domain for PSCA is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013 (2013), article ID 839831 (scFv C5-II); and U.S. Patent Publication No. 20090311181.

[0267] In some embodiments, the antigen-binding domain for VEGFR2 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010).

[0268] In some embodiments, the antigen-binding domain for Lewis Y is the antigen-binding portion (e.g., CDR) of an antibody described, for example, Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab(scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10 scFv).

[0269] In some embodiments, the antigen-binding domain for CD24 is the antigen-binding portion of an antibody (e.g., CDR) as described, for example, in Malial et al., Gastroenterology 143(5):1375-1384 (2012).

[0270] In some embodiments, the antigen-binding domain for PDGFR-β is the antigen-binding portion of the antibody Abcam ab32570 (e.g., CDR).

[0271] In some embodiments, the antigen-binding domain for SSEA-4 is the antigen-binding portion (e.g., CDR) of the antibody MC813 (Cell Signaling) or another commercially available antibody.

[0272] In some embodiments, the antigen-binding domain for CD20 is the antigen-binding portion (e.g., CDR) of the antibody rituximab, ofatumumab, ocrelizumab, vertuzumab, or GA101.

[0273] In some embodiments, the antigen-binding domain for folate receptor α is the antigen-binding portion (e.g., CDR) of the antibody IMGN853, or the antibody described in US20120009181; U.S. Patent No. 4,851,332, LK26: U.S. Patent No. 5,952,484.

[0274] In some embodiments, the antigen-binding domain for ERBB2(Her2 / neu) is the antigen-binding portion of the antibody trastuzumab or pertuzumab (e.g., CDR).

[0275] In some embodiments, the antigen-binding domain for MUC1 is the antigen-binding portion of the antibody SAR566658 (e.g., CDR).

[0276] In some embodiments, the antigen-binding domain for EGFR is the antigen-binding portion (e.g., CDR) of the antibody cetuximab, panitumumab, zaltumumab, nimotuzumab, or matuzumab. In some embodiments, the antigen-binding domain for EGFRvIII may be, for example, the antigen-binding domain of an antibody, antigen-binding fragment, or CAR described in PCT Publication WO2014 / 130657 (e.g., CDR, scFv, or VH and VL) or derived therefrom (in one embodiment, the CAR is a CAR described in WO2014 / 130657, the contents of which are incorporated herein in whole).

[0277] In some embodiments, the antigen-binding domain for NCAM is the antigen-binding portion (e.g., CDR) of antibody clone 2-2B:MAB5324 (EMD Millipore).

[0278] In some embodiments, the antigen-binding domain for ephrin B2 is the antigen-binding portion of an antibody (e.g., CDR) as described, for example, Abengozar et al., Blood 119(19):4565-4576 (2012).

[0279] In some embodiments, the antigen-binding domain for the IGF-I receptor is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in U.S. Patent No. 8,344,112B2;EP2322550 A1;WO 2006 / 138315 or PCT / US2006 / 022995.

[0280] In some embodiments, the antigen-binding domain for CAIX is the antigen-binding portion (e.g., CDR) of antibody clone 303123 (R&D Systems).

[0281] In some embodiments, the antigen-binding domain for LMP2 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in U.S. Patent No. 7,410,640 or U.S. 20050129701.

[0282] In some embodiments, the antigen-binding domain for gp100 is the antigen-binding portion (e.g., CDR) of the antibody HMB45, NKIbetaB, or the antibody described in WO2013165940 or US20130295007.

[0283] In some embodiments, the antigen-binding domain for tyrosinase is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in U.S. Patent No. 5,843,674 or U.S. Patent Application No. 19 / 950,504048.

[0284] In some embodiments, the antigen-binding domain for EphA2 is the antigen-binding portion of an antibody (e.g., CDR) as described, for example, Yu et al., Mol Ther 22(1):102-111 (2014).

[0285] In some embodiments, the antigen-binding domain for GD3 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in U.S. Patent No. 7,253,263, U.S. Patent No. 8,207,308, U.S.20120276046;EP1013761 A3;20120276046;WO2005035577 or U.S. Patent No. 6,437,098.

[0286] In some embodiments, the antigen-binding domain for fucosyl GM1 is the antigen-binding portion (e.g., CDR) of an antibody described, for example, US20100297138 or WO2007 / 067992.

[0287] In some embodiments, the antigen-binding domain for sLe is the antigen-binding portion of antibody G193 (in the case of Lewis Y) (e.g., CDR). See Scott AM et al, Cancer Res 60: 3254-61 (2000). Also described in Neeson et al, J Immunol May 2013 190 (Meeting Abstract Supplement) 177.10.

[0288] In some embodiments, the antigen-binding domain for GM3 is the antigen-binding portion (e.g., CDR) of the antibody CA 2523449 (mAb 14F7).

[0289] In some embodiments, the antigen-binding domain for HMWMAA is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Kmiecik et al., OncoImmunology 3(1):e27185 (2014) (PMID: 24575382) (mAb9.2.27), U.S. Patent No. 6,528,481, WO2010033866, or U.S. 20140004124.

[0290] In some embodiments, the antigen-binding domain for o-acetyl-GD2 is the antigen-binding portion of antibody 8B6 (e.g., CDR).

[0291] In some embodiments, the antigen-binding domain for TEM1 / CD248 is the antigen-binding portion (e.g., CDR) of an antibody described, for example, Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J Immunol Methods 363(2):221-232 (2011).

[0292] In some embodiments, the antigen-binding domain for CLDN6 is the antigen-binding portion of the antibody IMAB027 (Ganymed Pharmaceuticals) (e.g., CDR) (see, for example, clinicaltrial.gov / show / NCT02054351).

[0293] In some embodiments, the antigen-binding domain for TSHR is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in U.S. Patent No. 8,603,466, U.S. Patent No. 8,501,415, or U.S. Patent No. 8,309,693.

[0294] In some embodiments, the antigen-binding domain for GPRC5D is the antigen-binding portion (e.g., CDR) of the antibody FAB6300A (R&D Systems) or LS-A4180 (Lifespan Biosciences).

[0295] In some embodiments, the antigen-binding domain for CD97 is the antigen-binding portion (e.g., CDR) of an antibody described in, for example, U.S. Patent No. 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009), or an antibody from R&D:MAB3734.

[0296] In some embodiments, the antigen-binding domain for ALK is the antigen-binding portion (e.g., CDR) of an antibody, as described, for example, Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010).

[0297] In some embodiments, the antigen-binding domain for polysialic acid is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Nagae et al., J Biol Chem 288(47):33784-33796 (2013).

[0298] In some embodiments, the antigen-binding domain for PLAC1 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002 / bab.1177.

[0299] In some embodiments, the antigen-binding domain for globoH is the antigen-binding portion of the antibody VK9, or, for example, the antibody described in Kudryashov V et al., Glycoconj J.15(3):243-9 (1998), Lou et al., Proc Natl Acad Sci USA 111(7):2482-2487 (2014); MBr1: Bremer EG et al. J Biol Chem 259:14773-14777 (1984).

[0300] In some embodiments, the antigen-binding domain for NY-BR-1 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

[0301] In some embodiments, the antigen-binding domain for WT-1 is the antigen-binding portion of an antibody (e.g., CDR) as described, for example, Dao et al., Sci Transl Med 5(176):176ra33 (2013), or WO2012 / 135854.

[0302] In some embodiments, the antigen-binding domain for MAGE-A1 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv).

[0303] In some embodiments, the antigen-binding domain for sperm protein 17 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Song et al., Target Oncol 2013 Aug. 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012).

[0304] In some embodiments, the antigen-binding domain for Tie 2 is the antigen-binding portion (e.g., CDR) of the antibody AB33 (Cell Signaling Technology).

[0305] In some embodiments, the antigen-binding domain for MAD-CT-2 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in PMID:2450952, U.S. Patent No. 7,635,753.

[0306] In some embodiments, the antigen-binding domain for Fos-related antigen 1 is the antigen-binding portion of antibody 12F9 (Novus Biologicals) (e.g., CDR).

[0307] In some embodiments, the antigen-binding domain for MelanA / MART1 is the antigen-binding portion (e.g., CDR) of an antibody described in EP2514766 A2 or U.S. Patent No. 7,749,719.

[0308] In some embodiments, the antigen-binding domain for sarcoma translocation breakpoints is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Luo et al, EMBO Mol. Med. 4(6):453-461 (2012).

[0309] In some embodiments, the antigen-binding domain for TRP-2 is, for example, the antigen-binding portion of an antibody (e.g., CDR) as described in Wang et al, J Exp Med. 184(6):2207-16 (1996).

[0310] In some embodiments, the antigen-binding domain for CYP1B1 is, for example, the antigen-binding portion (e.g., CDR) of an antibody described in Maecker et al, Blood 102 (9): 3287-3294 (2003).

[0311] In some embodiments, the antigen-binding domain for RAGE-1 is the antigen-binding portion (e.g., CDR) of the antibody MAB5328 (EMD Millipore).

[0312] In some embodiments, the antigen-binding domain for human telomerase reverse transcriptase is the antigen-binding portion (e.g., CDR) of antibody catalog number LS-B95-100 (Lifespan Biosciences).

[0313] In some embodiments, the antigen-binding domain for intestinal carboxylesterase is the antigen-binding portion (e.g., CDR) of antibody 4F12: catalog number: LS-B6190-50 (Lifespan Biosciences).

[0314] In some embodiments, the antigen-binding domain for mut hsp70-2 is the antigen-binding portion (e.g., CDR) of the antibody Lifespan Biosciences: Monoclonal: Catalog No.: LS-C133261-100 (Lifespan Biosciences).

[0315] In some embodiments, the antigen-binding domain for CD79a is the antigen-binding portion (e.g., CDR) of the antibody Anti-CD79a antibody [HM47 / A9](ab3121) (available from Abcam), the antibody CD79A Antibody #3351 (available from Cell Signaling Technology), or the antibody HPA017748-Anti-CD79a antibody (produced in rabbits, available from Sigma Aldrich).

[0316] In some embodiments, the antigen-binding domain for CD79b is the antibody polatuzumab vedotin, as described in Dornan et al., “Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma” Blood. 2009 Sep. 24;114(13):2721-9. doi: 10.1182 / blood-2009-02-205500. Epub 2009 Jul. 24, or “4507 Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b / CD3 As a Potential Therapy for B Cell Malignancies” Abstracts of 56 th This is the antigen-binding portion (e.g., CDR) of the bispecific antibody Anti-CD79b / CD3, described at the ASH Annual Meeting and Exposition, San Francisco, Calif. Dec. 6-9, 2014.

[0317] In some embodiments, the antigen-binding domain for CD72 is the antigen-binding portion (e.g., CDR) of the antibody J3-109 described in Myers, and Uckun, “An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 June;18(1-2):119-22, or the anti-CD72 (10D6.8.1, mIgG1) described in Polson et al., “Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection” Cancer Res Mar. 15, 2009 69;2358.

[0318] In some embodiments, the antigen-binding domain for LAIR1 is the antigen-binding portion (e.g., CDR) of the antibody ANT-301 LAIR1 antibody available from ProSpec, or the anti-human CD305(LAIR1) Antibody available from BioLegend.

[0319] In some embodiments, the antigen-binding domain for the FCAR is the antigen-binding portion (e.g., CDR) of the antibody CD89 / FCARAntibody (catalog number 10414-H08H) available from Sino Biological Inc.

[0320] In some embodiments, the antigen-binding domain for LILRA2 is the antigen-binding portion (e.g., CDR) of the antibody LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or the Mouse Anti-LILRA2 antibody, monoclonal (2D7), available from Lifespan Biosciences.

[0321] In some embodiments, the antigen-binding domain for CD300LF is the antigen-binding portion (e.g., CDR) of the antibody Mouse Anti-CMRF35-like molecule 1 antibody, monoclonal [UP-D2], available from BioLegend, or the Rat Anti-CMRF35-like molecule 1 antibody, monoclonal [234903], available from R&D Systems.

[0322] In some embodiments, the antigen-binding domain for CLEC12A is described in Noordhuis et al., “Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody” 53 rd This refers to the antibody bispecific T cell engager (BiTE) scFv antibody and ADC described in ASH Annual Meeting and Exposition, Dec. 10-13, 2011, as well as the antigen-binding portion (e.g., CDR) of MCLA-117 (Merus).

[0323] In some embodiments, the antigen-binding domain for BST2 (also known as CD317) is the antigen-binding portion (e.g., CDR) of the antibody Mouse Anti-CD317 antibody, monoclonal [3H4] available from Antibodies-Online, or Mouse Anti-CD317 antibody, monoclonal [696739] available from R&D Systems.

[0324] In some embodiments, the antigen-binding domain for EMR2 (also known as CD312) is the antigen-binding portion (e.g., CDR) of the Mouse Anti-CD312 antibody, monoclonal [LS-B8033] available from Lifespan Biosciences, or the Mouse Anti-CD312 antibody, monoclonal [494025] available from R&D Systems.

[0325] In some embodiments, the antigen-binding domain for LY75 is the antigen-binding portion (e.g., CDR) of the Mouse Anti-Lymphocyte Antigen 75 Antibody, Monoclonal [HD30] available from EMD Millipore, or the Mouse Anti-Lymphocyte Antigen 75 Antibody, Monoclonal [A15797] available from Life Technologies.

[0326] In some embodiments, the antigen-binding domain for GPC3 is the antigen-binding portion (e.g., CDR) of the antibody hGC33, described in Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs. 2010 November;21(10):907-916, or MDX-1414, HN3, or YP7 (all three of which are described in Feng et al., “Glypican-3 antibodies: a new therapeutic target for liver cancer.” FEBS Lett. 2014 Jan. 21;588(2):377-82).

[0327] In some embodiments, the antigen-binding domain for FcRL5 is the antigen-binding portion (e.g., CDR) of an anti-FcRL5 antibody described by Elkins et al., “FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma” Mol Cancer Ther. 2012 October;11(10):2222-32.

[0328] In some embodiments, the antigen-binding domain for IGLL1 is the antigen-binding portion (e.g., CDR) of the antibody Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, monoclonal [AT1G4] available from Lifespan Biosciences, or Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, monoclonal [HSL11] available from BioLegend.

[0329] In some embodiments, the antigen-binding domain includes one, two, or three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3 derived from the antibodies listed above, and / or one, two, or three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3 derived from the antibodies listed above. In some embodiments, the antigen-binding domain includes the heavy chain variable region and / or the variable light chain region of the antibodies listed above.

[0330] In some embodiments, the antigen-binding domain includes a humanized antibody or antibody fragment. In some embodiments, a non-human antibody is humanized, and a specific sequence or region of the antibody is modified to increase its similarity to an antibody or fragment naturally produced in humans. In some embodiments, the antigen-binding domain is humanized.

[0331] Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patents No. 5,225,539, 5,530,101 and 5,585,089, each incorporated herein by reference as a whole), veneering, or resurfacing (see, for example, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.) See, for example, al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in whole), chain shuffling (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in whole), and, for example, U.S. Patent Publication No. US2005 / 0042664, U.S. Patent Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol.This includes techniques disclosed in 235(3):959-73 (1994), each of which is incorporated herein by reference as a whole. Often, framework residues within a framework region are substituted with corresponding residues of a CDR donor antibody to alter (e.g., improve) antigen binding. These framework substitutions are identified by methods well known in the art, including modeling of CDR-framework residue interactions to identify framework residues crucial for antigen binding, and sequence comparison to identify abnormal framework residues at specific locations. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, both incorporated herein by reference as a whole).

[0332] Humanized antibodies or antibody fragments retain one or more amino acid residues from a non-human source. These non-human amino acid residues are often referred to as “imported” residues and are typically obtained from “imported” variable domains. As provided herein, humanized antibodies or antibody fragments include one or more CDRs and framework regions derived from a non-human immunoglobulin molecule, wherein the amino acid residues constituting the framework are derived entirely or largely from the human germline. Several techniques for humanizing antibodies or antibody fragments are well known in the art and essentially involve replacing rodent CDRs or CDR sequences with corresponding sequences of human antibodies, following the methods of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), i.e., CDR grafts (EP 239,400, PCT publication number WO This can be carried out by U.S. Patents 91 / 09967, and U.S. Patents 4,816,567, 6,331,415, 5,225,539, 5,530,101, 5,585,089, and 6,548,640 (the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, substantially only a portion of the intact human variable domain is replaced with corresponding sequences derived from non-human species. Humanized antibodies are often human antibodies in which some CDR residues, and possibly some framework (FR) residues, are replaced with residues from similar sites in rodent antibodies.Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the details of which are incorporated herein by reference as a whole.

[0333] The selection of human variable domains (both light and heavy chains) used in the production of humanized antibodies is intended to reduce antigenicity. The sequences of variable domains from rodent antibodies are screened against the entire library of known human variable-domain sequences according to a so-called "best-fit" method. The human sequence most closely resembling the rodent sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference as a whole). Alternatively, a specific framework is used, derived from the consensus sequences of all human antibodies from a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993). The contents of these publications are incorporated herein by reference in their entirety). In some embodiments, the framework regions of the heavy chain variable region (e.g., all four framework regions) are derived from the VH4_4-59 germline sequence. In some embodiments, the framework regions may include one, two, three, four, or five modifications (e.g., substitutions) from the amino acids in the corresponding murine sequence, for example. In some embodiments, the framework regions of the light chain variable region (e.g., all four framework regions) are derived from the VK3_1.25 germline sequence. In some embodiments, the framework region may include one, two, three, four, or five modifications (e.g., substitutions) from the amino acids in the corresponding murine sequence.

[0334] In some embodiments, the portion of the CAR containing the antibody fragment is humanized while retaining high affinity for the target antigen and other desirable biological properties. In some embodiments, the humanized antibody and antibody fragment are prepared by a process that analyzes the parent sequence and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. By examining these displays, it is possible to analyze the likely roles of residues in the function of the candidate immunoglobulin sequence, for example, the residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this way, by selecting and combining FR residues from the recipient and import sequences, desired antibody or antibody fragment properties (such as improved affinity for the target antigen) can be achieved. Generally, CDR residues are most directly and significantly involved in influencing antigen binding.

[0335] In some embodiments, the humanized antibody or antibody fragment may retain antigen specificity similar to the original antibody (e.g., the ability to bind to human cancer-related antigens as described herein). In some embodiments, the humanized antibody or antibody fragment may have improved affinity and / or specificity for binding to human cancer-related antigens as described herein.

[0336] In some embodiments, the antigen-binding domain is characterized by specific functional features or properties of the antibody or antibody fragment. For example, in some embodiments, the portion of the CAR containing the antigen-binding domain specifically binds to tumor antigens as described herein.

[0337] In some embodiments, the binding domain of an anti-cancer-related antigen, as described herein, is a fragment (e.g., a single-chain variable fragment (scFv)). In some embodiments, the binding domain of an anti-cancer-related antigen, as described herein, is Fv, Fab, (Fab′)2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In some embodiments, the antibody and its fragments bind to the cancer-related antigen protein, as described herein, with wild-type affinity or enhanced affinity.

[0338] In some cases, scFv can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be produced by linking the VH and VL regions together using a flexible polypeptide linker. The scFv molecule contains a linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. The linker length can significantly influence how the variable regions of the scFv fold and interact. In fact, employing a short polypeptide linker (e.g., 5-10 amino acids) prevents intrachain folding. Interchain folding is also necessary for the two variable regions to come together and form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publications WO2006 / 020258 and WO2007 / 024715 (these are incorporated herein by reference).

[0339] scFv may contain a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may contain any native amino acids. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains a set of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer of 1 or greater (SEQ ID NO: 22). In some embodiments, the linker may be (Gly4Ser)4 (SEQ ID NO: 29) or (Gly4Ser)3 (SEQ ID NO: 30). Varying the linker length may preserve or enhance activity, potentially leading to superior efficacy in activity studies.

[0340] In some embodiments, the antigen-binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single-chain TCR (scTCR). Methods for constructing such TCRs are known in the art. See, for example, Willemsen RA et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (these references are incorporated herein by their entirety). For example, scTCRs containing Vα and Vβ genes from T cell clones linked by a linker (e.g., a flexible peptide) can be manipulated. This approach is very useful for cancer-related targets that are themselves intracellular, but fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC.

[0341] Bispecific CAR

[0342] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for two or fewer antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In one embodiment, the first and second epitopes are located on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are located on different antigens (e.g., different proteins (or different subunits of a multimeric protein)). In one embodiment, the bispecific antibody molecule includes a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule includes a half antibody having binding specificity to a first epitope and a half antibody having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule includes a half antibody or a fragment thereof having binding specificity to a first epitope and a half antibody or a fragment thereof having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule includes an scFv or a fragment thereof having binding specificity to a first epitope and an scFv or a fragment thereof having binding specificity to a second epitope.

[0343] In some embodiments, the antibody molecule is a multispecific (e.g., bispecific or tripspecific) antibody molecule. Protocols for generating bispecific or heterodimeric antibody molecules are known in the art and are not limited to, but include, for example, the “knob in a hole” approach described in U.S. Patent No. 5,731,168; electrostatic steering Fc pairing as described in WO 09 / 089004, WO 06 / 106905 and WO 2010 / 129304; chain exchange domain (SEED) heterodimerization as described in WO 07 / 110205; WO 08 / 119353, WO 2011 / 131746 and WO Fab arm exchange as described in 2013 / 060867, etc.; bi-antibody conjugates by, for example, antibody crosslinking and generating a bispecific structure using a heterobifunctional reagent having amine and sulfhydryl reactive groups as described in U.S. Patent No. 4,433,059, etc.; bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fab) from different antibodies through a reduction-oxidation cycle of the disulfide bond between two heavy chains, as described in U.S. Patent No. 4,444,878, etc.; as described in U.S. Patent No. 5,273,743, etc. Trifunctional antibodies such as those described in U.S. Patent No. 5,534,254 (examples include pairs of scFv crosslinked via C-terminal tails, preferably via disulfide or amine-reactive chemical crosslinks); bifunctional antibodies such as those described in U.S. Patent No. 5,582,996 (examples include Fab fragments with different binding specificities, dimerized by leucine zippers (e.g., c-fos and c-jun) that replace the constant domain); U.S. Patent No. 5,591,Bispecific and oligospecific monovalent and oligovalent receptors, as described in U.S. Patent No. 828, etc. (examples include the VH-CH1 regions of two antibodies (two Fab fragments) linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically having associated light chains); bispecific DNA antibody conjugates, as described in U.S. Patent No. 5,635,602, etc. (examples include crosslinking of antibodies or Fab fragments via double-stranded DNA fragments); bispecific fusion proteins, as described in U.S. Patent No. 5,637,481, etc. (examples include expression constructs containing two scFvs with a hydrophilic helical peptide linker in between, and a complete constant region); multivalent and multispecific binding proteins, as described in U.S. Patent No. 5,837,242, etc. (examples include polypeptide dimers (commonly called diabodies) having a first domain with a binding region for the Ig heavy chain variable region and a second domain with a binding region for the Ig light chain variable region) (as described in U.S. Patent No. 5,837,242, etc.) This also includes higher-order structures that create bispecific, tripspecific, or CD5raspecific molecules; minibody constructs having linked VL and VH chains further connected to the hinge and CH3 regions of the antibody by peptide spacers, as described in U.S. Patent No. 5,837,821, etc. (which can dimerize to form bispecific / polyvalent molecules); VH and VL domains linked by short peptide linkers (e.g., 5 or 10 amino acids) or without linkers in either direction (which can dimerize to form bispecific dibody structures); trimers and tetramers, as described in U.S. Patent No. 5,844,094, etc.; strings of VH domains (or VL domains in family members) linked by peptide bonds to a C-terminal crosslinkable group, as described in U.S. Patent No. 5,864,019, etc. (which can further associate with VL domains to form a series of FV (or scFv)); and U.S. Patent No. 5,869,This includes single-chain linked polypeptides having both VH and VL domains linked by a peptide linker, as described in Patent No. 620, etc. (linked to a polyvalent structure by non-covalent bonding or chemical crosslinking, using both scFV and diabody type forms to form structures such as homodivalent, heterodivalent, trivalent, and CD5ravalent). Further exemplary multispecific and bispecific molecules, as well as methods for producing them, are described in, for example, U.S. Patents 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, and 6,47 U.S. Patent No. 6,198, U.S. Patent No. 6,511,663, U.S. Patent No. 6,670,453, U.S. Patent No. 6,743,896, U.S. Patent No. 6,809,185, U.S. Patent No. 6,833,441, U.S. Patent No. 7,129,330, U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,No. 181, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US200 4242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, U S2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1, US200708738 1A1, US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US2008152645A1, US20081 71855A1, US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A1, US 2009162360A1, US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087A2, WO The following applications are described in 0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, and WO9964460A1. The contents of the above applications are incorporated herein by reference in their entirety.

[0344] Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, VH can be located upstream or downstream of VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is located upstream of its VL (VL1) along with its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is located upstream of its VH (VH2) along with its VL (VL2), resulting in the overall bispecific antibody molecule having the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is located upstream of its VH (VH1) along with its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is located upstream of its VL (VL2) along with its VH (VH2), resulting in the overall bispecific antibody molecule having the configuration VL1VH1-VH2-VL2. Optionally, a linker is placed between two antibodies or antibody fragments (e.g., scFv), for example, between VL1 and VL2 if the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 if the construct is arranged as VL1-VH1-VH2-VL2. The linker may be any linker as described herein, an example of which is a (Gly4-Ser)n linker (where n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQ ID NO: 29)). In general, the linker between two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, a linker is placed between VL and VH of the first scFv. Optionally, a linker is placed between VL and VH of the second scFv. In a structure having multiple linkers, any two or more linkers may be identical or different. Therefore, in some embodiments, a dual-specific CAR includes a VL, a VH, and optionally one or more linkers in an arrangement as described herein.

[0345] Stability and mutation

[0346] The stability of antigen-binding domains (e.g., scFv molecules (e.g., soluble scFv)) to cancer-associated antigens as described herein can be evaluated by reference to the biophysical properties (e.g., thermal stability) of conventional control scFv molecules or full-length antibodies. In some embodiments, humanized scFv has higher thermal stability than control binding molecules (e.g., conventional scFv molecules) in the described assays at temperatures of approximately 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 degrees Celsius.

[0347] The improved thermal stability of the antigen-binding domain for cancer-related antigens (e.g., scFv) described herein is subsequently conferred to the entire CAR construct, leading to improved therapeutic properties of the CAR construct. The thermal stability of the antigen-binding domain of cancer-related antigens (e.g., scFv) described herein can be improved by at least about 2°C or 3°C compared to conventional antibodies. In some embodiments, the antigen-binding domain of cancer-related antigens (e.g., scFv) described herein exhibits 1°C improved thermal stability compared to conventional antibodies. In another embodiment, the antigen-binding domain of cancer-related antigens (e.g., scFv) described herein exhibits 2°C improved thermal stability compared to conventional antibodies. In yet another embodiment, scFv exhibits 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15°C improved thermal stability compared to conventional antibodies. For example, a comparison can be made between the scFv molecules disclosed herein and scFv molecules or Fab fragments of antibodies from which scFv VH and VL are derived. Thermal stability can be measured using methods known in the art. For example, in some embodiments, Tm can be measured. Methods for measuring Tm, and other methods for determining protein stability, are described in more detail below.

[0348] Mutations in scFv (caused by humanization of soluble scFv or direct mutagenesis) can alter the stability of scFv and improve the overall stability of scFv and CAR constructs. The stability of humanized scFv is compared to that of murine scFv using measurements such as Tm, temperature denaturation, and temperature aggregation.

[0349] The binding ability of mutant scFv can be determined using assays known in the art and described herein.

[0350] In some embodiments, the antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein includes at least one mutation resulting from humanization, and as a result, the mutated scFv confers improved stability to the CAR construct. In another embodiment, the antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations resulting from humanization, and as a result, the mutated scFv confers improved stability to the CAR construct.

[0351] Methods for evaluating protein stability

[0352] The stability of the antigen-binding domain may be evaluated, for example, using the methods described below. Such methods allow for the determination of multiple thermal unfolding transitions, where either the least stable domain unfolds first or the cooperative unfolding limits the overall stability threshold of the multi-domain unit (e.g., a multi-domain protein exhibiting a single unfolding transition). The least stable domain can be identified in several additional ways. Mutagenesis can be performed to investigate the domain that limits overall stability. Furthermore, protease resistance of multi-domain proteins can be tested under conditions where the least stable domain is known to be essentially unfolded by DSC or other spectroscopic methods (Fontana, et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol. 393: 672-692). Once the least stable domain is identified, the sequence encoding this domain (or a portion thereof) may be adopted as the test sequence in this method.

[0353] thermal stability

[0354] The thermal stability of the composition may be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In some embodiments, thermal stability is evaluated by analytical spectroscopy.

[0355] An exemplary analytical spectroscopy technique is differential scanning calorimetry (DSC). DSC employs a calorimeter sensitive to the thermal absorption associated with the unfolding of most proteins or protein domains (see, e.g., Sanchez-Ruiz et al., Biochemistry, 27: 1648-52, 1988). To determine the thermal stability of a protein, a sample of the protein is inserted into a calorimeter and the temperature is increased until the Fab or scFv unfolds. The temperature at which the protein unfolds indicates the overall stability of the protein.

[0356] Another exemplary analytical spectroscopy technique is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures the difference in absorption between left-handed and right-handed polarized light, which arises from structural asymmetry. Disordered structures, i.e., unfolded structures, produce CD spectra that differ significantly from those of regular structures, i.e., folded structures. The CD spectrum reflects the sensitivity of proteins to denaturation effects due to increasing temperature and is therefore an indicator of the thermal stability of proteins (see van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000).

[0357] Another exemplary analytical spectroscopy method for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, op. cit.). Yet another exemplary analytical spectroscopy method for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma, op. cit.).

[0358] The thermal stability of a composition can be measured biochemically. An exemplary biochemical method for evaluating thermal stability is a thermal challenge assay. In a thermal challenge assay, the composition is exposed to a series of high temperatures over a set period of time. For example, in some embodiments, a test scFv molecule or a molecule containing scFv molecules is exposed to a series of temperature increases for, for example, 1 to 1.5 hours. The activity of the protein is then assayed by the relevant biochemical assay. For example, if the protein is a binding protein (e.g., scFv or scFv-containing polypeptide), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

[0359] Such assays may be performed in a high-throughput format and as disclosed in the examples using E. coli and high-throughput screening. Libraries of antigen-binding domains (e.g., including antigen-binding domains for cancer-related antigens (e.g., scFv variants) described herein) may be prepared using methods known in the art. Expression of antigen-binding domains (e.g., antigen-binding domains for cancer-related antigens (e.g., scFv) described herein) may be induced, and antigen-binding domains (e.g., antigen-binding domains for cancer-related antigens (e.g., scFv) described herein) may be subjected to thermal challenge. Challenged test samples may be assayed for binding, and those antigen-binding domains that are stable for cancer-related antigens (e.g., scFv) described herein may be scaled up and further characterized.

[0360] Thermal stability is assessed by measuring the melting temperature (Tm) of the composition using one of the techniques described above (e.g., analytical spectroscopy). The melting temperature is the temperature at the midpoint of the temperature transition curve where 50% of the composition's molecules are folded (see, e.g., Dimasi et al. (2009) J. Mol Biol. 393: 672-692). In some embodiments, the Tm values ​​for the antigen-binding domain for cancer-related antigens described herein (e.g., scFv) are approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C The temperatures are 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C. In some embodiments, the Tm value for IgG is approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C. The temperatures are C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C.In some embodiments, the Tm value for polyvalent antibodies is approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C. The temperatures are 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C.

[0361] Thermal stability can also be assessed by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetry techniques (such as DSC). The specific heat of a composition is the energy (e.g., kcal / mol) required to raise the temperature of 1 mole of water by 1°C. A large Cp is characteristic of a denatured protein composition or an inactive protein composition. The change in heat capacity (ΔCp) of a composition is measured by determining the specific heat of the composition before and after a temperature transition. Thermal stability may also be assessed by measuring or determining other parameters of thermodynamic stability, including the unfolding Gibbs free energy (ΔG), unfolding enthalpy (ΔH), or unfolding entropy (ΔS). One or more of the above biochemical assays (e.g., thermal challenge assays) can be used to determine the temperature at which 50% of the composition retains its activity (e.g., binding activity) (i.e., T C Determine the value.

[0362] Furthermore, mutations in the antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein can be produced to alter the thermal stability of the antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein, compared to the non-mutated antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein. When the humanized antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein is incorporated into a CAR construct, the antigen-binding domain of the cancer-related antigen (e.g., humanized scFv) described herein confers thermal stability to the entire CAR. In some embodiments, the antigen-binding domain for the cancer-related antigen (e.g., scFv) described herein comprises a single mutation that confers thermal stability to the antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein. In another embodiment, the antigen-binding domain for the cancer-related antigen (e.g., scFv) described herein comprises multiple mutations that confer thermal stability to the antigen-binding domain of the cancer-related antigen (e.g., scFv) described herein. In some embodiments, multiple mutations in the antigen-binding domain for the cancer-related antigen (e.g., scFv) described herein have an additive effect on the thermal stability of the antigen-binding domain for the cancer-related antigen (e.g., scFv) described herein.

[0363] Agglomeration rate %

[0364] The stability of a composition can be determined by measuring its tendency to aggregate. Aggregation can be measured by several non-limiting biochemical or biophysical techniques. For example, the aggregation of a composition may be evaluated using chromatography such as size exclusion chromatography (SEC). SEC separates molecules based on size. The column is packed with semi-solid polymer gel beads that allow ions and small molecules to enter but large molecules cannot. When a protein composition is applied to the top of the column, compactly folded proteins (i.e., non-aggregated proteins) are dispersed through a larger amount of solvent than is available to large protein aggregates. As a result, large aggregates move faster through the column, thus allowing the mixture to be separated or fractionated into its components. Each fraction can be quantified individually upon elution from the gel (e.g., by light scattering). Thus, the aggregation rate % of a composition can be determined by comparing the concentration of the fraction with the total concentration of the protein applied to the gel. A stable composition will elute from the column as essentially a single fraction and appear as essentially a single peak in the elution profile or chromatogram.

[0365] binding affinity

[0366] The stability of a composition can be evaluated by determining its target binding affinity. A wide variety of methods for determining binding affinity are known in the art. Surface plasmon resonance is employed as an exemplary method for determining binding affinity. Surface plasmon resonance is an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0367] In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence homologous to the amino acid sequence of the antigen-binding domain described herein, and the antigen-binding domain retains the desired functional properties of the antigen-binding domain described herein.

[0368] In some embodiments, the CAR comprises an antibody fragment. In some embodiments, the antibody fragment comprises an scFv.

[0369] In some embodiments, the antigen-binding domain of the CAR is manipulated by modifying one or more amino acids within one or both of the variable regions (e.g., VH and / or VL) (e.g., within one or more CDR regions and / or one or more framework regions). In some embodiments, the CAR comprises an antibody fragment. In some embodiments, the antibody fragment comprises an scFv.

[0370] Those skilled in the art will understand that further modifications to antibodies or antibody fragments may alter their amino acid sequence (e.g., from the wild type) but prevent them from altering their desired activity. For example, additional nucleotide substitutions may be made to a protein, resulting in amino acid substitutions at "non-essential" amino acid residues. For instance, non-essential amino acid residues within a molecule may be replaced with other amino acid residues from the same side-chain family. In another embodiment, a string of amino acids may be replaced with a structurally similar string of side-chain family members with different order and / or composition, for example, a conservative substitution may be made in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

[0371] Families of amino acid residues having similar side chains have been defined in the art, and these include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0372] Percent identity in relation to two or more nucleic acid or polypeptide sequences refers to two or more sequences that are identical. Two sequences are "substantially identical" if they have identical amino acid residues or nucleotides in a specified proportion (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity in a specified region or across the entire sequence if not specified). However, the sequences shall be compared and aligned for the maximum match across the comparison window or specified region as measured by one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity shall exist over a region of at least approximately 50 nucleotides (or 10 amino acids), or more preferably over a region of 100 to 500 or 1000 nucleotides (or 20, 50, 200, or more amino acids).

[0373] For sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared to it. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, and subsequence coordinates and sequence algorithm program parameters are specified as needed. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence compared to the reference sequence based on the program parameters. Methods for sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using local homology algorithms (Smith and Waterman, (1970) Adv. Appl. Math. 2:482c), homology alignment algorithms (Needleman and Wunsch, (1970) J. Mol. Biol. 48:443), similarity search methods (Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444), computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0374] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST2.0 algorithm, described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0375] The percentage identicality between two amino acid sequences can also be determined using an algorithm (E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17). This is incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage identicality between two amino acid sequences can be determined using an algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453). This is incorporated into the GAP program of the GCG software package (available at www.gcg.com) using either the Blossom62 matrix or the PAM250 matrix, as well as gap weightings 16, 14, 12, 10, 8, 6, or 4, and length weightings 1, 2, 3, 4, 5, or 6.

[0376] In some embodiments, this embodiment intends to modify the amino acid sequence of an initiating antibody or fragment (e.g., scFv) to generate a functionally equivalent molecule. For example, the VH or VL of the antigen-binding domain for the cancer-associated antigen (e.g., scFv) contained in the CAR can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the identity of the initiating VH or VL framework region of the antigen-binding domain for the cancer-associated antigen (e.g., scFv) described herein. This embodiment aims to modify the entire CAR construct (e.g., modify one or more amino acid sequences in various domains of the CAR construct) in order to generate functionally homogeneous molecules. The CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the identity of the initial CAR construct.

[0377] transmembrane domain

[0378] With respect to the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain attached to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids related to the extracellular region of the protein from which the transmembrane originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 of the extracellular region) and / or one or more additional amino acids related to the intracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 of the intracellular region). In some embodiments, the transmembrane domain is a domain related to one of the other domains of the CAR; for example, in some embodiments, the transmembrane domain may originate from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain originates. In some embodiments, the transmembrane domain does not originate from the same protein from which any of the other domains of the CAR originate. In some cases, transmembrane domains can be selected or modified by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, for example, to minimize interaction with other members of the receptor complex. In some embodiments, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In some embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell.

[0379] The transmembrane domain may originate from either a native or recombinant source. If the source is native, the domain may originate from any membrane-bound or transmembrane protein. In some embodiments, whenever the CAR binds to a target, the transmembrane domain can signal to the intracellular domain(s). Particularly useful transmembrane domains may include, for example, the alpha, beta, or zeta chain of a T cell receptor, or at least the transmembrane region(s) of CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domains are, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL , CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM It may include at least one transmembrane region of (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C.

[0380] In some cases, the transmembrane domain can attach to the extracellular region of the CAR (e.g., the antigen-binding domain of the CAR) via a hinge (e.g., a hinge from a human protein). For example, in some embodiments, the hinge may be a human Ig (immunoglobulin) hinge (e.g., IgG4 hinge, IgD hinge), a GS linker (e.g., the GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In some embodiments, the hinge or spacer includes (e.g., is composed of) the amino acid sequence of SEQ ID NO: 4. In some embodiments, the transmembrane domain includes (e.g., is composed of) the transmembrane domain of SEQ ID NO: 12.

[0381] In some embodiments, the hinge or spacer includes an IgG4 hinge. For example, in some embodiments, the hinge or spacer includes an amino acid sequence hinge, which is ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 6). In some embodiments, the hinge or spacer includes a hinge encoded by a nucleotide sequence, which is GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO: 7).

[0382] In some embodiments, the hinge or spacer includes an IgD hinge. For example, in some embodiments, the hinge or spacer includes an amino acid sequence hinge, which is RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 8).In some embodiments, the hinge or spacer includes a hinge encoded by a nucleotide sequence, which is (SEQ ID NO: 9).

[0383] In some embodiments, the transmembrane domain may be recombinant, in which case the transmembrane domain mainly consists of hydrophobic residues (such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the recombinant transmembrane domain.

[0384] Optionally, short oligo or polypeptide linkers of 2 to 10 amino acids in length may form a linkage between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11).

[0385] In some embodiments, the hinge or spacer includes a KIR2DS2 hinge.

[0386] Cytoplasmic domain

[0387] The cytoplasmic domain or region of a CAR contains an intracellular signaling domain. Generally, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to a cellular specialization function. For example, the effector function of a T cell may be cytolytic activity or helper activity (including cytokine secretion). Therefore, the term "intracellular signaling domain" refers to the portion of the protein that transmits effector function signals and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be employed, it is often not necessary to use the entire chain. Insofar as a cleaved portion of the intracellular signaling domain is used, such a cleaved portion may be used in place of the intact chain, as long as it transmits effector function signals. Therefore, the term "intracellular signaling domain" means including any cleaved portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[0388] Examples of intracellular signaling domains used in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in coordination to initiate signaling after binding to antigen receptors, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0389] It is known that signals generated by the TCR alone are insufficient to fully activate T cells, and that secondary and / or costimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0390] The primary signaling domain controls the primary activation of the TCR complex either in a stimulative or inhibitory manner. Primary intracellular signaling domains that act in a stimulative manner may contain a signaling motif known as an immunoreceptor tyrosine activation motif or ITAM.

[0391] Examples of ITAM-containing primary intracellular signaling domains include, but are not limited to, CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, the CAR includes an intracellular signaling domain (e.g., the primary signaling domain of CD3 zeta).

[0392] In some embodiments, the primary signaling domain includes a modified ITAM domain (e.g., a mutant ITAM domain with altered activity (e.g., increased or decreased activity) compared to a native ITAM domain). In some embodiments, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain (e.g., an optimized and / or cleaved ITAM-containing primary intracellular signaling domain). In some embodiments, the primary signaling domain includes 1, 2, 3, 4, or more ITAM motifs.

[0393] The intracellular signaling domain of a CAR may consist solely of a CD3 zeta signaling domain, or it may be combined with any other desired intracellular signaling domain useful in relation to the CAR. For example, the intracellular signaling domain of a CAR may include a CD3 zeta chain portion and a costimulatory signaling domain. A costimulatory signaling domain refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand that is necessary for the efficient response of lymphocytes to an antigen. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. For example, CD27 co-stimulation has been shown to promote the proliferation, effector function, and survival of human CART cells in vitro, and to enhance the persistence and antitumor activity of human T cells in vivo (Song et al. Blood. 2012;119(3):696-706).Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), and SLAMF4. Includes (CD244, 2B4), CD84, CD96 (tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp and CD19a.

[0394] The intracellular signaling sequences within the cytoplasmic portion of the CAR may be linked to each other in a random or specified order. Optionally, short oligolinkers or polypeptide linkers, for example, with a length of 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), may form links between the intracellular signaling sequences. In some embodiments, a glycine-serine doublet can be used as a suitable linker. In some embodiments, a single amino acid (e.g., alanine, glycine) can be used as a suitable linker.

[0395] In some embodiments, the intracellular signaling domain is designed to contain two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signaling domains. In some embodiments, the two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signaling domains are separated by a linker molecule (e.g., the linker molecule described herein). In some embodiments, the intracellular signaling domain contains two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0396] In some embodiments, the intracellular signaling domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In some embodiments, the intracellular signaling domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain. In some embodiments, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 14. In some embodiments, the CD3-zeta signaling domain is the signaling domain of SEQ ID NO: 18.

[0397] In some embodiments, the intracellular signaling domain is designed to include a CD3-zeta signaling domain and a CD27 signaling domain. In some embodiments, the CD27 signaling domain includes an amino acid sequence, which is QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 16). In some embodiments, the CD27 signaling domain is encoded by a nucleic acid sequence, which is AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 17).

[0398] In some embodiments, the CAR-expressing cells described herein may further include a second CAR, for example, which includes a different antigen-binding domain for the same or different targets (e.g., targets other than the cancer-associated antigens described herein, or different cancer-associated antigens described herein). In some embodiments, the second CAR includes an antigen-binding domain for a target expressed in the same cancer cell type as the cancer-associated antigen. In some embodiments, the CAR-expressing cells include a first CAR that targets a first antigen and includes an intracellular signaling domain having a co-stimulatory signaling domain but lacking a primary signaling domain, and a second CAR that targets a different second antigen and includes an intracellular signaling domain having a primary signaling domain but lacking a co-stimulatory signaling domain. Without intending to be bound by theory, placing a co-stimulatory signaling domain (e.g., 4-1BB, CD28, CD27, or OX-40) in the first CAR and a primary signaling domain (e.g., CD3 zeta) in the second CAR can limit the activity of the CAR to cells expressing both targets. In some embodiments, CAR-expressing cells include a first cancer-associated antigen CAR comprising an antigen-binding domain, a transmembrane domain, and a costimulatory domain for binding to the target antigen described herein, and a second CAR that targets a different target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, CAR-expressing cells include a first CAR comprising an antigen-binding domain, a transmembrane domain, and a primary signaling domain for binding to the target antigen described herein, and a second CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen-binding domain, a transmembrane domain, and a costimulatory signaling domain for the antigen.

[0399] In some embodiments, CAR-expressing cells include the XCARs and inhibitory CARs described herein. In some embodiments, the inhibitory CAR includes an antigen-binding domain that binds an antigen, which is present in normal cells but not in cancer cells (e.g., normal cells that also express CLL). In some embodiments, the inhibitory CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of the inhibitory molecule. For example, the intracellular domain of the inhibitory CAR may be the intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF beta.

[0400] In some embodiments, if a CAR-expressing cell contains two or more different CARs, the antigen-binding domains of the different CARs may be such that they do not interact with each other. For example, a cell expressing a first CAR and a second CAR may have an antigen-binding domain of the first CAR that does not associate with the antigen-binding domain of the second CAR (e.g., as a fragment, e.g., scFv), while the antigen-binding domain of the second CAR is, for example, VHH.

[0401] In some embodiments, the antigen-binding domain comprises a single-domain antigen-binding (SDAB) molecule, the complementarity-determining region of this molecule being part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain variable domains, naturally devoid light-chain binding molecules, single domains derived from conventional four-chain antibodies, engineered domains other than those derived from antibodies, and single-domain scaffolds. The SDAB molecule may be any single-domain molecule in the Art or any future single-domain molecule. The SDAB molecule may originate from any species, including, but is not limited to, mice, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. The term also includes naturally occurring single-domain antibody molecules derived from species other than camelids and sharks.

[0402] In some embodiments, SDAB molecules may be derived from the variable region of immunoglobulins found in fish, such as, for example, from a novel immunoglobulin isotype known as a NAR (non-antigen receptor) found in shark serum. Methods for producing single-domain molecules ("IgNAR") derived from the variable region of NARs are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0403] In some embodiments, the SDAB molecule is a naturally occurring single-domain antigen-binding molecule known as a light-chain-deficient heavy-chain molecule. Such single-domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, this variable domain derived from a naturally occurring light-chain-deficient heavy-chain molecule is known herein as VHH or nanobody to distinguish it from conventional VHs of four-chain immunoglobulins. Such VHH molecules may originate from camelid species, examples of which include camels, llamas, dromedaries, alpacas, and guanacos. Other non-camelid species may also produce naturally occurring light-chain-deficient heavy-chain molecules, and such VHHs are within the scope of the embodiments described herein.

[0404] SDAB molecules may be recombinant, CDR grafted, humanized, camelidized, deimmunized, and / or generated in vitro (e.g., by selection via phage display).

[0405] Furthermore, it has been discovered that cells having multiple chimeric membrane-embedded receptors, including antigen-binding domains that interact with each other, may be undesirable. This is because, for example, one or more of the antigen-binding domains inhibit the ability of the antigen-binding domains to bind their congener antigens. Therefore, cells having first and second non-natural chimeric membrane-embedded receptors containing antigen-binding domains that minimize such interactions are disclosed herein. Also disclosed herein are nucleic acids encoding first and second non-natural chimeric membrane-embedded receptors containing antigen-binding domains that minimize such interactions, as well as methods for producing and using such cells and nucleic acids. In one embodiment, one antigen-binding domain of the first and second non-natural chimeric membrane-embedded receptors comprises an scFv, and the other comprises a single VH domain (e.g., a single VH domain from a camelid, shark, or lamprey), or a single VH domain derived from a human or mouse sequence.

[0406] In some embodiments, the CAR comprises a first CAR and a second CAR, and one of the antigen-binding domains of the first CAR or the second CAR does not contain a variable light domain and a variable heavy domain. In some embodiments, one of the antigen-binding domains of the first CAR or the second CAR is an scFv, and the other is not an scFv. In some embodiments, one of the antigen-binding domains of the first CAR or the second CAR comprises a single VH domain, examples of which include a single VH domain from a camelid, shark, or lamprey, or a single VH domain derived from a human or mouse sequence. In some embodiments, one of the antigen-binding domains of the first CAR or the second CAR comprises a nanobody. In some embodiments, one of the antigen-binding domains of the first CAR or the second CAR comprises a VHH domain from a camelid.

[0407] In some embodiments, one antigen-binding domain of the first or second CAR contains an scFv and the other contains a single VH domain, examples of which include a single VH domain from a camelid, shark, or lamprey, or a single VH domain derived from a human or mouse sequence. In some embodiments, one antigen-binding domain of the first or second CAR contains an scFv and the other contains a nanobody. In some embodiments, one antigen-binding domain of the first or second CAR contains an scFv and the other contains a VHH domain from a camelid.

[0408] In some embodiments, when present on the cell surface, the binding of the antigen-binding domain of the first CAR to its congener antigen is not substantially reduced by the presence of the second CAR. In some embodiments, the binding of the antigen-binding domain of the first CAR to its congener antigen in the presence of the second CAR is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen-binding domain of the first CAR to its congener antigen in the absence of the second CAR.

[0409] In some embodiments, when present on the cell surface, the antigen-binding domains of the first CAR and the second CAR associate with each other less than when both are scFv antigen-binding domains. In some embodiments, the antigen-binding domains of the first CAR and the second CAR associate with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than when both are scFv antigen-binding domains.

[0410] In some embodiments, the CAR-expressing cells described herein may further express another agent (e.g., an agent that enhances the activity of the CAR-expressing cells). For example, in some embodiments, the agent may be an agent that inhibits an inhibitory molecule. In some embodiments, the inhibitory molecule (e.g., PD1) can reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGF beta. In some embodiments, the agent that inhibits the inhibitory molecule may be, for example, a molecule described herein, an example of which is an agent comprising a first polypeptide, an example of which is an inhibitory molecule associated with a second polypeptide that provides a positive signal to the cell, an example of which is an intracellular signaling domain described herein. In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule (examples include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these)) and a second polypeptide of an intracellular signaling domain as described herein (e.g., including a co-stimulatory domain (e.g., 41BB, CD27, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein). In some embodiments, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). By inhibiting the local interaction between PD1 and PD-L1, immunosuppression can be reversed.

[0411] In some embodiments, the agent comprises the extracellular domain (ECD) of an inhibitory molecule (e.g., programmed death 1 (PD1)) fused to a transmembrane domain and an intracellular signaling domain (e.g., 41BB and CD3 zeta) (also referred to herein as PD1 CAR). In some embodiments, the PD1 CAR, when used in combination with an XCAR as described herein, improves T cell persistence. In some embodiments, the CAR is a PD1 CAR comprising the extracellular domain of PD1 shown underlined in SEQ ID NO: 26. In some embodiments, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 26. [ka]

[0412] In some embodiments, the PD1 CAR includes the amino acid sequence shown below (SEQ ID NO: 39). [ka]

[0413] In some embodiments, the agent includes a nucleic acid sequence encoding a PD1 CAR (e.g., the PD1 CAR described herein). In some embodiments, the nucleic acid sequence of the PD1 CAR is shown below, where PD1 ECD is underlined in Sequence ID No. 27 below. [ka]

[0414] In some embodiments, this embodiment provides a population of CAR-expressing cells (e.g., CART cells). In some embodiments, the population of CAR-expressing cells includes a mixture of cells expressing different CARs. For example, in some embodiments, the population of CART cells may include first cells expressing a CAR having an antigen-binding domain for a cancer-related antigen as described herein, and second cells expressing a CAR having a different antigen-binding domain, an example of which antigen-binding domains include different antigen-binding domains for different cancer-related antigens as described herein, an example of which antigen-binding domains for cancer-related antigens different from those to which the antigen-binding domain of the CAR expressed by the first cell binds. As another example, the population of CAR-expressing cells may include first cells expressing a CAR containing an antigen-binding domain for a cancer-related antigen as described herein, and second cells expressing a CAR containing an antigen-binding domain for a target other than a cancer-related antigen as described herein. In some embodiments, the population of CAR-expressing cells may include, for example, first cells expressing a CAR containing a primary intracellular signaling domain, and second cells expressing a CAR containing a secondary signaling domain.

[0415] In some embodiments, this embodiment provides a population of cells in which at least one cell expresses a CAR having an antigen-binding domain for a cancer-associated antigen as described herein, and a second cell expresses another agent (e.g., an agent that enhances the activity of the CAR-expressing cell). For example, in some embodiments, the agent may be an agent that inhibits an inhibitory molecule. The inhibitory molecule (e.g., PD-1) can, in some embodiments, reduce the ability of the CAR-expressing cell to initiate an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGF beta. In some embodiments, the agent that inhibits the inhibitory molecule is, for example, a molecule described herein, an example of which is an agent comprising a first polypeptide, an example of which is an inhibitory molecule associated with a second polypeptide that provides a positive signal to the cell, an example of which is an intracellular signaling domain described herein. In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule such as PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF beta, or a fragment thereof, and a second polypeptide which is an intracellular signaling domain as described herein (e.g., a co-stimulatory domain (e.g., 41BB, CD27, OX40, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein). In some embodiments, the agent comprises a first polypeptide of PD-1 or a fragment thereof and a second polypeptide which is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0416] In some embodiments, this embodiment provides a method comprising administering a population of CAR-expressing cells (examples include CART cells, and examples include a mixture of cells expressing different CARs) in combination with another agent (examples include a kinase inhibitor, and examples include the kinase inhibitors described herein). In some embodiments, this embodiment provides a method comprising administering a population of cells in which at least one cell expresses a CAR having an antigen-binding domain of a cancer-associated antigen described herein, and a second cell expresses another agent (e.g., an agent that enhances the activity of CAR-expressing cells) in combination with the other agent (examples include a kinase inhibitor, and examples include the kinase inhibitors described herein).

[0417] Controllable Chimeric Antigen Receptors In some embodiments, controllable CARs (RCARs) whose CAR activity can be controlled are desirable to optimize the safety and efficacy of CAR therapy. There are many ways to control CAR activity. For example, inducible apoptosis using a caspase fused to a dimerization domain (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3;365(18):1673-1683) can be used as a safety switch in CAR therapy. In some embodiments, the RCAR comprises a set of polypeptides (typically two in the simplest embodiment), and the components of a standard CAR described herein (e.g., antigen-binding domain and intracellular signaling domain) are divided into separate polypeptides or members. In some embodiments, the set of polypeptides comprises a dimerization switch that can couple polypeptides to each other in the presence of a dimerization molecule, for example, by coupling the antigen-binding domain to the intracellular signaling domain.

[0418] In some embodiments, the RCAR comprises two polypeptides or members, namely: 1) an intracellular signaling member containing an intracellular signaling domain (e.g., the primary intracellular signaling domain described herein) and a first switch domain; and 2) an antigen-binding member containing an antigen-binding domain (e.g., targeting a tumor antigen as described herein) and a second switch domain. Optionally, the RCAR comprises a transmembrane domain as described herein. In some embodiments, the transmembrane domain may be located on the intracellular signaling member, on the antigen-binding member, or both. (Unless otherwise indicated, when members or elements of the RCAR are described herein, the order may be as shown, but other orders are also included. In other words, in some embodiments the order is as presented herein, but in other embodiments the order may be different. For example, the order of elements on one side of the transmembrane region may differ from the example. For example, the arrangement of the switch domain relative to the intracellular signaling domain may differ (e.g., reversed)).

[0419] In some embodiments, the first and second switch domains can form an intracellular or extracellular dimerization switch. In some embodiments, the dimerization switch may be, for example, a homodimerization switch in which the first and second switch domains are the same, or a heterodimerization switch in which the first and second switch domains are different from each other.

[0420] In some embodiments, the RCAR may include a “multiswitch.” The multiswitch may include a heterodimerized switch domain or a homodimerized switch domain. The multiswitch may independently include a plurality of switch domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) on a first member (e.g., an antigen-binding member) and a second member (e.g., an intracellular signaling member). In some embodiments, the first member may include a plurality of first switch domains (e.g., FKBP-based switch domains), and the second member may include a plurality of second switch domains (e.g., FRB-based switch domains). In some embodiments, the first member may include first and second switch domains (e.g., FKBP-based switch domains and FRB-based switch domains), and the second member may include first and second switch domains (e.g., FKBP-based switch domains and FRB-based switch domains).

[0421] In some embodiments, the intracellular signaling member includes one or more intracellular signaling domains (e.g., a primary intracellular signaling domain and one or more co-stimulatory signaling domains).

[0422] In some embodiments, the antigen-binding member may include one or more intracellular signaling domains (e.g., one or more co-stimulatory signaling domains). In some embodiments, the antigen-binding member includes a plurality (e.g., two or three) co-stimulatory signaling domains described herein (e.g., selected from 41BB, CD28, CD27, ICOS, and OX40), and in some embodiments, it does not include a primary intracellular signaling domain. In some embodiments, the antigen-binding member includes the co-stimulatory signaling domains of 41BB-CD27, 41BB-CD27, CD27-41BB, 41BB-CD28, CD28-41BB, OX40-CD28, CD28-OX40, CD28-41BB, or 41BB-CD28, in an extracellular to intracellular direction. In such embodiments, the intracellular binding member includes a CD3 zeta domain. In one such embodiment, the RCAR comprises (1) an antigen-binding member comprising an antigen-binding domain, a transmembrane domain, and two costimulatory domains and one first switch domain, and (2) an intracellular signaling domain comprising a transmembrane domain or membrane CD5-hering domain and at least one primary intracellular signaling domain, and a second switch domain.

[0423] Some embodiments provide RCARs in which the antigen-binding member is not CD5hered on the surface of the CAR cell. This makes it possible to easily pair cells having an intracellular signaling member with one or more antigen-binding domains without transforming the cell with a sequence encoding the antigen-binding member. In such embodiments, the RCAR comprises 1) an intracellular signaling member comprising a first switch domain, a transmembrane domain, an intracellular signaling domain (e.g., a primary intracellular signaling domain), and a first switch domain, and 2) an antigen-binding member comprising an antigen-binding domain and a second switch domain, wherein the antigen-binding member does not include a transmembrane domain or a membrane CD5hering domain, and optionally does not include an intracellular signaling domain. In some embodiments, the RCAR may further include 3) a second antigen-binding member. This second antigen-binding member comprises a second antigen-binding domain (e.g., a second antigen-binding domain that binds a different antigen from the antigen bound by the antigen-binding domain), and a second switch domain.

[0424] Furthermore, RCARs in which the antigen-binding member includes bispecific activation and targeting capabilities are also provided herein. In this embodiment, the antigen-binding member may include a plurality of (e.g., 2, 3, 4, or 5) antigen-binding domains (e.g., scFv), each antigen-binding domain binding to a target antigen, examples of which are different antigens or the same antigen, and examples of which are the same or different epitopes on the same antigen. In some embodiments, the plurality of antigen-binding domains are arranged in series, and optionally, a linker or hinge region is placed between each antigen-binding domain. Preferred linker and hinge regions are described herein.

[0425] In some embodiments, RCARs are provided having a configuration that allows for switching of proliferation. In these embodiments, the RCAR includes 1) an intracellular signaling member comprising an optional transmembrane domain or membrane CD5 hering domain, one or more co-stimulatory signaling domains (e.g., selected from 41BB, CD28, CD27, ICOS, and OX40), and a switch domain; and 2) an antigen-binding member comprising an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain (e.g., a CD3 zeta domain), wherein the antigen-binding member does not include a switch domain or does not include a switch domain that dimerizes with a switch domain on the intracellular signaling member. In some embodiments, the antigen-binding member does not include a co-stimulatory signaling domain. In some embodiments, the intracellular signaling member includes a switch domain from a homodimerization switch. In some embodiments, the intracellular signaling member includes a first switch domain of a heterodimerization switch, and the RCAR includes a second intracellular signaling member including a second switch domain of a heterodimerization switch. In such embodiments, the second intracellular signaling member includes the same intracellular signaling domain as the intracellular signaling member. In some embodiments, the dimerization switch is intracellular. In some embodiments, the dimerization switch is located extracellularly.

[0426] In any of the RCAR configurations described herein, the first and second switch domains include FKBP-FRB based switches as described herein.

[0427] Cells containing RCAR as described herein are also provided herein. Any cells engineered to express RCAR can be used as RCARX cells. In one embodiment, RCARX cells are T cells and are called RCART cells. In one embodiment, RCARX cells are NK cells and are called RCARN cells.

[0428] Furthermore, nucleic acids and vectors containing sequences encoding RCAR are also provided herein. Sequences encoding various elements of RCAR can be located on the same nucleic acid molecule, examples of which include the same plasmid or vector, examples of which include viral vectors, and examples of which include lentiviral vectors. In some embodiments, (i) a sequence encoding an antigen-binding member and (ii) a sequence encoding an intracellular signaling member may be located on the same nucleic acid (e.g., a vector). Production of the corresponding protein can be achieved, for example, by the use of separate promoters or by the use of a bisistronic transcript (which may result in the production of two proteins by cleavage of a single translation product or by translation of two separate protein products). In some embodiments, a sequence encoding a cleavable peptide (e.g., a P2A or F2A sequence) is located between (i) and (ii). Examples of peptide cleavage sites include the following: GSG residues are optional.

[0429] In some embodiments, the sequence encoding the IRES (e.g., EMCV or EV71 IRES) is placed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In some embodiments, a first promoter is operably ligated to (i) and a second promoter is operably ligated to (ii), resulting in (i) and (ii) being transcribed as separate mRNAs.

[0430] Alternatively, sequences encoding various elements of RCAR can be placed on different nucleic acid molecules, examples of which include different plasmids or vectors, examples of which include viral vectors, and examples of which include lentiviral vectors. For example, sequence (i) encoding antigen-binding members can be located on a first nucleic acid (e.g., a first vector), and sequence (ii) encoding intracellular signaling members can be located on a second nucleic acid (e.g., a second vector).

[0431] Dimerization switch

[0432] Dimerization switches can be non-covalent or covalent. In non-covalent dimerization switches, the dimerizing molecule promotes non-covalent interactions between switch domains. In covalent dimerization switches, the dimerizing molecule promotes covalent interactions between switch domains.

[0433] In some embodiments, RCAR includes an FKBP / FRAP or FKBP / FRB-based dimerization switch. FKBP12 (FKBP, or FK506-binding protein) is an abundant cytoplasmic protein that serves as the primary intracellular target of the natural product immunosuppressant rapamycin. Rapamycin binds to FKBP and its large PI3K homolog, FRAP (RAFT, mTOR). FRB is the 93-amino acid portion of FRAP and is sufficient to bind the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA 92: 4947-51).

[0434] In some embodiments, the FKBP / FRAP (e.g., FKBP / FRB) based switch can use a dimerized molecule (e.g., rapamycin or a rapamycin analog). The amino acid sequence of FKBP is as follows: [ka]

[0435] In some embodiments, the FKBP switch domain may contain an FKBP fragment capable of binding to FRB or a fragment or analog thereof, in the presence of rapamycin or rapalog, an example of which is the underlined portion of Sequence ID No. 40, namely: VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLETS (Sequence ID 41) The amino acid sequence of FRB is as follows: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK(Sequence ID 42)

[0436] As used herein, the term “FKBP / FRAP, e.g., FKBP / FRB-based switch” refers to a dimerizing switch which, in the presence of rapamycin or rapalog (e.g., RAD001), comprises an FKBP fragment or analogue capable of binding to FRB or a fragment or analogue thereof, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with the FKBP sequence of SEQ ID NO: 40 or 55, or 30, 25, 20, 1 A first switch domain differing by only 5, 10, 5, 4, 3, 2, or 1 amino acid residue or less, and a second switch domain containing, in the presence of rapamycin or rapalog, a FRB fragment or analogue having the ability to bind to an FRB or a fragment or analogue thereof, and having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with the FRB sequence of SEQ ID NO: 42, or differing by only 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue or less. In some embodiments, the RCAR described herein includes one switch domain containing the amino acid residue disclosed in SEQ ID NO: 40 (or SEQ ID NO: 41) and one switch domain containing the amino acid residue disclosed in SEQ ID NO: 42.

[0437] In some embodiments, the FKBP / FRB dimerization switch comprises an FRB-based switch domain (e.g., a modified FRB switch domain) and a modified FRB switch domain that exhibits altered (e.g., enhanced) complex formation between the FKBP-based switch domain and a dimerization molecule (e.g., rapamycin or rapalog, e.g., RAD001). In some embodiments, the modified FRB switch domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations selected from mutations at amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is mutated to any other native amino acid. In some embodiments, the mutant FRB includes a mutation in E2032, where E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I) (e.g., SEQ ID NO: 43), or leucine (E2032L) (e.g., SEQ ID NO: 44). In some embodiments, the mutant FRB includes a mutation in T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L) (e.g., SEQ ID NO: 45). In some embodiments, the mutant FRB includes mutations in both E2032 and T2098, where E2032 is mutated to any amino acid and T2098 is mutated to any amino acid (e.g., SEQ ID NO: 46). In some embodiments, the mutant FRB includes the E2032I and T2098L mutations (e.g., SEQ ID NO: 47). In some embodiments, the mutant FRB includes the E2032L and T2098L mutations (e.g., SEQ ID NO: 48). [Table 5]

[0438] Other suitable dimerization switches include GyrB-GyrB-based dimerization switches, gibberellin-based dimerization switches, tag / binder dimerization switches, and halotag / snaptag dimerization switches. Such switches and associated dimerizing molecules will be apparent to those skilled in the art, provided that the guidance provided herein is followed.

[0439] dimerization molecule

[0440] The binding between switch domains is facilitated by dimerizing molecules. In the presence of dimerizing molecules, interaction or association between switch domains enables signal transduction between the polypeptide associated with the first switch domain (e.g., fused) and the polypeptide associated with the second switch domain (e.g., fused). In the presence of non-limiting levels of dimerizing molecules, signal transduction increases by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, and 100 times, for example, as measured in the system described herein.

[0441] Rapamycin and rapamycin analogs (sometimes called rapalogs) (e.g., RAD001) can be used as dimerizing molecules in the FKBP / FRB-based dimerizing switches described herein. In one embodiment, the dimerizing molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridafololimus), biolimus, and AP21967. Additional rapamycin analogs suitable for use with FKBP / FRB-based dimerizing switches are further described in the section titled “Combination Therapies” or the subsection titled “Exemplary mTOR Inhibitors.”

[0442] Split CAR In some embodiments, CAR-expressing cells utilize split CARs. The split CAR approach is described in detail in references WO2014 / 055442 and WO2014 / 055657, which are incorporated herein by reference as a whole. Briefly, the split CAR system comprises cells expressing a first CAR having a first antigen-binding domain and a costimulatory domain (e.g., 41BB), and the same cells also express a second CAR having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cells encounter the first antigen, the costimulatory domain is activated, and the cells proliferate. When the cells encounter the second antigen, the intracellular signaling domain is activated, and cell-killing activity begins. Thus, CAR-expressing cells are fully activated only in the presence of both antigens.

[0443] Nonviral delivery methods In some embodiments, nonviral methods can be used to deliver nucleic acids encoding heterogeneous nucleic acid molecules such as the CARs described herein to cells, tissues, or subjects. This can also be done using components of a gene editing system.

[0444] In some embodiments, nonviral methods involve the use of transposons (also called transposition factors). In some embodiments, a transposon is a DNA fragment that can insert itself into a specific location within the genome, for example, a DNA fragment that can self-replicate and insert copies of itself into the genome, or a DNA fragment that can be excised from a longer nucleic acid and inserted into another location within the genome. For example, a transposon may consist of a DNA sequence composed of reverse repeating flanking genes for transposition.

[0445] Exemplary methods of nucleic acid delivery using transposons include the Sleeping Beauty transposon system (SBTS) and the Piggyback (PB) transposon system. For example, Aronovich et al. Hum. Mol. Genet. 20.R1 (2011):R14-20;Singh et al. Cancer Res. 15 (2008):2961-2971;Huang et al. Mol. Ther. 16(2008):580-589;Grabundzija et al. Mol. Ther. 18 (2010):1200-1209; Kebriaei et al. Blood. 122.21 (2013):166; Williams. Molecular Therapy 16.9 (2008):1515-16; Bell et al. Nat. Protoc. 2.12 (2007):3153-65; and Ding et al. Cell. 122.3 (2005):473-83. All of these are incorporated herein by reference.

[0446] SBTS comprises two components: 1) a transposon containing the transgene, and 2) a source of the transposase enzyme. The transposase can transpose the transposon from a carrier plasmid (or other donor DNA) to target DNA (such as the chromosome / genome of a host cell). For example, the transposase binds to the carrier plasmid / donor DNA, excises the transposon (containing the transgene(s)) from the plasmid, and inserts it into the host cell's genome. See, for example, Aronovich et al. cited above.

[0447] Exemplary transposons include pT2-based transposons. See, for example, Grabundzija et al. Nucleic Acids Res. 41.3 (2013):1829-47; and Singh et al. Cancer Res. 68.8 (2008):2961-2971. All of these are incorporated herein by reference. Exemplary transposases include Tc1 / mariner-type transposases, examples of which include SB10 transposase or SB11 transposase (e.g., highly active transposases that can be expressed from a cytomegalovirus promoter). See, for example, Aronovich et al.; Kebriaei et al.; and Grabundzija et al. All of these are incorporated herein by reference.

[0448] The use of SBTS enables the efficient incorporation and expression of a transgene (e.g., nucleic acid encoding the CAR described herein). For example, a method for generating cells (e.g., T cells or NK cells) that stably express the CAR described herein using a transposon system such as SBTS is provided herein.

[0449] In some embodiments, according to the methods described herein, one or more nucleic acids (e.g., plasmids) containing SBTS components are delivered to cells (e.g., T cells or NK cells). For example, the nucleic acids are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, such as the methods described herein, and such methods include electroporation, transfection, or lipofection. In some embodiments, the nucleic acids contain a transposon containing a transgene (e.g., the nucleic acid encoding the CAR described herein). In some embodiments, the nucleic acids contain a transposon containing a transgene (e.g., the nucleic acid encoding the CAR described herein), and further contain a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a system having two nucleic acids (e.g., a dual plasmid system) is provided, for example, the first plasmid containing a transposon containing a transgene, and the second plasmid containing a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are delivered together to the host cell.

[0450] In some embodiments, cells expressing the CARs described herein (e.g., T cells or NK cells) are generated by using a combination of gene insertion using SBTS and gene editing using a nuclease (e.g., zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas system, or modified meganuclease re-modified homing endonuclease).

[0451] In some embodiments, the use of nonviral delivery methods enables the reprogramming of cells (e.g., T cells or NK cells) and their direct injection into the target. Advantages of nonviral vectors include, but are not limited to, the ease and relatively low cost of producing sufficient quantities to satisfy a patient population, their stability during storage, and their lack of immunogenicity.

[0452] Nucleic acid constructs that encode CARs This embodiment also provides nucleic acid molecules that encode one or more CAR constructs described herein.

[0453] Accordingly, in some embodiments, the embodiments relate to a nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain that binds to a tumor antigen as described herein, a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain as described herein), the intracellular signaling domain comprising a stimulatory domain (e.g., a co-stimulatory signaling domain as described herein) and / or a primary signaling domain (e.g., a primary signaling domain as described herein, and an example thereof, a zeta chain as described herein). In some embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.In some embodiments, the transmembrane domains are, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM It may include at least one transmembrane region of (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp.

[0454] In some embodiments, the transmembrane domain includes the sequence of SEQ ID NO: 12, or a sequence having 95-99% identity thereto. In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a hinge region (e.g., the hinge described herein). In some embodiments, the hinge region includes the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or a sequence having 95-99% identity thereto. In some embodiments, the isolated nucleic acid molecule further includes a sequence encoding a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, and DNAM1. This includes (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, and PAG / Cbp. In some embodiments, the co-stimulatory domain includes the sequence of Sequence ID No. 16, or a sequence having 95-99% identity thereto.In some embodiments, the intracellular signaling domain includes a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta. In some embodiments, the intracellular signaling domain includes the sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence having 95-99% identity thereto, and the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence having 95-99% identity thereto, and the sequence containing the intracellular signaling domain is expressed as a single polypeptide chain in the same frame.

[0455] In some embodiments, the embodiments relate to an isolated nucleic acid molecule encoding a CAR construct, the CAR construct comprising: a leader sequence of SEQ ID NO: 2; an scFv domain as described herein; a hinge region of SEQ ID NO: 4, 6, 8, or 10 (or a sequence having 95-99% identity thereto); a transmembrane domain having the sequence of SEQ ID NO: 12 (or a sequence having 95-99% identity thereto); a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 14; or a CD27 co-stimulatory domain having the sequence of SEQ ID NO: 16 (or a sequence having 95-99% identity thereto); and a CD3 zeta-stimulatory domain having the sequence of SEQ ID NO: 18 or 20 (or a sequence having 95-99% identity thereto).

[0456] In some embodiments, the embodiments relate to nucleic acid molecules encoding a chimeric antigen receptor (CAR) molecule, which comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain including a stimulating domain, wherein the antigen-binding domain is associated with CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PRSS21, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, regmine, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutation, prostain, sulbibin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutation, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutIt binds to tumor antigens selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0457] In some embodiments, the encoded CAR molecule further comprises a sequence encoding a co-stimulatory domain. In some embodiments, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In some embodiments, the co-stimulatory domain comprises the sequence of SEQ ID NO: 14. In some embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 12. In some embodiments, the intracellular signaling domain comprises the 4-1BB functional signaling domain and the zeta functional signaling domain. In some embodiments, the intracellular signaling domain includes the sequence of SEQ ID NO: 14 and the sequence of SEQ ID NO: 18, and the sequence containing the intracellular signaling domain is expressed within the same frame and as a single polypeptide chain. In some embodiments, the anti-cancer-related antigen binding domain, as described herein, is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region includes SEQ ID NO: 4. In some embodiments, the hinge region includes SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

[0458] Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as screening libraries from cells expressing the gene, inducing the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be synthesized rather than cloned.

[0459] This embodiment also provides vectors into which heterologous nucleic acid molecules are inserted. Retrovirus-derived vectors (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they enable long-term and stable integration of the transgene and its transmission in daughter cells. Lentiviral vectors have further advantages over vectors derived from oncoretroviruses such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. They also have the further advantage of lower immunogenicity. Retrovirus vectors may also be, for example, gamma retrovirus vectors. Gamma retrovirus vectors may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long-terminal repeat sequences (LTRs), and the target transgene (e.g., a gene encoding CAR). Gamma retrovirus vectors may lack viral structural genes (gag, pol, env, etc.). Exemplary gamma retroviral vectors include murine leukemia virus (MLV), splenic focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), as well as vectors derived therefrom. Other gamma retroviral vectors are described, for example, by Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 June;3(6): 677-713.

[0460] In another embodiment, the vector containing the nucleic acid encoding the desired heterologous nucleic acid molecule is an adenovirus vector (A5 / 35). In yet another embodiment, the expression of the heterologous nucleic acid molecule can be achieved using transposons (such as Sleeping Beauty, CRISPR, Cas9, and zinc finger nucleases). See June et al. 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.

[0461] In short, the expression of native or synthetic nucleic acids encoding CARs is typically achieved by operably ligating a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for eukaryotic replication and incorporation. Typical cloning vectors contain transcription and translation terminators, start sequences, and promoters useful for controlling the expression of a desired nucleic acid sequence.

[0462] Nucleic acids can be cloned into multiple types of vectors. For example, nucleic acids can be cloned into vectors including, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0463] Furthermore, the expression vector may be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other manuals on virology and molecular biology. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0464] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Several adenovirus vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0465] Additional promoter elements (e.g., enhancers) control the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although multiple promoters have been shown to also contain functional elements downstream of the initiation site. Often, the spacing between promoter elements is flexible, resulting in maintainable promoter function even if elements are relatively inverted or moved. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription. Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, and phosphoglycerokinase (PGK) promoters.

[0466] One example of a promoter capable of expressing CAR-encoding nucleic acid molecules in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from nucleic acid molecules cloned into lentiviral vectors. See, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In some embodiments, the EF1a promoter includes the sequence provided as Sequence ID No. 1.

[0467] Another example of a promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can activate high levels of expression of any operatively linked polynucleotide sequence. However, other constitutive promoter sequences may also be used, including, but are not limited to, the Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Roussarcoma virus promoter, and human gene promoters, and examples of these include, but are not limited to, actin promoters, myosin promoters, elongation factor 1α promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, embodiments should not be limited to the use of constitutive promoters. Inducible promoters are also intended. The use of inducible promoters provides a molecular switch that can turn on the expression of an operatively linked polynucleotide sequence when expression is desired, or turn off such expression when expression is not desired. Examples of inductive promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0468] The vector may also include, for example, signal sequences that promote secretion, polyadenylation signals and transcription terminators (e.g., derived from the bovine growth hormone (BGH) gene), elements that enable episomal replication and replication in prokaryotes (e.g., derived from SV40 and ColE1 or other elements known in the art), and / or elements that enable selection (e.g., ampicillin resistance genes and / or zeosin markers).

[0469] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells intended to be transfected or infected by a viral vector. In some embodiments, the selectable marker may be held on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0470] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by the recipient organism or tissue, and that encode polypeptides whose expression manifests itself through several readily detectable characteristics (e.g., enzymatic activity). Reporter gene expression is assayed at a suitable time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially obtained. Generally, a construct with a minimal 5' facile region exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions may be ligated to a reporter gene and used to evaluate the agent's ability to regulate promoter-driven transcription.

[0471] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0472] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particulate guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, ambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0473] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, are the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0474] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other cutting-edge methods of targeted delivery of nucleic acids are also available, including the delivery of polynucleotides by targeted nanoparticles or other suitable submicron-sized delivery systems.

[0475] When nonviral delivery systems are used, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the (in vitro, ex vivo, or in vivo) delivery of nucleic acids to host cells is intended. In some embodiments, the nucleic acids may be associated with lipids. The lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, taken up into liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles, complexed with or otherwise associated with lipids. The compositions relating to lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist in a bilayer structure (as micelles) or in a "broken-down" structure. They may also simply be dispersed in solution, or in some cases form aggregates of non-uniform size or shape. Lipids are fatty substances that may be naturally occurring or synthetic. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0476] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Missouri; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, New York); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes may be characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement, followed by the formation of a closed structure that encapsulates water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures different from the usual vesicle structure in solution are also included. For example, lipids can take on micelle structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0477] Regardless of the method used to introduce exogenous nucleic acids into the host, various assays may be performed to confirm the presence of recombinant DNA sequences within host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art (examples include Southern blotting, Northern blotting, RT-PCR, and PCR) and “biochemical” assays (examples include, for example, detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting) or assays described herein).

[0478] This embodiment further provides a vector comprising a nucleic acid molecule encoding a heterologous molecule of interest. In some embodiments, the vector can be transduced directly into cells (such as T cells or NK cells), for example. In some embodiments, the vector is a cloning vector or an expression vector, and examples include, but are not limited to, vectors comprising one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviruses, and lentiviral vector constructs. In some embodiments, the vector can express the heterologous molecular construct of interest in mammalian immune effector cells (e.g., T cells, NK cells). In some embodiments, mammalian T cells are human T cells. In some embodiments, mammalian NK cells are human NK cells.

[0479] Source of cells When editing cells ex vivo before proliferation and genetic or other modifications, a source of cells such as T cells or natural killer (NK) cells can be obtained from a subject. The term “subject” is intended to include organisms (e.g., mammals) that can induce an immune response. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from multiple sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors.

[0480] In some embodiments, immunoeffector cells (e.g., T cells) can be obtained from blood units collected from a subject using any number of techniques known to skilled artisans, such as Ficol® isolation. In some embodiments, cells derived from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells may be placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or lack many, if not all, divalent cations.

[0481] The initial activation step in the absence of calcium may lead to increased activation. As will be readily apparent to those skilled in the art, the washing step may be achieved by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other salines with or without buffer. Alternatively, undesirable components may be removed from the apheresis sample, and the cells may be resuspended directly in the culture medium.

[0482] It is recognized that the method of this application can utilize culture medium conditions containing 5% or less (e.g., 2%) of human AB serum, and that known culture medium conditions and compositions (e.g., those described in Smith et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038 / cti.2014.31) can be employed.

[0483] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation or countercurrent centrifugation using a Percoll® gradient.

[0484] The methods described herein may include, for example, selecting a specific subpopulation of immune effector cells (e.g., T cells) that is a regulatory T cell depletion population (CD25+ depleted cells) using negative selection techniques described herein. Preferably, the regulatory T cell depletion population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells.

[0485] In some embodiments, an anti-CD25 antibody or fragment thereof, or IL-2, a CD25-binding ligand, is used to remove regulatory T cells (e.g., CD25+ T cells) from a population. In some embodiments, the anti-CD25 antibody or fragment thereof, or the CD25-binding ligand, is conjugated to a substrate (e.g., beads) or otherwise coated onto a substrate (e.g., beads). In some embodiments, the anti-CD25 antibody or fragment thereof is conjugated to a substrate as described herein.

[0486] In some embodiments, regulatory T cells (such as CD25+ T cells) are removed from the population using Miltenyi® CD25 depletion reagent. In some embodiments, the cell ratio to the CD25 depletion reagent is 1e7 cells per 20 μL, or 1e7 cells per 15 μL, or 1e7 cells per 10 μL, or 1e7 cells per 5 μL, or 1e7 cells per 2.5 μL, or 1e7 cells per 1.25 μL. In some embodiments, for example, in the case of regulatory T cells (e.g., CD25+ depletion), concentrations greater than 500 million cells / ml are used. In some embodiments, cell concentrations of 600 million, 700 million, 800 million, or 900 million cells / ml are used.

[0487] In some embodiments, the population of immune effector cells to be depleted is approximately 6 × 10⁶ 9 It contains 1 CD25+ T cell. In some embodiments, the population of immune effector cells to be depleted is approximately 1 × 10⁶ 9 ~1 × 10 10 This includes 10 CD25+ T cells and any integer value between them. In some embodiments, the resulting population of regulatory T-depleted cells is 2 × 10⁶ 9 It has fewer than 10 regulatory T cells (e.g., CD25+ cells) (for example, 1 × 10⁶ 9 , 5×10 8 , 1 x 10 8 , 5×10 7 , 1 x 10 7 The following CD25+ cells are present.

[0488] In some embodiments, regulatory T cells (e.g., CD25+ cells) are removed from a population using a CliniMAC system equipped with, for example, a depletion tube set (an example of which is tube 162-01). In some embodiments, the CliniMAC system is run in a depletion setting such as DEPLETION2.1.

[0489] While I don't want to be tied to any particular theory, I would like to reduce the levels of negative regulators of immune cells in the target before apheresis or during the production of CAR-expressing cell products (e.g., unwanted immune cells, e.g., T cells).REG By reducing the number of cells, the risk of recurrence in the target can be reduced. For example, T REG Methods for depleting cells are known in this field. REG Methods for reducing CD25 include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies as described herein), CD25 depletion, and combinations thereof.

[0490] In some embodiments, the manufacturing method involves T before the production of CAR-expressing cells. REG This includes reducing the number of cells (e.g., depleting them). For example, the manufacturing method involves using CAR-expressing cells (e.g., T cells, NK cells) before manufacturing the product, for example, T REG To deplete cells, the method involves contacting a sample (e.g., an apheresis sample) with an anti-GITR antibody and / or an anti-CD25 antibody (or a fragment thereof, or a CD25-binding ligand).

[0491] In some embodiments, the subject is T before collecting cells for the production of CAR-expressing cell products. REG The subjects are pre-treated with one or more therapies that reduce the number of cells, thereby reducing the risk of recurrence of CAR-expressing cell treatment. In some embodiments, T REG Methods for reducing cell activity include, but are not limited to, administering one or more of the following: cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof. Administration of one or more of the following may occur before, during, or after the injection of CAR-expressing cell products.

[0492] In some embodiments, subjects are pre-treated with cyclophosphamide before collecting cells for the production of CAR-expressing cell products, thereby reducing the risk of subjects re-treating with CAR-expressing cells. In some embodiments, subjects are pre-treated with an anti-GITR antibody before collecting cells for the production of CAR-expressing cell products, thereby reducing the risk of subjects re-treating with CAR-expressing cells.

[0493] In some embodiments, the population of cells to be eliminated is neither regulatory T cells nor tumor cells, but rather cells that otherwise adversely affect the proliferation and / or function of CART cells (e.g., cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immunosuppressive cells). In some embodiments, such cells are assumed to be eliminated concurrently with regulatory T cells and / or tumor cells, or after such depletion, or in a different order.

[0494] The methods described herein may include more than one selection step (e.g., more than one depletion step). Enrichment of T cell populations by negative selection can be achieved, for example, by a combination of antibodies induced to surface markers specific to negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies induced to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0495] The methods described herein may further include removing cells expressing tumor antigens (examples include tumor antigens that do not contain CD25, and examples include CD19, CD30, CD38, CD123, CD20, CD14, or CD11b) from a population, thereby providing a population of regulatory T-cell depleted cells (e.g., CD25+ depleted) and tumor antigen depleted cells suitable for the expression of CARs (e.g., CARs as described herein). In some embodiments, tumor antigen-expressing cells are removed simultaneously with regulatory T cells (e.g., CD25+ cells). For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof can be attached to the same substrate (e.g., beads) and used to remove cells. Alternatively, an anti-CD25 antibody or fragment thereof, or an anti-tumor antigen antibody or fragment thereof, can be attached to separate beads and a mixture thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells (e.g., CD25+ cells) and the removal of tumor antigen-expressing cells are sequential and can occur in either order, for example.

[0496] Also provided is a method comprising removing cells expressing checkpoint inhibitors (e.g., checkpoint inhibitors as described herein) (e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells) from a population to thereby provide a population of regulatory T-cell depleted cells (e.g., CD25+ depleted cells) and checkpoint inhibitor depleted cells (e.g., PD1+, LAG3+, and / or TIM3+ depleted cells). Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In some embodiments, checkpoint inhibitor-expressing cells are removed simultaneously with regulatory T cells (e.g., CD25+ cells). For example, an anti-CD25 antibody or a fragment thereof and an anti-checkpoint inhibitor antibody or a fragment thereof can be attached to the same bead and used to remove cells. Alternatively, an anti-CD25 antibody or a fragment thereof and an anti-checkpoint inhibitor antibody or a fragment thereof can be attached to separate beads and a mixture thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells (e.g., CD25+ cells) and the removal of checkpoint inhibitor-expressing cells are sequential and can occur in either order, for example.

[0497] The methods described herein may include a positive selection step. For example, T cells can be isolated by incubating them with anti-CD3 / anti-CD28 (e.g., 3×28) conjugate beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a sufficient time to positively select the desired T cells. In some embodiments, the time is about 30 minutes. In a further embodiment, the time is in the range of 30 minutes to 36 hours or more and all integer values ​​in between. In a further embodiment, the time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time is 10 to 24 hours (e.g., 24 hours). In any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunodeficient individuals, longer incubation times may be used to isolate T cells. Furthermore, the use of longer incubation times can improve the capture efficiency of CD8+ T cells. Therefore, subpopulations of T cells can be preferentially selected or eliminated at the start of culture or at any other point during processing simply by shortening or lengthening the time that T cells can bind to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein). Furthermore, subpopulations of T cells can be preferentially selected or eliminated at the start of culture or at any other desired point in time by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces.

[0498] In some embodiments, a population of T cells expressing one or more of the following molecules can be selected: IFN-7, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules (e.g., other cytokines). The cell expression screening method can be determined, for example, by the method described in PCT Publication No.: WO 2013 / 126712.

[0499] To isolate a desired population of cells by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure that the contact between cells and beads is maximized. For example, in some embodiments, concentrations of 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml are used. In some embodiments, a concentration of 1 billion cells / ml is used. In some embodiments, cell concentrations of 75 million cells / ml, 80 mi...

Claims

1. A cell containing heterogeneous nucleic acid molecules, The aforementioned cells have reduced or absent CD5 expression and / or function. The aforementioned heterologous nucleic acid molecule is inserted at the CD5 gene locus in the cell.

2. The cell according to claim 1, wherein the heterologous nucleic acid molecule substitutes the CD5 gene locus.

3. The cell according to claim 1, wherein the heterologous nucleic acid molecule is inserted into the CD5 gene locus, and such insertion reduces or eliminates the expression and / or function of CD5.

4. The cell according to claim 3, wherein a cell containing a heterologous nucleic acid molecule inserted into the CD5 locus produces a cell having a CD5 locus that includes a frameshift CD5 locus.

5. The cell according to claim 1, wherein the cell is an immune cell.

6. The cell according to claim 5, wherein the immune cell is a T cell, an NK cell, or a B cell.

7. The cell according to claim 6, wherein the T cell is an αβ T cell.

8. The cell according to claim 6, wherein the B cell is a B1 B cell.

9. The cell according to claim 1, wherein the cell does not express CD5 or does not express functional CD5 from the native CD5 gene locus.

10. The cell according to claim 1, wherein the heterogeneous nucleic acid molecule encodes the target protein.

11. The cell according to claim 1, wherein the heterogeneous nucleic acid molecule encodes a chimeric antigen receptor (CAR).

12. The cell according to claim 11, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

13. The cell according to claim 12, wherein the CAR comprises an antigen-binding domain for CD5.

14. The cell according to claim 13, wherein the antigen-binding domain for CD5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 56, an HCDR2 containing the amino acid sequence of SEQ ID NO: 57, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 58, and the VL comprising a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 59, an LCDR2 containing the amino acid sequence of SEQ ID NO: 60, and an LCDR3 containing the amino acid sequence of SEQ ID NO:

61.

15. The cell according to claim 14, wherein VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 62, and VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

63.

16. The cell according to claim 11, wherein the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of Sequence ID No.

66.

17. The cell according to claim 1, wherein the heterogeneous nucleic acid molecule encodes an antibody, chemokine, hormone, cytokine, etc.

18. The cell according to claim 1, wherein the heterogeneous nucleic acid molecule encodes RNA molecules such as miRNA, siRNA, mRNA, and antisense molecules.

19. The cell according to claim 1, wherein the cell contains a deletion or mutation in one or more exons of the CD5 gene.

20. The cells according to claim 1, wherein the cells have altered expression and / or function of CD5-related genes.

21. A pharmaceutical composition comprising a plurality of cells as described in claim 1.

22. The composition is about 1×10 ６ to about 1×10 ９ ; about 1×10 ７ to about 1×10 ９ ; about 1×10 ８ to about 1×10 ９ ; about 2×10 ８ to about 1×10 ９ ; about 3×10 ８ to about 1×10 ９ ; about 3×10 ８ to about 1×10 ９ ; about 3×10 ８ to about 1×10 ９ ; about 4×10 ８ to about 1×10 ９ ; about 5×10 ８ to about 1×10 ９ ; about 6×10 ８ ; about 7×10 ８ to about 1×10 ９ ; about 8×10 ８ ; about 9×10 ８ to about 1×10 ９ The pharmaceutical composition according to claim 21, comprising the cells as described above.

23. A method for producing cells containing a heterologous nucleic acid molecule inserted at the CD5 locus, comprising contacting the cells with a gene editing system under conditions sufficient to produce the cells containing the heterologous nucleic acid molecule inserted at the CD5 locus.

24. The method according to claim 23, wherein the cells have reduced or absent CD5 expression and / or function.

25. The method according to claim 23, wherein the gene editing system is selected from the group consisting of the CRISPR / Cas9 system, the zinc finger nuclease system, the TALEN system, and the meganuclease system.

26. The method according to claim 23, wherein the heterologous nucleic acid molecule inserted into the CD5 locus replaces the CD5 locus.

27. The method according to claim 26, wherein the heterologous nucleic acid molecule inserted into the CD5 gene locus reduces or eliminates the expression and / or function of CD5.

28. The method according to claim 26, wherein the heterologous nucleic acid molecule inserted into the CD5 locus produces cells having a CD5 locus including a frameshift CD5 locus.

29. The method according to claim 23, wherein the cells are immune cells.

30. The method according to claim 29, wherein the immune cells are T cells, NK cells, or B cells.

31. The method according to claim 30, wherein the T cells are αβ T cells.

32. The method according to claim 30, wherein the B cell is a B1 B cell.

33. The method according to claim 23, wherein the cells do not express CD5 or do not express functional CD5 from the native CD5 gene locus.

34. The method according to claim 23, wherein the heterogeneous nucleic acid molecule encodes the target protein.

35. The method according to claim 23, wherein the heterogeneous nucleic acid molecule encodes a chimeric antigen receptor (CAR).

36. The method according to claim 23, wherein the heterogeneous nucleic acid molecule encodes an antibody, chemokine, hormone, cytokine, etc.

37. The method according to claim 23, wherein the heterogeneous nucleic acid molecule encodes an RNA molecule such as miRNA, siRNA, mRNA, or an antisense molecule.

38. The method according to claim 23, wherein the gene editing system includes a targeting molecule that binds to a target sequence in the early exon or intron of the CD5 gene or CD5-related gene.

39. The method according to claim 23, wherein the gene editing system includes a targeting molecule that binds to a target sequence in a late exon or intron of the CD5 locus.

40. The method according to claim 23, wherein the gene editing system comprises a targeting molecule that binds to a target sequence of the CD5 locus, the target sequence being downstream of the fourth to last exon, for example, in the third to last exon, the second to last exon, or the last exon of the CD5 locus.

41. The method according to claim 23, wherein the gene editing system is a CRISPR system.

42. The method according to claim 41, wherein the CRISPR system comprises a gRNA molecule that hybridizes to the target sequence of the CD5 gene locus.

43. The method according to claim 41, wherein the CRISPR system is a CRISPR / Cas system.

44. The method according to claim 23, wherein the gene editing system comes into contact with the cells ex vivo or in vivo.

45. The aforementioned antigen-binding domain binds to tumor antigens, including, for example, CD5, CD2, CD7, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, and ERBB2. (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, H AVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-AL legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutation, prostain, sulbibin and telomerase, PCTA-1 / galectin 8, Melan A / MART1, Ras mutation, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TESL, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe cells according to claim 12, which include hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1, or the method according to claim 35.

46. A method for treating a subject having cancer, comprising administering to the subject cells according to any one of claims 1 to 20, or a composition according to claim 17 or 18.

47. The method according to claim 46, wherein the cells containing the heterologous nucleic acid molecule inserted at the CD5 locus are more effective in treating the cancer than cells containing the heterologous nucleic acid molecule not inserted at the CD5 locus.

48. The method according to claim 46, wherein the cancer is a blood cancer or a solid tumor.

49. Blood cancers include chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastocyte plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, and small The method according to claim 48, wherein the lymphoma is a cell type or large cell type follicular lymphoma, a malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström type macroglobulinemia, or a preleukemia.

50. The aforementioned cancers include colorectal cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eyeball, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, and soft gland cancer. The method according to claim 46, selected from the group consisting of histological sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary lymphoma of the central nervous system, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced carcinoma, combinations of the said cancers, and metastatic lesions of the said cancers.

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