CN118234501A

CN118234501A - Synovial extracellular matrix-specific chimeric antigen receptor for targeting regulatory T cells for the treatment of autoimmune diseases

Info

Publication number: CN118234501A
Application number: CN202280061407.8A
Authority: CN
Inventors: J·马塔埃; A-R·范德乌斯特德弗里斯; J·贝尔克; V·马姆斯特罗姆; K·霍珀; R·约翰逊; L·克拉雷斯科格
Original assignee: Kurara Co; Sonoma Biotherapy Co
Current assignee: Kurara Co; Sonoma Biotherapy Co
Priority date: 2021-07-29
Filing date: 2022-07-29
Publication date: 2024-06-21

Abstract

Disclosed herein are chimeric antigen receptors ("CARs") comprising an antigen binding site that recognizes a citrullinated polypeptide. Citrullinated polypeptides such as citrullinated vimentin, fibrinogen, and filaggrin are expressed in the synovium of subjects suffering from rheumatoid arthritis. T cells and in particular Treg cells expressing these CARs are further disclosed. Administration of these CAR-T cells can be used to treat rheumatoid arthritis and other diseases associated with citrullinated peptides.

Description

Synovial extracellular matrix-specific chimeric antigen receptor for targeting regulatory T cells for the treatment of autoimmune diseases

Cross Reference to Related Applications

The application claims the benefit of U.S. provisional application No. 63/227,320 filed on 7.29 of 2021 and U.S. provisional application No. 63/339,361 filed on 5.6 of 2022, each of which is hereby incorporated by reference in its entirety.

Reference to an electronic sequence Listing

The contents of the electronic sequence listing (23775501040 seqlist. Xml; size: 50,843 bytes; and date of creation: 2022, 7, 28 days) are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to chimeric antigen receptors that react with citrullinated antigens and regulatory T cells expressing these receptors for use in the treatment of autoimmune diseases.

Background

Autoimmune diseases affect a large number of people. For example, rheumatoid Arthritis (RA) is a chronic inflammatory disease that targets peripheral joints, resulting in bone erosion, impaired mobility and reduced quality of life. It is affecting 0.5% -1% of the population worldwide, and the incidence is continuously rising. The pathogenesis of RA is mainly located in synovial joints, where immune cells composed of T cells, B cells, macrophages and dendritic cells impregnate the lubricating membrane. In addition, the fibroblast-like synoviocytes present in the synovial sub-layer proliferate and cause cartilage damage.

Synovial hyperplasia in rheumatoid arthritis results in immune cells soaking the lubricating film and subsequent cartilage damage and bone erosion.

There is currently no cure for RA and many other autoimmune disorders. RA patients often require life-long treatment, which, in addition to being extremely expensive, may also cause long-term serious side effects such as infection and rheumatoid arthritis risk.

Drawings

The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the relevant art to make and use the embodiments and other embodiments as are apparent to the person skilled in the art. The present invention will be described in more detail in connection with the following figures, wherein:

fig. 1: initial data containing MND promoter and EGFRt scaffold showed that CV CAR BVCA1 and SBT01G had the strongest response to plate-bound full-length CV (n=2).

Fig. 2: assays using soluble full-length CV showed only CV CAR BVCA a1 and SBT01G-HL dose response (n=1).

Fig. 3: the assay with soluble bead-binding peptides showed binding specificity (n=1).

Fig. 4: MND-SBT01G showed a stronger response to plate-bound, antibody-captured CV (but not soluble CV) than MND-BVCA 1.

Fig. 5: testing of the first synovial fluid from the innovative study showed that SBT01G had a stronger response than BVCA.

Fig. 6: further testing of 15 synovial samples from swedish patients showed SBT01G to be stronger than BVCA1 for the samples that produced the response.

Fig. 7: primary Treg responses to Synovial Fluid (SF) showed that SBT01G was more sensitive to SF from RA patients than BVCA a 1.

Fig. 8: SBT01G, instead of BVCA1, was also able to react to plate-bound full-length PAD2 citrullinated fibrinogen.

Figure 9 SBT01G CAR and Treg as effectors reacted to citrullinated fibrinogen, but BVCA a did not.

Fig. 10A-10B: both the EF1A and MND promoters demonstrate a functional response of the CV CAR to the soluble bead-binding peptide. Thus, the CAR promoter does not affect Treg phenotype.

Fig. 11: scFv linkers have little effect on SBT01G CAR-based function.

Fig. 12A-12B: SBT01G was expressed by a higher percentage of cells than BVCA a, but BVCA a and SBT01G CAR-T cells had similar FoxP3 and Helios characteristics.

Fig. 13: CV-CAR Treg cells (SBT 01G) are activated by Citrullinated Vimentin (CV) rather than non-transduced Treg cells. Activation was demonstrated by proliferation, CD71 expression and specific increase in target antigen secretion by IL-10.

Fig. 14: CV-CAR Treg cells respond to citrullinated proteins in synovial fluid from most RA patients. In contrast, CV-CAR Treg cells do not respond to synovial fluid from normal controls (subjects not suffering from RA).

Fig. 15: CV-CAR Treg cells (SBT 01G) from both donors, but not non-transduced Treg cells, were specifically activated by synovial fluid from RA patients.

Fig. 16A-16B: assessment of the inhibitory function of CV-CAR Treg cells. Figure 16A shows that CV-CAR Treg cells were able to inhibit proliferation of CD3/CD28 pre-activated Teff cells in the presence (but not in the absence) of CV. Fig. 16B shows that CV-CAR Treg cells were able to inhibit proliferation of CD19-CAR Teff cells in the presence of CV, whereas non-transduced Treg cells were unable.

Fig. 17: a timeline of in vivo activation of human CV-CAR Treg cells in a Lipopolysaccharide (LPS) induced murine lung inflammation model is shown. Briefly, human CV-CAR Treg cells were administered Intravenously (IV) on day 0, human IL-2 was administered Intraperitoneally (IP) twice daily, and LPS was administered Intranasally (IN) on days 0, 1, 6, and 12. On day 13, mice were sacrificed and organs harvested to facilitate analysis of Treg cells.

Fig. 18A-18B: flow cytometry plots comparing Epidermal Growth Factor (EGFR) expression to CELL TRACE Violet (CTV) levels of human CV-CAR Treg cells are shown. Figure 18A shows how the proliferation rate of CV-CAR tregs (egfr+) is determined. Specifically, the proliferation ratio is equal to the% of EGFR+, CTV-cells divided by the% of EGFR+, CTV-cells. Fig. 18B shows how fold change in EGFR ratio is determined.

Fig. 19A-19B: CV-CAR Tregs proliferate in LPS-induced murine lung inflammation models but not in control recipients of PBS. Fig. 19A shows the absolute numbers of cd45+, cd3+ tregs in the lungs of the various study groups, and fig. 19B shows the proliferation rates of tregs in the lungs of the various study groups.

Disclosure of Invention

Regulatory T cells (tregs) are defective in RA patients and in mouse models. Thus, treg-based Adoptive Cell Therapy (ACT) represents a promising approach to RA. In fact, treg-based ACT reverses diseases in RA animal models. In this study, antibodies isolated from RA patients were used to engineer CARs specific for Citrullinated Vimentin (CV) and other post-translationally modified proteins found in large amounts and almost entirely in the synovial extracellular matrix (ECM) of the affected joint.

Disclosed herein are Chimeric Antigen Receptors (CARs) that specifically recognize antigens associated with autoimmune diseases. In particular, the CAR may be specific for a post-translationally modified antigen. In particular, CARs can specifically bind to citrullinated polypeptides including vimentin, citrullinated filaggrin, and citrullinated fibrinogen.

Chimeric Antigen Receptors (CARs) are engineered to specifically target post-translationally modified proteins (i.e., citrullinated vimentin, citrullinated filaggrin, and citrullinated fibrinogen) that are expressed in the extracellular matrix of inflamed joints in Rheumatoid Arthritis (RA) patients. In some embodiments, the single chain variable fragment (scFv) portion of the CAR is obtained from an antibody that has high specificity for citrullinated protein isolated from peripheral blood of RA patients. In one embodiment, a specific scFv chain is inserted into the second generation CAR construct. In some embodiments, the scFv chain is inserted into a CAR construct cloned in a lentiviral vector. In particular embodiments, references to antibodies are applicable to the antigen binding domains of the CARs of the present disclosure unless the context indicates otherwise.

Detailed Description

I. Definition of the definition

Unless otherwise indicated, terms and symbols of biochemistry, nucleic acid chemistry, molecular biology, developmental biology and molecular genetics follow terms and symbols of art standard treaties and literal materials such as: sambrook et al, molecular Cloning: A Laboratory Manual, version 2 (Cold Spring Harbor Press, 1989); alberts and Singer, developmental Biology, eighth edition (Sinauer Associates inc., sunderland, MA, 2006); kornberg and Baker, DNA Replication, second edition (W.H. Freeman, new York, 1992); gaits edit Oligonucleotide Synthesis: A PRACTICAL Approx (IRL Press, oxford, 1984); lehninger, biochemistry, second edition (Worth Publishers, new York, 1975); eckstein, eds., oligonucleotides and Analogs: A PRACTICAL Approx (Oxford University Press, new York, 1991); etc.

As used herein, the terms "antigen," "immunogen," and "antibody target" refer to a molecule, compound, or complex that is recognized by an antibody (i.e., can be bound by an antibody). The term may refer to any molecule that can be recognized by an antibody, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combination thereof (e.g., phosphorylated or glycosylated polypeptide, etc.). The skilled artisan will appreciate that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody.

As used herein, the term "epitope" refers to a localized site on an antigen that is recognized and bound by an antibody. An epitope may comprise several amino acids or multiple parts of several amino acids, e.g. 5 or 6 or more (e.g. 20 or more) amino acids or multiple parts of those amino acids. In some cases, an epitope includes a non-protein component, such as a component from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope may be composed of contiguous amino acids or amino acids from different portions of the protein that are accessed by protein folding (e.g., discontinuous epitopes).

As used herein, the term "antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene that specifically binds to and recognizes an antigen. Typically, the "variable region" contains the antigen binding region of an antibody (or functional equivalent thereof) and is most critical in terms of the specificity and affinity of binding. Exemplary immunoglobulin (antibody) structural units comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" (about 50-70 kD) chain.

Antibodies can be any of (i) five major classes of immunoglobulins based on the identity of their heavy chain constant domains-alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), and mu (IgM), or (ii) subclasses (isotypes) thereof (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2). The light chain may be lambda or kappa.

The following is a non-exhaustive list of different antibody forms, each retaining antigen binding activity:

(1) Intact immunoglobulins (also known as "intact" antibodies) (two light chains and two heavy chains, e.g., tetramers);

(2) Immunoglobulin polypeptides (light or heavy chain);

(3) Antibody fragments, such as Fv (monovalent or bivalent variable region fragments, and which may cover only the variable region (e.g., V _L and/or V _H))、Fab(V_LC_L V_HC_H)、F(ab')2、Fv(V_LV_H), scFv (single chain Fv)

(A polypeptide comprising V _L and V _H linked by a linker, such as a peptide linker), (scFv) 2, sc (Fv) 2, bispecific (scFv) 2, minibodies (sc (Fv) 2 fused to a CH3 domain), diabodies (non-covalent dimers of single chain Fv (scFv) fragments consisting of heavy chain Variable (VH) and light chain Variable (VL) regions linked by a small peptide linker), triabodies (trivalent sc (Fv) 3 or trispecific sc (Fv) 3);

(4) Multivalent antibodies (antibodies comprising binding regions that bind two different epitopes or proteins, e.g., a "scorpion" antibody);

(5) A fusion protein comprising a binding portion of an immunoglobulin fused to another amino acid sequence (e.g., a fluorescent protein); and

(6) Heavy chain-only antibodies or antibody fragments, which have only two heavy chains and lack two light chains typically found in antibodies.

The production and characterization of tandem scFv and diabodies is described, for example, in Asano et al (2011) J biol. Chem.286:1812; kenanova et al (2010) Prot ENG DESIGN SEL 23:789; asano et al (2008) Prot ENG DESIGN SEL 21:597.

As used herein, the phrase "CDR sequence set" refers to 3 heavy chain and/or 3 light chain CDRs of a particular antibody described herein. A "light chain" set of CDR sequences refers to the light chain CDR sequences. The "heavy chain" set of CDR sequences refers to the heavy chain CDR sequences. The "complete" set of CDR sequences refers to both heavy and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignment.

As used herein, the term "chimeric antibody" refers to an antibody having amino acid sequences derived from two or more species. In one embodiment, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies of desired specificity, affinity, and capacity derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous sequences derived from another species (typically in a subject receiving therapy, e.g., human) to avoid eliciting an immune response.

As used herein, the term "humanized antibody" refers to a chimeric antibody in which CDRs obtained from VH and VL regions of a non-human antibody having the desired specificity, affinity, and capacity are grafted onto human framework sequences. In one embodiment, the framework residues of the humanized antibodies are modified to improve and optimize antibody specificity, affinity, and capacity. Humanization (i.e., non-human CDR sequences substituted for the corresponding sequences of a human antibody) can be performed as described, for example, in U.S. patent nos. 5,545,806;5,569,825;5,633,425;5,661,016; riechmann et al Nature 332:323-327 (1988); marks et al, bio/technology 10:779-783 (1992); morrison, nature 368:812-13 (1994); the procedure described in Fishwild et al Nature Biotec hnology 14:845-51 (1996) was followed.

As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to the human produced antibody prepared by any technique known in the art.

The specificity of binding can be defined in terms of the comparative dissociation constant (Kd) of an antibody (or other targeting moiety) against a target as compared to the dissociation constant of antibodies and other materials or molecules not normally associated in the environment. The larger (higher) Kd is the Kd that describes the lower affinity interactions. Conversely, a smaller (lower) Kd is one that describes higher affinity interactions or tighter binding. By way of example only, the Kd of an antibody for specific binding to a target may be femtomolar, picomolar, nanomolar or micromolar, and the Kd of an antibody binding-independent material may be millimolar or higher. The binding affinity may be in the micromolar range (kd=10 ^-4 to 10 ^-6), nanomolar range (kd=10 ^-7 M to 10 ^-9 M), picomolar range (kd=10 ^-10 M to 10 ^-12 M), or femtomolar (kd=10 ^-13 M to 10 ^-15 M).

As used herein, an antibody "binds" or "recognizes" an antigen or epitope if it binds to the antigen or epitope with a Kd of less than 10 ^-4 M (i.e., in the micromolar range). The term "bind" (e.g., an antibody that binds to a cancer cell) with respect to a cell type typically indicates that the agent binds to most cells in a pure population of those cells. For example, an antibody that binds to a given cell type typically binds to at least 2/3 of the cells in a given cell population (e.g., 67%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). In some cases, binding to a polypeptide can be determined by comparing the binding of an antibody to a cell presenting the polypeptide to the binding (or lack thereof) of an antibody to a cell that does not express the polypeptide. Those skilled in the art will recognize that some variation will occur depending on the method and/or threshold of determining binding. The affinity of an antibody for a target can be determined according to methods known in the art (e.g., as reviewed in Ernst et al Determination of Equilibrium Dissociation Constants,Therapeutic Monoclonal Anti bodies(Wiley&Sons ed.2009)).

As used herein, the term "greater affinity" as used herein refers to the relative degree of antibody binding, wherein antibody X binds to target Y more strongly (Kon) and/or has a smaller dissociation constant (Koff) than to target Z, and in this context, antibody X has a greater affinity to target Y than to Z. Likewise, the term "smaller affinity" refers herein to the extent of antibody binding, wherein antibody X binds less strongly to target Y than to target Z and/or has a greater dissociation constant than it does, and in this context, antibody X has a smaller affinity to target Y than to Z. The affinity of the binding between an antibody and its target antigen can be expressed as KA equal to 1/KD, where KD is equal to kon/koff. Kon and koff values may be measured using surface plasmon resonance techniques, for example using molecular affinity screening system (MASS-1) (Sierra Sensors GmbH, hamburg Germany). An antagonist or blocking antibody is an antibody that partially or completely blocks, inhibits or neutralizes biological activity associated with a target antigen relative to activity under similar physiological conditions in the absence of the antibody. Antagonists may be competitive, non-competitive or irreversible. Competitive antagonists are substances that bind to the natural ligand or receptor at the same site as the natural ligand-receptor interaction or allosterically bind in a manner that induces alterations to prevent normal binding. Non-competitive antagonists bind at sites that are different from the natural ligand-receptor interaction, but reduce KD or the signal generated by the interaction. Irreversible inhibitors cause covalent modification of the receptor, thereby preventing any subsequent binding.

As used herein, the term "avidity" refers to the overall stability of the binding complex between an antibody and a target antigen. It is governed by three factors, namely (i) the intrinsic affinity of the antibody for the antigen, (2) the potency of the antibody, and (3) the geometric arrangement of the interacting components. Affinity is the strength of interaction between an antibody and a single target, while avidity is the cumulative strength of multiple affinities. In one embodiment, the antibodies provided herein are bivalent.

As used herein, an antibody "preferentially binds" to a first antigen relative to a second antigen if the antibody binds to the first antigen with greater affinity than to the second antigen. Preferential binding may be any of at least 2-fold, 5-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold affinity.

As used herein, an antibody "specifically binds" or is "specific for" a target antigen or group of target antigens if it binds to each member of the target antigen or group of target antigens with an affinity of at least any one of 1×10^-6M、1×10^-7M、1×10^-8M、1×10^-9M、1×10^-10M、1×10^-11M、1×10^-12M and, for example, binds to each member of the target antigen or group of target antigens with an affinity of at least twice that of the compared non-target antigens. Typically, specific binding is characterized by binding to an antigen with sufficient affinity such that the antibody can be used as a diagnostic agent for detecting an antigen or epitope and/or as a therapeutic agent targeting an antigen or epitope.

As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and/or unnatural 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 structure (e.g., a structure that is stable by hydrogen bonding, a relationship between the secondary structure and a structure formed from more than one protein). The protein may be further modified by other attached moieties such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

As used herein, an amino acid sequence is "composed" of only the amino acids in that sequence.

As used herein, a first amino acid sequence "consists essentially of" a second amino acid sequence if (1) comprises the second amino acid sequence and (2) is no more than 1, no more than 2, or no more than 3 amino acids longer than the second amino acid sequence.

As used herein, a first amino acid sequence is a "fragment" of a second amino acid sequence if the second amino acid sequence comprises the first amino acid sequence. In certain embodiments, the first amino acid sequence, which is a fragment of the second amino acid sequence, may have no more than 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer amino acids than the second amino acid sequence.

As used herein, a "functional equivalent" of a reference amino acid sequence is a sequence that is not identical to the reference sequence but contains minor changes (e.g., insertions, deletions, or substitutions of one or more amino acids). Functionally equivalent sequences retain the function (e.g., immunogenicity) of the reference sequence to which they are equivalent. If a functionally equivalent amino acid sequence contains one or more amino acid substitutions relative to the reference sequence, these are typically conservative amino acid substitutions.

As used herein, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with another amino acid residue without eliminating the desired properties of the protein. Suitable conservative amino acid substitutions may be made by substituting another amino acid with similar hydrophobicity, polarity, and R chain length. See, e.g., watson et al, "Molecular Biology of the Gene," 4 th edition, 1987,The Benjamin/Cummings pub. Co., menlo Park, calif., page 224. Examples of conservative amino acid substitutions include the following (note that certain classes are not mutually exclusive):

As used herein, the term "substantially identical" refers to identity between a first amino acid sequence that contains a sufficient or minimal number of i) identical amino acid residues as aligned in a second amino acid sequence or ii) amino acid residues that are conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences have a common domain and/or a common functional activity and/or a common immunogenicity. For example, amino acid sequences that contain a common domain or antigen domain that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are said to be substantially or essentially identical. In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first nucleotide sequence and the second nucleotide sequence encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode polypeptides having the same immunogenic properties.

As used herein, a chemical entity (e.g., a polypeptide) is "substantially pure" or "isolated" if the chemical entity is the primary chemical entity of its species (e.g., polypeptide) in the composition. This includes chemical entities representing more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.9% or more than 99.99% of the chemical entities of that kind in the composition. The substantially purified fraction is a composition in which the target material comprises at least about 50% (by mole) of all macromolecular materials present. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition are the purified species of interest. If the composition consists essentially of a single macromolecular substance, the target substance is purified to be essentially homogeneous (no contaminating substances in the composition can be detected by conventional detection methods). For the purposes of this definition, solvent species, small molecules, stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species.

The phrase "isolated antibody" refers to an antibody produced in vivo or in vitro, which has been removed from a source from which the antibody was produced (e.g., an animal, hybridoma, or other cell line such as a recombinant insect, yeast, or bacterial cell that produces the antibody).

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 percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal alignment purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence to achieve optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Where 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 molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity =number of identical overlapping positions/total number of positions multiplied by 100%). In one embodiment, the two sequences are of the same length. The determination of the percent identity between two sequences can also be accomplished using a mathematical algorithm. Preferred non-limiting examples of mathematical algorithms for comparing two sequences are those of Karlin and Altschul,1990, proc.Natl.Acad.Sci.U.S. A.87:2264-2268, as modified in Karlin and Altschul,1993, proc.Natl.Acad.Sci.U.S. A.90:5873-5877. Such algorithms are incorporated in the NBLAST and XBLAST programs of Altschul et al, 1990, J.mol. Biol. 215:403. A BLAST nucleotide search can be performed with the NB LAST nucleotide program parameter set (e.g., word length=12 for score=100) to obtain nucleotide sequences homologous to the nucleic acid molecules of the application. BLAST protein searches can be performed with an XBLAST program parameter set (e.g., score-50, word length = 3) to obtain amino acid sequences homologous to protein molecules described herein. To obtain a gap alignment for comparison purposes, gaps BLAST (Gapped BLAST) can be used as described in Altschul et al, (1997) Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to conduct iterative searches that detect long-range relationships between molecules (supra). When using BLAST, vacancy BLAST, and PSI-BLAST programs, default parameters (see, e.g., NCBI website) for each program (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller,1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When amino acid sequences are compared using the ALIGN program, a PAM120 weight residue table can be used with a gap length penalty of 12 and a gap penalty of 4. Techniques similar to those described above may be used to determine the percent identity between two sequences with or without allowing gaps. In calculating the percent identity, only exact matches are typically counted.

For antibodies, percent sequence identity can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the subject antibody region and the reference antibody region is the number of positions occupied by the same amino acids in both the subject antibody region and the reference antibody region divided by the total number of aligned positions of the two regions, multiplied by 100 to convert to a percent.

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)). NCBI-BLAST2 sequence comparison program is available from the national institutes of health (Besselda, malyland). NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmasked = yes, chain = all, expected number of occurrences = 10, minimum low complexity length = 15/5, multi-pass e value = 0.01, multiple Cheng Changliang = 25, attenuation of the final vacancy alignment = 25, and scoring matrix = BLOSUM62.

In the case of amino acid sequence comparison using NCBI-BLAST2, the amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B (to, with or agains t), which may alternatively be expressed in terms of the given amino acid sequence A having or comprising a certain amino acid sequence identity with a given amino acid sequence B (to, with or agains t), is calculated as follows: 100 by a score X/Y, where X is the number of amino acid residues that are assessed as identical matches in the alignment of A and B in the sequence alignment program NCBI-BLAST2, and where Y is the total number of amino acid residues in B. It will be appreciated that in the case where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and inter-sugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) 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 deaza adenine, guanine, cytosine, thymidine and uracil; xanthine and hypoxanthine. It will be appreciated that polynucleotides comprising non-transcribable nucleotide bases may be used as probes in, for example, hybridization assays. The nucleic acid may be double-stranded or single-stranded and represents the sense strand or the antisense strand. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences and optimized codons or synonymous codon equivalents.

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 techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. The isolated nucleic acid is also substantially free of sequences from which the nucleic acid is derived that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid).

Hybridization may occur with all or a portion of the nucleic acid sequence molecules. The hybridization portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (tm=81.5% -16.6 (Log 10[ na+ ]) +0.41 (% (g+c) -600/l), or a similar equation). Thus, parameters in the wash conditions that determine hybridization stability are sodium ion concentration and temperature. To identify molecules that are 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 about 1 ℃, e.g., if a nucleic acid molecule with >95% identity is sought, the final wash temperature will be reduced by about 5 ℃. Based on these considerations, one of skill in the art will be able to readily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. For example, stringent hybridization can be achieved using the following conditions: based on the above equation, hybridization was performed at Tm-5℃in 5 Xsodium chloride/sodium citrate (SSC)/5 XDenhardt's solution/1.0% SDS, followed by washing with 0.2 XSSC/0.1% SDS at 60 ℃. Moderately stringent hybridization conditions include a wash step in 3 XSSC at 42 ℃. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts and temperatures. Additional guidelines for hybridization conditions can be found in the following documents: current Protocols in Molecular Biology, john Wiley & Sons, N.Y.,2002, and Sambrook et al, molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory Press,2001.

As used herein, the term "expression construct" refers to a polynucleotide comprising an expression control sequence operably linked to a heterologous nucleotide sequence (i.e., a sequence to which the expression control sequence is not normally linked in nature) that is the subject of expression. As used herein, the term "expression vector" refers to a polynucleotide comprising an expression construct and sequences sufficient for replication in a host cell or insertion 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 modulates transcription and/or translation of a nucleotide sequence to which it is operably linked. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences) and ribosome binding sites (translational regulatory sequences).

As used herein, a nucleotide sequence is "operably linked" to an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes facilitating transcription of the nucleotide sequence by interaction between the polymerase and the promoter.

As used herein, the term "vector" includes any intermediate vector for a nucleic acid molecule that enables the nucleic acid molecule to be introduced into, and/or integrated into, a prokaryotic and/or eukaryotic cell, for example, and includes plasmids, phagemids, phages or viral vectors (e.g., retroviral-based vectors, lentiviral vectors, adeno-associated viral vectors, etc.). As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material (typically a circular DNA duplex) that can replicate independently of chromosomal DNA.

"Transfection" refers to the introduction of new genetic material into a cell. It includes transformation (direct uptake and incorporation of exogenous genetic material from its surrounding environment through the cell membrane), transduction (introduction of foreign DNA into the host cell by phage virus), and conjugation.

As used herein, "host cell" refers to a recombinant cell comprising an expression construct.

As used herein, the term "biological sample" refers to a sample containing cells (e.g., tumor cells) or biomolecules derived from cells.

As used herein, the terms "therapy," "treatment," "therapeutic intervention," and "amelioration" refer to any activity that results in a reduction in the severity of symptoms. The terms "treatment" and "prevention" are not intended to be absolute terms. Treatment and prevention may refer to any delay in onset, improvement in symptoms, improvement in patient survival, increase in survival time or survival rate, and the like. Treatment and prevention may be complete or partial. The treatment effect may be compared to an individual or collection of individuals who have not received treatment, or to the same patient at a different time prior to or during treatment. In some aspects, the severity of the disease is reduced by at least 10%, e.g., as compared to a subject prior to administration or a control subject not undergoing treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, is no longer detectable using standard diagnostic techniques. "Treatment" and "Treatment" may also mean an extension of survival compared to the expected survival when untreated. As used herein, "treatment" and "treatment" also include prophylactic treatment.

A composition or method that "comprises" or "comprises" one or more enumerated elements may include other elements not specifically enumerated (e.g., meaning open terms including but not limited to). For example, a composition "comprising" or "including" an antibody may contain the antibody alone or in combination with other components. In contrast, the phrase "consisting of … …" is inclusive, meaning that such embodiments do not include additional elements. The term "consisting essentially of … …" is meant to include the recited elements and other elements (e.g., partially enclosed terms) that do not materially affect the basic and novel characteristics of the claimed combination. It should be understood that aspects and embodiments described herein as "comprising" include "consisting of … …" and "consisting essentially of … …" embodiments.

As used herein, the following meanings apply unless otherwise indicated. The term "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The singular forms "a", "an" and "the" include plural referents. Thus, for example, reference to "an element" includes a combination of two or more elements, although other terms and phrases (e.g., "one or more") may be used with respect to one or more elements. The phrase "at least one of" includes "one of", "one of" or "one or more of" and "multiple of". The term "or" is non-exclusive, i.e., encompasses both "and" or "unless otherwise indicated. The term "any" between modifiers and sequences means that each member of the sequence is modified by the modifier. Thus, for example, the phrase "any of at least 1, 2, or 3" means "at least 1, at least 2, or at least 3".

Chimeric antigen receptor

A "chimeric antigen receptor" or "CAR" is an engineered molecule comprising an optional signal peptide, a target binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain, and an optional co-stimulatory domain. CARs are based on the structure of T cell receptors, which are expressed on T cells and are involved in cell-mediated immune responses. The "target binding domain" is also referred to herein as an "antigen binding domain" and thus the term "target" encompasses "antigen".

So-called "first generation" CARs have a targeting domain and a CD3 ζ signaling domain. So-called "second generation" CARs further include a co-stimulatory domain (e.g., a CD28 or 4-1BB domain). So-called "third generation" CARs comprise multiple co-stimulatory domains. So-called "fourth generation" CARs (also referred to as "keys") are engineered to release transgenic cytokines upon CAR signaling.

Chimeric antigen receptor ("CAR") includes the following elements: (1) an optional signal peptide, (2) a target binding domain, (3) an optional hinge region; (4) a transmembrane region; (5) an intracellular domain comprising a signal transduction domain. Optionally, the CAR may include any of the following: a cd3ζ signaling domain, an Fc receptor signaling domain, and a costimulatory (signaling) domain. That is, these optional elements may be included in addition to or in place of other optional elements. The target binding domain is heterologous to at least one of the other domains. That is, the target binding domain does not naturally occur on a T cell receptor, or at least one of the other domains is not in the same protein.

The "target binding domain" provides binding specificity for the CAR. "Signal peptides" direct polypeptides across the cell membrane. For so-called "universal CARs," the target binding domain may bind to the domain of an antibody that binds to the target antigen. A "hinge region" is a flexible connector region, such as a natural or synthetic polypeptide, or any other type of molecule, to provide structural flexibility and spacing from the flanking polypeptide regions. A "transmembrane domain" is a transmembrane protein domain, which is typically hydrophobic. After binding, the "signal transduction domain" or "signaling domain" transmits a signal into the cell via a signal transduction pathway. This signaling activates the activity of the cell. A "co-stimulatory domain" is an ancillary signaling domain that further transmits a signal.

In some embodiments, the CAR comprises:

(i) A target binding domain (also referred to herein as an antigen binding domain) such as VH-VL or VL-VH that reacts with a citrullinated protein or citrullinated fragment thereof, wherein the two variable domains are separated by a flexible linker of 15-25 amino acids in length;

(ii) A hinge domain;

(iii) A costimulatory domain; and

(Iv) An intracellular signaling domain (also referred to herein as an activation domain). That is, in some embodiments, the CAR comprises an antigen binding domain fused to a CAR platform comprising a hinge domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The CAR may further comprise a signal peptide (also referred to herein as a leader sequence) to direct expression of the CAR to the surface of a cell (e.g., treg).

In some embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequences of SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 28, and SEQ ID No. 19. In other embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequences of SEQ ID No. 30, SEQ ID No. 16, SEQ ID No. 28, and SEQ ID No. 19. In some embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequences of SEQ ID No. 30, SEQ ID No. 16, SEQ ID No. 29, and SEQ ID No. 19. In other embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequences of SEQ ID No. 30, SEQ ID No. 16, SEQ ID No. 29, and SEQ ID No. 19.

A. Signal peptides

The signal peptide may be any peptide having a function of allowing the polypeptide to pass through the cell membrane. The signal peptide may be derived from CD4, CD8, CD28, TLR or immunoglobulin receptor family.

For example, the signal peptide may comprise the following sequence:

MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 18); or (b)

MALPVTALLLPLALLLHAAR(SEQ ID NO:23)。

B. target binding domain

1. Structure of the

The target binding domain may comprise any polypeptide comprising a target binding function, e.g. an antibody as defined herein. In one embodiment, the target binding domain may comprise a form of antibody that retains antigen binding activity as defined herein. In one embodiment, the target binding domain may comprise a single chain antibody (scFV). The scFv can be linked to the transmembrane domain via a hinge domain whose length, flexibility and origin provide variability to the design of the CAR, and can, together with the transmembrane domain, facilitate interaction with antigen, facilitate construction of an immune synapse, and affect association of the CAR with additional proteins required to confer a robust activation signal.

2. Target/antigen

The chimeric antigen receptors disclosed herein comprise a target binding domain (also referred to herein as an antigen binding domain) that binds to a citrullinated antigen (e.g., those found in the synovium of a subject with rheumatoid arthritis). In particular, the target binding domain may bind one or more of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) citrullinated peptides thereof. In some embodiments, the target binding domain can bind to a citrullinated peptide fragment of (i) - (iii), wherein the peptide fragment is at least 10 amino acids in length, e.g., at least 12 amino acids, at least 14 amino acids, or at least 16 amino acids in length. In some embodiments, the target binding domain further binds tenascin-C. In some embodiments, the target binding domain may bind to two or more of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) tenascin-C, or a citrullinated peptide fragment thereof.

In some embodiments, the target domain is one or more citrullinated peptides selected from the group consisting of:

ST(Cit)SVSSSSY(Cit)(Cit)MFGG(SEQ ID NO:24)；

VYAT(Cit)SSAV(Cit)L(Cit)SSV(SEQ ID NO:25)；

(Cit)PAPPPISGGGY(Cit)A(Cit)(SEQ ID NO:26)；

SHQEST(Cit)GRSRGRSGRSGS(SEQ ID NO:27)。

In some embodiments, the antigen binding domain binds to one or more citrullinated peptides, but not to non-citrullinated counterparts. In some embodiments, the antigen binding domain binds to the citrullinated vimentin peptide as shown in SEQ ID No. 24, but not STRSVSSSSYRRMFGG (SEQ ID No. 45). In some embodiments, the antigen binding domain binds to a citrullinated vimentin peptide as shown in SEQ ID No. 25, but not VYATRSSAVRLRSSV (SEQ ID No. 46). In some embodiments, the antigen binding domain binds to citrullinated fibrinogen peptide as shown in SEQ ID NO. 26, but not RPAPPPISGGGYRAR (SEQ ID NO: 47). In some embodiments, the antigen binding domain binds to the citrullinated filaggrin peptide as shown in SEQ ID NO. 27, but not SHQESTRGRSRGRSGRSGS (SEQ ID NO: 48).

The target binding domain may comprise sequences from antibody VH and VL domains. In some embodiments, the target binding domain may comprise sequences from a heavy chain-only antibody or antibody fragment having only two VH domains. This includes specific sets of CDRs from VH and VL domains. In some embodiments, the target binding domain comprises CDRs from the VH domain of SEQ ID NO. 1 or SEQ ID NO. 2. In some embodiments, the target binding domain comprises CDRs from the VL domain of SEQ ID NO. 3 or SEQ ID NO. 4. In some embodiments, the target binding domain comprises CDRs from the VH and VL domains of SEQ ID NO. 1 or SEQ ID NO. 3, respectively. In some embodiments, the target binding domain comprises CDRs from the VH and VL domains of SEQ ID NO. 1 or SEQ ID NO. 4, respectively. In some embodiments, the target binding domain comprises CDRs from the VH and VL domains of SEQ ID NO. 2 or SEQ ID NO. 3, respectively. In some embodiments, the target binding domain comprises CDRs from the VH and VL domains of SEQ ID NO. 2 or SEQ ID NO. 4, respectively.

In some embodiments, the target binding domain comprises Complementarity Determining Regions (CDRs) from the VH and VL domains of SEQ ID NO:1 (SBT 01 VH (M)) and SEQ ID NO:4 (SBT 01 VL (G)). In some embodiments, the VH domain of the target binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:34 and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

Region(s)	SEQ ID	Sequence fragments	Residues	Length of
					LFR1	NO:38	SYVLTQPPSVSVAPGKTARITC	1-22	22
CDR-L1	NO:39	GGNNIGSKSVH	23-33	11
					LFR2	NO:40	WYQQKPGQAPVLVIY	34-48	15
CDR-L2	NO:41	YDSDRPS	49-55	7
					LFR3	NO:42	GIPERFSGSNSGNTATLTISRVEAGDEADYYC	56-87	32
CDR-L3	NO:43	QVWDSSSDHQV	88-98	11
					LFR4	NO:44	FGTGTKVTV	99-108	11

In certain embodiments, the target binding domain comprises a VH sequence selected from the group consisting of:

(1)SBT01 VH(M)

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEW

IGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA

RLDPFDYWGRGTLVTVSS(SEQ ID NO:1)；

(2)SBT01 VH(G)

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWI

GSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARL

DPFDYWGRGTLVTVSS (SEQ ID NO: 2); or (b)

A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to the foregoing sequence, provided that the target binding domain binds to a citrullinated antigen as described herein.

The target binding region may comprise a VL sequence selected from the group consisting of:

(1)SBT01 VL(M)

SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFF

DTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQV

FGPGTTVTVL(SEQ ID NO:3)；

(2)SBT01 VL(G)

SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYY

DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVF

GTGTKVTVL (SEQ ID NO: 4); or (b)

In another embodiment, the antigen binding region comprises a scFV comprising one or more VH domains comprising the amino acid sequence of the VH domain of SEQ ID No. 1 or SEQ ID No. 2. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having at least any one of SEQ ID No. 1 or SEQ ID No. 2, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein.

In some embodiments, the scFv comprises the VL domain of SEQ ID NO. 3 or SEQ ID NO. 4. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having at least any one of SEQ ID No. 3 or SEQ ID No. 4, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein.

In some embodiments, the scFV domain comprises VH and VL domains of SEQ ID NO. 1 and SEQ ID NO. 3, respectively. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having at least any one of SEQ ID No. 1 or SEQ ID No. 3, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22 is disposed between the VH and VL domains.

In some embodiments, the scFV domain comprises VH and VL domains of SEQ ID NO. 1 and SEQ ID NO. 4, respectively. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having any one of SEQ ID No. 1 and at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No. 4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22 is disposed between the VH and VL domains.

In some embodiments, the scFV domain comprises VH and VL domains of SEQ ID NO. 2 and SEQ ID NO. 3, respectively. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having any one of SEQ ID No. 2 and at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No. 3, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22 is disposed between the VH and VL domains.

In some embodiments, the scFV domain comprises VH and VL domains of SEQ ID NO. 2 and SEQ ID NO. 4, respectively. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having any one of SEQ ID No. 2 and at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No. 4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22 is disposed between the VH and VL domains.

In another embodiment, the antigen binding region comprises a scFV comprising an amino acid sequence selected from the group consisting of seq id nos:

SBT01G-VHVL-GGGSx linker-pSB_0149

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEW

IGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA

RLDPFDYWGRGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAP

GKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGS

NSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR

(SEQ ID NO: 5) (GGGSx linker underlined);

SBT01G-VHVL-Whitlow 218 linker-pSB_0158

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEW

IGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA

RLDPFDYWGRGTLVTVSSGSTSGSGKPGSGEGSTKGSYVLTQPPSVSV

APGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFS

GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR

(SEQ ID NO: 6) (Whitlow 218 linker underlined);

SBT01G-VHVL-AB pur linker-pSB_0159

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEW

IGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA

RLDPFDYWGRGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARSYVLTQ

PPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPS

GIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTK

VTVLR (SEQ ID NO: 7) (AB pur linker underlined); or (b)

For example, the linker may comprise the following sequence:

GGGGSGGGGSGGGGS (SEQ ID NO: 20) GGGSx linker; or (b)

GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21) Whitlow 218 linker; or (b)

ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 22) AB pur linker.

Optionally, any of the foregoing sequences may include sets of CDRs from the VH and VL domains described above.

C. Hinge region

In some embodiments, the hinge region of the disclosed CARs can be selected from the CD8, CD4, or CD28 extracellular domain, the Fc region of an IgG1 antibody, or the extracellular domain of any TLR receptor as known to those of skill in the art, and can be found in the GenBank database.

For example, the hinge region may comprise the following sequence:

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(SEQ ID NO:30)(CD28)；

Or (b)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC

GVLLLSLVITLYC (SEQ ID NO: 15) (CD 8); or (b)

A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to the hinge region sequence described above.

D. Transmembrane domain

The transmembrane domain may comprise a transmembrane domain of an immunoglobulin family receptor (e.g., CD 8). The intracellular domain may be selected from any transmembrane molecule on a T cell. For example, the Transmembrane (TM) domain of a disclosed CAR can comprise the TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4, or CD 8.

For example, the transmembrane domain may comprise the following sequence:

FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 16); or (b)

IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 17); or (b)

E. signal transduction domains

Cd3ζ signaling domain

In some embodiments, the signaling domain comprises a CD3 zeta signaling domain. The CD3 zeta signaling domain of the disclosed CAR molecules can comprise a CD3 zeta amino acid sequence, e.g., a signaling domain of CD3 zeta.

For example, the cd3ζ signaling domain may comprise the sequence:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19); or (b)

80%, 85%, 90%, 95%, 97%, 98%, 99% Or more than the aforementioned CD3ζ signaling domain sequence

99.5% Sequence identity of at least any one sequence identity.

For example, the CD3 zeta signaling domain may comprise amino acids 21-163, 31-142, 68-89 and/or 138-158 of the sequence shown in SEQ ID NO. 19, or a functional variant thereof (e.g., having 1,2, 3,4, 5, 6, 7, 8, 9, 10 or 10-20 amino acid substitutions, deletions or additions).

Fc receptor Signal transduction Domain

In some embodiments, the signaling domain comprises an Fc signaling domain. The Fc signaling domain can be any of Fc-alpha, fc-gamma, fc-epsilon, fc-mu, and Fc-delta receptors. For example, an Fc receptor signaling domain may comprise amino acids (e.g., one or more ITAM domains) involved in interactions with Src (e.g., fgr, fyn, hck, lyn, yes and Src) and ZAP-70 family kinases (see, e.g., sanchez-Mejorada et al (1998) J. Leukocyte biol.63:531; garcia-Garcia et al (2002) J. Leukocyte biol.72:1092). In some embodiments, the Fc receptor signaling domain comprises at least one ITAM domain, e.g., at least one ITAM domain from any of Fc-a, fc- γ, fc-epsilon, fc- μ, and Fc- δ receptors, or substantially the same thereof.

The sequence can also be found as follows:

F. co-stimulatory domains

In addition to the signal transduction domain of CD3 ζ or Fc receptor, the CARs of the disclosure may also include one or more costimulatory domains. The costimulatory domain can 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. The CAR construct may contain two or more co-stimulatory signaling domains (e.g., CD28 and 4-1 BB).

One or more co-stimulatory domains may be located between the signaling domain and the transmembrane region.

In certain embodiments, the CD28 co-stimulatory domain may comprise the following sequence:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID

NO 29); or (b)

A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to the aforementioned sequence.

In certain embodiments, the 41BB co-stimulatory domain may comprise the following sequence:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID

NO: 28); or (b)

III nucleic acids

A. Nucleic acid encoding CAR

Disclosed herein are nucleic acid molecules (polynucleotides) comprising nucleotide sequences encoding the CARs of the present disclosure. The nucleic acid of the disclosed CARs can be in the form of DNA or in the form of RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. RNA includes mRNA, siRNA, sRNA, ssRNA and the like.

For example, the nucleic acid may comprise a nucleotide sequence encoding a polypeptide of any one of SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4. In certain embodiments, the nucleotide sequence encoding a VH domain comprises:

(1)SBT01 VH(M)

CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGA

GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATAC

CACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGG

AGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCG

TCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCG

ATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTA

TTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCC

TGGTCACTGTCTCGAGC(SEQ ID NO:8),

(2)SBT01 VH(G)

CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG

AGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTA

GTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG

GAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCC

GTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACC

AGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTG

TATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCAC

CCTGGTCACTGTCTCGAGC (SEQ ID NO: 9); or (b)

In certain embodiments, the nucleotide sequence encoding a VL domain comprises:

(1)SBT01 VL(M)

TCCTATGTCCTGACTCAGCCACCCTCAGTGTCGCTGGCCCCGGGAGA

GACGGCCACAATTACTTGTGGTGGAGACGACATTGAAAATCAAAATG

TCAACTGGTATCAGCAGAAGTCAGGTCAGGCCCCTATGCTGCTCATC

TTCTTTGATACCAGACGGCCCTCAGGGATCCCGGAGCGATTCTCTGG

CTCCAGGTCTGAGGACACGGCCAACCTGACCATCACCAGGGTCGAGG

CCGGGGATGACGCCGACTATTTCTGTCAGGTGTATGATAGGAAGACTGATCACCAAGTCTTCGGACCTGGGACCACGGTCACCGTCCTA(SEQ ID

NO:10)；

(2)SBT01 VL(G)

TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAA

GACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGT

GTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCAT

CTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTG

GCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAA

GCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA(SEQID NO:11); Or (b)

In another embodiment, the nucleic acid molecule encoding a VH domain comprises SEQ ID NO 8 or SEQ ID NO 9. In some embodiments, the nucleic acid molecule encoding a VH domain comprises a nucleic acid sequence having at least any one of SEQ ID No. 8 or SEQ ID No. 9, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% provided that the scFV domain binds to a citrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding a VL domain comprises SEQ ID NO 10 or SEQ ID NO 11. In some embodiments, the nucleic acid molecule encoding a VL domain comprises a nucleic acid sequence having at least any one of SEQ ID No. 10 or at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No. 11, provided that the scFV domain binds to a citrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding a scFV domain comprises SEQ ID NO 8 and SEQ ID NO 10. In some embodiments, a nucleic acid molecule encoding a scFV comprises an amino acid sequence having at least any one of SEQ ID No. 8 or SEQ ID No.10, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding a scFV domain comprises SEQ ID NO 8 and SEQ ID NO 11. In some embodiments, a nucleic acid molecule encoding a scFV comprises an amino acid sequence having at least any one of SEQ ID No. 8 or SEQ ID No. 11, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding a scFV domain comprises SEQ ID NO 9 and SEQ ID NO 10. In some embodiments, a nucleic acid molecule encoding a scFV comprises an amino acid sequence having at least any one of SEQ ID NO 9 or SEQ ID NO 10, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding a scFV domain comprises SEQ ID NO 9 and SEQ ID NO 11. In some embodiments, a nucleic acid molecule encoding a scFV comprises an amino acid sequence having at least any one of SEQ ID No. 9 or SEQ ID No. 11, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% with the proviso that the scFV domain binds a citrullinated antigen as described herein.

In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the nucleotide sequence:

SBT01G-VHVL-GGGSx linker-pSB_0149

CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGA

GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATAC

CACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGG

AGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCG

TCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCG

ATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTA

TTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCC

TGGTCACTGTCTCGAGCGGTGGCGGTGGCTCGGGCGGTGGTGGGT

CGGGTGGCGGCGGATCTTCCTATGTGCTGACTCAGCCACCCTCAGT

GTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAAC

AACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCC

AGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGG

ATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCT

GACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTC

AGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC (SEQ ID NO: 12) (GGGSx linker underlined);

Or (b)

SBT01G-VHVL-Whitlow 218 linker-pSB_0158

CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGA

GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATAC

CACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGG

AGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCG

TCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCG

ATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTA

TTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCC

TGGTCACTGTCTCGAGCGGAAGCACGAGTGGTTCAGGCAAACCGG

GTTCCGGTGAAGGTTCAACAAAAGGTTCCTATGTGCTGACTCAGCC

ACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTG

GGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAA

GCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGC

CCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACG

GCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACT

ATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC(SEQ ID NO:13)(Whitlow 218

Joint underline); or (b)

SBT01G-VHVL-AB pur linker-pSB_0159

CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGA

GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATAC

CACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGG

AGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCG

TCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCG

ATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTA

TTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCC

TGGTCACTGTCTCGAGCGCCTCTAGCGGGGGGAGCACATCAGGAA

GCGGCAAGCCCGGTAGCGGCGAAGGCTCCTCTGGCAGCGCCCG

CTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAA

AGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAG

TGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCA

TCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCT

GGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGA

AGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTA

GTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC

(SEQ ID NO: 14) (AB pur linker underlined); or (b)

Optionally, these may include sequences encoding sets of CDRs from the VH and VL domains described herein.

The polynucleotide variant may contain a change in the coding region, the non-coding region, or both. In some embodiments, the polynucleotide variant comprises an alteration that produces a silent substitution, addition, or deletion without altering the properties or activity of the encoded CAR polypeptide. In some embodiments, the polynucleotide variant comprises an alteration that does not produce any change in the amino acid sequence. In some embodiments, polynucleotide variants contain "silent" substitutions due to the degeneracy of the genetic code. Polynucleotide variants may be generated for a variety of reasons, for example, to optimize codon expression for a particular host.

In some embodiments, the polynucleotides as described herein are isolated.

Polynucleotides encoding the CAR are isolated molecules or may be included within a vector (e.g., a plasmid, cosmid, artificial chromosome, or virus). Such vectors can be used to transfect target cells.

B. Expression constructs and vectors

The polynucleotide encoding the CAR of the present disclosure may include a regulatory element operably linked to the nucleotide sequence encoding the CAR. For example, a polynucleotide may include one or more transcriptional regulatory elements (e.g., promoters or enhancers) that, when present in a cell, cause expression of a sequence encoding the CAR in the cell.

The nucleic acids disclosed herein may be incorporated into vectors capable of transfecting cells. Such vectors include, but are not limited to, viral vectors, plasmids, and microbubbles, such as liposomes. Exemplary viral vectors are adenovirus vectors Ad, AAV, lentivirus, and Vesicular Stomatitis Virus (VSV) and retroviruses. Lentiviruses are a genus of the retrovirus family and include HIV, SIV and FIV. Lentiviruses can deliver large amounts of genetic material into the DNA of host cells. They are capable of infecting non-dividing cells.

IV. cells

In some embodiments, the recombinant (host) cell has a nucleic acid molecule encoding the disclosed CAR, wherein the nucleic acid molecule can further comprise an expression control sequence operably linked to the nucleotide sequence encoding the CAR. The assembled CAR (by synthesis, site-directed mutagenesis, or another method as known to those skilled in the art), a nucleic acid molecule encoding the disclosed CAR, may be inserted into an expression vector and operably linked to expression control sequences suitable for expressing the disclosed CAR in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and/or expression of the CAR polypeptide in a suitable host. As is well known in the art, in order to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that function in the chosen expression host.

The disclosure also provides nucleic acid molecules comprising the encoded CAR and/or cells expressing the CAR (e.g., recombinant cells).

The nucleic acid molecules encoding the disclosed CARs can be delivered to host cells, including but not limited to T cells, B cells, myeloid progenitor cells, macrophages, and the like, by plasmids or viral vectors as known to those of skill in the art. The resulting recombinant (host) cells may include, but are not limited to, T cells, CD 4T cells, treg cells, CD8 αt cells, CD8 βt cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells, T memory stem cells, and cells expressing class I or class II MHC as known to those of skill in the art. In some embodiments, the recombinant (host) cell having a nucleic acid molecule encoding the disclosed CAR can be a myeloid progenitor cell as known to those of skill in the art selected from the group consisting of: myeloid-derived co-progenitor cells, granulocyte macrophage progenitor cells, megakaryocyte erythrocyte progenitor cells, granulocyte progenitor cells and monocyte progenitor cells. In some embodiments, the myeloid cells are autologous or allogeneic cells.

In some embodiments, the cell expressing the CAR of the present disclosure is a Treg cell. "regulatory T cells" or "T _reg cells" are cells belonging to a subset of specialized T cells, whose function is to suppress the immune response, thereby maintaining homeostasis and self-tolerance. T _reg is capable of inhibiting T cell proliferation and cytokine production and plays a key role in preventing autoimmunity. T _reg is characterized by the expression of FoxP 3. Surface markers for T _reg include CD4, CD25 high (high molecular density) and CD127 low (low molecular density). Mouse and human tregs express GITR/AITR and CTLA-4. Human cd4+foxp3+ Treg cells can be divided into three subpopulations: (1) cd45ra+cd25+foxp3l0w resting Treg cells, (2) cd45ro+cd25high foxp3 high activated Treg cells, and (3) cd45ro+cd25+foxp3 low non-inhibitory effector T cells (Teff) that produce pro-inflammatory cytokines.

The cell to be transformed with a nucleic acid disclosed herein may be a cell taken from a subject to which the recombinant cell is to be administered. In this way, the problem of an allogeneic immune response may be alleviated.

The cells may be expanded ex vivo prior to administration to a subject.

Treg cells that have incorporated nucleic acids expressing the CARs of the present disclosure can express these CARs and be used in the methods described herein to treat rheumatoid arthritis.

Furthermore, the protein produced by the transformed/recombinant host may be purified according to any suitable method. Such methods include chromatography (e.g., ion exchange, affinity fractionation (sizing) column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags (such as hexahistidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase) can be attached to proteins to allow easy purification by appropriate affinity columns. In some embodiments, the proteins may also be physically characterized using techniques such as proteolysis, high Performance Liquid Chromatography (HPLC), nuclear magnetic resonance, and x-ray crystallography.

V. composition

Also disclosed are pharmaceutical compositions comprising recombinant cells having nucleic acid molecules encoding and/or expressing the disclosed CAR polypeptides and a pharmaceutically acceptable carrier; and methods for treating rheumatoid arthritis.

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

As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of the pharmaceutical composition and that can be safely administered to a subject. The term is used synonymously with "physiologically acceptable" and "pharmacologically acceptable". Pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art in light of the present disclosure. For a detailed list of suitable pharmacological compositions and techniques for their administration, reference may be made to articles such as: remington's Pharmaceutical Sciences, 17 th edition 1985; brunton et al ,"Goodman and Gilman'sThe Pharmacological Basis of Therapeutics,"McGraw-Hill,2005;University of the Sciences in Philadelphia(), "Remington: THE SCIENCE AND PRACTICE of Pharmacy," Lippincott Williams & Wilkins,2005; and University of THE SCIENCES IN PHILADELPHIA (incorporated), "Remington: THE PRINCIPLES of PHARMACY PRACTICE," Lippincott Williams & Wilkins,2008.

The pharmaceutically acceptable carrier is generally sterile, at least for human use. Pharmaceutical compositions generally comprise agents for buffering and preserving the deposit, and may include buffers and carriers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological (0.9%) saline, phosphate Buffered Saline (PBS), hank's balanced salt solution's balanced salt solution (HBSS), and various electrolyte solutions (e.g., PLASMALYTE ATM (Baxter)).

The pharmaceutical compositions may be formulated for any route of administration, including mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection (bolus or infusion)), oral, or transdermal.

An injectable (e.g., intravenous) composition can comprise a solution of the composition suspended in an acceptable carrier (e.g., an aqueous carrier). Any of a variety of aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoproteins, globulins, and the like. Typically, physiological buffer saline (135-150 mM NaCl) is used. The compositions may contain pharmaceutically acceptable auxiliary substances that approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. In some embodiments, the compositions may be formulated in a kit for intravenous administration.

Formulations suitable for parenteral administration (e.g., by intra-articular (in the joint), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes) include: aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. Injectable solutions and suspensions may also be prepared from sterile powders, granules and tablets. In the practice of the invention, the composition may be administered, for example, by intravenous infusion, external, intraperitoneal, intravesical or intrathecal. Formulations of the compositions may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

Cells can be cryopreserved. Cryopreservation may include formulating the cells with a cryopreservative (e.g., DMSO). Commercially available media include, for example, those available from Millipore SigmaAnd/>

The compositions may be formulated as dosage forms for administration. The term "dosage form" refers to a particular form of a drug and depends on the route of administration. Examples of dosage forms include, but are not limited to: a dispersing agent; a suppository; an ointment; cataplasm (cataplasm) (cataplasm); paste (paste); a powder; dressing; a cream; plaster; a solution; a patch; aerosols (e.g., nasal sprays or inhalers); a gelling agent; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; a liquid dosage form suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a patient.

The terms "dose" and "dose (dosage)" are used interchangeably herein. Dosage refers to the amount of active ingredient administered to an individual at each administration. The dosage will vary depending on a number of factors including the frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; route of administration; and an imaging mode in which the marker (if present) is detectable. One skilled in the art will recognize that the dosage may be modified according to the factors described above or based on the progress of the treatment.

Pharmaceutical formulations may be packaged or prepared in unit dosage forms. In this form, the formulation is subdivided into unit doses containing appropriate quantities of the active component depending, for example, on the dosage of the therapeutic agent or concentration of the composition. The unit dosage form may be a packaged formulation, the package containing discrete amounts of the formulation. The composition may also contain other compatible therapeutic agents, if desired.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising Complementarity Determining Regions (CDRs) from a VH domain of SEQ ID No.1 or SEQ ID No. 2. In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from a VL domain of SEQ ID No. 3 or SEQ ID No. 4. In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID No.1 and SEQ ID No. 3. In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID No.1 and SEQ ID No. 4. In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID No. 2 and SEQ ID No. 3. In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID No. 2 and SEQ ID No. 4. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID No. 1 (SBT 01 VH (M)) and SEQ ID No. 4 (SBT 01 VL (G)). In some embodiments, the VH domain of the CAR polypeptide comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:34 and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and the VL domain of the CAR polypeptide comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cells are T cells, CD4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In another embodiment, the composition of the invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide comprising a scFV comprising one or more VH domains comprising the amino acid sequence of the VH domain of SEQ ID No. 1 or SEQ ID No. 2. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises a scFV comprising an amino acid sequence having at least any one of SEQ ID No. 1 or SEQ ID No. 2, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% provided that the scFV domain binds a citrullinated antigen as described herein. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a scFV comprising a VL domain of SEQ ID No. 3 or SEQ ID No. 4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises a scFV comprising an amino acid sequence having at least any one of SEQ ID No. 3 or SEQ ID No. 4, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% provided that the scFV domain binds a citrullinated antigen as described herein. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD8 a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a scFV comprising VH and VL domains of SEQ ID No. 1 and SEQ ID No. 3. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises a scFV comprising an amino acid sequence having at least any one of SEQ ID No. 1 or SEQ ID No. 3, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% provided that the scFV domain binds a citrullinated antigen as described herein. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a scFV comprising VH and VL domains of SEQ ID No. 1 and SEQ ID No. 4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises a scFV comprising an amino acid sequence having any one of SEQ ID No. 1 and at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No.4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a scFV comprising VH and VL domains of SEQ ID No. 2 and SEQ ID No. 3. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises a scFV comprising an amino acid sequence having any one of SEQ ID No. 2 and at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No. 3, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

In some embodiments, the compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a scFV comprising VH and VL domains of SEQ ID No. 2 and SEQ ID No. 4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises a scFV comprising an amino acid sequence having any one of SEQ ID No. 2 and at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID No.4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cells are T cells, CD 4T cells, treg cells, CD 8a T cells, CD8 β T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells or T memory stem cells. In some embodiments, the cells are Treg cells.

VI methods of use

T cells, and in particular Treg cells, expressing the CARs disclosed herein are useful for treating rheumatoid arthritis. Methods of use include administering to a subject in need thereof (e.g., a subject having rheumatoid arthritis) an effective amount of a pharmaceutical composition of the present disclosure.

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 (e.g., doctor or nurse). Subjects include mammals, such as humans and non-human primates (e.g., monkeys), as well as dogs, cats, horses, cows, rabbits, rats, mice, goats, pigs, and other mammalian species. The subject may also include an avian. The patient may be an individual seeking treatment, monitoring, adjusting or modifying an existing treatment regimen, or the like. The term "rheumatoid arthritis subject" refers to an individual who has been diagnosed with rheumatoid arthritis. Rheumatoid arthritis patients may include those individuals who have not received treatment, are currently receiving treatment, have received treatment, and have discontinued treatment.

As used herein, the terms "effective amount," "effective dose," and "therapeutically effective amount" refer to an amount of an agent sufficient to produce a desired response (e.g., reduce or eliminate signs or symptoms of a condition or ameliorate a disorder). In some examples, an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or root causes of any disorder or disease and/or prevents the progression of the disease. For example, for a given parameter, a therapeutically effective amount will exhibit an increase or decrease in any of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100% of the therapeutic effect. Treatment efficacy may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can have an effect of any of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more as compared to a control.

The pharmaceutical composition may be administered by any suitable route including, but not limited to, intravenous, subcutaneous, intramuscular, or intraperitoneal routes. Examples of administration of the pharmaceutical composition include storing the composition at 10mg/ml in a sterile isotonic aqueous saline solution for injection at 4 ℃ and diluting it in 100ml or 200ml of 0.9% sodium chloride for injection prior to administration to a patient. The pharmaceutical composition is administered by intravenous infusion at a dose between 0.2 and 10mg/kg over a1 hour period. In other embodiments, the pharmaceutical composition is administered by intravenous infusion over a period of time between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via subcutaneous bolus injection.

The dosage of the composition is selected to provide effective therapy to the patient and is in the range of less than 0.1mg/kg body weight to about 25mg/kg body weight or in the range of 1mg-2 g/patient. In some cases, the dosage is in the range of 1-100mg/kg or about 50mg-8000 mg/patient. Dosages may be repeated at an appropriate frequency, which may range from once daily to once every three months, depending on the pharmacokinetics (e.g., half-life of the composition in the circulation) and pharmacodynamic response (e.g., duration of therapeutic effect of the composition) of the composition. In some embodiments, the in vivo half-life and administration of the composition between about 7 and about 25 days is repeated between once a week and once every 3 months.

The administration may be periodic. Depending on the route of administration, the dose may be administered, for example, once every 1,3,5, 7, 10, 14, 21, or 28 days or more (e.g., once every 2, 3, 4, or 6 months). In some cases, the administration is more frequent, for example 2 or 3 times daily. As will be appreciated by those skilled in the art, the patient may be monitored to adjust the dose and frequency of administration according to the progress of the treatment and any adverse side effects.

Thus, in some embodiments, additional administration is dependent on the progress of the patient, e.g., the patient is monitored between administrations. For example, after a first administration or a round of administration, the patient's tumor growth rate, recurrence (e.g., in the case of a post-operative patient), or general disease-related symptoms (e.g., weakness, pain, nausea, etc.) may be monitored.

In certain embodiments, T cells described herein are administered to the synovium of a joint of a subject suffering from rheumatoid arthritis. Such administration may be directly into the joint.

An exemplary method of the present disclosure includes isolating T lymphocytes from a biological sample obtained from a subject. Such T cells may be isolated by immunoaffinity (e.g., using a derivatized solid support and an anti-CD 4 antibody). Cd4+ regulatory T cells (tregs) can be separated from non-Treg cells based on their marker characteristics. Treg cells are cd4+, cd25+, CD127lo. Non Treg cells are: cd4+, cd25+, and cd127+. The isolated Treg cells are then transfected with an expression vector encoding a Chimeric Antigen Receptor (CAR) of the present disclosure. The transfected cells are expanded. The expanded cells are administered to a subject.

VII kit

As used herein, the term "kit" refers to a collection of items intended for use together. The kit may optionally include a reference agent and/or instructions for its use. The kit may further comprise a shipping container suitable for holding a container (e.g., vial) containing a composition as disclosed herein. The kit may comprise a container containing a collection of items.

Kits of the present disclosure may comprise a pharmaceutical composition as described herein contained in a container (e.g., a pouch or bottle for intravenous administration). The kit may also include a fluid conduit, such as a plastic tube, having a drip chamber. The drip chamber may be in communication with the intravenous needle through a fluid conduit. The fluid conduit may also contain one or more Y-sites and a rolling clip.

Examples

Abbreviations: CAR (chimeric antigen receptor); CF (citrullinated fibrinogen); CFSE (carboxyfluorescein succinimidyl ester); CV (citrullinated vimentin); EGFR (epidermal growth factor receptor); IN (intranasal); IV (intravenous); LPS (lipopolysaccharide); PAD2 (peptidyl arginine deiminase 2); PBMCs (peripheral blood mononuclear cells); PBS (phosphate buffered saline); RA (rheumatoid arthritis); scFv (single chain variable fragment); SF (synovial fluid); teff (effector T cells); treg (regulatory T cells); and UTD (untransduced). Example 1: SBT01G consistently performed as well as BVCA in the luciferase system or better than BVCA1

Jurkat-FF-luciferase transduction-50,000 cells per well in RPMI with 2X protamine sulfate in each of 2 flat bottom 96 well plates. The virus was diluted in RPMI such that the MOI of 100 μl was approximately 1. Mu.l was then added to one column on each 96-well plate. The plates were then rotated and placed in an incubator. The samples were pooled the next day. Transduced cells were resuspended in culture medium and 100 μl was transferred to the U-bottom plate in duplicate. Cells were stained with CV-AF488 and CV-AF647 and 1:100 anti-EGFR-PE at 4℃for 20min, washed once, and then analyzed by Novocyte. The samples were then scaled up into 6-well plates by adding 3ml of fresh medium.

Plate coating-Citrullinated Vimentin (CV) was first diluted 1:100 into 4ml PBS. 5 4-fold serial dilutions were obtained by transferring 1ml into 3 ml. Mu.l was then added to a 96-well plate. The plates were placed at 4 ℃ to coat overnight.

Luciferase assay-standardized transduced cells were pelleted and resuspended in 1ml RPMI. CV coated plates were washed three times with PBS. Mu.l of cells were added to the plate. Mu.l of 3 XPMA/Iono was added and the plates were placed in a 37℃incubator. After about 24 hours, the luciferase plates were read by adding 75 μ l BioGlo reagent, incubating for 2-3min in the dark and reading the plates.

FIG. 1 shows the response of virus-transduced Jurkat-FF-luciferase cells to different concentrations of full CV coated plates. The results show that BVCA and STB01 have the strongest response to the full-length CV for plate binding.

FIG. 2 shows the response of virus-transduced Jurkat-FF-luciferase cells to various concentrations of soluble CV. The results showed that only BVCA1 and SBT01G-HL had a dose-dependent response to soluble full-length CV.

FIG. 3 shows that BVCA1 and SBT01 demonstrate binding specificity for CV in assays with soluble bead-bound peptides.

FIG. 4 shows the reactions of BVCA1 and SBT01G at different protein concentrations for plate-bound CV, antibody (V9) captured CV and soluble CV. The results show that SBT01G has a stronger response to plate-bound CV and antibody (V9) captured CV (but not soluble CV) than BVCA a.

Example 2: SBT01 and BVCA1 response to synovial fluid from rheumatoid arthritis patients

Jurkat-FF-luciferase transduction-24X 10 ⁶ Jurkat-FF-luc cells were pelleted and resuspended in 24ml RPMI containing protamine sulfate and virus (MOI 3) to express the CARs of pSB_0147, pSB-0149 and pSB 0139. The cells were mixed and 4ml was aliquoted into each well of a 6-well plate. The plates were then rotated and placed in an incubator overnight. Cells were pelleted and re-seeded in 25ml RPMT of the T75 flask.

Synovial stimulation-transduced cells were pelleted and resuspended in RPMI and placed in the wells of a black/white plate. Synovial Fluid (SF) samples from RA patients were thawed, vortexed, and diluted in RPMI, and then added to plates containing transduced cells. Cells were incubated with SF at 37 ℃.

Luciferase assay-after about 24 hours, 75 μ l BioGlo reagent was added to each well and the plates incubated in the dark for 2-3min. The luminescence is then read on a plate reader.

Treg isolation and tissue culture-primary human Treg cells are derived from healthy donors, from leukopenia chamber residues or enriched leukopenia apheresis products (leukopaks). Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque Plus. Cd25+ cells were enriched by positive selection. Treg cells were then isolated by gating on cd4+cd25+cd127lo cells using FACS. After isolation, cells were stimulated with CTSDynabeads Treg Xpander (Gibco) at a 1:1 bead to cell ratio and cultured with recombinant human IL-2 (300 IU/mL) at a density of 25-30 ten thousand cells/mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and beta-mercaptoethanol. On day 9 of amplification, fresh CTSDynabeads Treg Xpander was added at a 1:1 bead to cell ratio.

Primary Treg transduction-primary Treg was transduced with CV-CAR construct via spin-occulat ion on day 2 of amplification in the presence of protamine sulfate.

Treg activation-Treg cells expressing CV-CAR are cultured in vitro with a synovial fluid sample dilution ranging from 1:5 to 1:160. Treg activation was assessed by measuring CD71 expression.

Flow cytometry and FACS analysis-activated cultures were collected and centrifuged at 300×g for 5min and then resuspended in 1X flow staining (Flowstain) buffer (Invitrogen) with vital dye (Invitrogen), anti-EGFR and CD71 surface staining antibodies. Tregs were incubated at 4 ℃ for 30min, then centrifuged and washed with 1X flow staining buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

FIG. 5 shows the response of SBT01G and BVCA1 to synovial fluid from RA patients. The data shows that SBT01G produces a stronger response to synovial fluid from several RA patients than BVCA a.

Figure 6 shows that SBT01G was again stronger than BVCA a1 for synovial samples from swedish RA patients that triggered the response.

Figure 7 shows the response of primary Treg cells transduced with CV-CAR to synovial fluid from multiple RA patients. The primary Treg cells expressing SBT01G CAR are more sensitive to synovial fluid from RA patients than the primary Treg cells expressing BVCA CAR.

Example 3: reaction of SBT01 and BVCA1 on citrullinated fibrinogen

Plate coating-citrullinated protein was diluted in PBS and added to wells of black/white isoplate. The plates were placed at 4 ℃ overnight to coat the plate wells.

Luciferase assay-Jurkat-FF-luc cell lines stably transduced and not transduced (UTD) were pelleted and resuspended in RPMI containing 10% FBS. Citrullinated protein coated plates were washed three times with PBS. 50,000 cells/well in 75 μl were added to the coated plates. Mu.l of 15 XPMA/Iono in RPMI was added to each well and the plate placed in a 37℃incubator. After about 24 hours, 75 μ l BioGlo reagent was added to each well and the plate incubated in the dark for 2-3min. The luminescence is then read on a plate reader.

Treg isolation and tissue culture-primary human Treg cells are derived from healthy donors, from leukopenia chamber residues or enriched leukopenia apheresis products. Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque Plus. Cd25+ cells were enriched by positive selection. Treg cells were then isolated by gating on cd4+cd25+cd127lo cells using FACS, and Teff was isolated by gating on cd4+cd25lobcd127 pos using FACS. After isolation, cells were stimulated with CTSDynabeads Treg Xpander (Gibco) at a 1:1 bead to cell ratio and cultured with recombinant human IL-2 (300 IU/mL) at a density of 25-30 ten thousand cells/mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and b-mercaptoethanol. On day 9 of amplification, fresh CTSDynabeads Treg Xpander was added at a 1:1 bead to cell ratio.

Primary Treg and Teff transduction-primary Treg and Teff were transduced with CD19-CAR and CV-CAR constructs via spin-occulation on day 2 of amplification in the presence of protamine sulfate.

Treg activation-the CAR-expressing tregs and Teff were marked with proliferation dye CFSE before activation of the culture began. Treg cells expressing CD19-CAR and CV-CAR were cultured in vitro with Citrullinated Vimentin (CV) or Citrullinated Fibrinogen (CF) at a dose ranging from 30ng/ml to 10 μg/ml. Treg activation was assessed by measuring the percentage of proliferating cells and by CD71 expression.

Flow cytometry and FACS analysis-activated cultures were collected and centrifuged at 300×g for 5min and then resuspended in 1X flow staining buffer (In vitrogen) with vital dye (Invitrogen), anti-EGFR and CD71 surface staining antibodies. Tregs were incubated at 4 ℃ for 30min, then centrifuged and washed with 1X flow staining buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

FIG. 8 shows the reaction of SBT01G and BVCA1 on plate-bound full-length peptidyl arginine deiminase 2 (PAD 2) -citrullinated fibrinogen. The results show that SBT01G, instead of BVCA a1, is also able to react to plate-bound full-length PAD2 citrullinated fibrinogen.

Figure 9 shows that SBT01G CARs expressed on Teff and Treg cells respond to Citrullinated Vimentin (CV) and Citrullinated Fibrinogen (CF). In contrast, BVCA1 CARs expressed on Teff cells and Treg cells reacted to CV but not CF.

Thus, fig. 1-9 show that SBT01G consistently performs as well as BVCA a or better than BVCA a in all assay systems.

Example 4: CV CARS showed functional response when using different promoters and linkers

Treg isolation and tissue culture-primary human Treg cells are derived from healthy donors, from leukopenia chamber residues or enriched leukopenia apheresis products. Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque Plus. Cd25+ cells were enriched by positive selection. Treg cells were then isolated by gating on cd4+cd25+cd127lo cells using FACS. After isolation, cells were stimulated with CTSDynabeads Treg Xpander (Gibco) at a 1:1 bead to cell ratio and cultured with recombinant human IL-2 (300 IU/mL) at a density of 25-30 ten thousand cells/mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and b-mercaptoethanol. On day 9 of amplification, fresh CTSDynabeads Treg Xpander was added at a 1:1 bead to cell ratio.

Treg activation-CAR-expressing tregs were marked with proliferation dye CFSE before activation of the culture began. Treg cells expressing CV-CAR were cultured in vitro with CV peptides captured by beads. Treg activation was assessed by measuring the percentage of proliferating cells.

Flow cytometry and FACS analysis activation assay-activation cultures were collected and centrifuged at 300×g for 5min and then resuspended in 1X flow staining buffer (Invitrogen) with vital dye (Invitrogen) and anti-EGFR surface staining antibody. Tregs were incubated at 4 ℃ for 30min, then centrifuged and washed with 1X flow staining buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

Flow cytometry and FACS analysis of Treg phenotype-on day 14 of expansion of non-transduced (UTD) cells, treg expressing CD19-CAR and CV-CAR were stained for transcription factors FoxP3 and Helios. Cells were fixed and permeabilized using eBiosciences FoxP transcription factor buffer kit (eBiosciences).

Treg cultures were resting-on day 14 of expansion, tregs expressing CD19-CAR and CV-CAR were harvested, debulked using magnets, and cultured with 300IU/ml IL-2 only at 50 ten thousand cells/ml.

Flow cytometry and FACS analysis of CAR expression-Treg expressing CD19-CAR and CV-CAR were stained with a vital dye (Invitrogen) and anti-EGFR surface staining antibody after day 14 and 2 or 5 days of amplification. CV-CAR expression was detected by incubating the cells with 1 μg/ml CV followed by incubation with FITC-labeled anti-vimentin (clone V9, invitrogen).

Plate coating-Citrullinated Vimentin (CV) was diluted in PBS and added to the wells of black/white isoplate. The plates were placed at 4 ℃ overnight to coat the plate wells.

Figure 10A shows that both EF1A and MND promoters drive expression of CV-CAR T cells and functional response to soluble bead-bound citrullinated peptide (pCV). Figure 10B shows that the phenotype of CV-CAR Treg cells was not altered using different promoters.

Figure 11 shows that selection of scFv linkers hardly affected the response of SBT01G CAR T cells to full-length CV.

Figure 12A shows that transduction with SBT01G CAR vector resulted in CV-CAR expression in a higher percentage of cells than in the case of transduction with BVCA CAR vector. Fig. 12B shows that SBT01G CAR and BVCA CAR Treg cells have similar FoxP3 and Helios characteristics.

Thus, figures 10-12 show that SBT01G shows a higher percentage of CAR-positive tregs, and that SBT01G shows a higher level of CAR expression.

Example 5: in vitro assessment of activation of CV-CAR Treg by citrullinated vimentin

Treg isolation and tissue culture-primary human Treg cells are derived from healthy donors, from leukopenia chamber residues or enriched leukopenia apheresis products. Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque Plus. Cd25+ cells were enriched by positive selection. Treg cells were then isolated by gating on cd4+cd25+cd127lo cells using FACS. After isolation, cells were stimulated with CTSDynabeads Treg Xpander (Gibco) at a 1:1 bead to cell ratio and cultured with recombinant human IL-2 (300 IU/mL) at a density of 25-30 ten thousand cells/mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and penicillin/streptomycin.

Treg activation-untransduced and CV-CAR expressing Treg cells are cultured in vitro with citrullinated vimentin antigen at a dose ranging from 10ng/mL to 10 μg/mL. Treg activation was assessed by measuring the percentage of proliferating cells, CD71 expression and the level of IL-10 secretion.

Flow cytometry and FACS analysis: the untransduced and CV-CAR expressing Treg cells were collected and centrifuged at 300×g for 5min, then resuspended in 1X RoboSep buffer (StemCell Technologies) with CD71 surface staining antibody and CELL TRACE Violet (CTV) vital dye mixture (Invitrogen). Tregs were incubated at 4 ℃ for 30min, then centrifuged and washed with 1X RoboSep buffer. The stained cells were then analyzed by flow cytometry.

Results

Treg activation in response to Citrullinated Vimentin (CV) was assessed in vitro. Figure 13 shows dose-dependent, CV-induced activation of CV-CAR Treg cells, as demonstrated by proliferation of cells, CD71 expression, and an increase in IL-10 secretion. In contrast, non-transduced Treg cells were not activated by CV. These results demonstrate that Treg activation following stimulation with CV is characteristic of CV-CAR expressing tregs.

The amount of CV present in synovial fluid of healthy donors and Rheumatoid Arthritis (RA) patients was assessed by ELISA, as shown in fig. 14. Jurkat reporter cells expressing one of three different CV-CARs or control CD 19-CARs were incubated with increasing amounts of synovial fluid and the response was measured as an indicator of CV-CAR binding and T cell activation signaling. Figure 14 shows that Jurkat cells (immortalized human T lymphocytes) expressing CV-CAR1 (BVCA 1) and CV-CAR2 (SBT 01G), but not CV-CAR3 (C03) or CD19-CAR, show an increased response to Synovial Fluid (SF). Figure 14 also shows that SF samples from more RA patients activate CV-CAR2 (SBT 01G) T cells compared to CV-CAR1 (BVCA 1) T cells.

To demonstrate that the CV-CAR expressing tregs are activated by citrullinated antigen in the synovial fluid of RA patients, the untransduced and CV-CAR2 tregs are incubated with RA synovial fluid or vehicle and proliferation and the level of CD71 expression are measured. Figure 15 shows that CV-CAR2 Treg cells from two donors are activated in a RA synovial specific manner.

Taken together, these results demonstrate that CV-CAR Treg cells are able to bind to citrullinated antigen present in RA synovial fluid, resulting in activation necessary for inhibitory function.

Example 6: assessment of CV-induced inhibition of Teff cells by CV-CAR Treg

Treg inhibition assay-CV-CAR expressing Treg cells were prepared from freshly prepared human enriched white blood cell apheresis product in 96 well U-bottom plates as described above. Tregs were incubated with CV as an antigen stimulator or with vehicle controls. Tregs were co-cultured with CD3/CD28 activated or CD19-CAR specific Teff cells to assess Treg inhibition of target cells. The Teff cells were co-cultured at the Teff ratio: (CD 3/CD28 activation) 1:4=0.25, 1:2=0.5, 1:1=1, 2:1=2 and 4:1=4, or (CD 19 specific) 1:4, 1:2, 1:1, 2:1 and 4:1.

Results

Figure 16A shows that CV-CAR tregs stimulated with CV were able to inhibit proliferation of CD3/CD28 activated Teff cells at low Treg to Teff ratio. Figure 16B shows that CV-CAR tregs stimulated with CV were able to inhibit proliferation of CD19-CAR Teff cells at low Treg to Teff ratios.

Example 7: analysis of CV-CAR Treg in rodent models of CV-related pulmonary inflammation

In human Rheumatoid Arthritis (RA) patients, a consistently high concentration of Citrullinated Vimentin (CV) in synovial fluid makes identification of CV as a relevant biomarker for human RA. However, the RA mouse model of the invention shows inconsistent CV levels, which complicates in vivo analysis of CV-CAR tregs. To induce CV production and release into the extracellular matrix, a mouse model of LPS-induced pulmonary inflammation was developed. The model is characterized by an acute inflammatory response including pulmonary neutrophilia and increased secretion of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1 beta (IL-1 b) and tumor necrosis factor-alpha (TNF-alpha) and CV accumulation in lung tissue within days after intranasal LPS challenge. Chimeric Antigen Receptors (CARs) specific for CV have been shown to react with accumulated CV proteins in lung tissue, which results in increased survival, proliferation and inhibitory activity of the corresponding CV-CAR Treg cells in an antigen-specific manner.

Method of

Immune compromised NCG mice six to eight weeks old were obtained from charles river Laboratories (CHARLES RIVER Laboratories). Mice were randomly grouped by body weight and divided into groups of 5-10 animals. On days 0, 1, 6 and 12, mice were treated Intranasally (IN) with LPS (5 mg/kg) to induce pulmonary inflammation and release and accumulate Citrullinated Vimentin (CV) IN the affected tissues. On day 0, CV-CAR Treg cells were harvested from the actively growing culture, the stimulatory beads were magnetically removed, followed by labeling the tregs with CELL TRACE Violet (CTV) (Invitrogen). CTV-labeled tregs were adjusted to 25x 10 ⁶ cells/mL in 0.9% sterile saline (NaCl) approximately 4 hours after initial LPS challenge, and injected Intravenously (IV) with a 200 μl dose such that each mouse received a total of 5x 10 ⁶ cells. After adoptive cell transfer of CV-CAR tregs or control CD19-CAR tregs, all mice received Intraperitoneal (IP) injection of IL-2 (160,000IU) on days 1,2, 5, 6, 7, 8, 9 and 12. Mice were monitored daily for any clinical signs of affliction and body weight was measured twice weekly. Mice were euthanized on day 13, and spleens and lungs were harvested and processed into single cell suspensions for flow cytometry analysis of human CV-CAR and CD19-CAR tregs.

Results

Figure 17 shows an exemplary timeline for inducing CV-related pulmonary inflammation and CV-CAR Treg cell activation in vivo in mice.

Results

Fig. 18A shows how proliferation rates are calculated from flow cytometry data, and fig. 18B shows how fold changes in EGFR rates (CAR markers) are calculated from the same data.

Figure 19A shows that the absolute number of CV-CAR tregs is significantly enriched in lung tissue from mice with LPS-induced pulmonary inflammation. Figure 19B shows that the proliferation rate of CV-CAR tregs was significantly increased in lung tissue from mice with LPS-induced lung inflammation. These results demonstrate that CV-CAR tregs have improved ability to target and proliferate in inflamed lung tissue characterized by citrullinated vimentin accumulation, as compared to control tregs. Thus, this murine model of pulmonary inflammation allows for in vivo testing of human CV-CAR tregs.

Exemplary embodiments

1. A Chimeric Antigen Receptor (CAR), comprising an antigen binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain, wherein the antigen binding domain specifically binds to a citrullinated polypeptide, optionally wherein the antigen binding domain specifically binds to three or more different citrullinated proteins or citrullinated fragments thereof.

2. The CAR of embodiment 1, wherein the antigen binding domain binds to a plurality of different citrullinated proteins or citrullinated fragments thereof.

3. The CAR of embodiment 1, wherein the antigen binding domain binds to one or more of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) citrullinated peptide fragments of these, e.g. such fragments having any of at least 10, 12, 14 or 16 amino acids.

4. The CAR of embodiment 1, wherein the antigen binding domain binds to at least two of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof.

5. The CAR of embodiment 1, wherein the antigen binding domain binds to all three of: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof.

6. A CAR according to embodiment 5, which further binds citrullinated tenascin-C.

7. The CAR of embodiment 1, wherein the antigen-binding domain comprises a VH domain and a VL domain, wherein:

(i) The VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 36, and

(Ii) The VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

8. The CAR of embodiment 1, wherein the intracellular signaling domain is derived from cd3ζ.

9. The CAR of embodiment 1, wherein the at least one co-stimulatory domain is derived from a member of the group consisting of: fceR1g, fcg, CD28, 4-1BB, CTLA-4/CD-28 hybrid, DAP10, CD27, and 2B4, optionally wherein the at least one co-stimulatory domain comprises a CD28 and/or 4-1BB co-stimulatory domain.

10. The CAR of embodiment 1, wherein the antigen binding domain comprises an antibody, an antibody fragment, a camelidae nanobody, a heavy chain-only antibody, or an aptamer.

11. The CAR of any one of embodiments 1 to 10, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain; and the hinge domain is a CD8 hinge domain or a CD28 hinge domain; and/or further comprising a signal peptide, optionally wherein the signal peptide is a CD8 signal peptide or a GM-CSF signal peptide.

12. The CAR of embodiment 11, wherein the antigen-specific binding domain comprises a single-chain fragment.

13. The CAR of embodiment 12, wherein the single-chain fragment comprises a single-chain variable fragment (scFv), optionally wherein the scFv fragment comprises:

(a) A VH domain comprising the amino acid sequence of SEQ ID No. 1; and

(B) A VL domain comprising the amino acid sequence of SEQ ID No. 4.

14. The CAR of embodiment 13, wherein the scFv fragment comprises:

(a) VH selected from:

(1)SBT01 VH(M)

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLE

WIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYY

CARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1); or (b)

(2)SBT01 VH(G)

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEW

IGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA

RLDPFDYWGRGTLVTVSS (SEQ ID NO: 2); and

(B) VL selected from:

(1)SBT01 VL(M)

SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFF

DTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQ

VFGPGTTVTVL (SEQ ID NO: 3) or

(2)SBT01 VL(G)

SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIY

YDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQ

VFGTGTKVTVL(SEQ ID NO:4)。

15. The CAR of embodiment 14, wherein the VH-VL fragments are linked by a linker selected from the group consisting of: (i) GGGSx (SEQ ID NO: 20), (ii) Whitlow 218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22).

16. The CAR of embodiment 13, wherein the scFv comprises the amino acid sequence of:

(a) SBT01G-VHVL-GGGSx linker-pSB_0149

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLE

WIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLD

PFDYWGRGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGKTARITC

GGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISR

VEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(SEQ ID NO:5)(GGGSx3

Joint underline); or (b)

(B) SBT01G-VHVL-Whitlow 218 linker-pSB_0158

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLE

WIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLD

PFDYWGRGTLVTVSSGSTSGSGKPGSGEGSTKGSYVLTQPPSVSVAPGKTARI

TCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTIS

RVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(SEQ ID NO:6)(Whitlow

Joint 218 underlined); or (b)

(C) SBT01G-VHVL-AB pur linker-pSB_0159

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLE

WIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLD

PFDYWGRGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARSYVLTQPPSVSVAP

GKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(SEQ ID NO:7)

(AB pur linker underlined).

17. The CAR of any one of embodiments 1-16, wherein the co-stimulatory domain comprises a CD28, 41BB, OX40, CD40L, myD, CD40, CD27, ICOS, or RANK/trail-R co-stimulatory domain.

18. A nucleic acid encoding the CAR of any one of embodiments 1-17.

19. The nucleic acid of embodiment 18, comprising DNA or RNA.

20. The nucleic acid of embodiment 18, wherein

The VH is encoded by a nucleic acid sequence comprising:

CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAG

ACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTT

ACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTG

GGAGTATCTATTACCGGGGGAACACCCACTACAATTCGTCCCTGAGGAGTC

GCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCAC

TTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC, or

CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAG

ACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTT

ACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTG

GGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTC

GAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGA

GCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACTCGACC

CATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC; and the VL is encoded by a nucleic acid sequence comprising:

TCCTATGTCCTGACTCAGCCACCCTCAGTGTCGCTGGCCCCGGGAGAGA

CGGCCACAATTACTTGTGGTGGAGACGACATTGAAAATCAAAATGTCAACT

GGTATCAGCAGAAGTCAGGTCAGGCCCCTATGCTGCTCATCTTCTTTGATAC

CAGACGGCCCTCAGGGATCCCGGAGCGATTCTCTGGCTCCAGGTCTGAGGA

CACGGCCAACCTGACCATCACCAGGGTCGAGGCCGGGGATGACGCCGACTA

TTTCTGTCAGGTGTATGATAGGAAGACTGATCACCAAGTCTTCGGACCTGGGACCACGGTCACCGTCCTA, or

TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAG

ACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCAC

TGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGAT

AGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGG

AACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGA

CTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA。

21. The nucleic acid of embodiment 20, wherein the VH and VL fragments are connected by a linker and comprise the nucleic acid sequence of (i) SEQ ID No. 12, (ii) SEQ ID No. 13, or (iii) SEQ ID No. 14.

22. An expression vector comprising an expression control sequence operably linked to a nucleic acid sequence according to any one of embodiments 16-21.

23. The expression vector of embodiment 22, wherein the expression control sequence comprises a regulatory region, wherein the regulatory region is selected from the group consisting of: promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 'and 3' untranslated regions, transcription initiation sites, termination sequences, polyadenylation sequences, nuclear localization, signals, and introns.

24. The expression vector of embodiment 23, wherein the vector is an adenovirus vector, a lentiviral vector, or a plasmid.

25. A host cell comprising the expression vector of embodiment 22, 23 or 24.

26. A modified T cell that has been modified to express the Chimeric Antigen Receptor (CAR) of any one of embodiments 1-17.

27. The modified T cell of embodiment 26, wherein the T cell is a mammalian T cell.

28. The modified T cell of embodiment 27, wherein the T cell is a Treg cell.

29. The modified T cell of embodiment 28, wherein the T cell is a human T cell.

30. The modified T cell of embodiment 29, wherein the T cell is a primary T cell.

31. The modified T cell of embodiment 30, wherein the T cell is cd4+, cd25+ and CD127lo.

32. A pharmaceutical composition comprising a plurality of modified T cells according to any one of embodiments 26 to 31 and a pharmaceutically acceptable carrier.

33. A method of treating a subject having rheumatoid arthritis, the method comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 32.

34. The method of embodiment 33, wherein the subject is a human.

35. The method of embodiment 33 or 34, wherein the pharmaceutical composition is administered into the synovium of the subject.

36. The method of embodiment 33 or 34, wherein the pharmaceutical composition is administered intravenously.

The method of embodiment 33, wherein administering comprises:

(a) Isolating T cells from a biological sample obtained from the subject;

(b) Enriching regulatory T cells (tregs) in said T cells;

(c) Transfecting the enriched Treg cells with an expression vector comprising an expression control sequence operably linked to a nucleotide sequence encoding a Chimeric Antigen Receptor (CAR) comprising

Comprising an antigen binding domain that binds to a citrullinated polypeptide;

(d) Expanding the transfected Treg cells; and (e) administering the expanded Treg cells to the subject.

A method of treating a subject having rheumatoid arthritis, the method comprising:

(a) Isolating T cells from a biological sample obtained from the subject;

(b) Enriching regulatory T cells (tregs) in said T cells;

(c) Transfecting the enriched with an expression vector encoding a CAR according to any one of embodiments 1-17

A collection of Treg cells;

(d) Expanding the transfected Treg cells; and

(E) Administering the expanded Treg cells to the subject.

38. The method of embodiment 37, wherein the amplifying comprises using anti-CD 3/CD28 coated beads.

39. The method of embodiment 37, wherein the amplifying does not use anti-CD 3/CD28 coated beads.

40. The method of embodiment 37, wherein the transfection is performed by use of viral vectors, electroporation, heat shock, phage, sonication, or calcium phosphate.

41. The method of any one of embodiments 37-40, further comprising administering one or more anti-inflammatory and/or therapeutic agents to the subject.

42. The method of embodiment 41, wherein the anti-inflammatory agent comprises an antibody that inhibits a pro-inflammatory cytokine.

43. The method of embodiment 42, wherein the anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof.

44. A kit comprising a container containing the pharmaceutical composition of embodiment 32 in communication with a drip chamber through a fluid conduit, wherein the drip chamber is in communication with an intravenous needle through a fluid conduit.

45. The kit of embodiment 44, wherein the container comprises a bag.

46. The kit of embodiment 44, wherein the fluid conduit between the container and the needle comprises one or more Y-sites and a rolling clip.

It should be understood that the description and drawings are not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, the specification and drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain, wherein the antigen binding domain specifically binds to three or more different citrullinated proteins or citrullinated fragments thereof.

2. The CAR of claim 1, wherein the antigen binding domain binds to all three of: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof.

3. A CAR according to claim 2, wherein the antigen binding domain further binds citrullinated tenascin-C.

4. The CAR of claim 1, wherein the antigen binding domain comprises a VH domain and a VL domain, wherein:

5. The CAR of claim 1, wherein the intracellular signaling domain is derived from CD3- ζ.

6. The CAR of claim 1, wherein the at least one co-stimulatory domain comprises a co-stimulatory domain of a member of the group consisting of: fceR1g, fcg, CD28, CD134 (OX 40), CD137 (4-1 BB), CTLA-4/CD-28 hybrids, DAP10, CD27, 2B4, and combinations thereof, optionally wherein the at least one co-stimulatory domain comprises a CD28 and/or 4-1BB co-stimulatory domain.

7. The CAR of claim 1, wherein the antigen binding domain comprises an antibody, an antibody fragment, a camelidae nanobody, a heavy chain-only antibody, or an aptamer.

8. The CAR of claim 1, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain.

9. The CAR of claim 1, wherein the hinge domain is a CD8 hinge domain or a CD28 hinge domain.

10. The CAR of claim 1, further comprising a signal peptide.

11. The CAR of claim 10, wherein the signal peptide is a CD8 signal peptide or a GM-CSF signal peptide.

12. The CAR of claim 1, wherein the antigen-specific binding domain comprises a single-chain fragment.

13. The CAR of claim 12, wherein the single-chain fragment comprises a single-chain variable fragment (scFv).

14. The CAR of claim 13, wherein the scFv fragment comprises:

(a) A VH domain comprising the amino acid sequence of SEQ ID No. 1; and

(B) A VL domain comprising the amino acid sequence of SEQ ID No. 4.

15. The CAR of claim 13, wherein the scFv fragment comprises:

(a) VH selected from:

(1)SBT01 VH(M)

HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSI

YYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1); or (b)

(2)SBT01 VH(G)

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIY

YSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYW

GRGTLVTVSS (SEQ ID NO: 2); and

(B) VL selected from:

(1)SBT01 VL(M)

SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL(SEQ ID NO:3) Or (b)

(2)SBT01 VL(G)

SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL(SEQ ID NO:4).

16. The CAR of claim 15, wherein the VH-VL fragments are linked by a linker selected from the group consisting of: (i) GGGSx (SEQ ID NO: 20), (ii) Whitlow 218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22).

17. The CAR of claim 16, wherein the scFv comprises the amino acid sequence of:

(a) The SBT01G-VHVL-GGGSx linker of SEQ ID NO. 5; or (b)

(B) The SBT01G-VHVL-Whitlow 218 linker of SEQ ID NO. 6; or (b)

(C) SBT01G-VHVL-AB pur linker of SEQ ID NO. 7.

18. A nucleic acid encoding the CAR of any one of claims 1-17.

19. The nucleic acid of claim 18, comprising DNA or RNA.

20. The nucleic acid of claim 18, wherein the VH is encoded by a nucleic acid sequence comprising SEQ ID No. 8 or SEQ ID No. 9; and the VL is encoded by a nucleic acid sequence comprising SEQ ID NO. 10 or SEQ ID NO. 11.

21. The nucleic acid of claim 20, wherein the VH and VL fragments are connected by a linker and comprise the nucleic acid sequence of SEQ ID No. 12, or (ii) SEQ ID No. 13, or (iii) SEQ ID No. 14.

22. An expression vector comprising an expression control sequence operably linked to a nucleic acid sequence according to claim 21.

23. The expression vector of claim 22, wherein the expression control sequence comprises a regulatory region, wherein the regulatory region is selected from the group consisting of: promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 'and 3' untranslated regions, transcription initiation sites, termination sequences, polyadenylation sequences, nuclear localization, signals, and introns.

24. The expression vector of claim 23, wherein the vector is an adenovirus vector, a lentiviral vector, or a plasmid.

25. A host cell comprising the expression vector of claim 24.

26. A modified T cell that has been modified to express the Chimeric Antigen Receptor (CAR) of any one of claims 1-17.

27. The modified T cell of claim 26, wherein the T cell is a mammalian T cell.

28. The modified T cell of claim 27, wherein the T cell is a Treg cell.

29. The modified T cell of claim 28, wherein the T cell is a human T cell.

30. The modified T cell of claim 29, wherein the T cell is a primary T cell.

31. A modified T cell according to claim 30, wherein the T cell is cd4+, cd25+ and CD127lo.

32. A pharmaceutical composition comprising a plurality of modified T cells according to claim 31 and a pharmaceutically acceptable carrier.

33. A method of treating a subject suffering from rheumatoid arthritis, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 32.

34. The method of claim 33, wherein the subject is a human.

35. The method of claim 33, wherein the pharmaceutical composition is administered into the synovium of the subject.

36. The method of claim 33, wherein the pharmaceutical composition is administered intravenously.

37. A method of treating a subject suffering from rheumatoid arthritis, the method comprising:

(a) Isolating T cells from a biological sample obtained from the subject;

(b) Enriching regulatory T cells (tregs) in said T cells;

(c) Transfecting the enriched Treg cells with an expression vector encoding the CAR of any one of claims 1-17;

(d) Expanding the transfected Treg cells; and

(E) Administering the expanded Treg cells to the subject.

38. The method of claim 37, wherein the amplifying comprises using anti-CD 3/CD28 coated beads.

39. The method of claim 37, wherein the amplifying does not include the use of anti-CD 3/CD28 coated beads.

40. The method of claim 37, wherein the transfection is performed by use of a viral vector, electroporation, heat shock, phage, sonication, or calcium phosphate.

41. The method of claim 37, further comprising administering one or more anti-inflammatory and/or therapeutic agents to the subject.

42. The method of claim 41, wherein the anti-inflammatory agent comprises an antibody that inhibits a pro-inflammatory cytokine.

43. The method of claim 42, wherein the anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof.

44. A kit comprising a container containing the pharmaceutical composition of claim 32, the container in communication with a drip chamber through a fluid conduit, wherein the drip chamber is in communication with an intravenous needle through a fluid conduit.

45. The kit of claim 44, wherein the container comprises a bag.

46. The kit of claim 44, wherein the fluid conduit between the container and the needle comprises one or more Y-sites and a rolling clip.