Antigen conjugates specific for IL-23R and uses thereof

Abstract

IL23R overexpression has been described as a common feature of pathogenic inflammatory cells involved in the development and maintenance of autoimmune diseases and chronic inflammation. The present disclosure relates to novel antigen conjugates directed against IL-23R, compositions comprising said antigen conjugates, and uses of said antigen conjugates. The present disclosure further relates to fusion proteins, such as chimeric antigen receptors, comprising said antigen conjugates.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 214,950 and European Patent Application No. 21181801.8, both filed on June 25, 2021. The disclosures of both priority applications are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on June 24, 2022, is named 025297_WO038_SL.txt and is 48,578 bytes in size. [Background technology]

[0003] Interleukin-23 (IL-23), a member of the IL-12 cytokine family, is composed of two subunits, p19 and p40. The IL-23 receptor (IL-23R) consists of a complex of the IL-23Rα subunit and the IL-12Rβ1 subunit, which is a common subunit of the IL-12 receptor and interacts with tyrosine kinase 2 (Tyk2).

[0004] IL-23R is expressed primarily on immune cells, particularly T cells (e.g., Th17 and γδT cells), macrophages, dendritic cells, and NK cells (Duvallet et al., Ann Med. (2011) 43(7):503-11). The IL-23 / IL-23R signaling pathway mediates the expression of IL-17-secreting immune cells, particularly CD4 + It has been described as important in promoting the proliferation and differentiation of Th17 and γδ T cells. IL23R overexpression has been described as a common feature of pathogenic inflammatory cells involved in the development and maintenance of autoimmune diseases and chronic inflammation. Cell surface expression of IL-23R is induced by IL-23 exposure and differs depending on the level of inflammation. Summary of the Invention

[0005] The inventors herein have developed a novel tool for activating immune cells at sites of inflammation based on binding of IL-23R overexpressed at the site of inflammation. More specifically, the inventors herein disclose novel antibodies capable of binding to IL-23R. In particular, the inventors herein disclose novel antibodies capable of binding to IL-23R with high affinity. Advantageously, the IL-23R-binding antibodies of the present invention have been found to be capable of binding to both human and mouse IL-23R. By including the antigen-binding portion of the antibody in a chimeric antigen receptor (CAR) expressed on the cell surface of immune cells, binding to IL-23R allows activation of these cells at sites of inflammation. The inventors disclose stable signaling and low background activation of the antigen-binding portion. Without being bound by theory, the inventors hypothesize that the advantage of the CAR of the present invention may reside in part in the ability of the CAR to reduce tonic signaling in immune cells (e.g., T cells) expressing the CAR, thereby enhancing the therapeutic potential of the cells. Therefore, these engineered immune cells may be valuable therapeutic tools for treating autoimmune and / or inflammatory diseases or disorders.

[0006] In one aspect, the present disclosure provides an isolated anti-IL-23 receptor (IL-23R) antibody or antigen-binding fragment thereof, wherein a heavy chain variable region (VH) of the antibody or fragment comprises complementarity determining regions (HCDRs) 1 to 3 comprising SEQ ID NOs: 1 to 3, respectively, or any CDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs: 1 to 3, and a light chain variable region (VL) of the antibody or fragment comprises complementarity determining regions (LCDRs) 1 to 3 comprising SEQ ID NOs: 4 to 6, respectively, or any CDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs: 4 to 6. In one aspect, the disclosure provides an isolated anti-IL-23 receptor (IL-23R) antibody or antigen-binding fragment thereof, wherein the heavy chain variable region (VH) of the antibody or fragment comprises complementarity determining regions (HCDRs) 1 to 3 having amino acid sequences sharing at least about 90% identity with SEQ ID NOs: 1 to 3, respectively, and the light chain variable region (VL) of the antibody or fragment comprises complementarity determining regions (LCDRs) 1 to 3 having amino acid sequences sharing at least about 90% identity with SEQ ID NOs: 4 to 6, respectively. In one aspect, the disclosure provides an isolated anti-IL-23 receptor (IL-23R) antibody or antigen-binding fragment thereof, wherein the heavy chain variable region (VH) of the antibody or fragment comprises complementarity determining regions (HCDRs) 1 to 3 having amino acid sequences sharing at least about 90% identity with SEQ ID NOs: 1 to 3, respectively, and the light chain variable region (VL) of the antibody or fragment comprises complementarity determining regions (LCDRs) 1 to 3 having SEQ ID NOs: 4 to 6, respectively. In some embodiments, the antibody or antigen-binding fragment can bind mouse and human IL-23R. In some embodiments, the antibody or antigen-binding fragment can bind human IL-23R alpha subunit with an EC50 of less than 40 nM. In some embodiments, the antibody or antigen-binding fragment can bind mouse IL-23R alpha subunit with an EC50 of less than 60 nM.In some embodiments, the VH comprises SEQ ID NO:7 or an amino acid sequence that is at least about 90% identical to SEQ ID NO:7, and the VL comprises SEQ ID NO:8 or any amino acid sequence that is at least about 90% identical to SEQ ID NO:8. In some embodiments, the VH comprises SEQ ID NO:7 and the VL comprises SEQ ID NO:8. In certain embodiments, the antibody or antigen-binding fragment is an scFv comprising SEQ ID NO:15 or any amino acid sequence that is at least about 95% identical to SEQ ID NO:15. In some embodiments, the antibody or antigen-binding fragment is an scFv comprising SEQ ID NO:15.

[0007] The present disclosure also provides a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an anti-IL-23R antibody or antigen-binding fragment thereof described herein, a transmembrane domain, and a cytoplasmic domain comprising an intracellular signaling domain. In some embodiments, the CAR further comprises a leader sequence. In some embodiments, the extracellular domain comprises an scFv comprising SEQ ID NO: 15. In some embodiments, the intracellular signaling domain comprises a human CD28 costimulatory signaling domain (e.g., comprising SEQ ID NO: 32 or an amino acid sequence that is at least about 90% identical to SEQ ID NO: 32) and / or a human CD3 zeta domain (e.g., comprising SEQ ID NO: 30 or an amino acid sequence that is at least about 90% identical to SEQ ID NO: 30). In some embodiments, the transmembrane domain is derived from human CD8 (e.g., comprising SEQ ID NO: 22 or an amino acid sequence that is at least about 90% identical to SEQ ID NO: 22). In certain embodiments, the CAR comprises the extracellular domain, the transmembrane domain, and / or the intracellular signaling domain. In some embodiments, the leader sequence comprises an amino acid sequence derived from a CD8 leader sequence, optionally comprising SEQ ID NO: 40 or an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98% or 99%) identical to SEQ ID NO: 40. In some embodiments, the leader sequence comprises an amino acid sequence derived from a CD25 leader sequence, optionally comprising SEQ ID NO: 58 or an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98% or 99%) identical to SEQ ID NO: 58. In a specific embodiment, the CAR comprises an anti-IL-23R scFv (e.g., comprising SEQ ID NO: 15), a hinge domain from human CD8 (e.g., comprising SEQ ID NO: 20), a transmembrane domain from human CD8 (e.g., comprising SEQ ID NO: 22), an intracellular signaling domain comprising a human CD28 costimulatory signaling domain (e.g., comprising SEQ ID NO: 32), and a human CD3 zeta domain (e.g., comprising SEQ ID NO: 30). The CAR may also comprise a tag and / or a leader sequence.In a specific embodiment, the CAR comprises an anti-IL-23R scFv (e.g., comprising SEQ ID NO: 15), an intracellular signaling domain comprising a hinge domain from human CD8 (e.g., comprising SEQ ID NO: 20), a transmembrane domain from human CD8 (e.g., comprising SEQ ID NO: 22), a human CD28 costimulatory signaling domain (e.g., comprising SEQ ID NO: 32), a human CD3 zeta domain (e.g., comprising SEQ ID NO: 30), and a leader sequence from CD8 (e.g., comprising SEQ ID NO: 40). In a specific embodiment, the CAR comprises an anti-IL-23R scFv (e.g., comprising SEQ ID NO: 15), an intracellular signaling domain comprising a hinge domain from human CD8 (e.g., comprising SEQ ID NO: 20), a transmembrane domain from human CD8 (e.g., comprising SEQ ID NO: 22), a human CD28 costimulatory signaling domain (e.g., comprising SEQ ID NO: 32), a human CD3 zeta domain (e.g., comprising SEQ ID NO: 30), and a leader sequence from CD25 (e.g., comprising SEQ ID NO: 58).

[0008] In one aspect, the present disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding fragment described herein or a CAR described herein. The present disclosure also provides a vector (e.g., an expression vector) comprising the nucleic acid molecule. Furthermore, the present disclosure provides a cell comprising the nucleic acid molecule or vector described herein. Furthermore, the present disclosure provides an immune cell comprising the nucleic acid molecule or vector described herein.

[0009] The present disclosure also provides a composition comprising the cells described herein. The present disclosure also provides a composition comprising the immune cells described herein. The present disclosure also provides a cell or cell population described herein for use as a medicament. The present disclosure also provides an immune cell or immune cell population described herein for use as a medicament. In some embodiments, the cell or immune cell population is for use in treating a disease or disorder mediated by IL-23R expressing cells in a subject in need of treatment. In some embodiments, the immune cell or immune cell population is for use in treating a disease or disorder mediated by IL-23R expressing cells in a subject in need of treatment. The present disclosure also provides a method for treating a disorder or disease in a subject in need of treatment, comprising administering to the patient a cell described herein, or a cell population described herein. The present disclosure also provides a method for treating a disorder or disease in a subject in need of treatment, comprising administering to the patient an immune cell described herein, or a cell population described herein. The disease or disorder can be a disease or disorder mediated by IL-23R expressing cells in a subject in need of treatment. The disease or disorder can be an autoimmune or inflammatory disease or disorder. In certain embodiments, the disease or disorder is an autoimmune or inflammatory disease or disorder.The disease or disorder can be selected from, for example, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), lupus (e.g., systemic lupus erythematosus), arthritis (e.g., rheumatoid arthritis or juvenile idiopathic arthritis), Sjogren's syndrome, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, type 1 diabetes, autoimmune thyroid disorder, myasthenia gravis, psoriasis, psoriatic arthritis, skin disease, and uveitis.In a specific embodiment, the disease is Crohn's disease. [Brief description of the drawings]

[0010] [Figure 1]1 shows a schematic diagram of the anti-IL-23R chimeric antigen receptor (CAR) construct (CAR#2) of the present disclosure. The anti-IL23R CAR comprises an scFv directed against human / mouse (hm) IL-23R (αIL-23R; IL23RLamS4-G3), a hinge domain (CD8 linker), a transmembrane domain from human CD8 (CD8 TM), an intracellular signaling domain from human CD28 (CD28), and a CD3 zeta (CD3ζ or CD3Z). The CAR construct further comprises a GFP coding sequence and an HA tag coding sequence. This construct is compared to the CAR#1 construct, which is derived from scFv 14-11-D07 and also targets IL-23R. [Figure 2A] Graphs monitoring the regulatory T cell (Treg) phenotype of non-transduced cells and cells transduced with CAR#2 or CAR#1 at the end of the first expansion cycle. Treg cells were labeled with antibodies directed against human CD4, CD25, CD127, and CTLA-4. Nuclear labeling was performed for detection of FOXP3 and Helios transcription factors (A). Helios and FOXP3 were also assessed after 11 days of CAR binding (B). Error bars represent the mean ± SEM from three independent experiments including a total of five Treg donors. [Figure 2B] Graphs monitoring the regulatory T cell (Treg) phenotype of non-transduced cells and cells transduced with CAR#2 or CAR#1 at the end of the first expansion cycle. Treg cells were labeled with antibodies directed against human CD4, CD25, CD127, and CTLA-4. Nuclear labeling was performed for detection of FOXP3 and Helios transcription factors (A). Helios and FOXP3 were also assessed after 11 days of CAR binding (B). Error bars represent the mean ± SEM from three independent experiments including a total of five Treg donors. [Diagram 3]Graph showing Treg activation status (measured by CD69 expression gated on GFP expression) either in the absence of activation (No Activation) or after 24 h of stimulation via CAR (by addition of IL-23R-coated beads; IL-23R) or TCR (by anti-CD3 and anti-CD28 coated beads; 3 / 28) from three independent experiments involving a total of five Treg donors. [Figure 4] Figure 1 is a combination of graphs showing that Treg cells expressing CAR#2 exhibit efficient CAR-mediated suppressive activity. Contact-dependent suppression mediated by IL-23R-CAR in the absence of activation (dotted curve) or after IL-23R-induced CAR activation (black line) or TCR-induced activation (grey curve) was assessed by measuring the proliferation of conventional T cells (Tconv) using flow cytometry. Error bars represent the mean ± SEM from three independent experiments including a total of four Treg donors. [Diagram 5] Schematic diagram of the mouse version (mCAR#2b) of the anti-IL-23R chimeric antigen receptor (CAR) construct of the present disclosure. This mouse version contains a scFv directed against human / mouse IL-23R (αIL-23R; IL23RLamS4-G3, which is human / mouse cross-reactive), the CD8 transmembrane domain from mouse hinge (mCD8 TM), the intracellular domain from mouse CD28 (mCD28), and mouse CD3 zeta (mCD3Z). This construct is compared to mouse CAR#2a (mCAR#2a), which corresponds to mCAR#2b but has the mouse CD28 transmembrane domain (mCD28 TM), and mouse CAR#1 (mCAR#1), which is derived from a published scFv (14-11-D07) targeting IL-23R. [Figure 6A] FIG. 1 depicts a schematic diagram illustrating the design of a short model of dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD). [Figure 6B] The set of graphs shows measurements of CTLA-4 positive Treg cells in mesenteric lymph nodes (left), colon (middle), and spleen (right). [Figure 7]A depicts a schematic showing the design of the efficacy model for DSS-induced IBD. B depicts graphs showing measurements of disease activity indexes for three groups of mice receiving control treatment (saline), non-transduced Treg cells ("Poly Treg"), or CAR-Treg cells ("CAR#2b Treg"). [Figure 8] Graph showing consistent transduction efficiency (approximately 40-50%) for CAR#2, CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7. Results are the mean ± SEM of nine different Treg donors. [Figure 9] 1 depicts a graph showing consistent survival and fold expansion of CAR-Tregs for CAR#2, CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7. Results are the mean ± SEM of 9 different Treg donors. [Figure 10] 1 depicts a graph showing CAR-Treg activation for CAR#2, CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7. CAR#3 showed the lowest activation background and the highest signal-to-noise after CAR activation. CAR Treg activation was assessed by flow cytometry by measuring CD69 expression on the cell surface of CAR Tregs 24 hours after inducing activation through the TCR (gray) with anti-CD3 / anti-CD28 coated beads (gray) or through the CAR with either IL-23R coated beads or Jurkats expressing low levels of IL-23R on the cell surface (Jurkat 572) or high levels of IL-23R on the cell surface (Jurkat 573). Results are the mean ± SEM of six Treg donors. Two-way ANOVA and Sidak's multiple comparison test. *=0.05, **=0.01, ***=0.001, and ****=0.0001. [Figure 11A]Figure 1 depicts a graph showing CAR-mediated suppressive activity of CAR#2, CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7. Suppression mediated by CAR Tregs was assessed by flow cytometry by measuring inhibition of Tconv proliferation after 3 days of co-culture. CAR Tregs are activated 24 hours prior to co-culture through the TCR using anti-CD3 / anti-CD28 coated beads (closed circles) or through the CAR using either IL-23R coated beads (closed squares) or Jurkat cell lines expressing high levels of IL-23R at the cell surface (Jurkat 573, grey squares) or low levels of IL-23R (Jurkat 572, open squares). Results are mean ± SEM from 4 independent experiments involving 6 Treg donors. Two-way ANOVA and Sidak's multiple comparison test. *=0.05, **=0.01. [Figure 11B] Figure 1 depicts a graph showing CAR-mediated suppressive activity of CAR#2, CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7. Suppression mediated by CAR Tregs was assessed by flow cytometry by measuring inhibition of Tconv proliferation after 3 days of co-culture. CAR Tregs are activated 24 hours prior to co-culture through the TCR using anti-CD3 / anti-CD28 coated beads (closed circles) or through the CAR using either IL-23R coated beads (closed squares) or Jurkat cell lines expressing high levels of IL-23R at the cell surface (Jurkat 573, grey squares) or low levels of IL-23R (Jurkat 572, open squares). Results are mean ± SEM from 4 independent experiments involving 6 Treg donors. Two-way ANOVA and Sidak's multiple comparison test. *=0.05, **=0.01. [Figure 12]The area under the curve (AUC) for CAR-mediated suppressive activity of CAR#2 and CAR#3 is shown. The AUC calculation reveals that only CAR#3 showed the same CAR-mediated suppression when Tregs were bound to Jurkats expressing high levels of IL-23R on the cell surface (Jurkat 573), while CAR-mediated suppression was low when CAR Tregs were bound to Jurkats expressing low levels (Jurkat 572). This exemplary result supports the reduction of off-target effects by CAR#3. [Figure 13] Figure 1 depicts a graph showing the phenotypic stability of the Treg phenotype. Major Treg markers (CD4, CD25, CD127, FoxP3, Helios and CTLA-4) were measured by flow cytometry. Results are the mean ± SEM of 9 different Treg donors. [Figure 14] 1 shows the affinity (EC50) results of CAR#1 and CAR#2 binding to mouse and human IL23R. [Figure 15] Shown are four different constructs (CAR#8, CAR#9, CAR#10 and CAR#11) made with different expression cassettes (PGK or EF1a promoter, + / -WPRE Mut6). [Figure 16] 1 depicts a graph showing the stability of scFv signaling with various CAR constructs of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] definition In this disclosure, the following terms have the following meanings.

[0012] "About" when referring to a measurable value, such as an amount, a period of time, or the like, is meant to encompass a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the particular value, where such variations are appropriate for performing the disclosed methods.

[0013] "Affinity" is used to define the strength of an antibody-antigen complex. Affinity indicates the strength of the interaction between an epitope and an antigen-binding site on an antibody. It is defined as the affinity constant K a or the dissociation constant K D It can be expressed as: K D An antibody is said to specifically bind an antigen when its K is less than 1 μM, preferably less than 100 nM or less than 10 nM. D can be measured, for example, by surface plasmon resonance (SPR) (BIAcore®) or biolayer interferometry, using, for example, an IBIS MX96 SPR system from IBIS Technologies, a ProteOn® XPR36 SPR system from Bio-Rad, or an Octet® system from ForteBio.

[0014] As used herein, an "antibody" or "immunoglobulin" refers to a tetramer comprising two heavy chains and two light chains interconnected by disulfide bonds. Each light chain is composed of a light chain variable domain or region (VL) and a light chain constant region (CL), and may be a kappa (κ) light chain or a lambda (λ) light chain. Each heavy chain is composed of a heavy chain variable domain or region (VH) and a heavy chain constant region (CH). Based on the amino acid sequence of the CH, antibodies may be assigned to different isotypes: IgA, IgD, IgE, IgG, or IgM. The IgG and IgA isotypes are further divided into subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The pairing of the VH and VL forms a single antigen-binding site. In one embodiment, the anti-IL-23R antibody of the present disclosure is an IgG antibody.

[0015] "Antigen-binding fragment" as used herein refers to a portion or region of an antibody or derivative that contains fewer amino acid residues than a whole antibody but is still capable of binding to the antigen of the whole antibody (e.g., IL-23R). Antigen-binding fragments include, but are not limited to, single chain antibodies, Fvs (e.g., scFvs), Fabs, Fab's, Fab'-SH, F(ab)'2, Fds, defucosylated antibodies, diabodies, triabodies, and tetrabodies.

[0016] "Chimeric antigen receptor" or "CAR" refers to a protein, such as a fusion protein, that when expressed in an immune cell provides the cell with specificity for a target ligand and intracellular signal generation. In some embodiments, a CAR comprises a set of polypeptides that includes a dimerization switch that can link the polypeptides together in the presence of a dimerization molecule, e.g., linking a ligand binding domain to an intracellular signaling domain. In one embodiment, a CAR comprises an optional leader sequence at the N-terminus that is cleaved during intracellular processing and localization of the chimeric antigen receptor to the cell membrane.

[0017] "Complementarity determining region" or "CDR" refers to the non-contiguous antigen-binding sites found within the heavy chain variable region (VH) and the light chain variable region (VL). The exact amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those set forth in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., JMB (1997) 273:927-948 ("Chothia" numbering scheme), or a combination thereof. More recently, a universal numbering system has been developed and has been widely adopted, the ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. (1999) 27:209-212). In one embodiment, the CDR boundaries herein are defined according to Kabat et al. (1991).

[0018] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. A costimulatory signaling domain may be the intracellular portion of a costimulatory molecule. Costimulatory molecules may be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors.

[0019] "Epitope" refers to a specific sequence of amino acids located on a protein(s) to which an antibody or antigen-binding fragment thereof binds. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear (or continuous) or structural, i.e., comprising two or more amino acid sequences (not necessarily contiguous) in different regions of the antigen.

[0020] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0021] "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., from about 230 amino acids (aa) to about 450 aa of a human gamma heavy chain, or corresponding sequences in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies) or naturally occurring allotypes thereof.

[0022] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains generates six hypervariable loops (three loops each from the H chain and L chain), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0023] "Identity" or "identical" as used herein to describe the relationship between two or more amino acid sequences or two or more nucleic acid sequences refers to the degree of sequence relatedness between the sequences being compared. "Identity" refers to the smaller percentage of identity matches between two or more sequences, with gap alignments (if any) being addressed by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related amino acid or nucleic acid sequences can be easily calculated by known methods. Such methods include, but are not limited to, those described in: Lesk AM (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov MR & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carrillo et al., SIAM J Appl Math. (1988) 48(5):1073-82. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two sequences include the GCG program package, such as GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., Nucleic Acids Res. (1984) 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., J Mol Biol. (1990) 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). Identity can also be determined using the well-known Smith Waterman algorithm.

[0024] As used herein, "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of the chimeric receptor-containing cell. Examples of immune effector function in chimeric receptor-T cells may include cytolytic activity, suppressive activity, regulatory activity, and helper activity, such as secretion of cytokines.

[0025] A "subject" is intended to include an organism (e.g., a mammal, a human) in which an immune response can be elicited. In one embodiment, the subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, awaiting or receiving medical care, or being / being / being the subject of medical treatment, or being monitored for the development of a disease or condition, such as an inflammatory or autoimmune condition, that is targeted. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is suffering from, and preferably diagnosed with, an autoimmune and / or inflammatory disease or disorder. In one embodiment, the subject is at risk of developing an autoimmune and / or inflammatory disease or disorder. Examples of risk factors include, but are not limited to, a genetic predisposition or family history of an autoimmune and / or inflammatory disease or disorder.

[0026] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are linked contiguously, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and the scFv can comprise a VL-linker-VH or a VH-linker-VL. In one embodiment, the antigen-binding fragment of the invention is a single-chain Fv (scFv).

[0027] "Therapeutically effective amount" refers to a level or amount of an antibody described herein that is intended to (1) delay or prevent the onset of a disease, disorder, or condition, (2) slow or stop the progression, progression, or worsening of one or more symptoms of a disease, disorder, or condition, (3) bring about an improvement in the symptoms of a disease, disorder, or condition, (4) reduce the severity or incidence of a disease, disorder, or condition, or (5) cure a disease, disorder, or condition, without causing significant negative or harmful side effects to the target. A therapeutically effective amount may be administered prior to the onset of a disease, disorder, or condition for a prophylactic or preventative effect. Alternatively or additionally, a therapeutically effective amount may be administered after the onset of a disease, disorder, or condition for a therapeutic effect.

[0028] "Treating" or "treatment" or "alleviating" refers to both therapeutic treatment and prophylactic or preventative treatment, the purpose of which is to prevent or slow down (alleviate) the targeted condition or disorder. Those in need of treatment include those who already have the disorder, and those who are prone to have the disorder, or those whose disorder should be prevented. In one embodiment, a subject is successfully "treated" against a disease or disorder if, after administration of a therapeutic amount of an antibody or cell according to the present disclosure, the subject shows at least one of the following: a reduction in the number or percentage (%) of pathogenic cells, some alleviation of one or more symptoms associated with the disease or disorder being treated, a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing therapeutic success and improvement in a disease are easily measurable by routine procedures familiar to physicians.

[0029] "Zeta" or alternatively "zeta chain", "CD3-zeta", or "TCR-zeta" is defined as the protein provided under GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, which are sufficient to functionally transmit the initial signal required for T cell activation. In one embodiment, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., that are functional orthologs thereof.

[0030] I. Antibodies and Antigen-Binding Fragments The present disclosure relates first to an isolated monoclonal antibody or antigen-binding fragment thereof, which binds to at least one IL-23R (eg, an IL-23R alpha subunit).

[0031] An "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to IL-23R is substantially free of antibodies that specifically bind to antigens other than IL-23R). However, an isolated antibody that specifically binds to IL-23R may have cross-reactivity to other antigens, such as IL-23R molecules from other species. In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals, particularly those that may interfere with the therapeutic use of the antibody (including, but not limited to, enzymes, hormones, and other proteinaceous or non-proteinaceous components). An isolated antibody herein may be an IgG antibody, such as an IgG1, IgG2, or IgG4 antibody.

[0032] I.1 Antigen specificity and affinity In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is capable of recognizing and binding to IL-23R expressed on the cell surface.

[0033] In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention are capable of recognizing and binding to soluble IL-23R (ie, not membrane bound).

[0034] Advantageously, it has been found that the IL-23R binding antibodies or antigen-binding fragments of the invention can bind to both human and mouse IL-23R with particularly high affinity (Figure 14). This cross-reactivity is beneficial for extrapolating the results of preclinical studies in mice to human clinical trials for the drug approval process.

[0035] In certain embodiments, an antibody or antigen-binding fragment of the invention, such as an scFv according to the invention, can bind both mouse and human IL-23R (Figure 14). Thus, the cross-reactivity of the scFv with mouse and human IL-23R is an advantageous feature.

[0036] In one embodiment, the antibody or antigen-binding fragment of the present invention recognizes and binds to the human IL-23R alpha subunit. Preferably, the antibody or antigen-binding fragment of the present invention binds to the human IL-23R alpha subunit with high affinity (Figure 14). The human IL-23R alpha subunit is a protein (Genbank accession number: NM_144701) encoded by a 2.8 kb long mRNA containing 11 exons.

[0037] In one embodiment, the antibody or antigen-binding fragment thereof of the invention is capable of recognizing and binding to an IL-23R variant, such as a variant of human IL-23R.

[0038] In one embodiment, a variant of IL-23R refers to an altered IL-23R in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids have been deleted, added, or substituted compared to the original (wild-type) IL-23R.

[0039] A splice variant of human IL-23R has already been identified (Kan et al, Genes and Immunity (2008) 9(7):631-639). Specifically, 24 different isoforms of IL-23R have been described: isoform_v1 (encoded by the mRNA with Genbank accession number AM990313), isoform_v2 (encoded by the mRNA with Genbank accession number AM990314), isoform_v3 (encoded by the mRNA with Genbank accession number AM990315), isoform_v4 (encoded by the mRNA with Genbank accession number AM990316), isoform_v5 (encoded by the mRNA with Genbank accession number AM990317), isoform_v6 (encoded by the mRNA with Genbank accession number AM990318), isoform_v7 (encoded by the mRNA with Genbank accession number AM990319), isoform_v8 (encoded by the mRNA with Genbank accession number AM990319), isoform_v9 (encoded by the mRNA with Genbank accession number AM990310), isoform_v10 (encoded by the mRNA with Genbank accession number AM990311), isoform_v11 (encoded by the mRNA with Genbank accession number AM990312), isoform_v12 (encoded by the mRNA with Genbank accession number AM990313), isoform_v13 (encoded by the mRNA with Genbank accession number AM990314), isoform_v14 (encoded by the mRNA with Genbank accession number AM990315), isoform_v15 (encoded by the mRNA with Genbank accession number AM990316), isoform_v16 (encoded by the mRNA with Genbank accession number AM990317), isoform_v17 (encoded by the mRNA with Genbank accession number AM990318), isoform_v18 (encoded by the mRNA with Genbank accession number AM99 isoform_v9 (encoded by the mRNA with Genbank accession number AM990320), isoform_v9 (encoded by the mRNA with Genbank accession number AM990321), isoform_v10 (encoded by the mRNA with Genbank accession number AM990322), isoform_v11 (encoded by the mRNA with Genbank accession number AM990323), isoform_v12 (encoded by the mRNA with Genbank accession number AM990324), isoform_v13 (encoded by the mRNA with Genbank accession number AM990325), isoform_v14 (encoded by the mRNA with Genbank accession number AM990326), isoform_v15 (encoded by the mRNA with Genbank accession number AM990327),isoform_v16 (encoded by the mRNA having Genbank accession number AM990328), isoform_v17 (encoded by the mRNA having Genbank accession number AM990329), isoform_v18 (encoded by the mRNA having Genbank accession number AM990330), isoform_v19 (encoded by the mRNA having Genbank accession number AM990331), isoform_v20 (encoded by the mRNA having Genbank accession number AM990332), isoform_v21 (encoded by the mRNA having Genbank accession number AM990333), isoform_v22 (encoded by the mRNA having Genbank accession number AM990334), isoform_v23 (encoded by the mRNA having Genbank accession number AM990335) and isoform_v24 (encoded by the mRNA having Genbank accession number AM990336). ,

[0040] Thus, in one embodiment, an antibody or antigen-binding fragment thereof of the invention is capable of recognizing and binding to a splice variant of human IL-23R selected from the group including isoform_v1, isoform_v2, isoform_v3, isoform_v4, isoform_v5, isoform_v6, isoform_v7, isoform_v8, isoform_v9, isoform_v10, isoform_v11, isoform_v12, isoform_v13, isoform_v14, isoform_v15, isoform_v16, isoform_v17, isoform_v18, isoform_v19, isoform_v20, isoform_v21, isoform_v22, isoform_v23, and isoform_v24.

[0041] Furthermore, single nucleotide polymorphisms in the alpha subunit of human IL-23R have already been described (Kan et al, supra; and Sivanesan et al., J Biol Chem. (2016) 291(16):8673-85).

[0042] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is capable of recognizing and binding to a human IL-23R variant comprising a single nucleotide polymorphism (SNP) in the alpha subunit, wherein the SNP is selected from the group comprising R381Q, G149R, V362I, and combinations thereof.

[0043] In one embodiment, the antibody or antigen-binding fragment of the present invention recognizes and binds to the mouse IL-23R alpha subunit. Preferably, the antibody or antigen-binding fragment of the present invention binds to the mouse IL-23R alpha subunit with high affinity (Figure 14). The mouse IL-23R alpha subunit is a protein (Genbank accession number: NM_144548) encoded by a 2.5 kb long mRNA containing 11 exons.

[0044] A splice variant of the mouse IL-23R has already been identified. Specifically, seven different isoforms of IL-23R have been described: isoform 1 (Uniprot Q5VWK5-1; NM_144701.3), isoform 2 (Uniprot Q5VWK5-2; XP_005270573), isoform 3 (Uniprot Q5VWK5-3), isoform 4, (Uniprot Q5VWK5-4), and isoform 5 (Uniprot Q5VWK5-5) (Zhang, et al., Immunogenetics 57:934-943 (2006)), and isoform 6, (Uniprot Q5VWK5-6; XP_005270574), and isoform 7 (Uniprot Q5VWK5-7) (Genome Res. 14:2121-2127 (2004)). In some embodiments, an anti-IL23R antibody or antigen-binding fragment thereof of the disclosure binds to one or more of isoforms 1-7. In some embodiments, an anti-IL23R antibody or antigen-binding fragment thereof of the disclosure binds to at least one of isoform 1 and isoform 3. In some embodiments, an anti-IL23R antibody or antigen-binding fragment thereof of the disclosure binds to at least one of isoform 4 and isoform 6.

[0045] I.2.1 CDR sequences In one embodiment, the CDRs are determined according to the Kabat CDR definition system.

[0046] In one embodiment, the heavy chain of an antibody or antigen-binding fragment of the invention has the following heavy chain CDRs (HCDRs): HCDR1: SSNYYWG (SEQ ID NO: 1) HCDR2: GSIYYSGNTYYNPSL (SEQ ID NO: 2) HCDR3: REWSPYESEGFDY (SEQ ID NO: 3) at least one, preferably at least two, and more preferably all three of

[0047] In a specific embodiment, an antibody or antigen-binding fragment of the invention comprises all of SEQ ID NOs: 1-3. In one embodiment, any of HCDR1, HCDR2, and / or HCDR3 may comprise one, two, three or more amino acid modifications (e.g., substitutions) compared to SEQ ID NOs: 1-3, respectively. In one embodiment, any of HCDR1, HCDR2, and / or HCDR3 has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 1-3, respectively.

[0048] In one embodiment, the light chain of an antibody or antigen-binding fragment of the invention has the following light chain CDRs (LCDRs): LCDR1: TGSSSNIGAGYDVH (SEQ ID NO: 4) LCDR2: GNNNRPS (SEQ ID NO: 5) LCDR3: QSYDTGLSAW (SEQ ID NO: 6) at least one, preferably at least two, and more preferably all three of

[0049] In a specific embodiment, an antibody or antigen-binding fragment of the invention comprises all of SEQ ID NOs: 4 to 6. In one embodiment, any of LCDR1, LCDR2, and / or LCDR3 may comprise one, two, three, four, five or more amino acid alterations (e.g., substitutions) compared to SEQ ID NOs: 4 to 6, respectively. In one embodiment, any of LCDR1, LCDR2, and / or LCDR3 has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 4 to 6, respectively.

[0050] In one embodiment of the antibody or antigen-binding fragment of the present invention, at least one, preferably at least two, and more preferably all three of its HCDRs 1 to 3 comprise SEQ ID NOs: 1 to 3, respectively, and at least one, preferably at least two, and more preferably all three of its LCDRs 1 to 3 comprise SEQ ID NOs: 4 to 6, respectively.

[0051] In one embodiment, the antibody or antigen-binding fragment of the invention comprises HCDR1-3 and LCDR1-3 having the sequences of SEQ ID NOs: 1-6, respectively.

[0052] In one embodiment of the antibody or antibody-binding fragment of the invention, any of HCDR1, HCDR2, and / or HCDR3 has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 1 to 3, respectively, and any of LCDR1, LCDR2, and / or LCDR3 has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 4 to 6, respectively.

[0053] I.2.2 VH and VL sequences In one embodiment, the antibody or antigen-binding fragment of the invention has a VH amino acid sequence comprising or consisting of SEQ ID NO:7.

[0054] In one embodiment, the VH amino acid sequence comprises or consists of SEQ ID NO:7 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid alterations (e.g., substitutions).

[0055] In one embodiment, the VH amino acid sequence comprises the HCDRs described above (e.g., SEQ ID NOs: 1-3) and shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 7.

[0056] In one embodiment, the antibody or antigen-binding fragment of the invention has a VL amino acid sequence comprising or consisting of SEQ ID NO:8.

[0057] In one embodiment, the VL amino acid sequence comprises or consists of SEQ ID NO:8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid alterations (e.g., substitutions).

[0058] In one embodiment, the VL amino acid sequence comprises the LCDRs described above (e.g., SEQ ID NOs: 4-6) and shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 8.

[0059] In one embodiment, the VH comprises or consists of SEQ ID NO:7 and / or the VL comprises or consists of SEQ ID NO:8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications (e.g., substitutions).

[0060] In one embodiment, the VH and VL comprise the above CDRs (e.g., SEQ ID NOs: 1-6) and share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs: 7 and 8, respectively.

[0061] In one embodiment, the amino acid modification may be an insertion, deletion, or substitution. In one embodiment, the amino acid modification does not significantly affect the binding properties of the antibody or antigen-binding fragment thereof that contains the modification. The designated variable region and CDR sequences may include insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids.

[0062] In one embodiment, the amino acid modification is preferably a substitution with a conservative amino acid. A conservative amino acid is an amino acid with a side chain that has similar physicochemical properties as the original amino acid. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDRs and / or variable regions of an antibody or antigen-binding fragment of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody or antigen-binding fragment can be tested for retained function (e.g., binding to IL-23R) using the assays described herein. In another embodiment, strings of amino acids in the CDRs and / or variable regions of an antibody or antigen-binding fragment of the invention can be replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0063] In one embodiment, the antibody or antigen-binding fragment of the invention has the following: - a VH comprising at least one (preferably three) HCDRs as defined herein and comprising or consisting of an amino acid sequence having SEQ ID NO: 7 or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7; - a VL comprising at least one (preferably three) LCDRs as defined herein and comprising or consisting of an amino acid sequence of SEQ ID NO: 8 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 8; Includes.

[0064] In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH comprising or consisting of SEQ ID NO:7 and a VL comprising or consisting of SEQ ID NO:8.

[0065] I.2.3 Linker In one embodiment, an antigen-binding fragment of the invention comprises a linker linking its VH and VL. In one embodiment, an antigen-binding fragment comprises, from N-terminus to C-terminus, a VL, a linker, and a VH. In another embodiment, an antigen-binding fragment comprises, from N-terminus to C-terminus, a VH, a linker, and a VL.

[0066] In one embodiment, the linker is a peptide linker having a length in the range of, for example, 2-20 or 2-15 amino acids.

[0067] For example, a glycine-serine doublet provides a particularly suitable linker (GS linker). In one embodiment, the linker is a GS linker. Examples of GS linkers include, but are not limited to, GS linkers, G2S linkers (e.g., GGS and (GGS)2), G3S linkers, and G4S linkers.

[0068] The G3S linker has the amino acid sequence (Gly-Gly-Gly-Ser). n or (GGGS) n (SEQ ID NO: 9), where n is a positive integer equal to or greater than 1 (e.g., n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10). An example of a G3S linker includes, but is not limited to, GGGSGGGSGGGSGGGS (SEQ ID NO: 10).

[0069] Examples of G4S linkers include, but are not limited to, (Gly4-Ser) corresponding to GGGGS (SEQ ID NO: 11); (Gly4-Ser)2 corresponding to GGGGSGGGGS (SEQ ID NO: 12); (Gly4-Ser)3 corresponding to GGGSGGGGSGGGGGS (SEQ ID NO: 13); and (Gly4-Ser)4 corresponding to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14). In one embodiment, the linker is a (G4S)3 linker (SEQ ID NO: 13).

[0070] I.2.4 Types of antibodies and fragments In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure is a humanized antibody.

[0071] In one embodiment, the antibody or antigen-binding fragment thereof according to the present disclosure is an antigen-binding fragment of an antibody, such as a single chain antibody, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, a defucosylated antibody, a diabody, a triabody, or a tetrabody.

[0072] In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure binds to human IL-23R alpha subunit with high affinity. In an embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR defined herein and binds to human IL-23R alpha subunit with high affinity.

[0073] In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure binds to the murine IL-23R alpha subunit with high affinity. In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR defined herein and binds to the murine IL-23R alpha subunit with high affinity.

[0074] In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure can bind to human IL-23R alpha subunit with an EC50 of less than 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM or 30 nM, particularly less than 100 nM, particularly less than 40 nM. In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure can bind to mouse IL-23R alpha subunit with an EC50 of less than 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM or 30 nM, particularly less than 100 nM, particularly less than 60 nM.

[0075] In one embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure can cross-react with both human and mouse IL-23R. In an embodiment, an antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR defined herein and can cross-react with both human and mouse IL-23R.

[0076] I.2.5 scFv complete sequence In one embodiment, the antigen-binding fragment of the invention is an scFv comprising the CDRs defined herein and comprises or consists of SEQ ID NO:15 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:15.

[0077] In one embodiment, the scFv binds to human IL-23R alpha subunit with high affinity. In certain embodiments, the scFv comprises the CDRs defined herein and binds to human IL-23R alpha subunit with high affinity. In certain embodiments, the scFv comprises or consists of SEQ ID NO: 15 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15 and binds to human IL-23R alpha subunit with high affinity.

[0078] In one embodiment, the scFv binds to mouse IL-23R alpha subunit with high affinity. In an embodiment, the scFv comprises the CDRs defined herein and binds to mouse IL-23R alpha subunit with high affinity. In an embodiment, the scFv comprises or consists of SEQ ID NO: 15 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15 and binds to mouse IL-23R alpha subunit with high affinity.

[0079] In one embodiment, an scFv according to the disclosure can bind to human IL-23R alpha subunit with an EC50 of less than 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM or 30 nM, particularly less than 100 nM, particularly less than 40 nM. In one embodiment, an scFv according to the disclosure can bind to mouse IL-23R alpha subunit with an EC50 of less than 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM or 30 nM, particularly less than 100 nM, particularly less than 60 nM.

[0080] In one embodiment, the scFv is capable of cross-reacting with both human and mouse IL-23R. In certain embodiments, the scFv comprises the CDRs defined herein and is capable of cross-reacting with both human and mouse IL-23R. In certain embodiments, the scFv comprises or consists of SEQ ID NO: 15 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15 and binds to both human and mouse IL-23R.

[0081] I.3 Nucleic acids Another object of the disclosure is an isolated nucleic acid encoding an isolated anti-IL-23R antibody, or antigen-binding fragment thereof, of the invention.

[0082] "Isolated nucleic acid", as used herein, is intended to refer to a nucleic acid that is substantially separated from other genomic DNA sequences, as well as proteins or complexes that naturally accompany the native sequence, such as ribosomes and polymerases. The term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, including recombinant or cloned DNA isolates, and chemically synthesized analogs, or biologically synthesized analogs by heterologous systems. Substantially pure nucleic acid includes isolated forms of nucleic acid. This refers to a nucleic acid that is originally isolated, and does not exclude genes or sequences that are later artificially added to the isolated nucleic acid.

[0083] In one embodiment, the nucleic acid encodes at least the VH or VL of an antibody or antigen-binding fragment of the invention. In one embodiment, the nucleic acid encodes the variable and constant regions of the heavy or light chain of an antibody or antigen-binding fragment of the invention. In one embodiment, the nucleic acid encodes both the heavy and light chains of the antibody or antigen-binding fragment.

[0084] In one embodiment, the nucleic acid herein comprises or consists of a nucleotide sequence encoding the VH of an antibody or antigen-binding fragment of the invention, which is SEQ ID NO:16 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:16.

[0085] In one embodiment, the nucleic acid herein comprises or consists of a sequence encoding the VL of an antibody or antigen-binding fragment of the invention, wherein the nucleotide sequence is SEQ ID NO:17 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:17.

[0086] In one embodiment, the nucleic acid herein comprises a nucleotide sequence encoding the VH and VL of an antibody or antigen-binding fragment of the invention. In a further embodiment, the nucleic acid herein comprises SEQ ID NOs: 16 and 17.

[0087] In one embodiment, the nucleic acid herein further comprises a linker nucleotide sequence between the VL coding sequence and the VH coding sequence. In a further embodiment, the linker nucleotide sequence comprises or consists of SEQ ID NO: 18.

[0088] In one embodiment, the nucleic acid herein comprises or consists of SEQ ID NO:19 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:19.

[0089] I.4. Production of Antibodies and Antigen-Binding Fragments The present disclosure also provides vectors for expressing the antibodies or antigen-binding fragments thereof of the invention, and methods of using the vectors to produce the antibodies or antigen-binding fragments thereof.

[0090] In general, a suitable vector contains an origin of replication functional in at least one host organism, a promoter sequence, convenient restriction endonuclease sites, one or more selectable markers and optionally an enhancer.

[0091] Examples of promoters and enhancers used in mammalian cell expression vectors include, but are not limited to, the SV40 early promoter and enhancer, the Moloney murine leukemia virus LTR promoter and enhancer, and the immunoglobulin H chain promoter and enhancer. For further examples of transcriptional regulatory sequences, see also Section II below.

[0092] Another object of the present disclosure is a method for producing and purifying the isolated antibodies or antigen-binding fragments thereof described herein. In one embodiment, the method comprises the steps of: - introducing an expression vector containing an expression cassette for the antibody or antigen-binding fragment into a competent host cell (e.g., a mammalian cell, such as a CHO cell or an NS0 cell), either in vitro or ex vivo; - culturing the transformed host cell in vitro or ex vivo under conditions suitable for expression of the antibody or antigen-binding fragment thereof; - optionally selecting cells that express and / or secrete the antibody or antigen-binding fragment; - recovering the expressed antibody or antigen-binding fragment from the cell culture; and - optionally purifying the recovered antibodies or antigen-binding fragments; Includes.

[0093] Methods for purifying proteins, particularly antibodies or antigen-binding fragments, are well known in the art and include, but are not limited to, protein A-sepharose, gel electrophoresis, and chromatography (e.g., affinity chromatography, such as affinity chromatography on protein L agarose).

[0094] II. Fusion Proteins The present disclosure further relates to a fusion protein comprising the antibody or antigen-binding fragment thereof of the present disclosure. In one embodiment, the fusion protein is a chimeric antigen receptor (CAR).

[0095] II.1 CAR Of interest to the present disclosure is a CAR specific for at least one IL-23R. The CAR may comprise (i) an extracellular binding domain comprising an anti-IL-23R antigen-binding fragment as described herein, (ii) an optional extracellular hinge domain, (iii) a transmembrane domain, (iv) an intracellular signaling domain, and (v) an optional tag and / or leader sequence. In one embodiment, the CAR comprises one or more polypeptides, e.g., two polypeptides.

[0096] II.1.1. Extracellular Binding Domain In one embodiment, the extracellular binding domain of the CAR comprises or consists of an antigen-binding fragment of the present disclosure. In one embodiment, the antigen-binding fragment comprises at least one of HCDR1-3 having SEQ ID NOs: 1-3, respectively, and / or at least one of LCDR1-3 having SEQ ID NOs: 4-6, respectively. In a further embodiment, the antigen-binding fragment in the extracellular binding domain of the CAR comprises HCDR1-3 and LCDR1-3 having SEQ ID NOs: 1-6, respectively. In one embodiment, the extracellular binding domain of the CAR comprises an antigen-binding fragment comprising a VH having a sequence of SEQ ID NO: 7 or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO: 7, and a VL having a sequence of SEQ ID NO: 8 or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO: 8.

[0097] In one embodiment, the extracellular binding domain of the CAR comprises or consists of an anti-IL-23R scFv with a peptide linker between the VH and VL, wherein the peptide linker comprises SEQ ID NO:13 or a sequence having at least about 90%, 95% or more identity to SEQ ID NO:13.

[0098] In one embodiment, the extracellular binding domain of the CAR comprises or consists of an anti-IL-23R scFv comprising SEQ ID NO:15 or a sequence having at least about 90%, 95% or more identity to SEQ ID NO:15.

[0099] II.1.2. Hinge domain In one embodiment, the extracellular IL-23R binding domain is connected to the transmembrane domain by a hinge domain.

[0100] In one embodiment, the hinge domain comprises or consists of about 2 to about 100 amino acids, for example, about 2 to about 75 amino acids.

[0101] In one embodiment, the hinge domain is a peptide linker as described herein, for example having a length ranging from 2-20 or 2-15 amino acids.

[0102] In one embodiment, the hinge domain comprises or consists of an amino acid sequence derived from a CD8 hinge (e.g., SEQ ID NO:20) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 20. In one embodiment, the hinge domain is a CD8 hinge encoded by SEQ ID NO:21 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:21.

[0103] II.1.3. Transmembrane Domains Examples of transmembrane domains that can be used in the CARs of the invention include the transmembrane domains of the alpha or beta chains of the T cell receptor (TCR); or CD28, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, PD1, I TGAX, CDl1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT These include, but are not limited to, the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0104] In one embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from a CD8 transmembrane domain (e.g., SEQ ID NO: 22) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 22. In one embodiment, the transmembrane domain is a CD8 transmembrane domain encoded by SEQ ID NO: 23 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 23.

[0105] In another embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from a CD28 transmembrane domain (e.g., SEQ ID NO:24) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:24. In one embodiment, the transmembrane domain is a CD28 transmembrane domain encoded by SEQ ID NO:25 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:25.

[0106] In another embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from a 4-1BB (CD137) transmembrane domain (e.g., SEQ ID NO: 26) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 26. In one embodiment, the transmembrane domain is a 4-1BB transmembrane domain encoded by SEQ ID NO: 27 or a nucleotide sequence having at least about 95% (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO: 27.

[0107] In another embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from the TNFR2 transmembrane domain (e.g., SEQ ID NO: 28) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 28. In one embodiment, the transmembrane domain is a TNFR2 transmembrane domain encoded by SEQ ID NO: 29 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 29.

[0108] In one embodiment, the transmembrane domain may be entirely artificial and may, for example, comprise primarily hydrophobic amino acids, such as valine and leucine.

[0109] II.1.4. Intracellular Signaling Domains In one embodiment, the intracellular signaling domain of a CAR of the invention may comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0110] In one embodiment, the intracellular signaling domain comprises a T cell primary signaling domain (or a sequence derived therefrom) and, optionally, one or more intracellular domains (or sequence(s) derived therefrom) of a T cell costimulatory molecule.

[0111] In some embodiments, the intracellular signaling domain comprises or consists of a primary signaling domain.

[0112] In one embodiment, the intracellular signaling domain comprises or consists of one or more intracellular domains of a T cell costimulatory molecule. In one embodiment, the intracellular signaling domain consists of one or more intracellular domains of a T cell costimulatory molecule.

[0113] In another embodiment, the intracellular signaling domain comprises or consists of at least one costimulatory domain and a primary signaling domain.

[0114] In another embodiment, the intracellular signaling domain comprises or consists of at least two costimulatory domains and a primary signaling domain.

[0115] In one embodiment, the T cell primary signaling domain comprises or consists of a functional signaling domain of CD3 zeta.

[0116] In one embodiment, the T cell primary signaling domain comprises or consists of the amino acid sequence of the CD3 zeta intracellular domain of SEQ ID NO: 30, or an amino acid sequence having at least about 95% (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO: 30. In one embodiment, the CD3 zeta primary signaling domain comprises or consists of an amino acid sequence having at least one, two or three modifications (but not more than 20, 10 or 5 modifications) of SEQ ID NO: 30.

[0117] In one embodiment, the CD3 zeta primary signaling domain is encoded by SEQ ID NO:31 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:31.

[0118] The stimulatory T cell primary signaling domain may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMS). In one embodiment, the T cell primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, the primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0119] In one embodiment, the intracellular signaling domain of a CAR of the invention comprises a T cell primary signaling domain (e.g., a CD3 zeta signaling domain) combined with one or more costimulatory signaling domains.

[0120] The costimulatory signaling domain may be derived from the intracellular domain of a T cell costimulatory molecule or from other cell surface molecules expressed on immune cells. Examples of costimulatory signaling domains include CD28, CD27, 4-1BB (CD137), MHC class I molecules, BTLA, Toll ligand receptor, OX40, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand specifically binding to CD83, CDS, ICAM-1, GITR, ARHR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44 , NKp30, NKp46, CD160(BY55), CD19, CD19a, CD4, CD8 alpha, CD8 beta, IL2ra, IL6Ra, IL2R beta, IL2R gamma, IL7R alpha, IL-13RA1 / RA2, IL-33R(IL1RL1), IL-10RA / RB, IL-4R, IL-5R(CSF2RB), IL-21R, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD 103, ITGAL, CD11a / CD18, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, CTLA-4(CD152), CD95, TNFR1(CD12 0a / TNFRSF1A), TNFR2(CD120b / TNFRSF1B), TGFbR1 / 2 / 3, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACA M1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, common gamma chain, ligand that specifically binds CD83, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.

[0121] In one embodiment of the present disclosure, the CAR of the present invention comprises at least one intracellular domain of a T cell costimulatory molecule selected from the group including CD28, TNFR2, 4-1BB, ICOS, CD27, OX40, CTLA4 and PD-1.

[0122] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from the CD28 intracellular domain (e.g., SEQ ID NO:32), or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:32. In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence having at least one, two or three modifications (but not more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO:32. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:33, or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:33.

[0123] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from the 4-1BB intracellular domain (e.g., SEQ ID NO:34) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:34. In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence having at least one, two or three modifications (but not more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO:34. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:35 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:35.

[0124] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from the CD27 intracellular domain (e.g., SEQ ID NO:36), or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:36. In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence having at least one, two or three modifications (but not more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO:36. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:37, or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:37.

[0125] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from the TNFR2 intracellular domain (e.g., SEQ ID NO: 38) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 38. In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence having at least one, two or three modifications (but not more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO: 38. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO: 39 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 39.

[0126] In one embodiment, the intracellular signaling domain of the CAR of the invention is - an amino acid sequence of the CD28 intracellular domain of SEQ ID NO: 32 or an amino acid sequence having at least about 95% (e.g. about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 32; - an amino acid sequence of the CD3 zeta intracellular domain of SEQ ID NO: 30 or an amino acid sequence having at least about 95% (e.g. about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 30; Includes.

[0127] In one embodiment, the intracellular signaling domain of a CAR of the invention comprises at least two different domains (e.g., a primary signaling domain and at least one intracellular domain of a T cell costimulatory molecule) that can be linked to each other in a random or specific order.

[0128] Optionally, a peptide linker may be used to connect separate signaling domains. In one embodiment, a glycine-serine doublet (GS) is used as a suitable linker. In one embodiment, a single amino acid, such as alanine (A) or glycine (G), is used as a linker. Other examples of peptide linkers are described in Section I above.

[0129] In some embodiments, the intracellular signaling domain of a CAR of the invention comprises two or more (e.g., 2, 3, 4, 5 or more) costimulatory signaling domains. In one embodiment, the two or more costimulatory signaling domains are separated by a peptide linker as described herein.

[0130] In one embodiment, the intracellular signaling domain of a CAR of the invention comprises the primary signaling domain of CD3 zeta (e.g., SEQ ID NO: 30) and the costimulatory signaling domain of CD28 (e.g., SEQ ID NO: 32).

[0131] II.1.5. Leader Sequence In one embodiment, the CAR of the present disclosure further comprises a leader sequence located N-terminal to the IL-23R-specific extracellular binding domain, which allows cell surface expression of the CAR protein after the protein is secreted from the Golgi complex.

[0132] Suitable leader sequences for the CAR of the present invention include a CD8 leader sequence, a CD25 leader sequence, or an Igk leader sequence.

[0133] A non-limiting example of a leader sequence is the leader sequence of CD8, which may comprise or consist of SEQ ID NO: 40. In one embodiment, the leader sequence comprises or consists of an amino acid sequence derived from a CD8 leader sequence (e.g., SEQ ID NO: 40) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 40. Preferably, the leader sequence consists of SEQ ID NO: 40.

[0134] In one embodiment, the nucleotide sequence encoding the leader sequence comprises or consists of a nucleotide sequence encoding a CD8 leader sequence (e.g., SEQ ID NO: 41) or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 41. Preferably, the leader sequence is encoded by a sequence consisting of SEQ ID NO: 41.

[0135] A further non-limiting example of a leader sequence is the leader sequence of CD25, which may comprise or consist of SEQ ID NO: 58. In one embodiment, the leader sequence comprises or consists of an amino acid sequence derived from the CD25 leader sequence (e.g., SEQ ID NO: 58) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 58. Preferably, the leader sequence consists of SEQ ID NO: 58.

[0136] In one embodiment, the nucleotide sequence encoding the leader sequence comprises or consists of a nucleotide sequence encoding a CD25 leader sequence (e.g., SEQ ID NO:59) or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 59. Preferably, the leader sequence is encoded by a sequence consisting of SEQ ID NO:59.

[0137] II.1.6. Tags In one embodiment, the CAR further comprises a tag, for example for quality control, enrichment and tracking in vivo. The tag can be located at the N-terminus or C-terminus of the CAR or can be present inside the CAR polypeptide. Examples of tags include, but are not limited to, hemagglutinin tag, polyarginine tag, polyhistidine tag, Myc tag, Strep tag, S-tag, HAT tag, 3xFlag tag, calmodulin binding peptide tag, SBP tag, chitin binding domain tag, GST tag, maltose binding protein tag, fluorescent protein tag, T7 tag, V5 tag and Xpress tag.

[0138] In one embodiment, a CAR of the disclosure comprises an HA tag (SEQ ID NO: 42). In one embodiment, the tag is encoded by SEQ ID NO: 43 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 43.

[0139] II.1.7. Exemplary CARs According to one embodiment, a CAR of the present disclosure comprises an IL-23R binding domain (e.g., a domain comprising or consisting of SEQ ID NO: 15), optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule, and a T cell primary signaling domain. Preferably, a CAR of the present disclosure further comprises a leader sequence.

[0140] In one embodiment, a CAR of the disclosure comprises an IL-23R binding domain (e.g., SEQ ID NO: 15), a transmembrane domain of CD8 (e.g., SEQ ID NO: 22), an intracellular domain of CD28 (e.g., SEQ ID NO: 32), and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 30). In one embodiment, a CAR of the disclosure further comprises a leader sequence.

[0141] In one embodiment, a CAR of the disclosure comprises an IL-23R binding domain (e.g., SEQ ID NO: 15), a hinge domain of CD8 (e.g., SEQ ID NO: 20), a transmembrane domain of CD8 (e.g., SEQ ID NO: 22), an intracellular domain of CD28 (e.g., SEQ ID NO: 32), and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 30).

[0142] In one embodiment, a CAR of the disclosure comprises an IL-23R binding domain (e.g., SEQ ID NO: 15), a hinge domain of CD8 (e.g., SEQ ID NO: 20), a transmembrane domain of CD8 (e.g., SEQ ID NO: 22), an intracellular domain of CD28 (e.g., SEQ ID NO: 32), a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 30), and a leader sequence. In one embodiment, the leader sequence comprises or consists of SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62. Preferably, the leader sequence comprises or consists of SEQ ID NO: 40 or SEQ ID NO: 58. Preferably, the leader sequence comprises or consists of SEQ ID NO: 58.

[0143] In one embodiment, a CAR of the disclosure comprises an anti-IL-23R scFv (e.g., an scFv comprising or consisting of SEQ ID NO: 15), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human CD3ζ. In one embodiment, a CAR of the disclosure further comprises a leader sequence.

[0144] In one embodiment, the CAR comprises or consists of SEQ ID NO:44 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:44.

[0145] The CAR of the present disclosure is a novel cross-reactive CAR that can bind mouse and human IL-23R (Example 3 and FIG. 14). In these exemplary results, the CAR of the present disclosure exhibits a similar binding profile between the human and mouse homologs of IL23R. In these exemplary results, CAR#2 of the present disclosure exhibits an approximately 1.4-fold difference in binding affinity between human IL23R and mouse IL23R, while CAR#1 exhibits an approximately 3-fold difference in binding affinity between human IL23R and mouse IL23R. These exemplary results show that CAR#2 of the present disclosure binds to human and mouse IL23R with more similar affinity compared to CAR#1. This similarity suggests that the use of CAR#2 in mouse preclinical models may be more representative of its interaction with human IL23R.

[0146] Advantageously, the CARs of the present disclosure demonstrated stable signaling and low background activation of the antigen-binding portion (Example 4 and FIG. 16).

[0147] II.1.8. Mouse CAR The present disclosure also provides a murine CAR comprising an IL-23R binding domain (e.g., a domain comprising or consisting of SEQ ID NO: 15), optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule, and a T cell primary signaling domain.

[0148] In one embodiment, a murine CAR of the present disclosure comprises an IL-23R binding domain (e.g., SEQ ID NO: 15), a transmembrane domain of mouse CD8 (e.g., SEQ ID NO: 50) or a transmembrane domain of mouse CD28 (SEQ ID NO: 52), an intracellular domain of mouse CD28 (e.g., SEQ ID NO: 54), and a murine CD3 zeta primary signaling domain (e.g., SEQ ID NO: 56). In certain embodiments, a murine CAR may also comprise a hinge domain of mouse CD8 (e.g., SEQ ID NO: 46) or a hinge domain of mouse CD28 (e.g., SEQ ID NO: 48). A murine CAR having any combination of the above-mentioned domains is contemplated.

[0149] II.2. Nucleic Acid Encoding CAR The present disclosure also relates to a nucleic acid sequence encoding the CAR described herein. An example of such a nucleic acid sequence is SEQ ID NO: 45 or a degenerate or codon-optimized version thereof.

[0150] II.3. Vectors for expressing CAR Another object of the disclosure is an expression vector comprising a nucleic acid encoding the CAR herein.

[0151] In one embodiment, the nucleic acid encoding CAR is DNA.In one embodiment, the nucleic acid encoding CAR is RNA.The examples of vectors that can be used in this disclosure include, but are not limited to, DNA vectors, RNA vectors, plasmids, episomes, and viral vectors (e.g., animal viruses).

[0152] In one embodiment, the expression vector may include regulatory elements, such as promoters, enhancers, and transcription terminators, for expressing or inducing the expression of a transgene (e.g., CAR) on the expression vector in a host cell. The vector may also include one or more selectable markers.

[0153] Examples of promoters and enhancers used in expression vectors for animal cells include, but are not limited to, SV40 early promoter and enhancer, Moloney murine leukemia virus LTR promoter and enhancer, immunoglobulin H chain promoter and enhancer, etc. Other examples of suitable constitutive promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter sequence, elongation factor lα (EF-lα) promoter, phosphoglycerate kinase (PGK) promoter, FOXP3-derived promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0154] Examples of suitable inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, a cumate promoter, and a tetracycline promoter.

[0155] Examples of suitable bidirectional promoters include, but are not limited to, those described by Luigi Naldini in U.S. Pat. No. 8,501,464 (hereby incorporated by reference), which discloses a bidirectional promoter comprising: i) a first minimal promoter sequence derived from the cytomegalovirus (CMV) or mouse mammary tumor virus (MMTV) genome; and ii) a complete efficient promoter sequence derived from an animal gene.

[0156] Examples of suitable vectors include, but are not limited to, pAGE107, pAGE103, pHSG274, pKCR, pSG1beta d2-4, and the like.

[0157] Examples of plasmids include, but are not limited to, replicating plasmids that contain an origin of replication, or integrating plasmids, such as pUC, pcDNA, pBR, and the like.

[0158] Many virus-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, and adeno-associated virus (AAV) vectors.

[0159] Retroviruses can provide a convenient platform for gene delivery systems.Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art.Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo.Many retroviral systems are known in the art.

[0160] In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art.

[0161] In one embodiment, a lentiviral vector is used.

[0162] In one embodiment, an AAV vector is used. As used herein, the term "AAV" encompasses all serotypes and variants, both naturally occurring and engineered. For example, the term encompasses AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8), and AAV type 9 (AAV-9). In one embodiment, the vector is an AAV6 vector. In one embodiment, the AAV is a pseudotyped AAV, such as an AAV with an AAV6 capsid, and a recombinant genome derived from another AAV serotype (e.g., with ITRs from AAV2).

[0163] Recombinant virus can be produced by known techniques in the art, such as transfection of packaging cells or transient transfection with helper plasmid or virus.Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, 293T cells, etc.Detailed protocols for producing such replication-defective recombinant viruses can be found in the art.Insect cells can also be used to produce recombinant viruses, such as recombinant AAV.

[0164] II.4 Cells expressing CAR The present disclosure further relates to immune cells and immune cell populations engineered to express the CARs described herein on their cell surface.

[0165] In one embodiment, the immune cells are regulatory T cells (Treg), CD8 + T cells, CD4 + The T cell may be a T cell, such as a T cell or a NK T cell.

[0166] In one embodiment, the immune cells are tumor infiltrating lymphocytes (TILs).

[0167] The present disclosure also relates to isolated and / or substantially purified immune cell populations, preferably T cell populations, comprising or consisting of immune cells engineered to express a CAR described herein on the cell surface.

[0168] In one embodiment, the immune cell, preferably a T cell, is inhibitory to a cell expressing on its surface the IL-23R that is recognized by the CAR.

[0169] In one embodiment, the immune cell, preferably a T cell, is cytotoxic to a cell expressing on its surface the IL-23R that is recognized by the CAR.

[0170] In one embodiment, the immune cell population, preferably the T cell population, is a Treg cell, CD8 + T cells, CD4 + It comprises or consists of T cells, and / or NK T cells.

[0171] In one embodiment, the T cells of the present disclosure are Treg cells.

[0172] In one embodiment, all Treg cells in the cell population of the present disclosure express a CAR as described herein and can therefore be defined as CAR monospecific (i.e., all Treg cells recognize the same antigen (IL-23R)). In one embodiment, the Treg cell population is TCR monospecific (i.e., all Treg cells recognize the same antigen as their TCR). In another embodiment, the Treg cell population is TCR multispecific (i.e., Treg cells can recognize antigens different from their TCR).

[0173] In one embodiment, a CAR of the present disclosure, when expressed by a T (e.g., Treg) cell, confers on the T cell the ability to bind to cells expressing IL-23R on their cell surface and become activated upon binding to IL-23R.

[0174] Examples of cells that express IL-23R include, but are not limited to, Th17 cells, αβT cells, neutrophils, γδT cells, NK cells, NK T cells, dendritic cells, and macrophages.

[0175] Thus, an immune cell population of the present disclosure (e.g., a T (e.g., Treg) cell population of the present disclosure) may be defined as a redirected immune cell population. As used herein, the term "redirected" refers to an immune cell bearing a CAR described herein that confers to the immune cell the ability to bind to and be activated by a ligand that is distinct from the ligand for which the immune cell is or would have become specific, or is activated by the immune cell.

[0176] In one embodiment, the Treg cells of the present disclosure are not cytotoxic, hi another embodiment, the Treg cells of the present disclosure are cytotoxic.

[0177] In one embodiment, the Treg cells of the present disclosure are CD4 + CD25 + FOXP3 + Treg cells, Tr1 cells, TGF-β-secreting Th3 cells, regulatory NK T cells, regulatory γδT cells, regulatory CD8 + T cells, and double negative regulatory T cells.

[0178] In one embodiment, the immune cells are CD4 + In one embodiment, the Treg cells are thymus-derived Tregs or adaptive or inducible Tregs. In one embodiment, the Treg cells are CD4 + FOXP3 + Treg cells or CD4 + FOXP3 - They are regulatory T cells (Tr1 cells).

[0179] In one embodiment, the immune cells are CD8 + In one embodiment, the CD8 + Treg cells are CD8 +CD28 - Treg cells, CD8 + CD103 + Treg cells, CD8 + FOXP3 + Treg cells, CD8 + CD122 + In one embodiment, the regulatory cells are selected from the group consisting of INFγ + IL10 + IL34 + CD8 + CD45RC low They are Treg cells.

[0180] In one embodiment, the immune cells of the present disclosure are human Treg cells.

[0181] In one embodiment, the immune cells (eg, T cells or Treg cells) are derived from stem cells, such as induced pluripotent stem cells (iPSCs).

[0182] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a pluripotent stem cell derived from a non-pluripotent cell (e.g., an adult somatic cell) by dedifferentiation or reprogramming. Specifically, an iPSC can be obtained by introducing a specific set of pluripotency-associated genes (reprogramming factors) into a cell. The reprogramming factors can be, for example, transcription factors Oct4 (Pou5f1), Sox2, c-Myc, and Klf4.

[0183] In one embodiment, Treg cells have the following phenotype: CD4 + CD25 + , e.g. CD4 + CD25 + CD127 - and CD4 + CD25 + CD127 - CD45RA + In one embodiment, the Treg cells have the following phenotype: CD4 + CD25 + , e.g. CD4 +CD25 + CD127 low and CD4 + CD25 + CD127 low CD45RA + In one embodiment, the Treg cells have the following phenotype: CD4 + CD25 + , e.g. CD4 + CD25 + CD127 low / - and CD4 + CD25 + CD127 low / - CD45RA + In one embodiment, the Treg cells have the following phenotype: FOXP3 + CD4 + CD25 + , e.g. FOXP3 + CD4 + CD25 + CD127 - and FOXP3 + CD4 + CD25 + CD127 - CD45RA + In one embodiment, the Treg cells have the following phenotype: FOXP3 + CD4 + CD25 + , e.g. FOXP3 + CD4 + CD25 + CD127 low and FOXP3 + CD4 + CD25 + CD127 low CD45RA + In one embodiment, the Treg cells have the following phenotype: FOXP3 + CD4 + CD25 + , e.g. FOXP3 + CD4 + CD25 + CD127 low / - and FOXP3 + CD4 + CD25 + CD127 low / -CD45RA + has.

[0184] In one embodiment, Treg cells have the following phenotype: CD4 + CD25 high , e.g. CD4 + CD25 high CD127 - and CD4 + CD25 high CD127 - CD45RA + In one embodiment, the Treg cells have the following phenotype: CD4 + CD25 high , e.g. CD4 + CD25 high CD127 low and CD4 + CD25 high CD127 low CD45RA + In one embodiment, the Treg cells have the following phenotype: CD4 + CD25 high , e.g. CD4 + CD25highCD127 low / - and CD4 + CD25highCD127 low / - CD45RA + In one embodiment, the Treg cells have the following phenotype: FOXP3 + CD4 + CD25 high , e.g. FOXP3 + CD4 + CD25 high CD127 - and FOXP3 + CD4 + CD25 high CD127 - CD45RA + In one embodiment, the Treg cells have the following phenotype: FOXP3 + CD4 + CD25 high , e.g. FOXP3 + CD4 + CD25 high CD127 lowand FOXP3 + CD4 + CD25 high CD127 low CD45RA + In one embodiment, the Treg cells have the following phenotype: FOXP3 + CD4 + CD25 high , e.g. FOXP3 + CD4 + CD25highCD127 low / - and FOXP3 + CD4 + CD25highCD127 low / - CD45RA + has.

[0185] In one embodiment, Treg cells have stable FOXP3 expression associated with selective demethylation and / or hypomethylation in the Treg-specific demethylated region (TSDR), a conserved region within intron 1 of the FOXP3 locus.

[0186] The expression level of a molecule can be determined by flow cytometry, immunofluorescence, or image analysis. To detect intracellular proteins, cells can be fixed and permeabilized prior to flow cytometry analysis.

[0187] In one embodiment, the expression level of a molecule in a cell population is indicated by the percentage of cells in the cell population that express the molecule (i.e., cells "+" for the molecule). The percentage of cells that express the molecule can be measured by FACS. The expression level of a cell marker of interest can be determined by comparing the median fluorescence intensity (MFI) of cells from a cell population stained with a fluorescently labeled antibody specific for this marker to the fluorescence intensity (FI) of cells from the same cell population stained with a fluorescently labeled antibody of different specificity but of the same isotype, same fluorescent probe, and from the same species (referred to as an isotype control). Cells from the population that are stained with a fluorescently labeled antibody specific for this marker and that show an MFI equal to or lower than cells stained with an isotype control do not express this marker and are designated (-) or negative. Cells from the population that are stained with a fluorescently labeled antibody specific for this marker and that show an MFI value higher than cells stained with an isotype control express this marker and are designated (+) or positive.

[0188] The terms "expressing" (i.e., "positive" or "+") and "not expressing" (i.e., "negative" or "-") refer to the expression level of a cell marker of interest, where "+" corresponds to a high or intermediate (also referred to as "+ / -") expression level of the cell marker, and "-" corresponds to zero expression level of the cell marker. The terms "low" or "lo" or "lo / -" or "low / -" refer to a cell marker of interest whose expression level is low compared to the expression level of that cell marker in the cell population analyzed as a whole. More specifically, the term "lo" refers to a distinct cell population expressing the cell marker at a lower level than one or more other distinct cell populations. The terms "high" or "hi" or "bright" refer to a cell marker of interest whose expression level is high compared to the expression level of that cell marker in the cell population analyzed as a whole. Generally, cells in the top 2, 3, 4, or 5% of staining intensity are designated "hi" and cells falling in the top half of the population are classified as "+". Cells with below 50% fluorescence intensity are designated "lo" cells, and cells with below 5% fluorescence intensity are designated "-" cells.

[0189] Advantageously, in addition to exhibiting good activity (high signal to background ratio) and good inhibitory activity, immune cells expressing the CARs of the present disclosure were found to have hypotonic signaling (Figures 3 and 10).

[0190] The term "tonic signaling" as used herein refers to a background of antigen-independent activity. Methods for measuring tonic signaling are well known to those skilled in the art and include, but are not limited to, measuring the metabolic activity of CAR-expressing cells, measuring one or more indicators of cell activation in the absence of stimulation by the antigen recognized by the receptor, measuring one or more phenotypic changes associated with cell aging or cell senescence, determining cell cycle progression in the absence of antigenic stimulation, and measuring the size of cells expressing a receptor compared to the size of unmodified cells.

[0191] Spontaneous expression of CD69 by CAR Treg cells compared to non-transduced Treg cells can be monitored to determine tonic signaling strength.

[0192] As demonstrated herein, engineered T cells and engineered Treg cells expressing the CAR constructs of the invention exhibit hypotonic signaling, and following CAR engagement, engineered Treg cells exhibit highly effective suppressive activity against T effector cell proliferation, thereby demonstrating the advantages of these Treg cells for cell therapy.

[0193] In one embodiment, the CAR of the present disclosure, when expressed by Treg cells, allows for a reduction in the background activation of Treg cells compared to other CAR constructs directed against IL-23R.

[0194] In addition, immune cells expressing the disclosed CARs were found to have stronger suppressive activity when the CAR-expressing cells bound to cells with high levels of IL-23R expression compared to cells with low levels of IL-23R expression (Figure 12). Thus, the disclosed CARs are expected to reduce off-target activity.

[0195] In one embodiment, the CAR of the present disclosure, when expressed by Treg cells, allows for reduced off-target activity of Treg cells compared to other CAR constructs directed against IL-23R.

[0196] III. Compositions, Pharmaceutical Compositions, and Medicaments Another object of the present disclosure is a composition comprising, consisting essentially of, or consisting of at least one antibody that binds to IL-23R as described herein, or at least one antigen-binding fragment of said antibody, or at least one nucleic acid or vector encoding an antibody or antigen-binding fragment of said antibody according to the present disclosure.

[0197] Another object of the present disclosure is a composition comprising, consisting essentially of, or consisting of at least one immune cell or at least one population of immune cells comprising a CAR according to the present disclosure.

[0198] In one embodiment, the composition is a pharmaceutical composition and further comprises at least one pharma- ceutically acceptable excipient.

[0199] Accordingly, another object of the present disclosure is a pharmaceutical composition comprising, consisting essentially of, or consisting of at least one immune cell or at least one population of immune cells comprising a CAR according to the present disclosure and at least one pharma- ceutically acceptable excipient.

[0200] As used herein, "consisting essentially of" means, with respect to a composition, that at least one antibody or antigen-binding fragment thereof, nucleic acid or expression vector, or at least one immune cell or population of immune cells is the only therapeutic or agent having biological activity within the composition.

[0201] The term "pharmaceutically acceptable excipient" refers to solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, isotonicity agents, stabilizers, preservatives, absorption delaying agents, etc. that do not produce adverse, allergic, or other untoward reactions when administered to a subject, such as a human.

[0202] Examples of pharma- ceutically acceptable excipients that may be used in the compositions of the present disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffers (e.g., phosphoric acid), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., sodium chloride, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, and zinc salts), and polyethylene glycol.

[0203] In one embodiment, the pharmaceutical composition according to the present disclosure comprises a vehicle suitable for injection. These may be, for example, isotonic sterile saline (including, for example, monosodium phosphate or disodium phosphate; sodium chloride, potassium chloride, calcium chloride or magnesium chloride; or mixtures of these salts), or a dry (e.g., lyophilized) composition that allows the constitution of an injectable solution when a suitable carrier such as sterile water or saline is added.

[0204] Another object of the present disclosure is a medicament comprising, consisting essentially of, or consisting of at least one antibody that binds to IL-23R as described herein, or at least one antigen-binding fragment of said antibody, or at least one nucleic acid or vector encoding an antibody or an antigen-binding fragment of said antibody according to the present disclosure.

[0205] Another object of the present disclosure is a medicament comprising, consisting essentially of, or consisting of a population of immune cells expressing a CAR of the present disclosure.

[0206] IV. Route of Administration In one embodiment, a composition, pharmaceutical composition, or medicament according to the present disclosure is administered parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir.

[0207] In one embodiment, the composition, pharmaceutical composition, or medicament is administered by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0208] Examples of forms suitable for injection include, but are not limited to, liquid solutions, such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for use in preparing a solution or suspension by the addition of a liquid prior to use, such as powders, liposomal forms, etc.

[0209] V. Dosage In one embodiment, an isolated antibody or antigen-binding fragment thereof, nucleic acid, expression vector, immune cell or immune cell population, composition, pharmaceutical composition, or medicament according to the present disclosure is administered in a therapeutically effective amount to a subject in need thereof.

[0210] However, it will be understood that the therapeutically effective amount and frequency of administration will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient may depend on a variety of factors, including the disease and severity of the disease being treated; the activity of the isolated antibody or its antigen-binding fragment, nucleic acid, expression vector, or immune cell used; the age, weight, general health, sex, and diet of the subject; the administration time, route of administration, and excretion rate of the specific therapeutic agent used; the duration of treatment; drugs used in combination or simultaneously with the specific therapeutic agent used; and similar factors well known in the medical field. For example, it is within the skill of the art to start administering the compound at a level lower than the amount required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered in multiple doses or in one dose.

[0211] In one embodiment, a subject (e.g., a human) receives a single administration of an immune cell or population of immune cells of the present disclosure.

[0212] In one embodiment, a subject (e.g., a human) receives at least two administrations of an immune cell or population of immune cells of the present disclosure.

[0213] In one embodiment, the immune cell populations are administered to a subject once a week, once a month, or once a year.

[0214] In one embodiment, the number of immune cells administered to a subject is about 10 2 pieces ~ about 10 9 pieces, about 10 3 pieces ~ about 10 8 pieces, about 10 4 pieces ~ about 10 7 Pieces, or about 10 5 pieces ~ about 10 6 It is a range of pieces.

[0215] In one embodiment, the immune cells are administered to a subject in need thereof in combination with at least one other active agent, which in one embodiment is an agent that can be used to treat an IL-23R-associated disease or disorder. Examples of other active agents include glucocorticoids (including, but not limited to, dexamethasone, prednisone, prednisolone, methylprednisolone, betamethasone, bedomethasone, tixocortol, triamcinolone, hydrocortisone, budesonide or fludrocortisone), antibodies or antagonists of human cytokines or growth factors (e.g., anti-TNF such as infliximab, adalimumab, certolizumab, etanercept; anti-IL1, anti-IL-6, anti-IL-12, anti-IL-17 and anti-IL-23 (e.g., brazikumab, guselkumab, mirikizumab, risankizumab), anti-IL-12 / IL-23 (e.g., ustekinumab); or IL -1 receptor antagonist analogues (e.g. anakinra), antibodies against cell surface molecules (e.g. anti-α4 integrin (e.g. natalizumab), anti-integrin β7 (e.g. etrolizumab), anti-α4-β7 integrin (e.g. vedolizumab), anti-CD2, anti-CD3 (e.g. visiluzumab), anti-CCR9, anti-LFA1 or anti-ICAM1); JAK inhibitors (e.g. filgotinib, upadacitinib), S1PR modulators (e.g. etrasimod, ozanimod), 5-aminosalicylic acid and its analogues (e.g. mesalazine, sulfazaline, olsalazine, or balsalazide); probiotics (e.g. Saccharomyces boulardii), antibiotics (e.g., metronidazole, ampicillin, ciprofloxacin, RHB-104), immunomodulators (e.g., tacrolimus, cyclosporine, methotrexate, thalidomide, leflunomide, and purine analogs such as azathioprine and 6-mercaptopurine), and stem cell therapy (e.g., dalvadostrocel).

[0216] In one embodiment, administration of the immune cells or populations of the present disclosure allows a subject to reduce the amount of at least one other active agent administered.

[0217] According to one embodiment, at least one immune cell population is administered prior to, simultaneously with, or following administration of at least one other active agent.

[0218] VI. Therapeutic Use The present disclosure further relates to at least one isolated antibody that binds IL-23R, or at least one antigen-binding fragment of said antibody, as described herein, for use as a medicament or for use in treating a disease, disorder, or condition in a subject in need of treatment.

[0219] The present disclosure relates to at least one nucleic acid or vector as described herein for use as a medicament, or for use in treating a disease, disorder, or condition in a subject in need of treatment.

[0220] The present disclosure further relates to a method for treating a disease, disorder, or condition in a subject in need of treatment comprising administering to the subject an isolated antibody or antigen-binding fragment thereof, nucleic acid or vector, or composition, pharmaceutical composition, or medicament described herein.

[0221] In one embodiment, an isolated antibody or antigen-binding fragment thereof, or a nucleic acid or vector according to the present disclosure may be used to treat a disease or disorder mediated by IL-23R-expressing cells (also referred to herein as an IL-23R-associated disease or disorder) in a subject in need of treatment.

[0222] Another object of the present disclosure is a method of cell therapy for treating a disease or disorder mediated by IL-23R-expressing cells in a subject in need of treatment, the method comprising administering to the subject an immune cell as described herein, such as a Treg cell as described herein.

[0223] In one embodiment, the administered immune cells are autologous cells, in other words, the cell therapy is autologous cell therapy. As used herein, the term "autologous" refers to any material derived from the same individual into which it is subsequently reintroduced.

[0224] In one embodiment, the cell therapy is an allogeneic cell therapy. As used herein, the term "allogeneic" refers to any material that is not derived from the subject being treated, but from an external source, such as induced pluripotent stem cells (iPSCs) or cells of cadaveric origin.

[0225] In one embodiment, the cell therapy is xenogeneic. As used herein, the term "xenogeneic" refers to any material derived from a subject of a different species than the subject to which the material is introduced.

[0226] In another embodiment, the immune cells administered are allogeneic cells, in other words, the cell therapy is allogeneic cell therapy.As used herein, the term "allogeneic" refers to any material that is derived from a different subject of the same species as the subject into which the material is introduced.Two or more subjects are said to be allogeneic to each other when the genes at one or more loci are not identical.In a further embodiment, the immune cells are derived from a healthy human donor.

[0227] In some embodiments, the genetically modified immune cells of the present invention can be allogeneic immune cells. In such cases, the cells can be engineered to reduce host rejection of the cells (graft rejection) and / or potential attack of the cells against the host (graft-versus-host disease). By way of example, cells can be engineered to be genotypically deficient in one or more of the following: (i) T cell receptor (TCR alpha or beta chain); (ii) polymorphic major histocompatibility complex (MHC) class I or II molecules (e.g., HLA-A, HLA-B or HLA-C; HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ or HLA-DR; or β2-microglobulin (B2M)); (iii) transporter associated with antigen processing (e.g., TAP-1 or TAP-2); (iv) MHC class II transactivator (CIITA); (v) minor histocompatibility antigen (MiHA; e.g., HA-1 / A2, HA-2, HA-3, HA-8, HB-1H or HB-1Y); and (vi) any combination thereof. Allogeneic engineered cells can also express invariant HLA or CD47 to protect engineered Treg cells from host rejection. These further genetic modifications can be carried out by gene editing techniques known in the art.

[0228] Moreover, edited allogeneic cells are particularly useful because they can be used in multiple patients without compatibility issues. Therefore, the allogeneic cells can be called "universal" and can be used "off-the-shelf". The use of "universal" cells will significantly improve the efficiency and reduce the cost of the employed cell therapy.

[0229] In certain embodiments, allogeneic immune cells can be engineered to not express any functional TCR on their surface, to not express one or more subunits that comprise a functional TCR, or to produce very little functional TCR on their surface.For example, immune cells described herein can be engineered to downregulate cell surface expression of TCR molecules.Alternatively, T cells can express substantially reduced TCR, for example, by expression of mutated or truncated forms of one or more subunits of TCR.The term "substantially reduced TCR" means that this TCR does not induce a harmful immune response in the host.

[0230] In certain embodiments, allogeneic immune cells can be engineered to not express functional HLA on their surface. For example, immune cells described herein can be engineered to downregulate cell surface expression of HLA, e.g., HLA class 1 and / or HLA class II and / or non-classical HLA molecules.

[0231] In certain embodiments, the T cells may lack a functional TCR as well as a functional HLA, e.g., HLA class I and / or HLA class II.

[0232] Modified immune cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knocking out or knocking down one or more subunits of TCR and / or HLA. For example, Treg cells can include knockdown of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription activator-like effector nucleases (TALENs), zinc finger endonucleases (ZFNs), meganucleases (mn, also known as homing endonucleases), or megaTALs (combination of TAL effectors with mn cleavage domains).

[0233] In some embodiments, a nucleic acid encoding a CAR described herein is inserted into a specific locus in the genome of an immune cell, such as the locus of a gene to be deleted. In some embodiments, a nucleic acid encoding a CAR described herein is inserted into a TCR and / or HLA locus, thereby resulting in inhibition of TCR and / or HLA expression.

[0234] Another object of the present disclosure is a method for treating an IL-23R-related disease or disorder in a subject in need of treatment, comprising administering to the subject at least one CAR as described herein or at least one nucleic acid or vector encoding a CAR as described herein. In one embodiment, the method is a method of gene therapy.

[0235] VII.IL-23R-related diseases In one embodiment, the IL-23R-related disease or disorder is an inflammatory cell-mediated disease or disorder, a Th17-mediated disease or disorder, or a γδT-mediated disease or disorder.

[0236] In one embodiment, the IL-23R-expressing cell-mediated disease is an autoimmune disease or disorder and / or an inflammatory disease or disorder.

[0237] Examples of IL-23R-associated diseases include, but are not limited to, autoimmune diseases or disorders, inflammatory diseases or disorders, allergic diseases or disorders, and cancer.

[0238] In one embodiment, the IL-23R-expressing cell-mediated disease or disorder is selected from inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), lupus (e.g., systemic lupus erythematosus), arthritis (e.g., rheumatoid arthritis and juvenile idiopathic arthritis), Sjogren's syndrome, systemic sclerosis, ankylosing spondylitis, type 1 diabetes, autoimmune thyroid disorders, multiple sclerosis, myasthenia gravis, psoriatic arthritis, skin diseases (e.g., psoriasis and atopic dermatitis), or uveitis.

[0239] In one embodiment, the IL-23R-expressing cell-mediated disease or disorder is Crohn's disease.

[0240] VIII. Manufactured products Another object of the present disclosure is an article of manufacture containing materials useful for the treatment of a disease or disorder mediated by IL-23R expressing cells.

[0241] The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bags, bottles, vials, syringes, pouches, etc. The containers may be formed from a variety of materials such as glass or plastic.

[0242] The article of manufacture, label, or package insert may further include instructions for administering the Treg cell populations of the present disclosure to a patient.

[0243] The present disclosure provides a kit comprising at least one immune cell population of the present disclosure. By "kit" is meant any article of manufacture (e.g., package or container) comprising at least one Treg cell population of the present disclosure. The kit may also include instructions for use.

[0244] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with cardiology, medicine, pharmaceutical and medicinal chemistry, cell biology, and molecular techniques described herein are those well known and commonly used in the art. Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. Throughout this specification and embodiments, the terms "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the general knowledge in the art.

[0245] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context.

[0246] In order that this disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only, and are not to be construed as limiting the scope of the disclosure in any way. EXAMPLES

[0247] The present disclosure is further illustrated by the following examples.

[0248] Example 1: Identification of IL-23R-CAR lead candidates Materials and Methods PBMC isolation Blood from healthy donors was collected by the Etablissement Francais du Sang (EFS). The day after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat by Ficoll gradient centrifugation, which allowed the removal of undesirable fractions of the blood product, such as granulocytes, platelets, and residual red blood cell contaminants. The cell populations of interest were then isolated as follows:

[0249] FOXP3 Tregs and CD4 + CD25 - Isolation of conventional T cells Human CD4 + CD127 low CD25 + CD4 T-regulatory T cell isolation kit (StemCell) was used according to the manufacturer's instructions. + CD25 + CD127 low Tregs were isolated. Briefly, CD25 + First, cells were diluted to 400-500 × 10 6 CD25 cells were isolated from PBMCs by column-free immunomagnetic positive selection using EasySep™ Releasable RapidSpheres™. + The bound magnetic particles were removed from the cells, and unwanted non-Tregs were targeted for depletion. The final isolated fraction contained highly purified CD4+ Tregs expressing high levels of FOXP3. + CD127 low CD25 + The cells were then immediately used for downstream applications. + CD25 - Conventional T cells were generated using kit #18063 (StemCell)+ CD25 - Responder T cells were isolated by choosing an optional protocol for isolating and used for functional studies in parallel with Tregs.

[0250] Activation and culture of isolated Tregs The isolated Treg cells were activated and cultured for 9 days. Briefly, on day 0, Treg cells (0.5 × 10 6 ) were cultured in 24-well plates (Costar) with Xvivo15 serum-free medium supplemented with 1000 U / ml IL-2 (Euromedex) + 100 nM rapamycin (Sigma-Aldrich) containing human transferrin (OZYME). CD3 / CD28 activation was then performed using Dynabeads® from Life Technology (0.5 × 10 per well). 6 The cells were fed with fresh culture medium supplemented with 1000 U / ml IL-2 on days 2, 4, and 7. Finally, on day 9, the cells were harvested, counted, and reactivated.

[0251] Production and titration of lentiviral vectors CAR-expressing lentiviral vectors (LV) were produced using a classical four-plasmid lentiviral system. Briefly, HEK293T cells (Lenti-X, Ozyme) were transfected with plasmids expressing the CAR-expressing transfer vector, HIV-1 Gag / pol (pMDLg / pRRE), HIV-1 Rev (pRSV.Rev), and VSV-G glycoprotein (pMD2.G) (Didier Trono, EPFL, Switzerland). 24 h after transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted and frozen at -80°C for long-term storage. Infectious titers, expressed in transducing units per milliliter (TU / ml), were obtained after transduction of Jurkat T cell lines using serial dilutions of viral supernatants, and transduction efficiency was evaluated 4 days later by monitoring GFP expression.

[0252] Transduction protocol Tregs were transduced with the chimeric receptor (see schematic of CAR construct in Figure 1) 2 days after activation. Briefly, each well was transduced with 2–5 × 10 6 Transduction was performed by loading transducing units (TU) / ml. After 6 hours at 37°C, viral particles were removed by washing. The plates were then incubated at 37°C with 5% CO2. Transduction efficiency was analyzed 5 days after transduction: (i) gene transfer efficiency was measured by analyzing the percentage of GFP positive cells in flow cytometry, and (ii) the percentage of transduced cells that expressed CAR on the cell surface was measured by analyzing hemagglutinin A (HA) tag expression or protein L staining in flow cytometry.

[0253] CAR constructs used for transduction An IL-23R CAR was designed that is composed of the CD8 transmembrane (TM) domain and the intracellular domain of CD28 linked to the intracellular domain of CD3 zeta, associated with an scFv directed against IL-23R. The constructs used in this study are listed and described in Figure 1.

[0254] Phenotypic analysis of transduced Tregs On day 9 of culture, Treg phenotype was analyzed by flow cytometry using the markers listed in Table 1. [Table 1]

[0255] CAR activation assay Activation assays were performed on day 9 of culture. Briefly, 0.05×10 6Tregs were seeded in a final volume of 200 μL in 96-well U-bottom plates either alone or in the presence of anti-CD3 / anti-CD28 coated beads (1:1 Treg to bead ratio) or in the presence of IL-23R coated beads (1:1 Treg to bead ratio). After 24 hours at 37° C., 5% CO2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry. Monitoring the spontaneous expression of CD69 on CAR Treg cells compared to non-transduced Treg cells allows for the determination of tonic signaling strength.

[0256] Inhibition of T cell proliferation assay Suppression analysis was performed on day 9 of culture. Briefly, Tregs were harvested, counted, and either activated through TCR with anti-CD3 / anti-CD28 coated beads (1:1 Treg to bead ratio) or through CAR IL-23R coated beads (1:1 Treg to bead ratio), or kept unactivated, and spontaneous suppressive activity was assessed. In parallel, allogeneic Tconvs were thawed, stained with Dye 450, and activated with anti-CD3 / anti-CD28 coated beads (3:1 Tconv to bead ratio). The next day, beads were removed from Tconvs and then co-cultured with unactivated or activated Tregs (untransduced or transduced). On day 3, cells were harvested and Tconv proliferation was assessed through measurement of dye450 dilution by flow cytometry. The % inhibition of Tconv proliferation was calculated as follows:

number

[0257] animal All procedures described in this study were reviewed and approved by the local ethical committee (CIEPAL). Experiments were performed on 8-week-old female LY5.1 mice. Mice were housed in groups of six and each mouse was uniquely identified. Animals were kept in ventilated cages (type II (16 × 19 × 35 cm, floor area = 500 cm)). 2)) under the following controlled conditions: (i) room temperature (22 ± 2 °C), (ii) humidity (55 ± 10%), (iii) photoperiod (12:12 h light:dark cycle 7 am:7 pm), and (iv) water and food (Ref. 2018, Harlan France) available ad libitum. Mice were allowed to acclimate for 5 days before the start of the experiment.

[0258] DSS-induced acute colitis DSS (dextran sulfate sodium, 40 kDa, MP Biomedicals) was administered to 8-week-old female LY5.1 mice on days 0-5 (D0-D5). DSS (two different concentrations) was added to drinking water (available ad libitum) that was replaced fresh every 2 days. Fresh water without DSS was provided during the recovery period (D5-D15). On day 5, 3 million CAR-positive Tregs isolated from the spleens of 10-week-old C57BL / 6J mice were injected via the tail vein. The CARs used to transduce mouse Tregs are described in Figure 5. Mice were scored daily based on four parameters: weight loss, stool consistency, presence or absence of blood, and stool collection time, as shown in Table 2 below. [Table 2]

[0259] Feces were collected for inflammatory marker analysis. On day 15, mice were euthanized by cervical dislocation and organs were harvested for ex vivo analysis.

[0260] ex vivo analysis Tissue digestion Colons were washed in PBS to remove feces and then incubated in PBS 5mM EDTA for 30 min at 37°C. Colons were digested with 2mg / mL collagenase D and 20μg / mL DNAse in RPMI medium for 30 min at 37°C. Colons were then disrupted through a cell strainer to obtain a single cell suspension. Cells were then washed and resuspended in 3mL PBS-2% FCS and 200μL was plated for antibody staining. Spleens and mesenteric lymph nodes were disrupted through a cell strainer to obtain a single cell suspension. Red blood cells were lysed with 1 mL of red blood cell lysis buffer for 2 min. Cells were then washed and resuspended in 5 mL and 1 mL of PBS-2% FCS, respectively, and 200 μL was plated for antibody staining. ELISA for detection of lipocalin in mouse feces Feces were collected daily and frozen at -20°C. Fecal samples were resuspended in 1 mL PBS Tween 0.1% and centrifuged at 4000 rpm for 20 min. Supernatants were collected and plated at 1 / 1000 for lipocalin detection using DuoSet ELISA Mouse Lipocalin 2 / NGAL (DY1857-05) according to the manufacturer's instructions. Multicolor flow cytometry analysis Cell subsets within the organs were stained for flow cytometric analysis as follows. [Table 3]

[0261] result Transduction efficiency and cell surface CAR expression Transduction efficiency was assessed by the percentage of GFP-positive cells, and CAR expression was monitored using antibodies directed against recombinant protein L, immunoglobulin kappa light chain binding protein, or HA tag. The results of the percentage of transduction efficiency and the percentage of transduced cells expressing CAR at the cell surface are provided in Table 4. Both constructs led to more than 95% CAR expression at the cell surface. Furthermore, the mean fluorescence intensity (MFI), which represents the number of CARs per cell, was comparable. [Table 4]

[0262] A stable Treg phenotype is observed in the presence of the novel anti-IL23R CAR#2 In general, a major challenge with engineered T cells is ensuring maintenance of the desired phenotype, especially since high expression of CAR has been shown to be associated with undesirable antigen-independent CAR activation (Frigault et al., Cancer Immunol Research (2015) 3(4):356-67). To assess whether the Treg phenotype changes during proliferation and CAR binding, a panel of markers associated with Treg identification was analyzed. Maintenance of expression of Helios and FOXP3, as well as other markers associated with the Treg phenotype, was analyzed using FOXP3. + IL-23R CAR-Tregs maintained high expression of FOXP3 and Helios at day 9 after expansion (Figure 2A), and remained stable up to 11 days after CAR conjugation with IL-23R-coated beads (Figure 2B).

[0263] Novel scFv-derived CARs maintain CAR-specific activation The CAR#2 construct reduced background activation of Treg cells compared to the CAR construct control (CAR#1). As shown in FIG. 3, CD69 in the absence of activation + The percentage of IL-23R CAR-Treg cells was 60% CD69 + The activation of IL-23R CAR-Treg cells using anti-CD3 / anti-CD28 coated beads was much lower in CAR#2 compared to CAR#1, which yielded IL-23R Treg cells. + This resulted in a 6.41-fold increase in cells compared with only a 1.52-fold increase in CAR#1.

[0264] Finally, activation of IL-23R CAR-Treg cells using IL-23R-coated beads was increased 3.6-fold for CAR#2 but only 1.33-fold for CAR#1. These results indicate superior CAR-mediated activation of Treg cells expressing CAR constructs containing the scFv of the present disclosure.

[0265] CAR#2 exhibits efficient CAR-mediated suppressive activity In CAR#2, induction of CAR-specific suppressive activity was observed compared to the IL-23R CAR construct with control scFv (CAR#1) (Figure 4). Indeed, as shown in Figure 4, the spontaneous suppressive activity of CAR#1-expressing Treg cells (in the absence of activation) was too strong to indicate specific TCR or CAR-mediated suppressive activity. In contrast, stimulation of CAR#2-expressing Treg cells resulted in strong suppressive activity, either after TCR binding or after CAR#2 activation with IL-23R ligand. These results demonstrate the superiority of the scFv of the present disclosure.

[0266] Tregs bearing a novel scFv-derived CAR are activated in vivo and reduce disease activity indices in a mouse model of DSS-induced IBD Mouse Treg cells were transduced with different constructs as described in Figure 5. Two versions of mouse CAR#2CD28 (mCAR#2) containing a novel scFv (cross-reactive with human and mouse) were constructed: (1) mCAR#2a, which contains the same TM domain as mCAR#1 (mouse CD28 TM), and (2) mCAR#2b, which contains the TM domain of mouse CD8 (more comparable to the human construct CAR#2).

[0267] IL-23R-CAR mouse Tregs transduced with either non-transduced (NT) or control scFv-derived mouse CAR constructs (mCAR#1) or scFv-derived mouse CAR constructs (mCAR#2a and 2b) of the present disclosure were injected into a mouse model of short dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD) (Figure 6A). This model was developed to measure in situ activation of injected cells and is a target binding model rather than an efficacy model. As shown in Figure 6B, IL-23R CAR Tregs (mCAR#2) containing scFvs of the present disclosure are found in vivo in the colon, mesenteric lymph nodes, and spleen. These Tregs are significantly more activated than non-transduced cells at the site of inflammation (in the colon and mesenteric lymph nodes) as measured by expression of CTLA-4 on Tregs using flow cytometry. Interestingly, mCAR#2b (containing a CD8 TM associated with the CD28 intracellular signaling domain) showed significant activation only in inflamed organs (colon and mesenteric lymph nodes) but not in non-inflamed organs (spleen).

[0268] Testing of Tregs in a DSS-induced IBD efficacy model (Figure 7A) showed that mCAR#2 induced a significant reduction in disease activity indicators (Figure 7B).

[0269] Consideration We identified CAR#2, an IL-23R-CAR lead candidate (LamS4G3). CAR#2 is a second-generation CAR composed of an anti-IL-23R-scFv fused to CD8TM / CD28 / CD3z, identified through extensive library screening that yielded over 100 potential hits. This CAR showed high specificity for IL-23R with no detectable tonic signaling and demonstrated specific CAR-dependent suppressive activity in vitro.

[0270] Example 2: In vitro selection of optimized IL-23R CAR constructs in human Tregs Starting from CAR#2 identified in Example 1, several optimizations were performed by varying the promoter, leader sequence, hinge length, and codon optimization. Six different optimized constructs were selected from a matrix of 32 constructs and produced in a clinical-ready backbone (without HA tag and GFP).

[0271] In this example, these different constructs were compared based on several parameters, including viability, fold expansion, stability of the Treg phenotype, CAR-mediated activation, and CAR-mediated suppression.

[0272] Materials and Methods Generation of IL-23R expressing Jurkat cell line The Jurkat IL23R cell line was generated by overexpressing IL23R in Jurkat cells via a puromycin-selectable lentiviral vector. The lentiviral vector was produced and transduced into Jurkat cells.

[0273] Generation of artificial APCs presenting rhuIL-23R Dynabeads (M-270 Epoxy; ThermoFisher Scientific) were conjugated with recombinant human IL-23R Fc chimeric protein (R&D, catalog number 1400-IR-050) using the Dynabeads antibody conjugation kit (Life technologies, Thermo fisher, catalog number 14311D) according to the manufacturer's instructions.

[0274] Isolation of PBMCs and regulatory T cells Tregs and Tconvs were freshly isolated from buffy coats obtained from the blood of healthy volunteers from the EFS (Marseille). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats by Ficoll gradient centrifugation the day after blood donation. CD4+ / CD25+ / CD127low nTreg cells were isolated as follows: CD25+ cells were isolated by column-free immunomagnetic positive selection using EasySep™ Releasable RapidSpheres™. The bound magnetic particles were then removed from the EasySep™ isolated CD25+ cells, and unwanted non-Tregs were targeted for depletion. The final isolated fraction contained highly purified CD4+CD127lowCD25+ cells expressing high levels of FOXP3.

[0275] Transduction The constructs used in this study are listed in Table 5. [Table 5]

[0276] Transduction was performed in 24-well plates. Briefly, 2 days after isolation and activation, 2 / 3 of the medium was removed from each well and 2-5 x 10E6TU / ml of each construct was added to each well. After 6 hours at 37°C, 2 / 3 fresh medium was added to each well, each well was homogenized, collected in a tube, and centrifuged. The supernatant was removed and each pellet was resuspended in fresh medium supplemented with 1000U / ml IL-2 before being seeded on a new plate. 4-5 days after transduction, efficacy was measured by analysis of the percentage of IgG* positive cells in flow cytometry.

[0277] On day 9 of culture, Treg phenotype was analyzed according to the procedure in STF-TRB-01-E009 V02. The markers used for this analysis are listed in the updated Table 6. [Table 6]

[0278] Activation assay On day 10 of culture, activation assays were performed. Briefly, 0.05x10^6 CAR-Tregs were seeded on PL96U bottoms alone or in the presence of anti-CD28 / anti-CD3 coated beads (1:1 Treg to bead ratio), or in the presence of IL-23R coated beads (1:1 Treg to bead ratio), or in the presence of Jurkat expressing low levels of IL-23R on the cell surface (Jurkat 572) or Jurkat expressing high levels of IL-23R (Jurkat 573). After 24 hours at 37°C, 5% CO2, cells were stained for CD4 and CD69 and then analyzed by flow cytometry.

[0279] Suppression assay Suppression assays were performed on day 10, when CAR-Tregs were harvested, counted, activated through the TCR using anti-CD28 / anti-CD3 coated beads (2:1 Treg to bead ratio), or through the CAR using IL-23R coated beads (1:1 Treg to bead ratio), or using Jurkat cell lines expressing low levels of IL-23R on the cell surface (Jurkat 572) or high levels of IL-23R (Jurkat 573), or kept unactivated, and their basal functional activity was assessed. In parallel, allogeneic Tconvs were thawed, stained with Dye 450, and activated with anti-CD28 / anti-CD3 coated beads (3:1 Tconv to bead ratio). The next day, beads were removed from Tconvs and then co-cultured with unactivated or activated Tregs. After 3 days of co-culture, Tconv proliferation was analyzed by flow cytometry.

[0280] result Similar transduction efficiency The selected CARs, covering low to high CAR expression levels, have no detectable background / tonic signaling and cover different biological elements (signal peptide / codon / linker / WPRE). The constructs used in the study were generated and produced in a clinical-ready backbone (without HA tag and GFP). Transduction efficiency was evaluated in flow cytometry using an anti-IgG antibody to measure the percentage (%) of cells positive for IgG, and CAR expression levels at the cell surface were evaluated by the mean fluorescence intensity (MFI) of IgG staining (Figure 8). The different constructs were transduced at 40-50%.

[0281] The different optimized constructs show the same survival and proliferation folds after the first cycle of culture. The effect of different alterations on the behavior of CAR-Tregs in culture was also evaluated. Regardless of the alteration, Tregs transduced with the different constructs showed the same viability levels (Figure 9, left) and fold expansion (Figure 9, right) at day 9.

[0282] CAR#3 demonstrated the lowest background activation and the highest signal to noise after CAR activation.

[0283] The effect of different optimizations on the activation of CAR-Tregs (Figure 10) was evaluated. The CAR-mediated activation profiles of CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7 were close to that of the non-optimized construct (CAR#2). However, the activation background of CAR#3 was slightly lower than that of CAR#2, which showed the best signal-to-noise.

[0284] Significant CAR-mediated suppression The CAR-mediated suppressive activity of the CAR constructs was measured (Figure 11). CAR binding was induced using either artificial APC (IL-23R-coated beads; bIL-23R) or low-expressing (Jurkat 572) or high-expressing (Jurkat 573) Jurkat cell lines. As a positive control, polyclonal activation (anti-CD3 / anti-CD28-coated beads; beads CD3 / 28) was used. Spontaneous suppression is measured without any activation (none). Similar to the activation data, the profiles of CAR#3, CAR#4, CAR#5, CAR#6, and CAR#7 are close to that of the non-optimized construct CAR#2. All constructs are able to mediate robust CAR-mediated suppressive activity when the CAR is bound using artificial APC (Figures 11A and 11B) or high-expressing IL-23R Jurkat cell lines (Figure 11A). All constructs showed very low spontaneous suppressive activity.

[0285] The area under the curve was calculated for CAR#2 and CAR#3 (Figure 12). The AUC calculation reveals that CAR#3 showed the same CAR-mediated suppression as CAR#2 when Tregs were bound to Jurkats expressing high levels of IL-23R on the cell surface (Jurkat 573). However, CAR#3 showed lower CAR-mediated suppression than CAR#2 when CAR Tregs were bound to Jurkats expressing low levels (Jurkat 572). This profile should reduce off-target effects.

[0286] Treg stability - all constructs showed good stability of the Treg phenotype Plasticity is a well-described characteristic of Tregs. The main markers for Treg identification are CD4, CD25, CTLA-4, FoxP3 and Helios. To measure the phenotypic stability of CAR Tregs, the expression of these markers was analyzed by flow cytometry at the end of the first expansion cycle (day 9) (Figure 13). No changes in the main Treg markers were observed after using the different CARs.

[0287] Consideration The differences between the different constructs were minimal, indicating that the standard CAR#2 is robust enough to accommodate different changes.

[0288] However, the CAR#3 construct stood out based on the following parameters: -Lower tonic signaling than CAR#2 resulting in higher signal-to-noise (Figure 10) -When Tregs are bound to Jurkats expressing high levels of IL-23R on the cell surface, CAR-mediated suppression is the same as CAR#2, but when CAR Tregs are bound to Jurkats expressing low levels of IL-23R, CAR-mediated suppression is lower than CAR#2 (Figure 12). This should reduce off-targeting.

[0289] Example 3: Characterization of IL23R-CAR:scFv(LamS4G3) affinity Materials and Methods The indicated scFvs were produced in mammalian 293t cells, purified, and biotinylated. Biotinylated scFvs were then incubated with mouse or human IL23Ra-coated beads in PBS for 30 min at 25°C. After low-temperature washing, bound IL23R-binding scFvs were detected by incubation with APC-labeled streptavidin and samples were analyzed on a flow cytometer. EC50s were calculated based on the median fluorescence intensity from each condition. Calculations, curve fitting, and EC50 calculations were performed via Graphpad Prism.

[0290] result Binding experiments revealed high affinity binding to human IL23R with an EC50 of 37.6 nM and similar affinity to mouse IL23Ra with an EC50 of 53.1 nM. In contrast, the benchmark scFv (also cross-reactive) bound to human IL23R with a high affinity of 4.4 Nm and a more significant difference of 13.1 nM to mouse IL23R (Figure 14).

[0291] In these exemplary results, the CARs of the present disclosure exhibit similar binding profiles between the human and mouse homologs of IL23R. In these exemplary results, CAR#2 of the present disclosure exhibits an approximately 1.4-fold difference in binding affinity between human IL23R and mouse IL23R, while CAR#1 exhibits an approximately 3-fold difference in binding affinity between human IL23R and mouse IL23R. These exemplary results show that CAR#2 of the present disclosure binds to human and mouse IL23R with more similar affinity compared to CAR#1. This similarity suggests that the use of CAR#2 in mouse preclinical models may be more representative of its interaction with human IL23R.

[0292] Example 4: Stability of scFv signaling Materials and Methods Four different constructs were generated with different expression cassettes (PGK or EF1a promoter, + / -WPRE Mut6) and transduced into primary human Tregs by lentiviral transduction (Figure 15).

[0293] result Selected IL-23R scFvs showed stable signaling and low background at similar expression levels in primary Treg cells (Figure 16).

[0294] Increased expression correlates with increased accumulation of CAR at the cell surface. While expression levels changed 5-fold, no background differences were observed relative to unmodified control cells (dark gray horizontal line in CAR tonic signaling graph) (Figure 16). Furthermore, all CAR expression conditions responded similarly strongly to CAR-dependent activation as well as to control activation with CD3 / CD28 beads (Figure 16). [Table 7] JPEG2024526201000010.jpg208159JPEG2024526201000011.jpg200159JPEG2024526201000012.jpg201159JPEG2024526201000013.jpg197159JPEG2024526201000014.jpg214159JPEG2024526201000015.jpg144159JPEG2024526201000016.jpg103159JPEG2024526201000017.jpg192159JPEG2024526201000018.jpg80159JPEG2024526201000019.jpg183159JPEG2024526201000020.jpg137159

Claims

1. An isolated anti-IL-23 receptor (IL-23R) antibody or antigen-binding fragment thereof, wherein the heavy chain variable region (VH) of said antibody or fragment comprises complementarity determining regions (HCDR) 1-3 each comprising SEQ ID NOs: 1-3, or any HCDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs: 1-3, and the light chain variable region (VL) of said antibody or fragment comprises complementarity determining regions (LCDR) 1-3 each comprising SEQ ID NOs: 4-6, or any LCDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs: 4-6, said antibody or antigen-binding fragment thereof.

2. An isolated anti-IL-23 receptor (IL-23R) antibody or antigen-binding fragment thereof, wherein the heavy chain variable region (VH) of said antibody or fragment comprises complementarity determining regions (HCDR) 1-3 each comprising SEQ ID NOs: 1-3, and the light chain variable region (VL) of said antibody or fragment comprises complementarity determining regions (LCDR) 1-3 each comprising SEQ ID NOs: 4-6, said antibody or antigen-binding fragment thereof.

3. The isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 1, which is capable of binding mouse and human IL-23R.

4. The isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 1 or 3, which is capable of binding to the human IL-23R alpha subunit with an EC50 of less than 40 nM.

5. The isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 1 or 2, which is capable of binding to the mouse IL-23R alpha subunit with an EC50 of less than 60 nM.

6. said VH comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7 or SEQ ID NO: 7, and said VL comprises any amino acid sequence that is at least about 90% identical to SEQ ID NO: 8 or SEQ ID NO: 8, the isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. said VH comprises SEQ ID NO: 7, and said VL comprises SEQ ID NO: 8, the isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 6.

8. The isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 1 or 2, which is an scFv comprising any amino acid sequence that is at least about 95% identical to SEQ ID NO:

15.

9. The isolated anti-IL-23R antibody or antigen-binding fragment thereof according to claim 8, which is an scFv comprising SEQ ID NO:

15.

10. (i) an extracellular domain comprising the anti-IL-23R antibody or antigen-binding fragment thereof according to claim 1, (ii) a transmembrane domain, (iii) a cytoplasmic domain comprising an intracellular signaling domain, and a chimeric antigen receptor (CAR) comprising the same.

11. The CAR according to claim 10, further comprising a leader sequence.

12. The CAR according to claim 10, wherein the extracellular domain comprises an scFv comprising SEQ ID NO:

15.

13. The intracellular signaling domain is a human CD28 co-stimulatory signaling domain, optionally comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 32 or SEQ ID NO: 32, and / or a human CD3 zeta domain, optionally comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 30 or SEQ ID NO: 30 and the CAR according to claim 10.

14. The CAR according to claim 10, wherein the transmembrane domain is derived from human CD8 and optionally comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 22 or SEQ ID NO:

22.

15. The CAR according to claim 10, wherein the leader sequence is a CD8 leader sequence and optionally comprises an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98% or 99%) identical to SEQ ID NO: 40 or SEQ ID NO: 40 and is derived from the CD8 leader sequence.

16. The CAR according to claim 10, wherein the leader sequence is a CD25 leader sequence and optionally comprises an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98% or 99%) identical to SEQ ID NO: 58 or SEQ ID NO: 58 and is derived from the CD25 leader sequence.

17. (i) an anti-IL-23R scFv, optionally comprising SEQ ID NO: 15, said anti-IL-23R scFv, and (ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO: 20, said hinge domain, and (iii) a transmembrane domain derived from human CD8, optionally comprising SEQ ID NO: 22, said transmembrane domain, and (iv) an intracellular signaling domain comprising a human CD28 co-stimulatory signaling domain, optionally comprising SEQ ID NO: 32, said intracellular signaling domain, and a human CD3 zeta domain, optionally comprising SEQ ID NO: 30, said human CD3 zeta domain, and (v) optionally a tag and / or a leader sequence, and A chimeric antigen receptor (CAR) comprising.

18. (i) an anti-IL-23R scFv, optionally comprising SEQ ID NO: 15, said anti-IL-23R scFv, and (ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO: 20, said hinge domain, and (iii) a transmembrane domain derived from human CD8, optionally comprising SEQ ID NO: 22, said transmembrane domain, and (iv) an intracellular signaling domain comprising a human CD28 co-stimulatory signaling domain, optionally comprising SEQ ID NO: 32, said intracellular signaling domain, and a human CD3 zeta domain, optionally comprising SEQ ID NO: 30, said human CD3 zeta domain, and (v) a leader sequence derived from CD8, optionally comprising SEQ ID NO: 40, said leader sequence, and A chimeric antigen receptor (CAR) comprising.

19. (i) an anti-IL-23R scFv, optionally comprising SEQ ID NO: 15, said anti-IL-23R scFv, and (ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO: 20, said hinge domain, and (iii) a transmembrane domain derived from human CD8, optionally comprising SEQ ID NO: 22, said transmembrane domain, and (iv) an intracellular signaling domain comprising a human CD28 co-stimulatory signaling domain, optionally comprising SEQ ID NO: 32, said intracellular signaling domain, and a human CD3 zeta domain, optionally comprising SEQ ID NO: 30, said human CD3 zeta domain, and (v) a leader sequence derived from CD25, optionally comprising SEQ ID NO: 58, said leader sequence, and A chimeric antigen receptor (CAR) comprising

20. A nucleic acid molecule encoding the antibody or antigen-binding fragment according to claim 1.

21. A vector comprising the nucleic acid molecule according to claim 20.

22. An immune cell that expresses the CAR according to claim 10, or comprises the nucleic acid molecule according to claim 20 or the vector according to claim 21.

23. A cell comprising the nucleic acid molecule according to claim 20 or the vector according to claim 21.

24. The cell according to claim 23, which is an immune cell.

25. A composition comprising the immune cell according to claim 22.

26. The immune cell according to claim 22 for use as a medicament.

27. The immune cell according to claim 22 for use in the treatment of a disease or disorder mediated by IL-23R-expressing cells in a subject in need of treatment, optionally wherein the disease or disorder is an autoimmune or inflammatory disease or disorder.

28. The disease or disorder is selected from the group consisting of inflammatory bowel disease (optionally Crohn's disease or ulcerative colitis), lupus (optionally systemic lupus erythematosus), arthritis (optionally rheumatoid arthritis or juvenile idiopathic arthritis), Sjögren's syndrome, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, type 1 diabetes, autoimmune thyroid disorder, myasthenia gravis, psoriasis, psoriatic arthritis, skin diseases, and uveitis, and optionally wherein the disease is Crohn's disease. The immune cell for use according to claim 27.

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