Lymphocyte activation gene 3 (LAG-3) targeted T cell silencing agents for treatment of autoimmune diseases

Abstract

Compositions and methods for preventing or treating autoimmune diseases are provided. The composition comprises a binding partner configured to bring LAG-3 and a T cell receptor closer to each other than in the absence of the binding partner, thereby providing an immunosuppressive effect on activated T cells.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 381,257, filed on October 27, 2022, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present disclosure generally relates to compositions and methods for preventing or treating autoimmune diseases, and more particularly, to a novel method of inhibiting the function of T cells that cause autoimmune diseases.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which is submitted in.xml format and incorporated herein by reference in its entirety. The.xml file is named "058636_00646_ST26.xml", created on October 27, 2023, and is 19,476 bytes in size.

[0006] Related Information

[0007] T cells are an important part of the human adaptive immune system. They can directly mediate tissue damage when recognizing self - antigens and can also regulate the responses of other immune cells, such as the production of auto - reactive antibodies by B cells through helper T cells or regulatory T cells. T cells have important functions in the pathogenesis and progression of many autoimmune diseases, such as multiple sclerosis, arthritis, diabetes, and systemic lupus erythematosus (SLE) as well as other diseases described herein. However, domesticating T cells for the treatment of autoimmune diseases requires delicate and well - coordinated control because the inhibition of T - cell immunity may simultaneously lead to severe immunodeficiency diseases. For example, the general immunosuppression by steroids only provides symptomatic relief without eliminating the pathological cause and is often associated with severe side effects and infections in multiple autoimmune diseases. Therefore, there remains an unmet need for compositions and methods that specifically target pathogenic autoreactive T cells while preserving other T - cell subsets that are beneficial for immune defense and homeostasis. The present disclosure is related to this need. Summary of the Invention

[0008] The present disclosure provides compositions and methods for treating or preventing autoimmune diseases. The composition comprises a binding partner having a first binding component and a second binding component, wherein the first binding component specifically binds to a T cell receptor (TCR) component or other proteins near the TCR component, and the second binding component specifically binds to LAG-3. When the T cell is involved in promoting one or more symptoms of an autoimmune disease, the binding of the binding partner inhibits the activity of the T cell. In some embodiments, the composition is provided in the form of a bispecific antibody-based T cell silencer (BiTS). It includes a polynucleotide encoding the bispecific binding partner. The polynucleotide can be DNA (including but not limited to cDNA) and can be present in any type of expression vector, or can be RNA. The present disclosure includes administering the binding partner or the polynucleotide encoding the binding partner to an individual suffering from an autoimmune disease. Administering a polynucleotide that results in the expression of the binding partner is also considered to be administering the binding partner itself. Administering the binding partner can be used for prevention and treatment. In some embodiments, administering the binding partner results in inhibiting the progression of an autoimmune disease, or inhibiting the development of an autoimmune disease, or inhibiting the recurrence of an autoimmune disease. The present disclosure includes cells engineered to express the binding partner. Such cells include but are not necessarily limited to T cells, natural killer cells, macrophages, T cell receptor-engineered cells, etc. In some embodiments, any modified cell expresses the binding partner in the form of a chimeric antigen receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 .Schematic diagram of LAG-3-mediated inhibition of T cell responses.

[0010] Figure 2. Panel A) Left: Schematic diagram of a classical in vitro assay to evaluate the function of LAG-3 on murine T cells. In this assay, in the presence of HEL peptide, MHC-II from murine LK35.2 B cells simultaneously binds to the murine T cell receptor (TCR) and murine LAG-3, and the 3A9 T cell hybridoma cells express a homologous TCR against the MHC-II / HEL complex. Right: Restoration of T cell activation using the known LAG-3 antagonist C9B7W. Panel B) Left: Schematic diagram of an artificial antigen-presenting cell (APC) method capable of dissociating the MHC-II / LAG-3 interaction. A membrane-anchored anti-mouse CD3 (145.2C11) single-chain variable fragment expressed on 293T cells is used to activate T cells, and MHC-II bound to a non-homologous OVA peptide binds only to LAG-3 and not to the TCR of 3A9 T cells. Right: Evaluation of the LAG-3 antagonist C9B7W in this artificial antigen-presenting cell (APC) method.

[0011] Figure 3 . Panel A) Schematic diagram of a murine artificial antigen-presenting cell (APC) method capable of activating murine 3A9 T cells containing an NFAT-GFP reporter. Panel B) Generation of artificial APCs containing membrane-anchored single-chain variable fragments (scFvs) of the anti-mouse CD3 antibody 145.2C11 with different expression levels. Median fluorescence intensity of three different membrane-anchored αCD3 293T clones. Panel C) IL-2 secretion after co-culture of different αCD3 clones with 3A9 T cells. Panel D) Evaluation of different αCD3 clones when co-cultured with T cells containing an NFAT-GFP reporter. Fluorescence reporting cell activation was quantified using images taken with a CellInsight CX7.

[0012] Figure 4 . Panel A) Evaluation of the function of MHC-II fused to a non-homologous peptide expressed on different αCD3 APC clones on LAG-3 + 3A9 T cells by IL-2 measurement after 24 hours. Panel B) Co-culture of αCD3 low APCs in the presence or absence of MHC-II (non-homologous) to evaluate the individual contribution of MHC-II to LAG-3 signaling (with or without anti-LAG-3 C9B7W).

[0013] Figure 5 . Panel A) Schematic diagram of the proposed rapamycin analogue (rapalog) system, in which the rapamycin analogue can induce aCD3e and MHC-II (非同源)heterodimers, thereby forcing the TCR-CD3 complex to approach LAG-3. Panel B) Evaluating the ability of the rapamycin analog heterodimer system to induce the proximity of the TCR-CD3 complex to LAG-3 and trigger LAG-3-mediated T cell inhibition. After 24 hours of co-culture, T cell activation was measured by IL-2.

[0014] Figure 6 . Panel A) Schematic diagram of the LAG-3-CAR-T system for evaluating the participation of membrane or soluble LAG-3 ligands in LAG-3 signaling. Panel B) Images obtained using CellInsight CX7 for quantifying the fluorescence generated by the putative ligands of LAG-3 activating LAG-3CAR-TNFκB GFP Jurkat reporter cells. Plasmids representing each ligand were transfected into HEK-293T cells, followed by co-culture with LAG-3CAR-T NFκB-GFP Jurkat reporter cells. Panel C) Two different forms of soluble FGL1 (oligomer and dimer) activate LAG-3CAR-T.

[0015] Figure 7 . Panel A) Localization of the extracellular domain of LAG-3 by anti-mouse LAG-3 antibody M8. Panel B) Binding epitope of anti-mouse LAG-3 antibody M8. The key residues of M8 that bind to domain 1 of LAG-3 are indicated (PDB: 7TZE).

[0016] Figure 8 . Panel A) Evaluating the MHC-II and FGL1 blocking abilities of M8 (10 ug / ml) in the LAG-3CAR-T assay. Panel B) Evaluating anti-LAG-3 antibodies in the LK35.2 / 3A9 co-culture assay. LK35.2 cells were pulsed with 1 μM HEL peptide and co-cultured with T cells in the presence of isotype or anti-mouse LAG-3 M8 or C9B7W antibodies (10 μg / mL). After 24 hours of co-culture, T cell activation was measured by IL-2. Panel C) Evaluating anti-LAG-3 antibodies C9B7W and M8 in the MC38 tumor model. From day 6, mice were treated with the indicated antibodies at 100 μg per mouse, for a total of 4 doses (twice a week).

[0017] Figure 9 . Antibody variable domain sequence of M8, with CDR regions shown in bold. The heavy chain sequence of M8 is SEQ ID NO:1. The light chain sequence of M8 is SEQ ID NO:2. A non-limiting embodiment of the present disclosure is shown using the bispecific form of the H57 antibody sequence (which also contains the M8 antibody sequence).

[0018] Figure 10. Panel A) Schematic of the TCR / LAG-3 (mouse / mouse) bispecific antibody (bispecific T cell silencer, BiTS) that induces proximity between MHC-II and LAG-3. A mutant version that retains binding to TCR but not to LAG-3 is shown as a control. Panel B) Binding of H57xM8 BiTS and mutants to TCR and LAG-3.

[0019] Figure 11 . H57xM8 BiTS and mutants were evaluated in the suppression of T cell responses. Panel A) CD4 + (3A9, carrying a TCR reactive to MHC-II-HEL peptide) or CD8 + (B3Z, carrying a TCR reactive to MHC-I-OVA peptide) T cell hybridoma cells were stimulated with membrane-anchored anti-CD3 in the presence or absence of LAG-3 overexpression and treated with isotype (human IgG1), H57xM8 BiTS, or H57xM8 BiTS (mut) . After co-culture for 24 hours, T cell activation was measured by IL-2. The percentage of suppression of T cell activation was calculated using the isotype control as a reference. Panel B) 3A9 or B3Z T cells were stimulated with cognate peptides MHC (3A9 with MHC-II-HEL or B3Z with MHC-I-OVA) respectively and treated with isotype (human IgG1), H57xM8 BiTS, or H57xM8 BiTS (mut) . After co-culture for 24 hours, T cell activation was measured by IL-2. The percentage of suppression of T cell activation was calculated using the isotype control as a reference. Panel C) H57xM8 BiTS and mutants were evaluated in an ex vivo CD8+ OT-I primary T cell system. Naïve OT-1 T cells carrying WT OVA TCR and either the OVA TCR in a Lag3 - / - background were isolated and stimulated with the mutuDC cell line pulsed with SIINFEKL peptide (1 ng / mL). T cell activation was measured by IL-2 after co-culture for 24 hours and by IFNγ after co-culture for 120 hours. Panel D) Dose-response curve of H57xM8 BiTS in this ex vivo OT-I T cell system. T cell activation was measured by IL-2 after co-culture for 48 hours.

[0020] Figure 12 . Panel A): Schematic of the TCR / LAG-3 (mouse / human) bispecific antibody that induces proximity between TCR and human LAG-3. In CD4 + (3A9) or CD8 +Evaluate H57xRE (relatlimab) BiTS (Panel B) in the (B3Z) T cell system. Human LAG-3+3A9 or B3Z T cells were stimulated with anti-CD3 or MHC-I-OVA respectively, and treated with BiTS (10 ug / mL). After co-culturing for 24 hours, T cell activation was measured by IL-2.

[0021] Figure 13 . Evaluate H57xM8 BiTS in the experimental autoimmune encephalomyelitis (EAE) model. Two treatment regimens were used: Panel A): prophylactic; Panel B): therapeutic. C57BL / 6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) and injected i.p. with pertussis toxin on days 0 and 2, and then treated i.p. with 1.25 mg / kg of PBS or BiTS. For prophylactic treatment: mice were treated daily from day 6 to day 10 before symptoms appeared. For therapeutic treatment: mice were treated every other day from day 13 to day 21.

[0022] Figure 14 . Evaluate H57xM8 BiTS in the co-receptor deletion system. Use CRISPR-Cas9 to generate mouse LAG-3 + CD4 KO 3A9 and CD8 KO B3Z cells. Panel A) LAG-3 + 3A9 CD4 KO cells were stimulated with membrane-anchored anti-CD3 and treated with BiTS (1 ug / ml). Panel B) LAG-3 + B3Z CD8 KO cells were stimulated with mutuDC cells with the indicated SIINFEKL peptide and treated with BiTS (1 ug / ml).

[0023] Figure 15 . Evaluate OKT3xRE (relatlimab) BiTS in the Jurkat NFκB GFP reporter system. Panel A) Schematic diagram of the Jurkat NFκB GFP reporter system. Panel B) Design of OKT3xRE BiTS. Panel C) LAG-3 + Jurkat cells were activated by MHC-I-gp100 expressed on 293T and treated with BiTS (5 ug / ml).

[0024] Figure 16 . Evaluate OnoxRE (relatlimab) BiTS in the Jurkat NFAT GFP reporter system. Panel A) Schematic diagram of the Jurkat NFAT GFP reporter system. Panel B) Design of OnoxRE BiTS. Panel C) LAG-3 (306-318) HA-specific TCR+ Jurkat cells were activated by DR1 HA expressed on 293T (306-318) and treated with BiTS (5 μg / ml).

[0025] Figure 17 . Panel A): Binding epitope of anti-mouse TCR antibody H57-597 (PDB: 1NFD). Key residues for the binding of H57-597 to the TCRβ constant chain are indicated. Panel B): Variable domain sequence of the H57-597 antibody, with CDR regions shown in bold. SEQ ID NO: 18 and 19 are shown in the figure.

[0026] Figure 18 . H57xNivo (nivolumab) BiTS was evaluated in the human PD-1 + LAG-3 - CD4+ T cell (3A9) or CD8+ T cell (B3Z) system. Panel A): Design of H57xNivo BiTS. Panel B): 3A9 cells and B3Z cells were stimulated with membrane-anchored anti-CD3 or mutuDC cells (2 ng / mL) loaded with SIINFEKL (SEQ ID NO: 17) peptide, respectively, and treated with BITS (1 μg / ml).

[0027] Figure 19 . H57xM8 BiTS was evaluated in the RIP-OVA autoimmune diabetes model. Panel A) RIP-OVA model experimental protocol. On day 0, in vitro-activated OT-I T cells (300,000) were injected i.v. into RIP-OVA mice, and from day 0 to day 8, they were treated every other day with 1.25 mg / kg of PBS, H57xM8 BiTS, or mutant H57xM8 BiTS. Diabetes was defined as blood glucose > 250 mg / dL for 3 consecutive days. Panel B) Diabetes incidence in RIP-OVA mice (n = 8) treated with PBS, H57xM8 BiTS (mut) or H57xM8 BiTS. Panel C) Representative histological images of islets, indicating no insulitis, peri-insulitis, and insulitis. Panel D) Histological assessment of insulitis in surviving RIP-OVA mice on day 14. At least 10 islets were graded in each mouse.

[0028] Figure 20. Evaluate H57xM8 BiTS in the anti-41BB autoimmune hepatitis model. Panel A) Experimental protocol for the hepatitis model. C57BL / 6 mice were injected i.p. with 100 μg of anti-41BB on days 0 and 7, and treated daily with 1.25 mg / kg of PBS or H57xM8 BiTS from day 6 to day 10. Liver sections, liver homogenates, and sera were collected for histological, cytokine, and ALT analyses, respectively. Panel B) Representative histological images of liver sections from healthy controls, PBS, and H57xM8 BiTS-treated groups. Panels C-F) Measurement of ALT, IFNγ, TNFα, and MCP-1 from healthy controls, PBS, and H57xM8 BiTS-treated groups (n = 4).

[0029] Figure 21 . Evaluate H57xM8 BiTS in different CD4 / CD8 subsets in the liver in the anti-41BB autoimmune hepatitis model. Intrahepatic lymphocytes were stained for several T cell surface markers to determine the depletion effect of H57xM8 BiTS compared to healthy controls, PBS-treated, and mutant H57xM8 BiTS-treated groups. Panel A) CD45, CD8, and CD4. Panel B) CD45, CD4, CD8, and LAG-3. Panel C) CD45, CD8, and PD-1.

[0030] Figure 22 . Evaluate H57xM8 BiTS in the anti-41BB autoimmune hepatitis model. C57BL / 6 mice were injected i.p. with 100 μg of anti-41BB on days 0 and 7, and treated daily with 1.25 mg / kg of PBS, H57xM8 BiTS, or mutant H57xM8 BiTS from day 6 to day 10. Panels A-D) Liver homogenates were collected for cytokine analysis of IFNγ, TNFα, MCP-1, and IL-12p70 (n = 5).

[0031] Figure 23 . Overview diagram. This figure provides a non-limiting schematic of the method provided by the present disclosure. Detailed implementation

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0033] Each numerical range given throughout this specification includes its upper and lower limits, as well as each narrower numerical range falling therein, as if these narrower numerical ranges were expressly written out.

[0034] As used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" associated with a numerical value encompasses a variation of + / - 10%, + / - 5% or + / - 1%.

[0035] The present disclosure includes each amino acid sequence described herein and all nucleotide sequences encoding such amino acid sequences. This includes each antibody sequence and its antigen-binding fragments. The invention includes polynucleotide and amino acid sequences having 80 - 99% similarity (including the end values and including all numerical values and numerical ranges therebetween) to the sequences provided herein. All amino acid sequences described herein may include amino acid substitutions, such as conservative substitutions, which do not adversely affect the function of the protein comprising such amino acid sequence.

[0036] The present disclosure reveals, among other aspects, previously unknown characteristics of T cells when LAG-3 is located adjacent to TCR components or adjacent to one or more proteins that are typically located near the TCR on the surface of T cells. The present disclosure demonstrates the inhibitory effect on the TCR involved in harmful immune responses using a bispecific binding partner that specifically binds to LAG-3 and specifically binds to the TCRβ chain simultaneously. Based on this demonstration, it is expected that binding partners that bind to other components of the TCR complex or other proteins near the TCR will have a similar effect on T cells. Accordingly, the present disclosure includes the use of a binding partner that comprises a first binding component that specifically binds to LAG-3 and a second binding component that binds to a component of the TCR complex (such as the TCRβ chain, TCRγ chain or δ chain) or a protein that is typically located near the TCR complex (including but not limited to CD3δ, CD3γ, CD3ε and CD3ζ, CD4, CD5, CD6, CD7 and CD8).

[0037] Non-limiting embodiments of the present disclosure are demonstrated using the H57 antibody as a component of the binding partner. The terms "H57" and "H57-597" as used in this specification refer to the same antibody construct that specifically binds to the mouse TCRβ chain. The H57 antibody is known in the art and is commercially available, for example, from BioXCell catalog number #BE0102. Other TCR-binding antibodies are known in the art, including the BMA-031 antibody, which has the BMA-031 heavy chain sequence

[0038]

[0039] and the BMA-031 light chain (κ) sequence

[0040]

[0041] and the JOVI-1 antibody, which has a heavy chain sequence

[0042]

[0043] and the JOVI-1 light chain (κ) sequence

[0044]

[0045] In some embodiments, the LAG-3-specific binding component of the construct comprises a LAG-3-specific antibody, examples of which are known in the art and include but are not necessarily limited to the antibodies designated IMP761, MGD013, BI754111, XmAb22841, Sym022, MK-4280, TSR-033, REGN3767, GSK2831781, LBL-007, LAG525, INCAGN02385, and Relatlimab sold under the trade name OPDUALAG. In some embodiments, use of the bispecific construct described herein exhibits improved immunosuppressive effects compared to use of a construct that binds only LAG-3 (such as that described in Angin et al. J Immunol (2020) 204(4):810–81) or a LAG-3 binder that does not bring LAG-3 into proximity to the TCR chain. In one non-limiting embodiment, use of the bispecific construct induces more inhibition of T cell activation compared to a construct that binds only LAG-3 or a LAG-3 binder that does not bring LAG-3 into proximity to the TCR chain.

[0046] In addition to known antibodies, the present disclosure includes the following heavy and light chain sequences of antibodies for use as anti-human LAG-3 components for the prevention and / or treatment of autoimmune diseases:

[0047] The heavy chain variable domain of H4-10

[0048]

[0049] The light chain variable domain (κ) of H4-10

[0050]

[0051] The heavy chain variable domain of H3-6

[0052]

[0053] H3-6 Light chain variable domain (κ)

[0054]

[0055] H8 Heavy chain variable domain

[0056]

[0057] H8 Light chain variable domain (κ)

[0058]

[0059] H12 Heavy chain variable domain

[0060]

[0061] H12 Light chain variable domain (κ)

[0062]

[0063] M7695 Heavy chain variable domain

[0064]

[0065] M7695 Heavy chain variable domain (κ)

[0066]

[0067] In some embodiments, the binding partner is a bispecific antibody, but other forms are also included within the scope of the present disclosure, such as trispecific antibodies. The term "antibody" includes all forms of binding partners that specifically bind to their cognate antigen, including but not limited to antibody fragments that specifically bind to a cognate antigen. Bispecific antibodies include all forms of bispecific antibodies, including but not necessarily limited to those described in / / doi.org / 10.3389 / fimmu.2021.626616, the description of which is incorporated herein by reference.

[0068] For the component of a bispecific antibody that binds to the TCR α-chain or β-chain, typically this component will bind to the constant region of one of these chains. However, customized bispecific antibodies that bind to variable regions (including but not necessarily limited to the complementarity-determining regions (CDRs) of the TCR α-chain or β-chain) are also included within the scope of the present disclosure, such as for use in personalized medicine approaches.

[0069] It is known that LAG-3 is a T cell checkpoint receptor that is mainly present on activated T cells and can be induced by cytokines such as interleukin (IL)-2, IL-7, and IL-12. LAG-3 negatively regulates the proliferation, activation, effector functions, and homeostasis of both CD8+ and CD4+ T cells. The immunosuppressive activity of LAG-3 is mediated by its intracellular signaling domain. The secreted protein fibrinogen-like protein 1 (FGL1) is a high-affinity LAG-3 ligand that is independent of MHC-II. FGL1 is a fibrinogen family protein secreted by hepatocytes, which has no clear association with fibrin clot formation but has been shown to be active in hepatocyte proliferation and liver metabolic functions.

[0070] Despite more than three decades of research, the exact mechanism by which LAG-3 inhibits T cells remains unclear. To fill this knowledge gap, the present disclosure provides a series of designed in vitro T cell function systems to clearly define the role of MHC-II / LAG-3 interactions in the absence of TCR engagement ( Figure 2-4 ). The present disclosure reveals that the proximity of LAG-3 to the TCR-CD3 complex, which is normally enforced by MHC-II, is crucial for the function of LAG-3 ( Figure 5 ). Based in part on this determination, the present disclosure provides representative and non-limiting bispecific antibodies that bring LAG-3 closer to the TCR complex. These antibodies exhibit strong LAG-3-dependent in vitro activity against both CD4 + and CD8 + T cells ( Figures 10-13 , 19-22). Given the potent immunosuppressive activity of this antibody approach, the antibodies of the present disclosure are referred to herein as bispecific T cell silencers (BiTS).

[0071] In a non-limiting example, the present disclosure demonstrates that the BiTS significantly improved disease symptoms in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) ( Figure 13 ), prevented the development of diabetes ( Figure 19 ), and prevented hepatitis in the anti-41BB autoimmune hepatitis model ( Figure 20 ). Other beneficial effects in reducing autoimmune effects are also shown in the figures. Thus, the methods provided herein demonstrate the use of BiTS as effective T cell checkpoint agonists for the treatment of MS and diabetes, and it is expected that this approach can be extended to other autoimmune diseases, as further described below.

[0072] It will be appreciated from the present specification and the accompanying drawings that the present disclosure provides a unique in vitro T cell function assay system for determining the specific role of MHC-II / LAG-3 interaction without simultaneously triggering the TCR. The importance of LAG-3 proximity to the TCR and the enhancement of LAG-3 function driven by small molecules are considered novel. In this regard, recent publications have shown that LAG-3 mediates T cell inhibition through tonic signaling, preventing Lck from interacting with the co-receptors CD8 and CD4 in the immune synapse (PMID: 35437325), which may occur in an MHC-II-independent manner, although FGL1 may still play a role in this system. In contrast, the data provided herein demonstrate that TCR proximity (rather than co-receptor engagement) is an important factor in LAG-3-mediated immunosuppression, and this mechanism can be used to design LAG-3-based checkpoint agonists, representative examples of which have been described and used in the figures and data of the present disclosure. Without being bound by any particular theory, it is also considered that ligand engagement of MHC-II is required since MHC-II is capable of binding both the TCR and LAG-3 simultaneously. Thus, the present disclosure supports the interpretation that MHC-II and possibly oligomeric forms of FGL1 enhance the proximity between the TCR-CD3 complex and LAG-3, and that LAG-3 acts on this complex to exert its inhibitory function. Accordingly, the present disclosure challenges the conventional dogma in the co-signaling receptor field that immune receptor signaling is only triggered upon trans interaction with a membrane ligand. Instead, the present disclosure reveals that receptor signaling requires an additional level of cis TCR proximity control. Previously available treatments for autoimmune diseases have mainly focused on cytokines or B cell modulators. Thus, the present disclosure provides an alternative to these approaches by demonstrating a T cell-based immunotherapy that targets activated T cells while sparing naive T cells. Accordingly, the BITS is considered a unique checkpoint agonist for treating autoimmune diseases, given its selective and potent activity in inhibiting CD4+ and CD8+ LAG-3-positive activated T cells (but not LAG-3-negative T cells), and the close association of LAG-3 with multiple autoimmune diseases. The method is different from the use of the T cell modulator CTLA-4-Ig abatacept, which does not activate the immune checkpoint pathway but inhibits the co-stimulatory pathway. Thus, the data provided in the present disclosure support the conversion of LAG-3 antibodies, which are typically antagonists, into agonists that trigger the inhibitory function of LAG-3 through the BiTS. The described BiTS method can be combined with other autoimmune immunotherapies, such as those targeting B cells or cytokines (in the form of multispecific antibodies or combination therapies).

[0073] On the one hand, the present disclosure provides a new functional system that involves artificial antigen-presenting cells (aAPCs) and murine T cell hybridomas for distinguishing the roles of MHC-II on TCR and LAG-3. For this system, we expressed a membrane-tethered anti-mouse CD3ε single-chain variable fragment (scFv), which serves as the TCR-CD3 activation signal, and used MHC-II covalently conjugated to a non-homologous peptide that does not trigger the TCR as the LAG-3 ligand ( Figure 2 B). However, in this assay, LAG-3-mediated T cell inhibition caused by LAG-3 antibody blockade was not observed ( Figure 2 B). Therefore, we also constructed aAPC clones with low, medium, and high levels of membrane-tethered anti-mouse CD3ε expression, which stimulated murine T cell activation with different potencies, as indicated by the NFAT GFP reporter signal ( Figure 3 A-B). Using this system, we found that overexpression of MHC-II fused to a non-homologous peptide did not trigger LAG-3-mediated immunosuppression of LAG-3-positive T cells under different intensities of TCR stimulation ( Figure 4 A). Similarly, anti-LAG-3 antibody was ineffective in the absence or presence of MHC-II / non-homologous peptide ( Figure 4 B). This unexpected result led us to determine that LAG-3 must be in proximity to the TCR-CD3 complex to exert its inhibitory function.

[0074] In the known system, MHC-II binds to both the TCR-CD3 complex and LAG-3 simultaneously, keeping LAG-3 and the TCR-CD3 complex in close contact. To test the importance of TCR-CD3 proximity in LAG-3 function, we generated an in vitro APC / T cell co-culture system with or without forced proximity between the TCR-CD3 complex and LAG-3. We utilized a rapamycin analog-induced heterodimer system by attaching the FRB domain of mTOR to MHCII (covalently conjugated to a non-homologous peptide) and FKBP12 to the membrane-tethered anti-CD3ε antibody, thereby bridging the TCR-CD3 complex and LAG-3 ( Figure 5 A). After incubation with a non-immunosuppressive rapamycin analog (a small molecule), a tight interaction with FRB and FKBP12 was formed, and it was speculated that this would promote the proximity of MHC-II to the membrane-tethered anti-CD3ε antibody, and the same for LAG-3 and the TCR-CD3 complex. Experiments showed that such heterodimers could trigger LAG-3-mediated inhibition ( Figure 5 B), indicating that LAG-3 indeed functions when in proximity to the TCR.

[0075] The present disclosure includes a unique LAG-3 antibody screening system, namely a murine LAG-3 chimeric antigen receptor-like (CAR) NFκB-GFP reporter cell assay, for validating functional LAG-3 ligands and screening LAG-3 antibodies with or without different ligand blocking capabilities( Figure 6 A). This system can be used, for example, to identify other bispecific binding partners that can be used in the methods. To develop this screening system, we combined the CD28 / 41BB / CD3ζ intracellular domains with the extracellular domain (ECD) and transmembrane (TM) domain of LAG-3 and overexpressed the chimera in Jurkat NFκB-GFP reporter cells. Among all known LAG-3 ligands, we found that only MHC-II and FGL1 (membrane-anchored or oligomeric forms) were able to trigger LAG-3-CAR activation( Figure 6 B-C). Using this LAG-3CAR reporter system, we have identified several antibodies that can function as the bispecific antibodies. In non-limiting examples, the data provided in the present disclosure show that a hybridoma-derived anti-mouse LAG-3 antibody (M8) can block MHC-II- and FGL1-mediated signal transduction( Figure 7 A-B). This domain 1 antibody binds to an epitope outside the representative loop region (68-91aa) of LAG-3 and exhibits a more potent function of reversing LAG-3-mediated immunosuppression than the C9B7W anti-mouse LAG-3 domain 2 antibody( Figures 7-8 ). Thus, the present disclosure includes the design and use of other BiTSs that anchor the LAG-3 receptor to the TCR complex on the same T cell. This enables the TCR complex to be inhibited after antigen-induced TCR activation, which is expected to promote the proximity of LAG-3 to the TCR (cis) and mediate LAG-3 + positive T cell inhibition. Based on this M8 anti-mouse LAG-3 antibody clone( Figure 9 ), we developed an anti-mouse TCRβ (clone H57-597, commercially available) / M8 bispecific single-chain variable fragment (ScFv) antibody (H57xM8 BiTS), which induces the proximity of LAG-3 to the TCR-CD3 complex( Figure 10 ). We found that H57xM8BiTS was able to inhibit CD4 and CD8 T cell activation in a LAG-3-dependent manner( Figure 11 ). In addition, we also found that an anti-mouse TCRβ (clone H57-597) / anti-human LAG-3 (relatlimab from BMS) BiTS (H57xRE BiTS) could also effectively inhibit human LAG-3-positive CD4 or CD8 T cells( Figure 12)。The results support that the present method converts the LAG-3 antagonist into a BiTS form of LAG-3 agonist by fusing with an anti-TCR antibody, and uses the anti-LAG-3 antagonist to enhance T cell activation( Figures 10-12 )。We further demonstrated that H57xM8 BiTS inhibited the TCR-CD3 complex and did not affect co-receptor signaling( Figure 14 )。When using BiTS form of anti-human CD3 antibody, we found that these BiTS activated rather than inhibited T cell activity, indicating that the H57-597 epitope is important( Figures 15-16 )。 Figure 15 A binding partner that binds to an epitope within the human CD3 complex and is not a LAG-3 binding partner was used. This binding partner is the OKT3 monoclonal antibody, which is known in the art and commercially available, for example, from BioXCell catalog number BE0001-2. Similarly, Figure 16 Another anti-CD3 antibody that binds to an epitope different from OKT3 was used. This binding partner is an anti-CD3 antibody from Ono Pharmaceutical Co., Ltd., described in US20220281977A1, the description of which is incorporated herein by reference.

[0076] The H57-597 antibody targets the FG loop within the constant chain of TCRβ( Figure 17 )。In addition, we found that anti-mouse TCRβ (H57-597 clone) / anti-human PD-1 (nivolumab) was unable to inhibit T cells, indicating that proximity induction via LAG-3 is very important( Figure 18 )。The present disclosure includes BiTS comprising a fusion of an anti-LAG-3 ScFv with other available TCR-CD3 ScFv antibodies other than targeting TCR, such as targeting CD3ε or CD6, and CD3ε or CD6 has been shown to be closely related to the TCR-CD3 complex. Generally, the present disclosure includes using anti-CD3ε antibodies with weak T cell activation or no T cell activation.

[0077] Given the potent function of anti-LAG-3 / TCR BiTS in inhibiting CD4 and CD8 T cell activation in a LAG-3-dependent manner, the present disclosure includes using the bispecific binding partner to treat a variety of autoimmune diseases, as further described below. In a non-limiting demonstration, we tested H57xM8 BiTS in a MOG peptide-induced CD4-driven experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. The BiTS significantly improved the EAE disease symptoms, even at a low dose (1 mg / kg) of 5 doses daily( Figure 13)。We tested H57xM8 BiTS in an autoimmune diabetes model and found that the BiTS prevented the development of diabetes( Figure 18 )。In an autoimmune hepatitis model induced by anti-41BB, we found that H57xM8 BiTS prevented the development of hepatitis( Figure 20 )。In addition, the BiTS did not deplete LAG-3 + T cells in this model( Figure 21 )。Compared with the mutant version of H57xM8 BiTS, H57xM8 BiTS was more effective in inhibiting the production of inflammatory cytokines in the liver( Figure 22 )。These data demonstrate the feasibility of using the BiTS approach in T cell-related autoimmune diseases. LAG-3 is closely related to the pathogenesis of EAE, diabetes, and arthritis, and the soluble LAG-3 ligand FGL1 has been shown to have therapeutic effects in murine arthritis. It is expected that the BiTS may also synergize with existing treatment regimens such as anti-BAFF blockers (for treating lupus) and CTLA-4-Ig (for treating arthritis).

[0078] Although the bispecific binding partners in the present disclosure are as described above, the binding partners can be further modified to be, for example, trispecific, so as to specifically bind other targets in addition to the TCR component and the LAG-3 component. Thus, the bispecific binding partner includes a binding partner that binds to at least two different said targets. Accordingly, the components of the bispecific binding partners in the present disclosure can be provided as intact immunoglobulins or fragments of immunoglobulins, including but not necessarily limited to antigen-binding (Fab) fragments, Fab' fragments, (Fab')2 fragments, Fd (N-terminal portion of the heavy chain) fragments, Fv fragments (two variable domains), diabodies (Dbs), dAb fragments, single-domain fragments or single monomeric variable antibody domains, single-chain diabodies (scDb), isolated complementarity-determining regions (CDRs), single-chain variable fragments (scFv), and other antibody fragments that retain antigen-binding function. In some embodiments, the chimeric antigen receptor (CAR) of the present disclosure comprises an scFv that comprises heavy and light chain variable regions. As is known in the art for the previously described CARs, the scFv is present in a continuous polypeptide that also comprises a CD3ζ chain and a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain. The CAR may also comprise a co-receptor hinge sequence, such as a CD8 co-receptor hinge sequence

[0079] In some embodiments, the binding partners of the present disclosure may include constant regions, such as Fc regions. Any isotype that can include a constant region may be used. However, the present disclosure includes the prerequisite that the methods described herein do not require Fc / Fcr interactions.

[0080] In certain embodiments, the bispecific binding partner may include a linker amino acid that links a first binding component that specifically binds a TCR component or other protein adjacent to the TCR to a second binding component that specifically binds LAG-3. Suitable amino acid linkers may consist mainly of relatively small neutral amino acids such as glycine, serine, and alanine, and may include multiple copies of a glycine- and serine-rich sequence. In a particular and non-limiting embodiment, the linker contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0081] The binding partner and the pharmaceutical composition comprising the binding partner can be administered to an individual in need thereof by any suitable route, examples of which include intravenous, intramuscular, intraperitoneal, intracerebrospinal fluid, subcutaneous, intra-articular, intra-synovial, oral, topical, or inhalation routes, depending on the specific condition being treated. The composition can be administered parenterally or enterically. The composition can be introduced in a single administration or multiple administrations, or can be introduced continuously over a period of time. For example, the administration can be a predetermined number of administrations, or daily, weekly, or monthly administrations, which can be continuous or intermittent, depending on the treatment indication.

[0082] The present disclosure includes binding partners for diagnostic and prophylactic methods. For therapeutic methods, in certain embodiments, the binding partner can be delivered in the form of an mRNA or DNA polynucleotide encoding the binding partner. Administration of DNA or RNA encoding any of the binding partners described herein is also considered a method of delivering such binding partners to an individual or one or more cells. Methods of delivering DNA and RNA encoding proteins are known in the art and can be adapted to deliver binding partners in view of the benefits of the present disclosure. In some embodiments, one or more expression vectors are used, which one or more expression vectors include viral vectors. Thus, in some embodiments, viral expression vectors are used. Viral expression vectors can use naked polynucleotides or can comprise any viral particles, including but not limited to defective interfering particles or other replication-defective viral constructs, as well as virus-like particles. In some embodiments, the expression vector comprises a modified viral polynucleotide, such as from an adenovirus, herpesvirus, or retrovirus.

[0083] In some embodiments, the present disclosure includes modified cells that are modified to express the binding partner. In some embodiments, the modified cells are modified lymphocytes. In some embodiments, the modified cells are T cells, natural killer cells, or macrophages. In some embodiments, the modified cells are modified stem cells. In some embodiments, the modified cells are totipotent stem cells, pluripotent stem cells, or multipotent stem cells. In some embodiments, the cells are used as therapeutic agents.

[0084] In some embodiments, an individual in need of the compositions of the present disclosure has been diagnosed with or is suspected of having an autoimmune disease. In some embodiments, the autoimmune disease is any of the following: systemic lupus erythematosus, rheumatoid arthritis, chronic inflammation, celiac disease, Crohn's disease, colitis, type 1 diabetes, inflammatory bowel disease, autoimmune encephalitis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic esophagitis, multiple sclerosis (MS), including but not limited to relapsing-remitting MS, secondary progressive MS, primary progressive MS, and progressive-relapsing MS, or gastritis, Graves' disease, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, thrombocytopenic purpura, myasthenia gravis, pernicious anemia, psoriasis, Sjogren's syndrome, ulcerative colitis, graft-versus-host disease (GVHD), or any autoimmune disease characterized by type II, III, or IV hypersensitivity reactions, polymyalgia rheumatica, Addison's disease, Behcet's disease, scleroderma (systemic sclerosis), autoimmune pancreatitis, autoimmune hemolytic anemia, hypoparathyroidism, Guillain-Barré syndrome, reactive arthritis, or sarcoidosis. In some embodiments, the individual has been diagnosed with or is suspected of having one or a combination of primary progressive multiple sclerosis (PPMS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), or progressive-relapsing MS (PRMS).

[0085] In some embodiments, an effective amount of one or more binding partners is administered to an individual in need thereof. In some embodiments, an effective amount refers to an amount that alleviates one or more signs or symptoms of a disease and / or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset or recurrence of a disease. The precise dosage can be selected by the individual physician depending on the patient to be treated. The dosage and administration can be adjusted to provide a sufficient level of the binding partner to maintain the desired effect. Other factors that can be considered include the severity and type of the disease state, the age, weight, and sex of the patient, the desired duration of treatment, the method of administration, the timing and frequency of administration, drug combinations, responsiveness, and / or tolerance / response to treatment.

[0086] In some embodiments, the binding partner is produced by a host cell via a recombinant expression vector and cell culture. In some embodiments, the cell culture includes prokaryotic cells or eukaryotic cells. In some embodiments, the cell culture is mammalian cells. In some embodiments, the cells are CHO cells. In some embodiments, the cells are HEK293 cells or derivatives thereof.

[0087] The present disclosure provides a kit comprising a binding partner and / or a cell culture expressing the binding partner. Generally, the kit comprises one or more sealed containers containing the binding partner or cells expressing the binding partner. Instructions for the use of the binding partner for therapeutic and / or prophylactic purposes may be included in the kit.

[0088] Those skilled in the art can understand other embodiments of the present disclosure by referring to the specification and practice of the disclosure disclosed herein. The specification and examples are considered to be merely exemplary.

Claims

1. A binding partner having a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a T cell receptor (TCR) moiety or another protein adjacent to the TCR chain, wherein the TCR moiety is optionally a TCR beta (TCRβ) chain, and the second binding moiety specifically binds to LAG-3.

2. The binding partner according to claim 1, wherein the first binding moiety and the second binding moiety are in the form of a bispecific antibody-based T cell silencer (BiTS).

3. The binding partner according to claim 2, wherein the first moiety and the second moiety are in the form of the BiTS, and wherein the binding of the BiTS to a T cell inhibits the activity of the T cell.

4. The binding partner according to any one of claims 1-3, wherein the TCR moiety is a TCRβ chain.

5. A method comprising administering to an individual suffering from an autoimmune disease a binding partner having a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a T cell receptor (TCR) chain or another protein adjacent to the TCR chain, and the second binding moiety specifically binds to LAG-3.

6. The method according to claim 5, wherein the first binding moiety and the second binding moiety are in the form of a bispecific antibody-based T cell silencer (BiTS).

7. The method according to claim 6, wherein the first binding moiety binds to a T cell receptor (TCR) chain.

8. The method according to claim 7, wherein the TCR chain is a TCRβ chain.

9. The method according to any one of claims 5-7, wherein the autoimmune disease is any one of the following: systemic lupus erythematosus, rheumatoid arthritis, chronic inflammation, celiac disease, Crohn's disease, colitis, type 1 diabetes, inflammatory bowel disease, autoimmune encephalitis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic esophagitis, multiple sclerosis (MS), including but not limited to relapsing-remitting MS, secondary progressive MS, primary progressive MS, and progressive-relapsing MS, or gastritis, Graves' disease, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, thrombocytopenic purpura, myasthenia gravis, pernicious anemia, psoriasis, Sjogren's syndrome, ulcerative colitis, graft-versus-host disease (GVHD) or any autoimmune disease characterized by type II, III or IV hypersensitivity reactions, polymyalgia rheumatica, Addison's disease, Behcet's disease, scleroderma (systemic sclerosis), autoimmune pancreatitis, autoimmune hemolytic anemia, hypoparathyroidism, Guillain-Barré syndrome, reactive arthritis or sarcoidosis.

10. The method according to claim 9, wherein the progression of the autoimmune disease is inhibited.

11. The method according to claim 9, wherein the severity of the autoimmune disease or the severity of one or more symptoms of the autoimmune disease is reduced.

12. A pharmaceutical composition comprising a binding partner according to any one of claims 1-3.

13. The pharmaceutical composition according to claim 12, wherein the first binding component and the second binding component are in the form of a bispecific antibody-based T cell silencer (BiTS).

14. The pharmaceutical composition according to claim 13, wherein the TCR component is a TCRβ chain or a TCR-CD3 complex.

15. A modified cell, which is modified to express a binding partner according to any one of claims 1-3.

16. The modified cell according to claim 15, wherein the binding partner is in the form of a bispecific antibody-based T cell silencer (BiTS).

17. The modified cell according to claim 16, wherein the binding partner is in the form of a bispecific antibody-based T cell silencer (BiTS).

18. The modified cell according to claim 17, wherein the TCR component is a TCR beta (TCRβ) chain.

19. A polynucleotide encoding a binding partner according to any one of claims 1-3.

20. The polynucleotide according to claim 19, wherein the binding partner is in the form of a bispecific antibody-based T cell silencer (BiTS), and wherein the polynucleotide is contained in an expression vector.

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