Chimeric antigen receptors containing antibody-inducible domains

Abstract

The present disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular helper domain, T cells expressing said CARs can be activated in an antigen-independent manner by said extracellular helper domain. In particular, said extracellular helper domain of said CARs of the present disclosure is an antibody-inducible domain that can be bound by an antibody or a polypeptide comprising an antigen-binding fragment thereof. In this way, T cells expressing said CARs can be activated by binding of said antibody to said antibody-inducible domain.

Description

Cross-references to related applications

[0001] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 559,795, filed February 29, 2024, the entire contents of which are incorporated herein by reference.

[0002] Reference to the electronic sequence list The contents of the electronic serial number (237752001240SEQLIST.xml; size: 45,978 bytes; creation date: February 26, 2025) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular helper domain, through which CAR-expressing T cells can be activated in an antigen-independent manner. Specifically, the extracellular helper domain is an antibody-inducible domain, through which CAR-expressing T cells can be activated upon antibody binding to the antibody-inducible domain. Background Technology

[0004] Chimeric antigen receptors (CARs), comprising portions of both antibodies and T-cell receptors (TCRs), were first described in the 1980s. First-generation CARs included an antigen-binding domain (ABD), a hinge, a transmembrane domain, and an intracellular signal transduction domain of CD3ζ. Subsequent generations of CARs have been engineered to include one or more intracellular co-stimulatory domains to enhance their ability to activate T cells expressing them (Smith and Shen, J. Transl. Med, 21:515, 2023).

[0005] Currently, four anti-CD19 CAR T-cell immunotherapies have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of various CD19+ leukemias and lymphomas. In fact, anti-CD19 CAR T-cell immunotherapy has resulted in significant clinical responses, including high levels of remission, in patients with poor prognoses. However, there are drawbacks in the manufacture and use of CAR T cells, including the need for antigen-dependent activation and the requirement for co-expression of tags for detecting and / or eliminating CAR T cells.

[0006] Therefore, there is a need in the art for CAR backbones that have been engineered to be activated in a CAR-specific, ABD-nonspecific manner. There is also a need in the art to identify CAR-expressing T cells in an ABD-nonspecific manner and in the absence of co-expression of a separate peptide tag. Summary of the Invention

[0007] This disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular helper domain, through which T cells expressing the CAR can be activated in an antigen-independent manner. Specifically, the extracellular helper domain of the CAR of this disclosure can be bound by an antibody or a peptide containing its antigen-binding fragment. Thus, T cells expressing the CAR can be activated by the binding of an antibody to the extracellular helper domain. Attached Figure Description

[0008] Figure 1 A schematic diagram (top) of a standard chimeric antigen receptor (CAR) expression cassette and a schematic diagram (bottom) of an exemplary expression cassette of the CAR of this disclosure containing domain 3 (D3) of vascular endothelial growth factor receptor 2 (VEGFR2) are shown. The promoter region (Prom.) and the truncated epidermal growth factor receptor (EGFRt) expression tag are shown at the 5' and 3' ends of the expression cassette, respectively. TM denotes a transmembrane domain.

[0009] Figure 2 The standard CAR is depicted, as well as an exemplary CAR containing VEGFR2 and D3 (CAR containing VEGF2R).

[0010] Figure 3A The percentage of various CAR-expressing Jurkat cells expressing CD25 after culture in the absence of antigen or in the presence of K562 cells expressing antigen is shown. Figure 3B The percentage of various CAR-expressing Jurkat cells expressing CD25 after incubation with anti-VEGFR2 antibody, anti-human epidermal growth factor receptor 2 (Her2) antibody, anti-interleukin 4 receptor (IL4Ra) antibody, or without antibody (no stimulation) is shown. In both figures, mock represents the untransduced control.

[0011] Figure 4A The timeline of transduction and expansion of Treg cells engineered to express CAR is shown. On day 3, Treg cells were transduced with lentivirus. The transduced Treg cells were then expanded in a medium containing interleukin-2 (IL-2). On day 9, the transduced Treg cells were separated and restimulated under one of four conditions. Figure 4B The study showed a fold increase in the number of Treg cells, which were engineered to express CARs containing VEGFR2 and subjected to restimulation on day 9 under one of four conditions: 1) anti-CD3 / anti-CD28 beads; 2) anti-VEGFR2 beads; 3) 1 µg / mL anti-VEGFR2 antibody; and 4) 3.5 µg / mL anti-VEGFR2 antibody.

[0012] Figure 5A schematic diagram of a standard CAR expression cassette (top) and a schematic diagram of an exemplary CAR expression cassette of this disclosure containing domain 4 (D4) of human epidermal growth factor receptor 2 (Her2) are shown (bottom). Prom. and EGFRt are shown at the 5' and 3' ends of the expression cassette, respectively.

[0013] Figure 6 The standard CAR is depicted, as well as an exemplary CAR containing Her2 and D4 (CAR containing Her2).

[0014] Figure 7A The percentage of various CAR-expressing Jurkat cells expressing CD25 after culture in the absence of antigen or in the presence of K562 cells expressing antigen is shown. Figure 7B The percentages of various CAR-expressing Jurkat cells expressing CD25 after incubation with anti-VEGFR2 antibody, anti-Her2 antibody, anti-IL4Ra antibody, or no antibody (no stimulation) are shown. In both figures, the simulation represents the untransduced control.

[0015] Figure 8 A schematic diagram of a standard CAR expression cassette (top) and a schematic diagram of an exemplary CAR expression cassette of this disclosure containing domains 3 to 5 of platelet-derived growth factor (PDGFR) α are shown (bottom). Prom. and EGFRt are shown at the 5' and 3' ends of the expression cassette, respectively.

[0016] Figure 9 The standard CAR is depicted, as well as exemplary CARs of D3-D5 containing PDGFRa (CARs expressing PDGFRa).

[0017] Figure 10 The percentage of various CAR-expressing Jurkat cells expressing CD25 after culture in the absence of antigen or in the presence of K562 cells expressing antigen is shown.

[0018] Figure 11 A schematic diagram of a standard CAR expression cassette (top) and a schematic diagram of an exemplary CAR expression cassette of this disclosure containing an extracellular domain (ECD) 1 and / or 2 of the interleukin-4 receptor α (IL4Ra) are shown (bottom). Prom. and EGFRt are shown at the 5' and 3' ends of the expression cassette, respectively.

[0019] Figure 12 A standard CAR is depicted, as well as exemplary CARs containing IL4Ra, specifically D1 and / or D2 (CARs containing IL4Ra).

[0020] Figure 13AThe percentage of various CAR-expressing Jurkat cells expressing CD25 after culture in the absence of antigen or in the presence of K562 cells expressing antigen is shown. Figure 13B The percentage of various CAR-expressing Jurkat cells expressing CD25 after incubation with anti-VEGFR2 antibody, anti-Her2 antibody, anti-IL4Ra antibody, or no antibody (no stimulation) is shown. Simulations represent untransduced controls.

[0021] Figure 14 A schematic diagram of a standard CAR expression cassette (top) and a schematic diagram of an exemplary CAR expression cassette of this disclosure containing an extracellular domain (ECD) 1 and / or 2 of differentiation cluster 4 (CD4) are shown (bottom). Prom. and EGFRt are shown at the 5' and 3' ends of the expression cassette, respectively.

[0022] Figure 15 A standard CAR is depicted, as well as an exemplary CAR containing CD4, specifically D1 and / or D2 (CAR containing CD4).

[0023] Figure 16 The percentage of various CAR-expressing Jurkat cells expressing CD25 after culture in the absence of antigen or in the presence of K562 cells expressing antigen is shown. Simulations represent untransduced controls.

[0024] Figure 17 A schematic diagram of a standard CAR expression cassette (top) and a schematic diagram of an exemplary CAR expression cassette of this disclosure containing an extracellular domain (ECD) 1 and / or 2 of differentiation cluster 2 (CD2) are shown (bottom). Prom. and EGFRt are shown at the 5' and 3' ends of the expression cassette, respectively.

[0025] Figure 18 A standard CAR is depicted, as well as an exemplary CAR containing CD2, D1, and / or D2 (CAR containing CD2).

[0026] Figure 19 The percentage of various CAR-expressing Jurkat cells expressing CD25 after transduction is shown, in the presence or absence of antigen and in the presence of K562 cells expressing antigen. Simulations represent untransduced controls.

[0027] Figure 20 The activity and amplification of CAR-Treg (SBT-1927) containing the VEGFR domain were shown under specified conditions after restimulation on day 9.

[0028] Figure 21 The activity and amplification of CAR-Treg (SBT-1934) containing the IL4R domain were shown under specified conditions after restimulation on day 9.

[0029] Figure 22 The activity and amplification of CAR-Treg (SBT-1936) containing the IL4R domain were shown under specified conditions after restimulation on day 9.

[0030] Figure 23 This demonstrates the amplification of CAR-Treg (SBT-1927) containing the VEGFR domain after restimulation with ramucirumab (VEGFR antibody conjugated) beads or CD3 / CD28 beads.

[0031] Figure 24 This demonstrates the amplification of CAR-Treg (SBT-1934) containing the VEGFR domain after restimulation with dupilumab (IL4R antibody conjugated) beads or CD3 / CD28 beads.

[0032] Figure 25 The amplification of CAR-Treg (SBT-1934) containing the VEGFR domain is shown after restimulation with dupilumab (IL4R antibody conjugated) beads or CD3 / CD28 beads. Detailed Implementation

[0033] This disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular helper domain, through which CAR-expressing T cells can be activated in an antigen-independent manner. Specifically, the extracellular helper domain is an antibody-inducible domain, wherein CAR-expressing T cells can be activated upon binding of an antibody to the antibody-inducible domain.

[0034] definition Unless otherwise stated, the practice of this disclosure will employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are within the skill level of those skilled in the art.

[0035] As used herein, the terms “antigen,” “immunogen,” and “antibody target” refer to a molecule, compound, or complex that is recognized (i.e., can be bound by an antibody). The term can refer to any molecule that can be recognized by an antibody, such as peptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated or glycosylated peptides). Those skilled in the art will understand that this term does not imply that the molecule is immunogenic in every case, but only that it can be targeted by an antibody or an antigen-binding domain derived therefrom.

[0036] As used herein, the term "epitope" refers to a local site on an antigen that is recognized and bound by an antigen-binding domain of an antibody or a fragment derived therefrom. An epitope may comprise a portion of several amino acids, such as five or six, or more, such as 20 or more amino acids, or portions of those amino acids. In some cases, an epitope includes non-protein components, such as components derived from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, when the target is a protein, an epitope may consist of consecutive amino acids or amino acids from different portions of a protein that are adjacent through protein folding (e.g., a discontinuous epitope).

[0037] As used herein, the term "antibody" refers to a polypeptide containing the framework region from an immunoglobulin gene that specifically binds to and recognizes an antigen. Typically, the "variable region" contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in terms of binding specificity and affinity. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" (approximately 25 kD) chain and a "heavy" (approximately 50-70 kD) chain.

[0038] Antibodies can belong to (i) any of the five major classes of immunoglobulins based on the identity of their heavy chain constant domains: α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), or (ii) their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The light chain can be λ or κ.

[0039] The following is a non-exhaustive list of different antibody forms, all of which retain antigen-binding activity: (1) Whole immunoglobulin (also known as “intact” antibody) (two light chains and two heavy chains, such as tetramer); (2) Immunoglobulin polypeptides (light chain or heavy chain); (3) Antibody fragments, such as Fv (monovalent or bivalent variable region fragments, and may only cover the variable region (e.g., V)). L and / or V H ), Fab (V L C L V H C H ), F(ab')2, Fv (V L V H ), scFv (single-chain Fv) (containing V linked by a linker (e.g., a peptide linker) L and V HThe peptides, (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, microantibodies (sc(FV)2 fused with the CH3 domain), bimeric antibodies (non-covalent dimers of single-chain Fv(scFv) fragments composed of heavy chain variable regions (VH) and light chain variable regions (VL) linked by small peptide linkers), and trimeric antibodies (trivalent sc(Fv)3 or trispecific sc(Fv)3); (4) Multivalent antibodies (including antibodies that bind to the binding regions of two different epitopes or proteins, such as "scorpion" antibodies); (5) Fusion proteins (such as fluorescent proteins) that include a binding portion of an immunoglobulin fused to another amino acid sequence; and (6) Antibodies or antibody fragments that have only heavy chains, which have only two heavy chains and lack the two light chains that are usually found in antibodies.

[0040] As used in this article, the phrase "CDR sequence set" refers to the three heavy chain and / or three light chain CDRs of a specific antibody or antigen-binding domain. The "light chain" CDR sequence set refers to light chain CDR sequences. The "heavy chain" CDR sequence set refers to heavy chain CDR sequences. The "complete" CDR sequence set refers to both heavy and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignment.

[0041] As used herein, the term "humanized antigen-binding domain" refers to a chimeric antigen-binding domain into a human frame region by transplanting a CDR obtained from the VH and VL regions of a nonhuman antibody having the desired specificity, affinity, and capability. In one embodiment, the frame residues of the humanized antigen-binding domain are modified to improve and optimize the specificity, affinity, and capability of the antigen-binding domain.

[0042] As used herein, the term "human antigen-binding domain" refers to the antigen-binding domain of an antibody produced by humans or an antibody having a corresponding amino acid sequence.

[0043] As used herein, if an antigen-binding domain binds to a first antigen with a greater affinity than the second antigen, then it "preferentially binds" to the first antigen relative to the second antigen. Preferential binding can be at least one of 2, 5, 9, 10, 20, 30, 40, 50, 100, 500, or 1000 times greater affinity.

[0044] As used in this article, if the antigen-binding domain is 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10-11 M, 1×10 -12 If at least one of M binds to each member of the target antigen or the target antigen group with an affinity at least twice that of a non-target antigen used as a comparison, then it "specifically binds to the target antigen or the target antigen group" or "is specific to the target antigen or the target antigen group". Typically, specific binding is characterized by binding to the antigen with sufficient affinity such that the antigen-binding domain can be used as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent targeting the antigen or epitope.

[0045] As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and / or non-natural amino acids linked by peptide bonds. The term "peptide" refers to a short polypeptide, typically no more than 30 amino acids in length. The amino acid sequence of a polypeptide is referred to as its "primary structure." The term "protein" refers to a polypeptide having secondary, tertiary, and / or quaternary structures, such as structures stabilized by hydrogen bonds, relationships between secondary structures, and structures formed by more than one protein. Proteins can be further modified by other attached components, such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0046] As used in this article, an amino acid sequence is "composed" only of the amino acids in that sequence.

[0047] As used herein, if a first amino acid sequence (1) contains a second amino acid sequence and (2) is longer than the second amino acid sequence by no more than one, two, or three amino acids, then the first amino acid sequence is "substantially composed" of the second amino acid sequence.

[0048] As used herein, if a second amino acid sequence contains a first amino acid sequence, then the first amino acid sequence is a “fragment” of the second amino acid sequence. In some embodiments, the first amino acid sequence, which is a fragment of the second amino acid sequence, may be no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer amino acids than the second amino acid sequence.

[0049] As used herein, a “functional equivalent” of a reference amino acid sequence is a sequence that differs from the reference sequence but contains minor alterations, such as, for example, the insertion, deletion, or substitution of one or more amino acids. The functionally equivalent sequence retains the function (e.g., immunogenicity) of its equivalent reference sequence. If the functionally equivalent amino acid sequence contains substitutions of one or more amino acids relative to the reference sequence, these are typically conserved amino acid substitutions.

[0050] As used herein, “conservative amino acid substitution” refers to the substitution of one amino acid residue for another without compromising the desired properties of the protein. Suitable conservative amino acid substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R-chain length. See, for example, Watson et al., “Molecular Biology of the Gene,” 4th ed., 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conserved amino acid substitutions include the following (note that some categories are not mutually exclusive):

[0051] As used herein, the term "substantially identical" refers to the identity between first amino acid sequences containing a sufficient or minimum number of amino acid residues that i) are identical to the matched amino acid residues in the second amino acid sequence, or ii) are conserved substitutions of the matched amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences have common structural domains and / or common functional activities and / or common immunogenicity. For example, amino acid sequences containing common structural or antigenic domains with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity are described as sufficiently or substantially identical. In the context of nucleotide sequences, the term "substantially identical" herein refers to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to the matched nucleotides in the second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with common functional activities, or encode common structural polypeptide domains or common functional polypeptide activities, or encode polypeptides with the same immunogenic properties.

[0052] As used herein, a chemical entity (such as a polypeptide) is considered "substantially pure" or "isolated" if it is the dominant chemical entity of its class (e.g., a polypeptide) in the composition. This includes chemical entities representing more than 50%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99% of their class in the composition. A substantially purified fraction is a composition in which the target class constitutes at least about 50% (on a molar basis) of all macromolecular classes present. Typically, a substantially pure composition means that about 80% to 90% or more of the macromolecular classes present in the composition are the class of interest to be purified. If the composition consists substantially of a single macromolecular class, the target class is purified to substantially homogeneity (contaminant classes are not detectable in the composition by conventional detection methods). Solvents, small molecules, stabilizers (e.g., BSA), and elemental ions are not considered macromolecular classes in this definition.

[0053] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid or nucleic acid sequence to allow for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecule is identical at that position. The percentage of identity between two sequences is a function of the number of shared positions (i.e., identity % = number of overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. Mathematical algorithms can also be used to determine the percentage of identity between two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, adapted as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set (e.g., score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of this disclosure. BLAST protein searches can be performed using the XBLAST procedure parameter set (e.g., up to score 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gap alignments for comparative purposes, gap BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be used. Alternatively, PSI-BLAST can be used for iterative searches to detect distance relationships (Id.) between molecules. When using BLAST, gap BLAST, and PSI-Blast procedures, the default parameters of each procedure (e.g., the default parameters for XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, allowing or disallowing gaps. Typically, only exact matches are calculated when determining the percentage of identity.

[0054] For antibody and antigen-binding domains, the percentage of sequence identity can be determined when their sequences are aligned to the maximum extent via IMGT. After alignment, if the subject antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the percentage of sequence identity between the subject antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in both the subject antibody region and the reference antibody region by the total number of aligned positions in both regions, multiplied by 100, and then converted to a percentage.

[0055] The percentage of amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program is available from the National Institutes of Health in Bethesda, Maryland. NCBI-BLAST2 uses several search parameters, all of which are set to their default values, including, for example, demasking = yes, chain = all, expected occurrences = 10, minimum low complexity length = 15 / 5, multiple pass e-value = 0.01, multiple pass constant = 25, final gap alignment decay = 25, and score matrix = BLOSUM62.

[0056] When using NCBI-BLAST2 for amino acid sequence comparison, the amino acid sequence identity % between a given amino acid sequence A and a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing specific amino acid sequence identity with a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program NCBI-BLAST2 in the A and B alignments of this program, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % between A and B is not equal to the amino acid sequence identity % between B and A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of natural bases, sugars, and inter-sugar (backbone) bonds, and includes cDNA. This term also includes modified or substituted sequences containing monomers or portions thereof that are not naturally occurring. The nucleic acid sequence of this application may be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence, and may include naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza- and deza-adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. It should be understood that polynucleotides containing non-transcribed nucleotide bases can be used as probes, for example, in hybridization assays. Nucleic acids may be double-stranded or single-stranded, and represent sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon optimizations or synonymous codon equivalents.

[0057] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemical substances when chemically synthesized. Isolated nucleic acids also substantially lack the naturally occurring side-joint sequences (i.e., the sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid originates.

[0058] Hybridization can occur in whole or in part with nucleic acid sequences. The length of the hybridized portion is typically at least 15 nucleotides (e.g., 20, 25, 30, 40, or 50). Those skilled in the art will recognize that the stability of the nucleic acid duplex or hybrid is determined by Tm, which, in a sodium-containing buffer, is a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar equation). Therefore, the parameters determining the stability of the hybrid in the washing conditions are sodium ion concentration and temperature. To identify molecules similar to but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch results in a decrease in Tm of approximately 1°C; for example, if searching for nucleic acid molecules with >95% identity, the final washing temperature will be reduced by approximately 5°C. Based on these considerations, those skilled in the art will be able to readily select suitable hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. As an example, stringent hybridization can be achieved under the following conditions: based on the equation above, hybridization is performed at Tm -5°C in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt solution / 1.0% SDS, followed by washing at 60°C with 0.2x SSC / 0.1% SDS. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures. Further guidance on hybridization conditions can be found in: Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0059] As used herein, the term "expression construct" refers to a polynucleotide containing an expression control sequence operatively linked to a heterologous nucleotide sequence that is the object of expression (i.e., a sequence that is not normally linked in nature). As used herein, the term "expression vector" refers to a polynucleotide containing an expression construct and sequence sufficient to replicate in a host cell or insert into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term "expression control sequence" refers to a nucleotide sequence that regulates transcription and / or translation of a nucleotide sequence operatively linked to it. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome-binding sites (translational regulatory sequences).

[0060] As used in this article, when the expression control sequence functions to regulate the transcription of nucleotide sequences in the cell, the nucleotide sequence is "operably linked" to the expression control sequence. This includes promoting the transcription of the nucleotide sequence through the interaction between the polymerase and the promoter.

[0061] As used herein, the term "vector" includes any intermediate medium for nucleic acid molecules that enables said nucleic acid molecules to be introduced, for example, into prokaryotic and / or eukaryotic cells and / or integrated into the genome, and includes plasmids, phage particles, viral phages, or viral vectors, such as retrovirus-based vectors, lentiviral vectors, adeno-associated virus vectors, etc. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, typically a circular DNA double helix, which can replicate independently of chromosomal DNA.

[0062] "Transfection" refers to the introduction of new genetic material into cells. It includes transformation (the direct uptake and incorporation of foreign genetic material from the surrounding environment through the cell membrane), transduction (the introduction of foreign DNA into host cells through viruses, bacteriophages, and conjugation).

[0063] As used in this article, “host cell” refers to a recombinant cell containing the expression construct.

[0064] As used herein, the term “biological sample” refers to a sample containing cells (e.g., cells) or biomolecules derived from cells.

[0065] As used herein, the terms “therapy,” “treatment,” “therapeutic intervention,” and “improvement” refer to any activity that results in a reduction in the severity of symptoms. The terms “treatment” and “prevention” are not absolute terms. Treatment and prevention can refer to any delay in onset, improvement in symptoms, increase in patient survival, increase in survival time or survival rate, etc. Treatment and prevention can be complete or partial. Treatment effects can be compared to untreated individuals or groups, or to the same patient before treatment or at different times during treatment. In some respects, the severity of the disease is reduced by at least 10% compared to, for example, individuals before administration or untreated controls. In some respects, the reduction in disease severity is at least 25%, 50%, 75%, 80%, or 90%, or in some cases, it is no longer detectable using standard diagnostic techniques. “Treatment” or “treatment” can also mean prolonged survival compared to expected survival without treatment. The term “treatment” as used herein also includes preventative treatment.

[0066] A composition or method that “comprising” or “includes” one or more of the listed elements may include other elements not specifically listed (e.g., an open-ended term means including, but not limited to). For example, a composition that “comprising” or “includes” an antibody may contain an antibody, alone or in combination with other components. Conversely, the phrase “composed of” is closed, indicating that such an embodiment does not include additional elements. The term “substantially composed of” refers to including the listed elements and other elements that do not materially affect the essential and novel features of the claimed combination (e.g., a partially closed term). It should be understood that aspects and embodiments described herein as “comprising” include embodiments that are “composed of” and “substantially composed of”.

[0067] As used herein, unless otherwise stated, the following meanings apply. The word “may” is used in a permissive sense (i.e., implying a possibility), not in a mandatory sense (i.e., implying a requirement). The singular forms “a / an” and “the” include plural pronouns. Thus, for example, a submission of “one element” includes a combination of two or more elements, although other terms and phrases such as “one or more” are used for one or more elements.

[0068] The phrase “at least one” includes “one,” “one or more,” “one or plurality,” and “multiple.” Unless otherwise stated, the term “or” is non-exclusive, meaning it encompasses both “and” and “or.” The term “any” refers to each member of the sequence modified by the modifier. Thus, for example, the phrase “at least one of 1, 2, or 3” means “at least 1, at least 2, or at least 3.” The term “multiple” in relation to a reference object (e.g., an object such as T cells) refers to 3 or more, preferably 10 or more, preferably 100 or more, preferably 1,000 or more, preferably 10 ... 6 One or more, preferably 10 7 One or more, preferably 10 8 One or more, preferably 10 9 One or more, but not an unlimited number of, references (e.g., preferably less than 10) 12 10 or less, preferably less than 10 11 10 or less, preferably less than 10 10 indivual).

[0069] As used in this document, the term “about” covers 90% to 110% of the value. For example, the phrase “about 20 residues (amino acids) long” used to describe a peptide linker refers to a peptide linker of 18 to 22 amino acids in length, and includes peptide linkers of 20 amino acids in length.

[0070] I. Chimeric antigen receptor (CAR) A chimeric antigen receptor (CAR) is an engineered molecule comprising an optional signal peptide, a target antigen-binding domain, an optional hinge region, a transmembrane domain, an intracellular signal transduction domain, and an optional co-stimulatory domain. CARs are based on the structure of T-cell receptors, which are expressed on T cells and participate in cell-mediated immune responses. The target-binding domain is also referred to herein as an antigen-binding domain or antigen recognition domain, and therefore the term "target" encompasses "antigen."

[0071] So-called "first-generation" CARs have a targeting domain and a CD3ξ signaling domain. So-called "second-generation" CARs further include co-stimulatory domains, such as CD28 or 4-1BB domains. So-called "third-generation" CARs contain multiple co-stimulatory domains. So-called "fourth-generation" CARs (also known as "TRUCKS") are engineered to release transgenic cytokines during CAR signaling.

[0072] A chimeric antigen receptor (“CAR”) comprises the following elements: (1) an optional signal peptide, (2) a target antigen-binding domain, (3) a linker; (4) a transmembrane region; and (5) an intracellular domain containing a signal transduction domain. Optionally, the CAR may include a co-stimulatory (signal transduction) domain. That is, in addition to the desired elements, these optional elements may be included. The target antigen-binding domain is heterologous to at least one of the other domains. That is, the antigen-binding domain is not natively present on a T cell receptor, or is not in the same protein as at least one of the other domains of the CAR.

[0073] The signal peptide guides the CAR polypeptide across the cell membrane. The antigen-binding domain provides the CAR with binding specificity. This domain can bind to the domain of an antibody, which then binds to the target antigen, forming a so-called "universal CAR." The connector, which can act as a hinge region, is a flexible linker, such as a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing for the side-attached polypeptide region. The transmembrane domain is a transmembrane protein domain. The intracellular signal transduction domain, following antigen binding by the antigen-binding domain, transmits the signal into the cell via the signal transduction domain. This signal transduction activates cellular activity. The co-stimulatory domain is an auxiliary signal transduction domain that further transmits the signal.

[0074] This document provides a chimeric antigen receptor (CAR) that also includes an extracellular helper domain, through which CAR-expressing T cells can be activated in an antigen-independent manner. Specifically, the extracellular helper domain of this disclosure is an "antibody-inducible domain," through which CAR-expressing T cells can be activated upon antibody binding to the antibody-inducible domain. In some embodiments, the adapter includes a "first adapter" located between the antigen-binding domain and the antibody-inducible domain. In some embodiments, the adapter further includes a "second adapter" located between the antibody-inducible domain and the transmembrane domain.

[0075] A. Signal peptide The signal peptide that may be present at the N-terminus of the nascent CAR of this disclosure during cellular expression can be the signal peptide of any human type I transmembrane protein or any other signal peptide suitable for transferring the nascent CAR to the surface of human cells. In some embodiments, the signal peptide is derived from a protein of the human immunoglobulin superfamily. In some embodiments, the signal peptide is derived from CD4, CD8, CD19, CD28, TCR, or an immunoglobulin chain.

[0076] One example signal peptide is the GMCSF signal peptide: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:1). Another example signal peptide is the CD8α signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO:2). In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:1 or SEQ ID NO:2.

[0077] B. Antigen-binding domain The antigen-binding domain (ABD) of the CAR disclosed herein can have any desired specificity and can be any suitable polypeptide form for binding the target antigen and transmitting the signal through the transmembrane domain to the intracellular domain of the CAR. In some embodiments, the antigen-binding domain (ABD) is a single-chain antibody (scFV). In some implementations, such as when ABD is scFV, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, wherein the heavy chain variable region comprises complementary regions (CDR)-H1, CDR-H2, and CDR-H3 of the antibody, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 of the antibody. The sequence of the antibody is stored in version 13.1 (August 2023) of the ABCD (Antibody Chemical Definition) database hosted by Expasy and operated by the Swiss Institute of Bioinformatics (see Lima et al., Nucleic Acids Research, 48:D2610264, 2020). In some implementations, the CDR, as defined using Kabat nomenclature, can be easily determined using online tools such as the abYsis annotation tool for a given sequence. In some implementations, the antigen-binding domain includes the light chain variable region and heavy chain variable region of the antibody disclosed in ABCD version 13.1 (August 2023), which includes 24,485 sequence antibodies against 4,171 different target antigens.

[0078] In some embodiments, the antigen comprises citrullinated vimentin (CV). In an exemplary embodiment, the antigen-binding domain is a CV-binding domain comprising a heavy chain variable region (VH) containing a heavy chain CDR of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and a light chain variable region (VL) containing a light chain CDR of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, the CV-binding domain comprises a VH containing an amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and a VL containing an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, the CV-binding domain comprises a VH containing an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and a VL containing an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0079] In other embodiments, the antigen comprises CD19 (also known as B lymphocyte surface antigen B4). In an exemplary embodiment, the antigen-binding domain is a CD19-binding domain comprising a light chain variable region (VL) containing the light chain CDR of SEQ ID NO:9 and a heavy chain variable region (VH) containing the heavy chain CDR of SEQ ID NO:10. In some embodiments, the CD19-binding domain comprises a VL containing the amino acid sequence of SEQ ID NO:9 and a VH containing the amino acid sequence of SEQ ID NO:10. In some embodiments, the CD19-binding domain comprises a VL containing an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:9, and a VH containing an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:10.

[0080] In further embodiments, the antigen includes B-cell maturation antigen (BCMA, also known as tumor necrosis factor receptor superfamily member 17 or TNFRSF17). In an exemplary embodiment, the antigen-binding domain is a BCMA-binding domain comprising a light chain variable region (VL) containing the light chain CDR of SEQ ID NO:11 and a heavy chain variable region (VH) containing the heavy chain CDR of SEQ ID NO:12. In some embodiments, the BCMA-binding domain comprises a VL containing the amino acid sequence of SEQ ID NO:11 and a VH containing the amino acid sequence of SEQ ID NO:12. In some embodiments, the BCMA-binding domain comprises a VL containing an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:11, and a VH containing an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:12.

[0081] The antigen-binding domain of the CAR disclosed herein comprises a VH region and a VL region, preferably separated by a peptide linker. In some embodiments, the antigen-binding domain is arranged as VH-linker-VL. In other embodiments, the antigen-binding domain is arranged as a VL-linker-VH structure. In some embodiments, the peptide linker is about 4 to about 24 amino acids in length. In an exemplary embodiment, the linker is a glycine linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 13). In other exemplary embodiments, the linker is a Whitlow linker comprising the following amino acid sequence: GSTGSGKPGSGEGSTKG (SEQ ID NO: 14). In yet another exemplary embodiment, the linker is an ABpur linker comprising the following amino acid sequence: ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 15).

[0082] In an exemplary embodiment, the antigen-binding domain (ABD) is a CV-binding domain comprising the amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:16. In other exemplary embodiments, the ABD is a CD19-binding domain comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:17 or SEQ ID NO:18. In another exemplary embodiment, ABD is a BCMA-binding domain comprising the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:19 or SEQ ID NO:20.

[0083] C. Connector The antigen-binding domain and antibody-inducible domain of the CAR disclosed herein are separated by a linker (first linker). The antibody-inducible domain and transmembrane domain of the CAR disclosed herein may also be separated by a linker (second linker).

[0084] The linker can be a "flexible peptide linker" with a length of 4 to 24 amino acids. In some embodiments, the flexible peptide linker comprises [XaXbXcXdXeXfXg]n, wherein Xa, Xb, Xc, and Xd are independently selected from G and S, Xe, Xf, and Xg are independently selected from G, S, and are absent, and n is 1, 2, or 3. That is, the flexible peptide linker is rich in glycine and serine residues and has a length of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and a length of at most 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 amino acids.

[0085] The linker can be a "hinge region" with a length of about 10 to about 20, 30, 40, or 50 amino acids. In some embodiments, the hinge region is a CD8a hinge region, a CD28 hinge region, or an IgG4 hinge region. In some embodiments, the hinge region is an IgG4 hinge region.

[0086] In some embodiments, the first connector is a flexible peptide connector. In an exemplary embodiment, the first connector is a flexible peptide connector and the second connector is a flexible peptide connector, an IgG4 hinge region, or is absent. In some embodiments, the first connector is an IgG4 hinge region. In an exemplary embodiment, the first connector is an IgG4 hinge region and the second connector is an IgG4 hinge region, a flexible peptide connector, or is absent.

[0087] In some embodiments, the hinge region is a CD8a hinge region. In an exemplary embodiment, the CD8a hinge region comprises the amino acid sequence TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 21.

[0088] In some embodiments, the hinge region is a CD28 hinge region. In an exemplary embodiment, the CD28 hinge region comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 22), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 22.

[0089] In some embodiments, the hinge region is an IgG4 hinge region. In an exemplary embodiment, the IgG4 hinge region comprises: the amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 23), or an amino acid sequence that differs from SEQ ID NO: 23 by one or two amino acids.

[0090] D. Antibody-inducible domain The antibody-inducible domain (AID) of the CAR disclosed herein is a polypeptide of about 60 to about 360 amino acids in length, which can be bound by an antibody (e.g., containing an antibody epitope). The AID is located between the antigen-binding domain and the transmembrane domain. In some embodiments, the AID is side-connected to a first linker or hinge domain at the N-terminus. In some embodiments, the AID is side-connected to a second linker or hinge at the C-terminus. In some preferred embodiments, the AID comprises a fragment of the extracellular region of a human cell surface protein, provided that it does not contain fragments of B cell or T cell antigen receptors. Specifically, the length of AID is at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 amino acids and at most 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250 or 240 amino acids.

[0091] When the AID of the CAR disclosed herein is bound by an antibody (e.g., a monoclonal antibody), the AID is able to transmit signals to the intracellular domain of the CAR via its transmembrane domain. Thus, the AID provides an alternative to the ABD for CAR-specific activation of T cells expressing the CAR. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is an IgG antibody. In some embodiments, the IgG antibody is an IgG1 antibody. In some preferred embodiments, the monoclonal antibody is a human antibody or a humanized antibody. In other embodiments, the monoclonal antibody is a rodent antibody, such as a mouse monoclonal antibody or a rat monoclonal antibody. In some embodiments, the monoclonal antibody is a human / rodent chimeric antibody.

[0092] In some implementations, the antibody-inducible domain includes members of the group consisting of: vascular endothelial growth factor receptor 2 (VEGFR2) domain, human epidermal growth factor receptor 2 (HER2) domain, platelet-derived growth factor receptor-α (PDGFRα) domain, interleukin-4 receptor-α (IL-4Rα) domain, CD4 domain, and CD2 domain. In an exemplary embodiment, the antibody-inducible domain comprises a fragment of a human protein selected from the group consisting of: domain 3 of vascular endothelial growth factor receptor 2 (VEGFR2-D3), domain 4 of human epidermal growth factor receptor 2 (HER2-D4), domains 3-5 of platelet-derived growth factor receptor α (PDGFRα-D3-D5), domains 1-2, 1, or 2 of interleukin-4 receptor α (IL-4Rα-D1-D2, IL-4Rα-D1, or IL-4Rα-D2), domains 1-2, 1, or 2 of CD4 (CD4-D1-2, CD4-D1, or CD4-D2), and domains 1-2, 1, or 2 of CD2 (CD2-D1-D2, CD2-D1, or CD2-D2).

[0093] In some embodiments, AID comprises a human VEGFR2 domain. In some embodiments, the VEGFR2 domain is capable of being bound by an anti-VEGFR2 antibody or a VEGFR2 binding fragment. In some embodiments, the anti-VEGFR2 antibody is ramucirumab. In some embodiments, the anti-VEGFR2 antibody is alacizumab. In an exemplary embodiment, the VEGFR2 domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:31.

[0094] In some embodiments, AID comprises a human HER2 domain. In some embodiments, the HER2 domain is capable of being bound by an anti-HER2 antibody or its HER2-binding fragment. In some embodiments, the anti-HER antibody is selected from the group consisting of trastuzumab, margetuximab, pertuzumab, and timigutuzumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In an exemplary embodiment, the HER2 domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:32.

[0095] In some embodiments, AID comprises a human PDGFRα domain. In some embodiments, the PDGFRα domain is capable of being bound by an anti-PDGFRα antibody or its PDGFRα binding fragment. In some embodiments, the anti-PDGFRα antibody is selected from the group consisting of olaratumab and tovetumab. In an exemplary embodiment, the PDGFRα domain comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:33.

[0096] In some embodiments, AID comprises a human IL-4Rα domain. In some embodiments, the IL-4Rα domain is capable of being bound by an anti-IL-4Rα antibody or its IL-4Rα binding fragment. In some embodiments, the IL-4Rα antibody is dupilumab. In some embodiments, the IL-4Rα antibody is pascolizumab. In exemplary embodiments, the IL-4Rα domain comprises: i) the amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.

[0097] In some embodiments, the AID comprises a human CD4 domain. In some embodiments, the CD4 domain is capable of being bound by an anti-CD4 antibody or a CD4 binding fragment. In some embodiments, the anti-CD4 antibody is selected from the group consisting of: cedelizumab, clenoliximab, ibaliizumab, keliximab, priliximab, tregalizumab, zanolimumab, and semzuvolimab. In an exemplary embodiment, the CD4 domain comprises: i) an amino acid sequence selected from SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0098] In some embodiments, AID comprises a human CD2 domain. In some embodiments, the CD2 domain is capable of being bound by an anti-CD2 antibody or its CD2-binding fragment, or a lymphocyte function-associated antigen-IgG fusion protein selected from the group consisting of siplizumab. In an exemplary embodiment, the CD2 domain comprises: i) an amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:40 or SEQ ID NO:41.

[0099] E. Transmembrane domain The antibody-inducible domain (and optionally a second linker or hinge) of the CAR disclosed herein is separated from the intracellular signal transduction domain by a transmembrane domain of about 20 to about 30 amino acids in length.

[0100] In some embodiments, the transmembrane domain is a CD8a transmembrane domain. In an exemplary embodiment, the CD8a domain comprises the amino acid sequence of IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:24), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:24.

[0101] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In an exemplary embodiment, the CD28 transmembrane domain is a wild-type CD28 transmembrane domain comprising the following amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:25). In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:25.

[0102] In some embodiments, the transmembrane domain is a modified CD28 transmembrane domain. In an exemplary embodiment, the amino acid sequence of the modified CD28 transmembrane domain comprises insertions, substitutions, and / or deletions relative to SEQ ID NO:25. In some embodiments, the amino acid sequence of the CD28 transmembrane domain comprises at least one substitution selected from the group consisting of: C165L, Y166L, S167L, T171L (these correspond to the C13L, Y14L, S15L, and T19L substitutions of the wild-type CD28™ sequence of SEQ ID NO:25). In some embodiments, the modified CD28 transmembrane domain comprises the following amino acid sequence: FWVLVVVGGVLALLLLLLLVLVAFIIFWV (SEQ ID NO:42). In some embodiments, the CD28™ domain comprises the following common amino acid sequence: FWVLVVVGGVLAX1X2X3LLVX4VAFIIFWV (SEQ ID NO:43), wherein X1 is C or L, X2 is Y or L, X3 is S or L, and X4 is T or L, and / or at least one of X1, X2, X3 and X4 is L.

[0103] F. Intracellular signal transduction domains The intracellular signal transduction domain of the CAR disclosed herein includes a CD3ζ signal transduction domain. In some preferred embodiments, the intracellular signal transduction domain further includes a co-stimulatory domain.

[0104] In some embodiments, the co-stimulatory domain may be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, OX40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4, etc. In some embodiments, the CAR of this disclosure comprises two or more co-stimulatory signal transduction domains (e.g., CD28 and 4-1BB).

[0105] In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. In an exemplary embodiment, the CD28 co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:26), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:26.

[0106] In some embodiments, the co-stimulatory domain is a 4-1BB co-stimulatory domain. In an exemplary embodiment, the 4-1BB co-stimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:27), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:27.

[0107] In an exemplary embodiment, the CD3ζ signal transduction domain comprises the amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:28), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:28.

[0108] In some embodiments, the intracellular signal transduction domain comprises a CD28 co-stimulatory domain and a CD3ζ signal transduction domain. In an exemplary embodiment, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:29, or amino acids having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:29.

[0109] In some embodiments, the intracellular signal transduction domain comprises a 4-1BB co-stimulatory domain and a CD3ζ signal transduction domain. In an exemplary embodiment, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:30, or amino acids having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:30.

[0110] II. Nucleic acids encoding CAR The nucleic acid molecule (polynucleotide) encoding the CAR disclosed herein can be a separate molecule or can be contained in a cassette or vector.

[0111] A. Nucleic acid This disclosure provides nucleic acid molecules (polynucleotides) comprising a nucleotide sequence encoding a CAR as described herein (e.g., containing a coding region of a CAR as described herein). The nucleic acid may be in the form of DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotides as described herein are isolated.

[0112] For example, the nucleic acid may contain a nucleotide sequence encoding the CAR polypeptide described herein or a portion thereof. In some embodiments, the nucleotide sequence encodes a polypeptide containing an amino acid sequence of a specific SEQ ID NO, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a specific SEQ ID NO.

[0113] In some embodiments, the polynucleotide is a variant comprising changes to a coding region, a non-coding region, or both. In some embodiments, the polynucleotide variant comprises a silent change. In other embodiments, the polynucleotide variant comprises substitutions, additions, or deletions, but does not alter the binding properties of the antigen-binding domain of the encoded CAR polypeptide. In some embodiments, the polynucleotide variant comprises one or more changes that do not alter the amino acid sequence of the CAR. In some embodiments, the polynucleotide variant contains a “silent” substitution due to the degeneracy of the genetic code. Polynucleotide variants can arise for a variety of reasons, such as to optimize codon expression in a particular host cell.

[0114] B. Expression box and carrier This disclosure further provides expression cassettes and vectors comprising a nucleic acid molecule encoding the CAR described herein. In some embodiments, the nucleic acid molecule may further comprise an expression control sequence operatively linked to a nucleotide sequence encoding the CAR. In some embodiments, the nucleic acid molecule encoding the disclosed CAR may be inserted into an expression vector in a operative combination with an expression control sequence suitable for expressing the disclosed CAR in desired host cells. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the CAR peptide in suitable host cells.

[0115] For example, a polynucleotide may contain one or more transcriptional regulatory elements (such as promoters or enhancers) that, when present in a cell, cause the expression of a sequence encoding a CAR in the cell.

[0116] The nucleic acids disclosed herein can be incorporated into vectors that can be introduced into host cells. Such vectors include, but are not limited to, viral vectors and plasmids. Exemplary viral vectors include, but are not limited to, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Lentivirals are particularly desirable because they are capable of delivering large amounts of genetic material into the host cell's genome and because they are capable of infecting non-dividing cells.

[0117] III. Host Cell This disclosure also provides a host cell (e.g., a recombinant cell) comprising a nucleic acid molecule, expression cassette, or expression vector encoding the CAR described herein. In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. In other embodiments, the host cell is a prokaryotic cell, such as a bacterial cell.

[0118] As is well known to those skilled in the art, the nucleic acid molecule encoding the disclosed CAR can be delivered to host cells via transformation, transfection, or transduction. The resulting recombinant (host) cells can be immune cells, including but not limited to T cells, such as CD4+ T cells, CD8α+ T cells, or CD8β+ T cells. In some embodiments, the T cells are Treg cells. In some embodiments, the T cells are T helper (Th) cells. In some embodiments, the T cells are cytotoxic T lymphocytes (CTLs). In some embodiments, the recombinant (host) cell containing the nucleic acid molecule encoding the disclosed CAR is a lymphocyte (e.g., T cells, B cells, or NK cells).

[0119] In some embodiments, the cells expressing the CAR of this disclosure are Treg cells. "Regulatory T cells" or "Treg cells" are cells belonging to a specific subset of T cells that suppress immune responses, thereby maintaining homeostasis and self-tolerance. Tregs can suppress T cell proliferation and cytokine production and play a key role in preventing autoimmunity. Tregs are characterized by the expression of FoxP3. Surface markers of Tregs include high CD4 and CD25 (high molecular density) and low CD127 (low molecular density). Mouse and human Tregs express GITR / AITR and CTLA-4. Human CD4+FoxP3+ Treg cells can be divided into three subsets: (1) CD45RA+CD25+FoxP3 low-quiet Treg cells (2) CD45RO+CD25 high FoxP3 highly activates Treg cells, and (3) CD45RO+CD25+FoxP3low non-inhibitory effector T cells (Teff) that produce pro-inflammatory cytokines.

[0120] In some embodiments, the host cells are autologous cells (e.g., recombinant progeny of cells obtained from the intended recipient). That is, the cells to be transformed with the nucleic acids disclosed herein can be cells taken from the subject receiving the recombinant cells. This mitigates the problem of allogeneic immune responses. Even so, in other embodiments, the host cells are allogeneic cells (e.g., recombinant progeny of cells obtained from an individual immunologically different from the intended recipient). Regardless of the source of the host cells, they can be expanded in vitro prior to administration to the subject.

[0121] Furthermore, proteins derived from transformed / recombinant hosts can be purified using any suitable method. These methods include chromatography (e.g., ion exchange, affinity and fractionation column chromatography), centrifugation, differential dissolution, or any other standard technique used for protein purification. Affinity tags (such as hexahistidine, maltose-binding domains, influenza virus capsid sequences, and glutathione S-transferases) can be attached to proteins to facilitate purification by passing them through a suitable affinity column. In some embodiments, the proteins can also be physically characterized using techniques such as proteolysis, high-performance liquid chromatography, nuclear magnetic resonance, and X-ray crystallography.

[0122] IV. Composition The pharmaceutical composition and method of use for treating diseases or conditions are also disclosed, the pharmaceutical composition comprising a host cell (e.g., a recombinant cell) containing a nucleic acid molecule encoding and expressing the disclosed CAR polypeptide and a pharmaceutically acceptable excipient.

[0123] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical agent (e.g., a drug as described herein or recombinant Treg cells) and a pharmaceutically acceptable excipient.

[0124] As used herein, the term “pharmaceutical acceptable” means a compound that is compatible with the other components of a pharmaceutical composition and can be safely administered to a subject. This term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable.” Based on this disclosure, pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art. For a detailed list of suitable pharmaceutical compositions and techniques for their administration, reference may be made to texts such as: Remington's Pharmaceutical Sciences, 17th edition, 1985; Brunton et al., “Goodman and Gilman's The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0125] Pharmaceutical compositions are generally sterile, at least for human use. They typically contain pharmaceutically acceptable excipients for buffering and preservation during storage, and may include buffers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable excipients include, but are not limited to, plain (0.9%) saline, phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), and various electrolyte solutions.

[0126] The pharmaceutical composition can be formulated for any route of administration. However, in most embodiments of this disclosure, the pharmaceutical composition is formulated for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection, whether by bolus or infusion) administration.

[0127] Injectable (e.g., intravenous) pharmaceutical compositions may comprise a solution of the drug suspended in a pharmaceutically acceptable excipient, such as an aqueous excipient. Any of a variety of aqueous excipients may be used, such as water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% glucose, etc., and may include glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, etc. Typically, a common buffered saline (135-150 mM NaCl) will be used. Pharmaceutically acceptable excipients may contain adjuvants for mimicking physiological conditions, such as pH adjusters and buffers, tonicotinic agents, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the composition may be formulated for intravenous administration.

[0128] Pharmaceutical compositions suitable for parenteral administration (such as intravenous administration) include aqueous and non-aqueous isotonic sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the composition isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injectable solutions and suspensions may also be prepared from sterile powders, granules, and tablets. In practice, the pharmaceutical compositions may be administered, for example, by intravenous infusion. The pharmaceutical compositions may be provided in single-dose or multi-dose sealed containers.

[0129] The host cells may be cryopreserved. Cryopreservation may involve preparing the host cells with a cryopreservation agent such as DMSO. Commercially available culture media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0130] The terms “dosage” and “volume” are used interchangeably herein. A dosage refers to the amount of a drug administered to an individual at each administration. Dosages will vary depending on many factors, including frequency of administration; individual size and tolerability; severity of disease; risk of side effects; route of administration; and the form of imaging of detectable markers (if present). Those skilled in the art will recognize that dosages can be modified based on the foregoing factors or on treatment progress.

[0131] Pharmaceutical compositions can be packaged or prepared in unit dose form. In this form, the pharmaceutical composition is subdivided into unit doses containing appropriate amounts of the pharmaceutical agent, for example, based on the dosage or concentration of the agent in the pharmaceutical composition. The unit dose form can be a packaged formulation containing discrete amounts of the pharmaceutical composition. If desired, the pharmaceutical composition may also contain other compatible therapeutic agents.

[0132] V. Usage Instructions Host cells expressing the CARs disclosed herein (e.g., recombinant T cells) can be used to treat a variety of diseases and conditions. Methods of use include administering an effective amount of the pharmaceutical composition of this disclosure containing cells expressing CARs (such as CAR-T cells) to a subject in need (e.g., an individual suffering from a disease or condition). In some methods of this disclosure where the subject has cancer, the host cells comprise CD3+ T cells expressing a CAR that binds to an antigen expressed by the cells of the cancer. In some methods of this disclosure where the subject has an autoimmune disease or inflammatory condition, the host cells comprise Treg cells expressing a CAR that binds to an antigen associated with the autoimmune disease or inflammatory condition. In some embodiments, the host cells are the subject's own cells.

[0133] As used herein, the term "subject" refers to an individual animal. As used herein, the term "patient" refers to a subject under the care or supervision of a healthcare provider, such as a doctor or nurse. Subjects include mammals, such as humans, and non-human primates, such as monkeys, as well as dogs, cats, horses, cattle, rabbits, rats, mice, goats, pigs, and other mammal species. Subjects may also include birds. In some embodiments, the subject is a human patient. A patient can be an individual seeking treatment, monitoring, adjustment, or modification of an existing treatment regimen, etc. Subjects suffering from a disease or condition can include individuals who have not yet received treatment, individuals who are currently receiving treatment, individuals who have previously received treatment, and individuals who have discontinued treatment.

[0134] As used herein, the terms “effective amount,” “effective dose,” and “therapeutic effective amount” refer to the amount of a drug sufficient to produce a desired response, such as reducing or eliminating signs or symptoms of a disease or improving the condition. In some instances, an “effective amount” is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any condition or disease and / or prevents disease progression. For example, for a given parameter, a therapeutic effective amount will show an increase or decrease in therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease of “-fold.” For example, a therapeutic effective amount may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more than a control.

[0135] The pharmaceutical composition may be administered via any suitable route, including but not limited to intravenous, subcutaneous, intramuscular, or intraperitoneal routes. Examples of administration include storing 10 mg / ml of the composition in a sterile isotonic saline solution at 4°C and diluting it in 100 ml or 200 ml of 0.9% sodium chloride for injection prior to administration to the patient. The pharmaceutical composition is administered via intravenous infusion over a 1-hour timeframe at a dose between 0.2 mg / kg and 10 mg / kg. In other embodiments, the pharmaceutical composition is administered via intravenous infusion over a period between 15 minutes and 2 hours. In still other embodiments, the administration procedure is a rapid subcutaneous concentration.

[0136] The dosage of the pharmaceutical composition is selected to provide effective treatment to the patient, and this dosage is in the range of less than 0.1 mg / kg body weight to about 25 mg / kg body weight, or in the range of 1 mg to 2 g / patient. In some cases, the dosage is in the range of 1 to 100 mg / kg, or about 50 mg to 8000 mg / patient. Depending on the pharmacokinetics of the composition (e.g., the half-life of the composition in circulation) and pharmacodynamic response (e.g., the duration of the therapeutic effect of the composition), it can be repeated at an appropriate frequency, ranging from once daily to once every three months. In some embodiments, the composition is repeated with an in vivo half-life of about 7 days to about 25 days and is administered weekly to once every 3 months.

[0137] Application can be periodic. Depending on the route of administration, the dosage can be given once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6 months). In some cases, administration is more frequent, such as twice or three times daily. As those skilled in the art will recognize, patients can be monitored to adjust the dosage and frequency of administration based on treatment progress and any adverse side effects.

[0138] Therefore, in some implementations, additional administration is based on patient progress, for example, by monitoring the patient between administrations. For instance, the patient's symptom relief rate can be monitored after the first administration or after the first round of administration.

[0139] VI. Reagent Kit As used herein, the term "kit" refers to a group of items intended to be used together. In some embodiments, the kit includes a reagent and instructions for use thereof. In some embodiments, the kit further includes a container, such as a vial, containing a composition as disclosed herein. For example, the kit may include a container containing a pharmaceutical composition, such as a bag or bottle for intravenous administration, the pharmaceutical composition containing multiple recombinant cells expressing the CAR of this disclosure. In some embodiments, the kit may further include a fluid conduit, such as a plastic tube, having an infusion lumen. The infusion lumen may be communicated with an intravenous injection needle via the fluid conduit. The fluid conduit may also include one or more Y-shaped tees and roller clamps.

[0140] VII. Listed Implementation Schemes Implementation Scheme 1. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain, a first linker, an antibody-inducible domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antibody-inducible domain is a polypeptide of about 60 to about 360 amino acids in length.

[0141] Implementation Scheme 2. The CAR as described in Implementation Scheme 1, wherein the antibody-inducible domain comprises members of the group consisting of: vascular endothelial growth factor receptor 2 (VEGFR2) domain, human epidermal growth factor receptor 2 (HER2) domain, platelet-derived growth factor receptor-α (PDGFRα) domain, interleukin-4 receptor-α (IL-4Rα) domain, CD4 domain, and CD2 domain.

[0142] Implementation Scheme 3. The CAR as described in Implementation Scheme 2, wherein the antibody-inducible domain comprises the VEGFR2 domain.

[0143] Implementation Scheme 4. The CAR as described in Implementation Scheme 3, wherein the VEGFR2 domain is capable of being bound by an anti-VEGFR2 antibody selected from the group consisting of ramucirumab and aracizumab, or a VEGFR2 binding fragment thereof, optionally wherein the VEGFR2 domain is capable of being bound by ramucirumab.

[0144] Implementation Scheme 5. The CAR as described in Implementation Scheme 4, wherein the VEGFR2 domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:31.

[0145] Implementation Scheme 6. The CAR as described in Implementation Scheme 2, wherein the antibody-inducible domain comprises the HER2 domain.

[0146] Implementation Scheme 7. The CAR as described in Implementation Scheme 6, wherein the HER2 domain is capable of being bound by an anti-HER2 antibody or its HER2-binding fragment selected from the group consisting of trastuzumab, maggotuximab, pertuzumab and telmituzumab, optionally wherein the HER2 domain is capable of being bound by trastuzumab.

[0147] Implementation Scheme 8. The CAR as described in Implementation Scheme 7, wherein the HER2 domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:32.

[0148] Implementation Scheme 9. The CAR as described in Implementation Scheme 2, wherein the antibody-inducible domain comprises the PDGFRα domain.

[0149] Implementation Scheme 10. The CAR as described in Implementation Scheme 9, wherein the PDGFRα domain is capable of being bound by an anti-PDGFRα antibody selected from the group consisting of olatuzumab and tovertumab, or a PDGFRα binding fragment thereof.

[0150] Implementation Scheme 11. The CAR as described in Implementation Scheme 10, wherein the PDGFRα domain comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:33.

[0151] Implementation Scheme 12. The CAR as described in Implementation Scheme 2, wherein the antibody-inducible domain comprises an IL-4Rα domain.

[0152] Implementation Scheme 13. The CAR as described in Implementation Scheme 12, wherein the IL-4Rα domain is capable of being bound by an anti-IL-4Rα antibody selected from the group consisting of polyumab and pacozumab or its IL-4Rα binding fragment.

[0153] Implementation Scheme 14. The CAR as described in Implementation Scheme 13, wherein the IL-4Rα domain comprises: i) an amino acid sequence of SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.

[0154] Implementation Scheme 15. The CAR as described in Implementation Scheme 2, wherein the antibody-inducible domain comprises a CD4 domain.

[0155] Implementation Scheme 16. The CAR as described in Implementation Scheme 15, wherein the CD4 domain is capable of being bound by an anti-CD4 antibody or its CD4 binding fragment selected from the group consisting of: cilizumab, crixacillin, ibalizumab, kelixim, prixizumab, trelizumab, zamumab, and sizulvolimumab.

[0156] Implementation Scheme 17. The CAR as described in Implementation Scheme 16, wherein the CD4 domain comprises: i) an amino acid sequence selected from SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39.

[0157] Implementation Scheme 18. The CAR as described in Implementation Scheme 2, wherein the antibody-inducible domain comprises a CD2 domain.

[0158] Implementation Scheme 19. The CAR as described in Implementation Scheme 18, wherein the CD2 domain is capable of being bound by an anti-CD2 antibody or its CD2 binding fragment, wherein the anti-CD2 antibody is ciplezumab.

[0159] Implementation Scheme 20. The CAR of Implementation Scheme 19, wherein the CD2 domain comprises: i) an amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:40 or SEQ ID NO:41.

[0160] Implementation Scheme 21. The CAR as described in any one of Implementation Schemes 1 to 20, wherein T cells expressing the CAR can be activated upon binding of an antibody or a fragment thereof to the antibody's inducible domain. Implementation Scheme 22. The CAR of any one of Implementation Schemes 1 to 21 further comprises a second linker located between the antibody-inducible domain and the transmembrane domain.

[0161] Implementation Scheme 23. The CAR as described in any one of Implementation Schemes 1 to 21, wherein the first adapter and / or the second adapter is a flexible polypeptide adapter with a length of 4 to 24 amino acids.

[0162] Implementation Scheme 24. The CAR of Implementation Scheme 23, wherein the flexible polypeptide linker comprises the amino acid sequence [XaXbXcXdXeXfXg]n, wherein Xa, Xb, Xc and Xd are independently selected from G and S, Xe, Xf and Xg are independently selected from G, S and absent, and n is 1, 2 or 3, optionally wherein the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46.

[0163] Implementation Scheme 25. The CAR as described in any one of Implementation Schemes 1 to 21, wherein the first connector and / or the second connector is a hinge region having a length of 10 to 50 amino acids.

[0164] Implementation Scheme 26. The CAR as described in Implementation Scheme 25, wherein the first connector is the flexible polypeptide connector, and the second connector is a hinge region having a length of 10 to 50 amino acids.

[0165] Implementation Scheme 27. The CAR as described in Implementation Scheme 25 or Implementation Scheme 26, wherein the hinge region is selected from the group consisting of the CD8a hinge, the CD28 hinge region and the IgG4 hinge region.

[0166] Implementation Scheme 28. The CAR as described in Implementation Scheme 27, wherein the hinge region is a CD8a hinge region comprising the amino acid sequence of SEQ ID NO:21.

[0167] Implementation Scheme 29. The CAR as described in Implementation Scheme 28, wherein the hinge region is a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:22.

[0168] Implementation Scheme 30. The CAR as described in Implementation Scheme 29, wherein the hinge region is an IgG4 hinge region comprising the amino acid sequence of SEQ ID NO:23.

[0169] Implementation Scheme 31. The CAR as described in any one of Implementation Schemes 1 to 30, wherein the transmembrane domain is a CD28 transmembrane domain or a CD8a transmembrane domain.

[0170] Implementation Scheme 32. The CAR of Implementation Scheme 31, wherein the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:43, optionally wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:42.

[0171] Implementation Scheme 33. The CAR as described in Implementation Scheme 31, wherein the transmembrane domain is a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO:24.

[0172] Implementation Scheme 34. The CAR of any one of Implementation Schemes 1 to 33, wherein the intracellular signal transduction domain comprises the CD3ζ signal transduction domain.

[0173] Implementation Scheme 35. The CAR as described in Implementation Scheme 34, wherein the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:28.

[0174] Implementation Scheme 36. The CAR as described in Implementation Scheme 34 or Implementation Scheme 35, wherein the intracellular signal transduction domain further comprises a co-stimulatory domain.

[0175] Implementation Scheme 37. The CAR as described in Implementation Scheme 36, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain.

[0176] Implementation Scheme 38. The CAR of Implementation Scheme 37, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain, optionally wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:26.

[0177] Implementation Scheme 39. The CAR as described in Implementation Scheme 38, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:29.

[0178] Implementation Scheme 40. The CAR of Implementation Scheme 37, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain, optionally wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:27.

[0179] Implementation Scheme 41. The CAR as described in Implementation Scheme 40, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:30.

[0180] Implementation Scheme 42. The CAR as described in any one of Implementation Schemes 1 to 41 further comprises a signal peptide.

[0181] Implementation Scheme 43. The CAR as described in Implementation Scheme 42, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.

[0182] Implementation Scheme 44. The CAR of any one of Implementation Schemes 1 to 43, wherein the antigen-binding domain comprises a single-chain antibody fragment (scFV) or a single-domain antibody (sdAb).

[0183] Implementation Scheme 45. The CAR as described in Implementation Scheme 44, wherein the antigen-binding domain comprises scFv.

[0184] Implementation Scheme 46. The CAR of Implementation Scheme 44, wherein the antigen-binding domain comprises an sdAb, and optionally wherein the sdAb comprises a heavy chain variable region fragment (VHH).

[0185] Implementation Scheme 47. The CAR of any one of Implementation Schemes 1 to 46, wherein the antigen-binding domain binds to an antigen selected from the group consisting of citrullinated vimentin (CV), CD19 and BCMA.

[0186] Implementation Scheme 48. The CAR of Implementation Scheme 47, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and the light chain CDR of the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; (ii) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; or (iii) The amino acid sequence of SEQ ID NO:16.

[0187] Implementation Scheme 49. The CAR of Implementation Scheme 47, wherein the antigen-binding domain binds to CD19, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:10 and the light chain CDR of the amino acid sequence of SEQ ID NO:9; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or (iii) The amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18.

[0188] Implementation Scheme 50. The CAR of Implementation Scheme 47, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:12 and the light chain CDR of the amino acid sequence of SEQ ID NO:11; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:11; or (iii) The amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20.

[0189] Implementation Scheme 51. A nucleic acid encoding a CAR as described in any one of Implementation Schemes 1 to 50.

[0190] Implementation Scheme 52. An expression vector comprising an operative combination of the nucleic acid described in Implementation Scheme 51 and an expression control sequence.

[0191] Implementation Scheme 53. The expression vector as described in Implementation Scheme 52, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector, a retroviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0192] Implementation Scheme 54. The expression vector as described in Implementation Scheme 53, wherein the viral vector is a lentiviral vector.

[0193] Implementation Scheme 55. The expression vector as described in Implementation Scheme 52, wherein the vector is a plasmid.

[0194] Implementation Scheme 56. A host cell comprising the expression vector described in any one of Implementation Schemes 52 to 55.

[0195] Implementation Scheme 57. A modified T cell engineered to express the CAR of any one of Implementation Schemes 1 to 50, wherein the modified T cell is a mammalian T cell.

[0196] Implementation Scheme 58. A pharmaceutical composition comprising a plurality of modified T cells as described in Implementation Scheme 57 and a pharmaceutically acceptable excipient.

[0197] Implementation Scheme 59. The pharmaceutical composition of Implementation Scheme 58, wherein the modified T cells are human T cells.

[0198] Implementation Scheme 60. The pharmaceutical composition as described in Implementation Scheme 59, wherein the human T cells are regulatory T cells (Tregs) as CD4+, CD25+, and CD12710.

[0199] Implementation Scheme 61. The pharmaceutical composition as described in Implementation Scheme 60, wherein the human Treg is FOXP3+ and HELIOS+.

[0200] Implementation Scheme 62. A method of treating a disease or ailment, comprising administering to a subject in need an effective amount of the pharmaceutical composition described in any one of Implementation Schemes 58 to 61.

[0201] Implementation Scheme 63. A method of treating a subject suffering from an autoimmune disease or inflammatory condition, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of Implementation Schemes 58 to 61, wherein the CAR expressed by the modified T cells comprises an antigen-binding domain that binds to an antigen expressed on the cells of the autoimmune disease or the inflammatory condition.

[0202] Implementation Scheme 64. A method of treating a subject with cancer, comprising administering to the subject an effective amount of the pharmaceutical composition of Implementation Scheme 58 or Implementation Scheme 59, wherein the CAR expressed by the modified T cells comprises an antigen-binding domain that binds to an antigen expressed on the cancer cells.

[0203] Implementation Scheme 65. A kit comprising a container containing a pharmaceutical composition according to any one of embodiments 58 to 61, the container being in communication with an infusion chamber via a fluid conduit, wherein the infusion chamber is configured to be in communication with an intravenous injection needle via a fluid conduit.

[0204] Example Abbreviations: Ab (antibody); ABD (antigen-binding domain); AID (antibody-inducible domain); CAR (chimeric antigen receptor); CD (differentiation cluster); CV (citrullinated vimentin); D (domain); EGFR (epidermal growth factor receptor); EGFRt (EGFR tag); extracellular domain (EC or ECD); FACS (fluorescence-activated cell sorting); HER2 (human epidermal growth factor receptor 2); IL-2 (interleukin 2); IL4Ra (interleukin 4 receptor); MOI (multiplicity of infection); PDGFRa (platelet-derived growth factor receptor α); Prom. (promoter); scFv (single-chain variable fragment); SR (serum substitute); TM (transmembrane domain); Treg (regulatory T cell); VEGFR2 (vascular endothelial growth factor receptor 2); and WT (wild-type).

[0205] The following embodiments are included for illustrative purposes only and are not intended to limit the scope of the invention, which is defined by the claims.

[0206] Example 1: Materials and methods for constructing and testing T cells engineered to express chimeric antigen receptors (CARs) with antibody-inducible domains (AIDs). CAR-specific activation requires the binding of a specific antigen to the antigen-binding domain of the CAR expressed on T cells. Furthermore, selective detection and elimination of CAR-expressing T cells typically require co-expression of a tag (such as EGFRt). To address one or more of these drawbacks, novel CAR scaffolds are constructed and tested. Specifically, the CAR disclosed herein is engineered to include an antigen-inducible domain (AID). The AID serves as a tool for activating, detecting, and eliminating CAR-T cells.

[0207] The creation of lentivirus constructs. Standard CAR constructs and various CAR constructs containing antibody-inducible domains were generated. Each construct contained a coding region for an EGFR tag, located after the P2A (ribosomal skipping) site for co-expression of CAR and EGFR5. The constructs were packaged into lentiviral particles for transduction into cells of interest. HEK293 FT suspension cells (Invitrogen) were loaded at 4.7 x 10⁻⁶ cells / mL. 6 Inoculate at / mL and transfect with LV_MAX transfection reagents using transfer and packaging plasmids (Aldevron pALD-VSV-G, pALD-GagPol, pALD-Rev) according to the manufacturer's protocol (Gibco). Collect viral supernatant and filter through a 0.45 µm polyvinylidene fluoride filter to remove cell debris. Centrifuge the viral supernatant at 10,000 xg overnight to concentrate the viral vector. After 12 to 16 hours, remove the supernatant and resuspend the viral vector pellet. Aliquot the resuspended viral vector and store at -80°C.

[0208] Functional titer determination. Functional titers were assessed to identify ideal transduction conditions for T cells. SupT1 cells (ATCC) were cultured in RPMI medium supplemented with 10% fetal bovine serum. On the day of transduction, 50 µL of SupT1 cells were inoculated at 4 x 10⁻⁶ cells / mL. 5Cells / mL were added to each well of a 96-well plate. The lentiviral stock solution was thawed at room temperature and diluted 1:20 in medium. Subsequently, three-fold serial dilutions were performed in medium, from 60 to 393,660 times. 50 µL of the diluted lentiviral solution was added to a 96-well plate containing 50 µL of cells. On day 2 of transduction, 100 µL of medium was added. On day 3, transduced cells were harvested and stained with an appropriate antibody to detect surface markers. The labeled cells were analyzed by flow cytometry. Untransduced SupT1 cells were used as a negative control to set the gating for flow cytometry analysis. The functional titer was calculated using a viral dilution that produced 5%–20% marker-positive cells according to the following formula: Jurkat-FF-luciferase transduction. The CAR of interest was expressed in a reporter cell line via transduction using a lentiviral vector prepared as described above. This reporter cell line (Jurkat-NFAT-FF-luc) expressed firefly luciferase under the control of the nuclear factor of activated T-cell (NFAT) response element. CAR expression and Jurkat cell activation were assessed by measuring bioluminescence and by flow cytometry.

[0209] Activation of Jurkat cells by target cells expressing CAR antigens. Jurkat cells expressing CAR were co-cultured overnight with K562 cells expressing CAR antigen at varying Jurkat / K562 cell ratios. Following co-culture, a fraction of these cells was used to assess luciferase expression, a biomarker for CAR-mediated activation. Luciferase expression was detected by measuring luminescence using a SpectraMax plate reader. The remaining fraction of cells was used for flow cytometry, including assessing the percentage of cells expressing CD25, another biomarker for CAR-mediated activation.

[0210] Antibody activation of Jurkat cells. Jurkat cells expressing CAR were incubated overnight in medium containing 3 µg / ml of monoclonal antibody as a stimulus or in a medium without a stimulus. The monoclonal antibodies tested included: ramucirumab (anti-VEGFR2 Ab); trastuzumab (anti-HER2 Ab); and dupilumab (anti-IL4Ra Ab). The percentage of cells expressing CD25, a marker of activation via CAR, was measured by flow cytometry.

[0211] Treg isolation and amplification.Primary human Treg cells were derived from leukoreduction chamber residuals or healthy donors obtained through apheresis. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll-PaquePlus. CD25+ T cells were enriched by positive selection. Treg cells were then isolated using FACS by gating CD4+ / CD25+ / CD127lo cells.

[0212] Treg cell transduction and expansion. The CAR of interest was expressed in Treg cells by transducing Tregs with the lentiviral vector prepared as described above. Following isolation, Tregs were stimulated on day 0 with CTS™ Dynabeads™ Treg Xpander reagent (Gibco) at a 1:1 bead-to-cell ratio in the presence of recombinant human IL-2 (300 IU / mL). On day 3, Tregs were transduced via rotational transfection by adding an appropriate volume of virus to achieve an MOI of approximately 12 based on the day 0 cell count. Tregs were amplified by adding fresh medium containing IL-2 on days 4, 5, and 7. On day 9, the amplified CAR+Tregs underwent secondary stimulation.

[0213] Secondary stimulation of CAR+Treg. Expanded CAR+ Tregs were separated and subjected to one of four different stimulation conditions in the presence of IL-2: 1) activation of granules with anti-CD3 and anti-CD28 antibodies at a cell:granule ratio of 1:1; 2) activation of granules with anti-VEGFR2 antibody at a cell:granule ratio of 1:3; 3) 1.0 µg / mL anti-VEGFR2 antibody (ramucirumab); and 4) 3.5 g / mL anti-VEGFR2 antibody (ramucirumab). After culturing the expanded cells under each condition, the fold change in Treg number was assessed on day 14. The Treg populations (CAR+=EGFRt+) and (CAR-=EGFRt-) were also assessed by flow cytometry.

[0214] flow Mode Cellular techniques. The expression of EGFRt, CAR, and CD25 on transduced cells was assessed by staining cells with anti-EGFRt, anti-scFV (CAR), and anti-CD25 antibodies, and the fluorescence staining was subsequently measured by flow cytometry.

[0215] Example 2: T cells engineered to express a chimeric antigen receptor (CAR) with a VEGFR2 antibody-inducible domain (AID). This embodiment describes a novel CAR backbone containing domain 3 of vascular endothelial growth factor receptor 2 (VEGFR2) as an antibody-inducible domain, and the expression and subsequent activation of the novel CAR on recombinant Jurkat cells (immortalized human T cells) and recombinant Treg cells (primary human T cells) using the materials and methods of Example 1.

[0216] Design coding standard CAR or CAR containing VEGFR2 and expression box with EGFRt tag ( Figure 1 Subsequently, a lentiviral vector was generated for CAR expression on transduced T cells. Figure 2 Cartoon diagrams of the standard CAR expressed on the cell surface and four different VEGFR2-containing CARs are shown. Combinations of the VEGFR2 domain with the first adapter and optional second adapter are shown in Table 2-1a.

[0217] Table 2-1a. Antibody-inducible domain and flanking sequences of VEGFR2-containing CARs

[0218] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies, and tag and CAR expression was assessed by flow cytometry. As shown in Tables 2-1a and 2-1b, more than 50% of the transduced Jurkat cell population expressed either the EGFRt tag or one of the standard CARs or VEGFR2-containing CARs.

[0219] Table 2-1b. Cells engineered to express VEGFR2-containing CARs

[0220] Then, the activation of transduced Jurkat cells was assessed by measuring CD25 (interleukin-2 receptor α chain) expression in transduced cells (K562 cells expressing the CAR antigen) at a target-to-effect cell ratio of 4:1 (K562:Jurkat) with or without the antigen. Figure 3A As shown, when Jurkat cells expressing CAR are co-cultured with K562 cells expressing antigen, Jurkat cells expressing either the standard CAR or a CAR containing VEGFR2 are specifically activated through the common antigen-binding domain of the CARs.

[0221] The activation of transduced Jurkat cells was further evaluated by measuring CD25 expression in transduced cells in the presence or absence of antibodies capable of binding to the VEGFR2 domain of some CARs disclosed herein. Figure 3BAs shown, only a very small percentage of untransduced Jurkat cells (simulated) and Jurkat cells expressing the standard CAR expressed CD25, with or without antibody stimulation. Conversely, Jurkat cells expressing the VEGFR2-containing CAR were activated by culturing these cells in the presence of an anti-VEGFR2 antibody. Neither the anti-HER2 nor the anti-IL4RA antibody induced CD25 expression at levels higher than the background levels observed in the absence of antibodies (no stimulation) on cells expressing the VEGFR2-containing CAR. Similarly, Figure 3B This demonstrates that T cells expressing VEGFR2-containing CARs can be activated in a CAR-specific manner upon contact with anti-VEGFR2 antibodies.

[0222] like Figure 4A The description describes the assessment of transduced Treg activation by measuring Treg amplification after subjecting Tregs to one of four restimulation conditions. In short, Tregs expressing a VEGFR2-containing CAR were stimulated in a CAR-nonspecific manner by culturing cells in the presence of CD3 / CD28 beads and IL-2, or in a CAR-specific (and antigen-binding domain nonspecific) manner by culturing cells in the presence of anti-VEGFR2 beads or anti-VEGFR2 antibody (ramucirumab) and IL-2. Figure 2 VEGFR2 CAR 1). For example... Figure 4B As shown, with the introduction of anti-VEGFR2 restimulators (beads or antibodies) on day 9, Tregs expressing VEGFR2-containing CARs amplified to equivalent levels by day 14, which was slightly lower than the amplification levels observed by day 14 with the introduction of anti-CD3 / anti-CD28 bead restimulators on day 9. Interestingly, the use of anti-VEGFR2 restimulators significantly induced selective amplification of CAR-expressing Tregs, as shown by the increased percentage of EGFRt-positive Tregs observed in the transduced Treg population restimulated with anti-VEGFR2 beads or anti-VEGFR2 antibodies compared to transduced Tregs restimulated with anti-CD3 / anti-CD28 beads (see Table 2-2).

[0223] Table 2-2. Percentage of Tregs expressing VEGFR2-CAR after restimulation

[0224] Example 3: T cells engineered to express a chimeric antigen receptor (CAR) with a HER2 antibody-inducible domain (AID). This embodiment describes a novel CAR backbone containing domain 4 of human epidermal growth factor receptor 2 (HER2) as an antibody-inducible domain, and the expression and subsequent activation of the novel CAR on recombinant Jurkat cells (immortalized human T cells) using the materials and methods of Example 1.

[0225] Design coding standard CAR or CAR containing HER2 and EGFRt tag expression box ( Figure 5 Subsequently, a lentiviral vector was generated for CAR expression on transduced T cells. Figure 6 Cartoon diagrams of the standard CAR expressed on the cell surface and three different HER2-containing CARs are shown. Combinations of the HER2 domain with the first adapter and optional second adapter are shown in Table 3-1a.

[0226] Table 3-1a. Antibody-inducible domain and flanking sequences of HER2-containing CARs

[0227] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies, and tag and CAR expression was then assessed by flow cytometry. As shown in Tables 3-1a and 3-1b, more than 50% of the transduced Jurkat cell population expressed either the EGFR tag or a standard CAR or a HER2-containing CAR.

[0228] Table 3-1b. Cells engineered to express HER2-containing CARs

[0229] Then, the activation of transduced Jurkat cells was assessed by measuring CD25 (interleukin-2 receptor α chain) expression in transduced cells (K562 cells expressing the CAR antigen) at a target-to-effect cell ratio of 4:1 (K562:Jurkat) with or without the antigen. Figure 7A As shown, when Jurkat cells expressing CAR are co-cultured with K562 cells expressing antigen, Jurkat cells expressing either the standard CAR or a HER2-containing CAR are specifically activated through the common antigen-binding domain of the CARs.

[0230] The activation of transduced Jurkat cells was further assessed by measuring CD25 expression in transduced cells in the presence or absence of antibodies capable of binding to the HER2 domain of some CARs disclosed herein. Figure 7BAs shown, only a very small percentage of untransduced Jurkat cells (simulated) and Jurkat cells expressing the standard CAR expressed CD25, with or without antibody stimulation. Conversely, Jurkat cells expressing the HER2-containing CAR were activated by culturing these cells in the presence of an anti-HER2 antibody. Neither anti-VEGFR2 nor anti-IL4RA antibodies induced CD25 expression at levels higher than the background levels observed in the absence of antibodies (no stimulation) on cells expressing the HER2-containing CAR. Similarly, Figure 7B This demonstrates that T cells expressing HER2-containing CARs can be activated in a CAR-specific manner upon contact with anti-HERR2 antibodies.

[0231] Example 4: T cells engineered to express a chimeric antigen receptor (CAR) with an antibody-inducible domain (AID) of PDGFRα. This embodiment describes a novel CAR backbone comprising platelet-derived growth factor receptor α (PDGFRa) domains 3 to 5 as antibody-inducible domains, and the expression and subsequent activation of the novel CAR on recombinant Jurkat cells (immortalized human T cells) using the materials and methods of Example 1.

[0232] Design coding standard CAR or CAR containing PDGFRa and expression cassette with EGFRt tag ( Figure 8 Subsequently, a lentiviral vector was generated for CAR expression on transduced T cells. Figure 9 Cartoon diagrams of the standard CAR expressed on the cell surface and three different PDGFRa-containing CARs are shown. Combinations of the PDGFRa domain with the first adapter and optional second adapter are shown in Table 4-1a.

[0233] Table 4-1a. Antibody-inducible domains and flanking sequences of CARs containing PDGFRa

[0234] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies, and tag and CAR expression was assessed by flow cytometry. As shown in Tables 4-1a and 4-1b, more than 20% of the transduced Jurkat cell population expressed either the EGFR tag or a standard CAR or a PDGFRa-containing CAR.

[0235] Table 4-1b. Cells engineered to express CARs containing PDGFRa

[0236] Then, the activation of transduced Jurkat cells was assessed by measuring CD25 (interleukin-2 receptor α chain) expression in transduced cells (K562 cells expressing the CAR antigen) at several effector-to-target cell ratios (Jurkat:K562) in the presence or absence of the antigen. Figure 10 As shown, when Jurkat cells expressing CAR are co-cultured with K562 cells expressing antigen, Jurkat cells expressing either the standard CAR or a CAR containing PDGFRa are specifically activated through the common antigen-binding domain of the CAR.

[0237] Example 5: T cells engineered to express a chimeric antigen receptor (CAR) with an IL4Rα antibody-inducible domain (AID). This embodiment describes a novel CAR backbone containing domains 1 and 2 of interleukin-4 receptor α (IL4Ra), with domain 1 or domain 2 as an antibody-inducible domain, and the expression and subsequent activation of the novel CAR on recombinant Jurkat cells (immortalized human T cells) using the materials and methods of Example 1.

[0238] Design coding standard CAR or CAR containing IL4Ra and expression box with EGFRt tag ( Figure 11 Subsequently, a lentiviral vector was generated for CAR expression on transduced T cells. Figure 12 Cartoon diagrams of the standard CAR expressed on the cell surface and five different IL4Ra-containing CARs are shown. Combinations of the IL4Ra domain with the first adapter and optional second adapter are shown in Table 5-1a.

[0239] Table 5-1a. Antibody-inducible domain and flanking sequences of CARs containing IL4Ra

[0240] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies, and tag and CAR expression was assessed by flow cytometry. As shown in Tables 5-1a and 5-1b, more than 50% of the transduced Jurkat cell population expressed either the EGFR tag or a standard CAR or an IL4Ra-containing CAR.

[0241] Table 5-1b. Cells engineered to express CAR containing IL4Ra

[0242] Then, the activation of transduced Jurkat cells was assessed by measuring CD25 (interleukin-2 receptor α chain) expression in transduced cells (K562 cells expressing the CAR antigen) at a target-to-effect cell ratio of 4:1 (K562:Jurkat) with or without the antigen. Figure 13A As shown, when Jurkat cells expressing CAR are co-cultured with K562 cells expressing antigen, Jurkat cells expressing either the standard CAR or an IL4Ra-containing CAR are specifically activated through the common antigen-binding domain of the CARs.

[0243] The activation of transduced Jurkat cells was further evaluated by measuring CD25 expression in transduced cells in the presence or absence of antibodies capable of binding to the IL4Ra domain of some CARs disclosed herein. Figure 13B As shown, only a very small percentage of untransduced Jurkat cells (simulated) and Jurkat cells expressing standard CARs expressed CD25, with or without antibody stimulation. Conversely, Jurkat cells expressing IL4Ra-containing CAR1-4 (containing domain 1 or domains 1 and 2) were activated by culturing these cells in the presence of anti-IL4Ra antibodies. Neither anti-VEGFR2 nor anti-HER2 antibodies induced CD25 expression at levels higher than the background levels observed in the absence of antibodies (no stimulation) on cells expressing IL4Ra-containing CARs. Similarly, Figure 13B This demonstrates that T cells expressing VEGFR2-containing CARs can be activated in a CAR-specific manner upon contact with anti-VEGFR2 antibodies.

[0244] Example 6: T cells engineered to express a chimeric antigen receptor (CAR) with an antibody-inducible domain (AID) of CD4 This embodiment describes a novel CAR backbone containing domains 1 and 2 of differentiation cluster 4 (CD4), domain 1 or domain 2 as an antibody-inducible domain, and the expression and subsequent activation of the novel CAR on recombinant Jurkat cells (immortalized human T cells) using the materials and methods of Example 1.

[0245] Design coding standard CAR or CAR containing CD4 and EGFRt tag expression box ( Figure 14 Subsequently, a lentiviral vector was generated for CAR expression on transduced T cells. Figure 15 Cartoons are shown illustrating the standard CAR expressed on the cell surface and seven different CD4-containing CARs. Combinations of the CD4 domain with the first adapter and optional second adapter are shown in Table 6-1a.

[0246] Table 6-1a. Antibody-inducible domain and flanking sequences of CD4-containing CARs

[0247] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies, and tag and CAR expression was then assessed by flow cytometry. As shown in Tables 6-1a and 6-1b, more than 20% of the transduced Jurkat cell population expressed either the EGFR tag or one of the standard CAR or CD4-containing CARs.

[0248] Table 6-1b. Cells engineered to express CD4-containing CARs

[0249] Then, the activation of transduced Jurkat cells was assessed by measuring CD25 (interleukin-2 receptor α chain) expression in transduced cells (K562 cells expressing the CAR antigen) at two different effector cell to target cell ratios (Jurkat:K562 ratio) in the presence or absence of the antigen. Figure 16 As shown, when Jurkat cells expressing CAR are co-cultured with K562 cells expressing antigen, Jurkat cells expressing either the standard CAR or a CD4-containing CAR are specifically activated through the common antigen-binding domain of the CARs.

[0250] Example 7: T cells engineered to express a chimeric antigen receptor (CAR) with an antibody-inducible domain (AID) of CD2. This embodiment describes a novel CAR backbone containing domains 1 and 2 of differentiation cluster 2 (CD2) or domain 1 as an antibody-inducible domain, and the expression and subsequent activation of the novel CAR on recombinant Jurkat cells (immortalized human T cells) using the materials and methods of Example 1.

[0251] Design coding standard CAR or CAR containing CD2 and EGFRt tag expression box ( Figure 17 Subsequently, a lentiviral vector was generated for CAR expression on transduced T cells. Figure 18 Cartoons are shown illustrating the standard CAR expressed on the cell surface and four different CD2-containing CARs. Combinations of the CD2 domain with the first adapter and optional second adapter are shown in Table 7-1a.

[0252] Table 7-1a. Antibody-inducible domain and flanking sequences of CD2-containing CARs

[0253] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies, and tag and CAR expression was then assessed by flow cytometry. As shown in Tables 7-1a and 7-1b, more than 30% of the transduced Jurkat cell population expressed either the EGFR tag or a standard CAR or a CD2-containing CAR.

[0254] Table 7-1b. Cells engineered to express CD2-containing CARs

[0255] Then, the activation of transduced Jurkat cells was assessed by measuring CD25 (interleukin-2 receptor α chain) expression in transduced cells (K562 cells expressing the CAR antigen) at different effector-to-target cell (K562:Jurkat) ratios, in the presence or absence of the antigen. Figure 19 As shown, when Jurkat cells expressing CARs are co-cultured with K562 cells expressing antigens, Jurkat cells expressing either the standard CAR or a CD2-containing CAR are activated via the common antigen-binding domain of the CARs. However, the percentage of Jurkat cells expressing CD2 CAR1, CD2 CAR2, or CD2 CAR3 (background level) in the absence of the antigen is higher than the percentage observed with Jurkat cells expressing CARs containing other antibody-inducible domains.

[0256] Example 8: Expansion and characterization of T cells engineered to express chimeric antigen receptors (CARs) with antibody-inducible domains (AIDs). This embodiment describes the evaluation of human regulatory T cells (Tregs) engineered to express CARs containing the antibody-inducible domain (AID) as shown in Table 8-1, as well as a first adapter and an optional second adapter.

[0257] Table 8-1. Antibody-inducible domains and flanking sequences of CAR^

[0258] ^SEQ ID: G4Sx3 (NO:46); G3SG3 (NO:45); VEGFR2 (NO:31); IL4Ra(D1-D2) (NO:36); IL4Ra(D1) (NO:34); IgG4 hinge (NO:23); CD28TM (NO:25); and CD28TM(M5) (NO:42).

[0259] All constructs in Table 8-1 include the IL5Ra tag, and all constructs except the control SBT-1026, which lacks the CAR, include a CAR with the same antigen-binding domain. The Treg expressing the CAR containing the VEGFR2 domain (SBT-1927) can be stimulated with ramucirumab (anti-VEGFR antibody). The Tregs expressing the CAR containing the IL4Ra domain (SBT-1934, SBT-1935, and SBT-1936) can be stimulated with dupilumab (anti-IL4R antibody).

[0260] On day 0, Tregs were isolated from healthy human donors and activated with CTS™ Dynabeads™ Treg Xpander reagent (Gibco) at a 1:1 bead-to-cell ratio in the presence of recombinant human IL-2 (300 IU / mL). On day 3, Tregs were transduced using a lentiviral expression vector. Transduced Tregs were amplified by adding fresh medium containing IL-2, restimulated on day 9, further amplified, and subsequently harvested. Various restimulation conditions were tested: IL-2 alone (i); CD3 / CD28 beads 1:1 (ii); VEGFR or IL4R Ab biotin-streptavidin beads 1:1 (iii) or 2:1 (iv); VEGFR or IL4R Ab biotin 3.5 mcg / ml (v) or 7 mcg / ml (vi).

[0261] like Figure 20 As shown, transduced Tregs (SBT-1927) expressing CARs with VEGFR2 antibody-inducible domains remained viable and expanded well when stimulated with CD3 / CD28 beads or VEGFR Ab beads at a 1:1 cell-to-bead ratio. When Tregs were stimulated in solution with VEGFR Ab (without bead conjugation), minimal expansion was observed.

[0262] Following restimulation with IL4R Ab, the activity of transduced Tregs (SBT-1934 or SBT-1936) expressing CARs with IL4R antibody-inducible domains showed greater variability (see [link to relevant documentation]). Figure 21 and Figure 22 Although Treg cells expanded at a 1:1 cell-to-bead ratio in the presence of IL4R beads, cell proliferation was delayed and the number of Treg cells was lower than when restimulated with CD3 / CD28 beads.

[0263] Notably, such as Figure 23 As shown in Table 8-2, stimulation with bead-bound VEGFR Ab resulted in the enrichment and proliferation of Treg (SBT-1927) expressing a CAR with a VEGFR2 antibody-inducible domain.

[0264] Table 8-2. Percentage of tag-positive Tregs (indicating VEGFR2-containing CAR expression SBT-1927)

[0265] like Figure 24 , Figure 25 As shown in Tables 8-3 and 8-4, stimulation with bead-bound IL4R Ab resulted in the enrichment and amplification of Tregs (SBT-1934 and SBT-1936) expressing CARs with IL4R antibody-inducible domains.

[0266] Table 8-3. Percentage of tag-positive Tregs (indicating IL4R-containing CAR expression SBT-1934)

[0267] Table 8-4. Percentage of tag-positive Tregs (indicating IL4R-containing CAR expression SBT-1936)

[0268] After staining, flow cytometry was used to further characterize the expression of CD4, CD25, CD27, CD70, CD71, IL5Ra tag (CAR tag), G4S (flexible linker), FOXP3 and Helios in Treg cells engineered to express CARs with antibody-inducible domains.

[0269] On day 9, prior to restimulation, the expression of the G4S flexible linker and CAR tag was measured. Furthermore, as shown in Tables 8-5, immunophenotypic analysis of Tregs was performed targeting the expression of CD27, CD70, and CD71 (TfR1, transferrin receptor protein 1).

[0270] Table 8-5. Translocators and CAR-tag expression on Tregs before restimulation

[0271] On day 14 of the expansion study (5 days after restimulation), Treg cells engineered to express CARs with antibody-inducible domains were evaluated by flow cytometry. Table 8-6 shows the results of SBT-1934 Tregs (expressing CARs with VEGFR domains) after restimulation under various conditions. Table 8-7 shows the results of SBT-1934 Tregs (expressing CARs with IL4R domains) after restimulation under various conditions. Table 8-8 shows the results of SBT-1936 Tregs (expressing CARs with IL4R domains) after restimulation under various conditions.

[0272] Table 8-6. Immunophenotypes of Tregs (SBT-1927) engineered to express CARs containing VEGFR domains

[0273] Table 8-7. Immunophenotypes of Tregs (SBT-1934) engineered to express CARs containing the IL4R domain

[0274] Table 8-8. Immunophenotypes of Tregs (SBT-1936) engineered to express CARs containing the IL4R domain

[0275] To evaluate the efficacy of Tregs (SBT-1934) engineered to express IL4R-domain-containing CARs by restimulation with IL4R antibody beads at a 1:1 cell-to-bead ratio, the expression of the CAR (via staining against the G4S linker) and activation marker (CD71) in Helios+ Tregs was assessed. As shown in Tables 8-9, Tregs expressing high levels of FOXP3 were enriched in activated cells (CD71+) expressing the IL4R-domain-containing CAR (G4S+). Conversely, Tregs expressing low levels of FOXP3 showed significantly fewer activated cells (CD71+) expressing the IL4R-domain-containing CAR (G4S+).

[0276] Table 8-9. Correlation between CAR expression levels and activation levels

[0277] In summary, the CAR backbone disclosed herein can be used to generate engineered CAR-T cells that can be stimulated via CAR binding to an antibody-inducible domain, rather than solely via CAR binding to an antigen-binding domain or via TCR cross-linking with CD3 and CD28. While stimulation via TCR cross-linking with anti-CD3 and CD28 does not result in changes in CAR-tagged positive T cells, stimulation via the antibody-inducible domain on the CAR backbone induces a highly enriched population of CAR-T cells. Similarly, the data presented herein indicate the benefits of using such antibodies targeting the extracellular helper domains of CAR for the in vitro production of CAR-expressing T cells, such as CAR-Tregs.

[0278] sequence >SEQ ID NO:1 (GMCSF signal peptide) Homo sapiens MLLLVTSLLLCELPHPAFLLIP >SEQ ID NO:2 (CD8a signal peptide) Homo sapiens MALPVTALLLPLALLLHAARP >SEQ ID NO:3 (SBT01VH = SBT01VHM) Homo sapiens - 116aa HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS >SEQ ID NO:4 (GVH = SBT01VHG) Homo sapiens - 116aa QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS >SEQ ID NO:5 (U01VH = UCSF_BVCA VH) Homo sapiens - 119aa EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSS >>SEQ ID NO:6 (SBT01VL = SBT01VLM) Homo sapiens - 109aa SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVLR >SEQ ID NO:7 (GVL = SBT01VLG) Homo sapiens - 109aa SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR >SEQ ID NO:8 (U01VL = UCSF BVCA VL) Homo sapiens - 111aa AIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA >SEQ ID NO:9 (FM63VL) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEIT >SEQ ID NO:10 (FM63VH) EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS >SEQ ID NO:11 (ida VL) DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK >SEQ ID NO:12 (ida VH) QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS SEQ ID NO:13 synthetic glycine linker GGGGSGGGGSGGGGS >SEQ ID NO:14 synthetic Whitlow linker GSTSGSGKPGSGEGSTKG >SEQ ID NO:15 synthetic ABpur linker ASSGGSTSGSGKPGSGEGSSGSAR >SEQ ID NO:16 – BVCA CV ABD (254aa) synthesis EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARAIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA >SEQ ID NO:17 -tisa CD19 ABD (242aa) synthesis DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS >SEQ ID NO:18 - liso CD19 ABD (245aa) synthesis DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS >SEQ ID NO:19 - ida BCMA ABD (246aa) synthesis DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS >SEQ ID NO:20 - cilta BCMA ABD (242aa) synthesis QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSS >SEQ ID NO:21 - CD8a hinge - Homo sapiens TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD >SEQ ID NO:22 – CD28 hinge - Homo sapiens IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP >SEQ ID NO:23 – IgG4 hinge - Homo sapiens ("P") ESKYGPPCPPCP >SEQ ID NO:24 - CD8a transmembrane – Homo sapiens IYIWAPLAGTCGVLLLSLVITLYC >SEQ ID NO:25 – CD28 transmembrane WT – Homo sapiens (27aa) FWVLVVVGGVLACYSLLVTVAFIIFWV >SEQ ID NO:26 (CD28 costimulatory domain) Homo sapiens RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >SEQ ID NO:27 (41BB costimulatory domain) Homo sapiens KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL >SEQ ID NO:28 (CD3ζ intracellular signal transduction domain) Homo sapiens RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Synthesis of SEQ ID NO:29 (CD28+CD3ζ) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Synthesis of SEQ ID NO:30 (41BB+CD3ζ) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:31 (VEGFR2 extracellular amino acid sequence - ramucirumab binding domain) GYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFV SEQ ID NO:32 (HER2 extracellular amino acid sequence - trastuzumab binding domain) CHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRAS SEQ ID NO:33 (PDGFRα extracellular amino acid sequence - olatozumab binding domain) FNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGFIEIKPTFSQLEAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLIENLTEITT DVEKIQEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELLTQVPSSILDLVDDHHGSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWTILANNVSNIITEIHSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRSELTVA SEQ ID NO:34 (IL4Rα D1 extracellular amino acid sequence) MKVLQEPTCVSDYMSISTCEWKMNGPTNCSTELRLLYQLVFLLSEAHTCIPENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQLLWKGSFKPSEHVKPRAPG SEQ ID NO:35 (IL4Rα D2 extracellular amino acid sequence) EHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQH SEQ ID NO:36 (IL4Rα D1-D2 extracellular amino acid sequence) MKVLQEPTCVSDYMSISTCEWKMNGPTNCSTELRLLYQLVFLLSEAHTCIPENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQH SEQ ID NO:37 (CD4 D1 extracellular amino acid sequence) KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGL SEQ ID NO:38 (CD4 D2 extracellular amino acid sequence) VQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLA SEQ ID NO:39 (CD4 D1-D2 extracellular amino acid sequence) KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLA SEQ ID NO:40 (CD2 D1 extracellular amino acid sequence) KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVS SEQ ID NO:41 (CD2 D1-D2 extracellular amino acid sequence) KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD SEQ ID NO:42 (CD28-modified transmembrane domain amino acid sequence) FWVLVVVGGVLALLLLLVLVAFIIFWV (C13L, Y14L, S15L, and T19L) SEQ ID NO:43 FWVLVVVGGVLAX1X2X3LLVX4VAFIIFWV (SEQ ID NO:43), Where X1 is C or L, X2 is Y or L, X3 is S or L, and X4 is T or L. Common sequences of flexible peptide linkers [XaXbXcXdXeXfXg]n, where Xa, Xb, Xc, and Xd are independently selected from G and S. Xe, Xf, and Xg are independently selected from G, S, and do not exist, and n is 1, 2, or 3.

[0279] SEQ ID NO:45 (Flexible peptide linker 1) GGGSGGG SEQ ID NO:46 (Flexible peptide linker 2) GGGGSGGGGSGGGGS.

Claims

1. A chimeric antigen receptor (CAR) comprising: Antigen-binding domain, First connector, Antibody-inducible domain, Transmembrane domains, and Intracellular signal transduction domains The antibody-inducible domain is a polypeptide with a length of about 60 to about 360 amino acids.

2. The CAR of claim 1, wherein the antibody-inducible domain comprises members of the group consisting of: vascular endothelial growth factor receptor 2 (VEGFR2) domain, human epidermal growth factor receptor 2 (HER2) domain, platelet-derived growth factor receptor-α (PDGFRα) domain, interleukin-4 receptor-α (IL-4Rα) domain, CD4 domain, and CD2 domain.

3. The CAR of claim 2, wherein the antibody-inducible domain comprises a VEGFR2 domain.

4. The CAR of claim 3, wherein the VEGFR2 domain is capable of being bound by an anti-VEGFR2 antibody selected from the group consisting of ramucirumab and aracizumab, or a VEGFR2 binding fragment thereof, optionally wherein the VEGFR2 domain is capable of being bound by ramucirumab.

5. The CAR of claim 4, wherein the VEGFR2 domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

31.

6. The CAR of claim 2, wherein the antibody-inducible domain comprises the HER2 domain.

7. The CAR of claim 6, wherein the HER2 domain is capable of being bound by an anti-HER2 antibody or its HER2-binding fragment selected from the group consisting of trastuzumab, maggotuximab, pertuzumab and telmituzumab, optionally wherein the HER2 domain is capable of being bound by trastuzumab.

8. The CAR of claim 7, wherein the HER2 domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

32.

9. The CAR of claim 2, wherein the antibody-inducible domain comprises a PDGFRα domain.

10. The CAR of claim 9, wherein the PDGFRα domain is capable of being bound by an anti-PDGFRα antibody selected from the group consisting of olatuzumab and tovertumab, or a PDGFRα binding fragment thereof.

11. The CAR of claim 10, wherein the PDGFRα domain comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

33.

12. The CAR of claim 2, wherein the antibody-inducible domain comprises an IL-4Rα domain.

13. The CAR of claim 12, wherein the IL-4Rα domain is capable of being bound by an anti-IL-4Rα antibody selected from the group consisting of polyumab and pacozumab, or an IL-4Rα binding fragment thereof.

14. The CAR of claim 13, wherein the IL-4Rα domain comprises: i) an amino acid sequence of SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:

36.

15. The CAR of claim 2, wherein the antibody-inducible domain comprises a CD4 domain.

16. The CAR of claim 15, wherein the CD4 domain is capable of being bound by an anti-CD4 antibody or its CD4-binding fragment selected from the group consisting of: cilizumab, crixacillin, ibalizumab, kelixim, prixim, trelizumab, zamumab, and sizulvolimumab.

17. The CAR of claim 16, wherein the CD4 domain comprises: i) an amino acid sequence selected from SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:

39.

18. The CAR of claim 2, wherein the antibody-inducible domain comprises a CD2 domain.

19. The CAR of claim 18, wherein the CD2 domain is capable of being bound by an anti-CD2 antibody or its CD2 binding fragment, wherein the anti-CD2 antibody is ciplezumab.

20. The CAR of claim 19, wherein the CD2 domain comprises: i) an amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41; or ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:40 or SEQ ID NO:

41.

21. The CAR of any one of claims 1 to 20, wherein T cells expressing the CAR can be activated when an antibody or a fragment thereof binds to the antibody-inducible domain.

22. The CAR of any one of claims 1 to 21, further comprising a second linker located between the antibody-inducible domain and the transmembrane domain.

23. The CAR of any one of claims 1 to 21, wherein the first linker and / or the second linker is a flexible polypeptide linker having a length of 4 to 24 amino acids.

24. The CAR of claim 23, wherein the flexible polypeptide linker comprises the amino acid sequence [XaXbXcXdXeXfXg]n, wherein Xa, Xb, Xc, and Xd are independently selected from G and S, Xe, Xf, and Xg are independently selected from G, S, and are absent, and n is 1, 2, or 3, optionally wherein the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:

46.

25. The CAR of any one of claims 1 to 21, wherein the first connector and / or the second connector is a hinge region having a length of 10 to 50 amino acids.

26. The CAR of claim 25, wherein the first connector is the flexible polypeptide connector, and the second connector is a hinge region having a length of 10 to 50 amino acids.

27. The CAR of claim 25 or claim 26, wherein the hinge region is selected from the group consisting of the CD8a hinge, the CD28 hinge region, and the IgG4 hinge region.

28. The CAR of claim 27, wherein the hinge region is a CD8a hinge region comprising the amino acid sequence of SEQ ID NO:

21.

29. The CAR of claim 28, wherein the hinge region is a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:

22.

30. The CAR of claim 29, wherein the hinge region is an IgG4 hinge region comprising the amino acid sequence of SEQ ID NO:

23.

31. The CAR of any one of claims 1 to 30, wherein the transmembrane domain is a CD28 transmembrane domain or a CD8a transmembrane domain.

32. The CAR of claim 31, wherein the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

43.

33. The CAR of claim 31, wherein the transmembrane domain is a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO:

24.

34. The CAR of any one of claims 1 to 33, wherein the intracellular signal transduction domain comprises the CD3ζ signal transduction domain.

35. The CAR of claim 34, wherein the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:

28.

36. The CAR of claim 34 or claim 35, wherein the intracellular signal transduction domain further comprises a co-stimulatory domain.

37. The CAR of claim 36, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain.

38. The CAR of claim 37, wherein the co-stimulatory domain comprises the CD28 co-stimulatory domain.

39. The CAR of claim 38, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:

29.

40. The CAR of claim 37, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain.

41. The CAR of claim 40, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:

30.

42. The CAR according to any one of claims 1 to 41, further comprising a signal peptide.

43. The CAR of claim 42, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.

44. The CAR of any one of claims 1 to 43, wherein the antigen-binding domain comprises a single-chain antibody fragment (scFV) or a single-domain antibody (sdAb).

45. The CAR of claim 44, wherein the antigen-binding domain comprises scFv.

46. ​​The CAR of claim 44, wherein the antigen-binding domain comprises an sdAb, and optionally wherein the sdAb comprises a heavy chain variable region fragment (VHH).

47. The CAR of any one of claims 1 to 46, wherein the antigen-binding domain binds to an antigen selected from the group consisting of citrullinated vimentin (CV), CD19, and BCMA.

48. The CAR of claim 47, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and the light chain CDR of the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; (ii) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; or (iii) The amino acid sequence of SEQ ID NO:

16.

49. The CAR of claim 47, wherein the antigen-binding domain binds to CD19, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:10 and the light chain CDR of the amino acid sequence of SEQ ID NO:9; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or (iii) The amino acid sequence of SEQ ID NO:17 or SEQ ID NO:

18.

50. The CAR of claim 47, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:12 and the light chain CDR of the amino acid sequence of SEQ ID NO:11; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:11; or (iii) The amino acid sequence of SEQ ID NO:19 or SEQ ID NO:

20.

51. A nucleic acid encoding a CAR as described in any one of claims 1 to 50.

52. An expression vector comprising an operative combination of the nucleic acid of claim 51 and an expression control sequence.

53. The expression vector of claim 52, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector, a retroviral vector, an adenovirus vector, or an adeno-associated virus vector.

54. The expression vector of claim 53, wherein the viral vector is a lentiviral vector.

55. The expression vector of claim 52, wherein the vector is a plasmid.

56. A host cell comprising the expression vector according to any one of claims 52 to 55.

57. A modified T cell engineered to express the CAR of any one of claims 1 to 50, wherein the modified T cell is a mammalian T cell.

58. A pharmaceutical composition comprising a plurality of modified T cells as described in claim 57 and a pharmaceutically acceptable excipient.

59. The pharmaceutical composition of claim 58, wherein the modified T cells are human T cells.

60. The pharmaceutical composition of claim 59, wherein the human T cells are regulatory T cells (Tregs) that are CD4+, CD25+, and CD12710.

61. The pharmaceutical composition of claim 60, wherein the human Treg is FOXP3+ and HELIOS+.

62. A method of treating a disease or ailment, comprising administering to a subject in need an effective amount of the pharmaceutical composition of any one of claims 58 to 61.

63. A method of treating a subject suffering from an autoimmune disease or inflammatory condition, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 58 to 61, wherein the CAR expressed by the modified T cells comprises an antigen-binding domain that binds to an antigen expressed on the cells of the autoimmune disease or the inflammatory condition.

64. A method of treating a subject with cancer, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 58 or claim 59, wherein the CAR expressed by the modified T cells comprises an antigen-binding domain that binds to an antigen expressed on the cancer cells.

65. A kit comprising a container containing a pharmaceutical composition according to any one of claims 58 to 61, the container being in communication with an infusion chamber via a fluid conduit, wherein the infusion chamber is configured to be in communication with an intravenous injection needle via a fluid conduit.

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