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

Abstract

Compositions and methods for preventing or treating autoimmune diseases are provided, which include a binding partner configured to bring LAG-3 and a T cell receptor closer together than would be the case in the absence of the binding partner, thereby providing an immunosuppressive effect on activated T cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 381,257, filed October 27, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates generally to compositions and methods for the prevention or treatment of autoimmune diseases, and more particularly to a new approach to suppressing T cell function that contributes to autoimmune diseases.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted in .xml format and is incorporated herein by reference in its entirety. The .xml file is named "058636_00646_ST26.xml", was created on October 27, 2023, and is 19,476 bytes in size.

[0004] Related Information T cells are an essential component of the human adaptive immune system, directly mediating tissue damage upon recognition of self-antigens and regulating other immune cell responses, such as B cell production of autoreactive antibodies, via helper T cells and regulatory T cells. T cells play a key role in the pathogenesis and progression of many autoimmune diseases, including multiple sclerosis, arthritis, diabetes, systemic lupus erythematosus (SLE), and other disorders described herein. However, conditioning T cells for the treatment of autoimmune diseases requires delicate and well-coordinated control, as attenuation of T cell immunity can simultaneously lead to severe immunodeficiency. For example, common immunosuppression with steroids only alleviates symptoms (symptomatic treatment) but does not eliminate the cause of the pathology, and is often associated with serious side effects and infections in various autoimmune diseases. Therefore, there remains an unmet need for compositions and methods that specifically target disease-causing autoreactive T cells while sparing other T cell subsets beneficial to immune defense and homeostasis. The present disclosure addresses this need. Summary of the Invention

[0005] The present disclosure provides compositions and methods for use in treating or preventing autoimmune diseases. The compositions include a binding partner having a first binding moiety that specifically binds to a T cell receptor (TCR) component or other protein in close proximity to the TCR component and a second binding moiety that specifically binds to LAG-3. Binding of the binding partners suppresses the activity of T cells to the extent that T cells are involved in promoting one or more symptoms of the autoimmune disease. In some embodiments, the compositions are provided as bispecific antibody-based T cell silencers (BiTS). A polynucleotide encoding the bispecific binding partner is included. The polynucleotide may be DNA, including but not limited to cDNA, present in any type of expression vector, or RNA. The present disclosure includes administering a described binding partner or a polynucleotide encoding the described binding partner to an individual with an autoimmune disease. Administration of a polynucleotide that results in expression of the binding partner is considered to be administration of the binding partner itself. Administration of the described binding partner can be used prophylactically and therapeutically. In embodiments, administration of the described binding partners results in inhibition of the progression of an autoimmune disorder, or inhibition of the onset of an autoimmune disorder, or inhibition of the recurrence of an autoimmune disorder. Included in the present disclosure are cells engineered to express the described binding partners. Such cells include, but are not necessarily limited to, T cells, natural killer cells, macrophages, T cell receptor engineered cells, and the like. In embodiments, any engineered cell expresses the described binding partner in a chimeric antigen receptor format. [Brief explanation of the drawings]

[0006] [Figure 1] Schematic diagram of LAG-3-mediated inhibition of T cell responses.

[0007] [Figure 2] Panel A) Left: Schematic of a classic in vitro assay to assess the function of LAG-3 on mouse T cells. In this assay, mouse LK35.2 B cell-derived MHC-II simultaneously binds both the mouse T cell receptor (TCR) and mouse LAG-3 in the presence of HEL peptide, and 3A9 T cell hybridoma cells express the cognate TCR for the MHC-II / HEL complex. Right: Restoration of T cell activation using C9B7W, a known LAG-3 antagonist. Panel B) Left: Schematic of an artificial antigen-presenting cell (APC) approach that can uncouple MHC-II / LAG-3 interactions. A membrane-tethered 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-cognate OVA peptide binds only to LAG-3 and not to the TCR of 3A9T cells. Right: Evaluation of the LAG-3 antagonist C9B7W in this artificial antigen-presenting cell (APC) approach.

[0008] [Figure 3] Panel A) Schematic of the murine artificial antigen-presenting cell (APC) approach capable of activating murine 3A9 T cells containing an NFAT-GFP reporter. Panel B) Generation of artificial APCs with different expression levels of membrane-tethered single-chain variable fragments (scFv) of the anti-mouse CD3 antibody 145.2C11. Three different membrane-tethered αCD3 Median fluorescence intensity of 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 the NFAT-GFP reporter. Fluorescence was measured using CellInsight Activation of reporter cells was quantified using images taken with CX7.

[0009] [Figure 4] Panel A) Different αCD3 Functionality of MHC-II fused to non-cognate peptides expressed on APC clones (LAG-3 + Assessment of function on 3A9 T cells (measured by IL-2 after 24 hours). Panel B) To assess the individual contribution of MHC-II to LAG-3 signaling, αCD3 low APCs were co-cultured (with or without anti-LAG-3 C9B7W) in the presence or absence of MHC-II (non-cognate).

[0010] [Figure 5] Panel A) Rapalogs inhibit aCD3e and MHC-II (非同族) Schematic of the proposed rapalog system, which can induce heterodimerization of LAG-3 and thereby force close proximity between the TCR-CD3 complex and LAG-3. Panel B) Evaluation of a rapalog heterodimer system that induces proximity between the TCR-CD3 complex and LAG-3, resulting in LAG-3-mediated T cell inhibition. T cell activation was measured with IL-2 after 24 hours of co-culture.

[0011] [Figure 6] Panel A) Schematic of the LAG-3-CAR-T system to assess the involvement of LAG-3 signaling by membrane or soluble LAG-3 ligands. Panel B) CellInsight LAG-3 with putative ligands of LAG-3, using images obtained with CX7. CAR-T NFκB GFP Quantification of fluorescence from activation of Jurkat reporter cells. HEK-293T cells were transfected with plasmids expressing each ligand, followed by LAG-3 activation. CAR-T NFκB-GFP Co-cultured with Jurkat reporter cells. Panel C) Activation of LAG-3 CAR-T by two different forms of soluble FGL1 (oligomers and dimers).

[0012] [Figure 7] Panel A) LAG-3 extracellular domain mapping with anti-mouse LAG-3 antibody M8. Panel B) Binding epitope of the anti-mouse LAG-3 antibody M8. Residues important for M8 binding to domain 1 of LAG-3 are indicated (PDB: 7TZE).

[0013] [Figure 8] Panel A) LAG-3 Evaluation of the MHC-II and FGL1 blocking ability of M8 (10ug / ml) in CAR-T assays. Panel B) Evaluation of anti-LAG-3 antibodies in an LK35.2 / 3A9 co-culture assay. LK35.2 cells were incubated at 1 μM Pulsed with HEL peptide and treated with isotype antibody or anti-mouse LAG-3 antibody They were co-cultured with T cells in the presence of M8 or C9B7W antibody (10 μg / mL). T cell activation was measured by IL-2 stimulatory activity 24 hours after co-culture. Panel C) Evaluation of anti-LAG-3 antibodies C9B7W and M8 in the MC38 tumor model. Mice were treated with the indicated antibodies at 100 ug per mouse four times (twice weekly) starting on day 6.

[0014] [Figure 9] Antibody variable domain sequences of M8 with CDR regions in bold. The sequence of the M8 heavy chain is SEQ ID NO: 1. The sequence of the M8 light chain is SEQ ID NO: 2. Non-limiting embodiments of the disclosure are exemplified using the H57 antibody sequence in a bispecific format that also includes the M8 antibody sequence.

[0015] [Figure 10] Panel A) Schematic of the TCR / LAG-3 (mouse / mouse) bispecific antibody (Bispecific T cell silencer: BiTS) used to induce proximity of MHC-II and LAG-3. As a control, a mutated version that retains binding to the TCR but not to LAG-3 is shown. Panel B) H57xM8 Binding of BiTS and mutants to TCR and LAG-3.

[0016] [Figure 11] H57xM8 in inhibiting T cell responses Assessment of BiTS and mutants. Panel A) CD4 with or without LAG-3 overexpression + (3A9, with TCR that reacts with MHC-II-HEL peptide) or CD8 + T cell hybridoma cells (B3Z, which has a TCR that reacts with the MHC-I-OVA peptide) were stimulated with membrane-tethered anti-CD3 and the isotype (human IgG1), H57xM8 BiTS, or H57xM8 BiTS (mut) After 24 hours of co-culture, T cell activation was measured with IL-2. The percentage of inhibition of T cell activation was calculated using an isotype control as a reference. Panel B) 3A9 or B3Z T cells were stimulated with their respective cognate peptide MHC (MHC-II-HEL for 3A9 and MHC-I-OVA for B3Z) and were immunized with isotype (human IgG1), H57xM8 BiTS, or H57xM8 BiTS (mut) After 24 hours of co-culture, T cell activation was measured with IL-2. The percentage of inhibition of T cell activation was calculated using an isotype control as a reference. Panel C) Ex vivo CD8 + H57xM8 in OT-I primary T cell line Evaluation of BiTS and mutants.OVA TCR (WT or Lag3 - / - Naive OT-1 with background T cells were isolated and stimulated with mutuDC cell lines pulsed with SIINFEKL peptide (1 ng / mL). T cell activation was measured by IL-2 after 24 hours of co-culture and IFNγ after 120 hours. Panel D) This ex vivo OT-I H57xM8 in T cell lines Dose-response curve of BiTS. T cell activation was measured with IL-2 after 48 hours of co-culture.

[0017] [Figure 12] Panel A) Schematic of the TCR / LAG-3 (mouse / human) bispecific antibody used to induce proximity of TCR and human LAG-3. CD4 + (3A9) or CD8 + (B3Z) Evaluation of H57xRE (relatlimab) BiTS in T cell lines (Panel B). Human LAG-3 + 3A9 or B3Z T cells were stimulated with anti-CD3 or MHC-I-OVA, respectively, and treated with BiTS (10 μg / mL). After 24 hours of co-culture, T cell activation with IL-2 was measured.

[0018] [Figure 13] H57xM8 in an experimental autoimmune encephalomyelitis (EAE) model Evaluation of BiTS. Two treatment regimens were employed: Panel A) prophylactic; Panel B) therapeutic. C57BL / 6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) and injected ip with pertussis toxin on days 0 and 2, followed by PBS or BITS ip at 1.25 mg / kg. For prophylactic treatment, mice were treated daily from days 6 to 10 before symptom onset. For therapeutic treatment, mice were treated every other day from days 13 to 21.

[0019] [Figure 14] H57xM8 in the absence of a coreceptor Evaluation of BiTS. Mouse LAG-3 + CD4 KO 3A9 and CD8 KO B3Z cells were generated using CRISPR-Cas9. Panel A) LAG-3 + 3A9 CD4 KO cells were stimulated with membrane-tethered anti-CD3 and treated with BiTS (1 ug / ml). Panel B) LAG-3 + B3Z CD8 KO cells were stimulated with mutuDC cells bearing the indicated SIINFEKL peptides and treated with BiTS (1 ug / ml).

[0020] [Figure 15] Jurkat NFκB Evaluation of OKT3xRE (leratolimab) BiTS in a GFP reporter system. Panel A) Jurkat NFκB Schematic diagram of the GFP reporter system. Panel B) OKT3xRE BiTS design Panel C) LAG-3 with gp100-specific TCR + Jurkat cells were activated with MHC-I-gp100 expressed on 293T cells and treated with BiTS (5 μg / ml).

[0021] [Figure 16] Jurkat NFAT Evaluation of OnoxRE (leratolimab) BiTS in a GFP reporter system. Panel A) Jurkat NFAT Schematic diagram of the GFP reporter system. Panel B) OnoxRE BiTS design. Panel C) HA (306-318) LAG-3 with specific TCR + Jurkat cells were cultured using 293T cells expressing DR1 HA (306-318) and treated with BiTS (5 μg / ml).

[0022] [Figure 17] Panel A) Anti-mouse TCR antibody H57-597 (PDB: Binding epitope of IFN-γ (IFN-γ-1NFD). H57-597 indicates the key residues for binding to the TCR β constant chain. Panel B) H57-597 antibody variable domain sequence with CDR regions in bold. SEQ ID NOs: 18 and 19 are shown.

[0023] [Figure 18] Human PD-1 + LAG-3 - CD4 + T cells (3A9) or CD8 + Evaluation of H57xNivo (nivolumab) BiTS in T cell (B3Z) lineage. Panel A) H57xNivo BiTS design. Panel B) 3A9 and B3Z cells were stimulated with membrane-tethered anti-CD3 or mutuDC cells (with SIINFEKL [SEQ ID NO: 17] peptide [2 ng / mL]), respectively, and treated with BITS (1 ug / ml).

[0024] [Figure 19] H57xM8 in the RIP-OVA autoimmune diabetes model BiTS evaluation. Panel A) Experimental scheme of the RIP-OVA model. In vitro activated OT-I T cells (300,000 cells) were injected intravenously into RIP-OVA mice on day 0, and then injected every other day from day 0 to day 8 with PBS, H57xM8 BiTS, mutant H57xM8 Diabetes was defined as a blood glucose level of >250 mg / dL for three consecutive days. Panel B) PBS, H57xM8 BiTS(mut), H57xM8 Diabetes incidence in RIP-OVA mice administered either BiTS (n=8). Panel C) Representative histological images of pancreatic islets showing 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 scored per mouse.

[0025] [Figure 20] H57xM8 in an anti-41BB autoimmune hepatitis model BiTS evaluation. Panel A) Experimental scheme of the hepatitis model. C57BL / 6 mice were injected ip with 100 μg of anti-41BB on days 0 and 7, and then daily with PBS or H57xM8 from days 6 to 10. BiTS was administered at 1.25 mg / kg. Liver sections, liver homogenates, and serum were collected and subjected to histological, cytokine, and ALT analyses, respectively. Panel B) Healthy control group, PBS-treated group, H57xM8 Representative histological images of liver sections from the BiTS-treated group. Panels C-F) Healthy control group, PBS-treated group, H57xM8 Measurement of ALT, IFNγ, TNFα, and MCP-1 in the BiTS-treated group (n=4).

[0026] [Figure 21] H57xM8 inhibits different CD4 / CD8 subsets in the liver in an anti-41BB autoimmune hepatitis model Evaluation of BiTS. Intrahepatic lymphocytes were stained for several T cell surface markers, and H57xM8 The effect of BiTS depletion was investigated (healthy control group, PBS-treated group, mutant H57xM8 (compared to the BiTS-treated group). Panel A) CD45, CD8, CD4 Panel B) CD45, CD4, CD8, LAG-3 Panel C) CD45, CD8, PD-1

[0027] [Figure 22] H57xM8 in an anti-41BB autoimmune hepatitis model Evaluation of BiTS. C57BL / 6 mice were injected ip with 100 μg of anti-41BB on days 0 and 7, and then daily from days 6 to 10 with PBS, H57xM8 BiTS, or mutant H57xM8 Either BiTS was administered at 1.25 mg / kg. Panels A-D) Liver homogenates were collected for IFNγ, TNFα, MCP-1, and IL-12p70 cytokine analysis (n=5).

[0028] [Figure 23] Overview: A diagram providing a non-limiting schematic of the approach provided by the present disclosure.

[0029] 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.

[0030] Every numerical range given throughout this specification includes not only its upper and lower limits, but also every narrower numerical range subsumed within that range, as if such narrower numerical ranges were all expressly written herein.

[0031] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural references 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, it is understood that the particular value forms another embodiment by use of the antecedent "about." The term "about" with respect to numerical values ​​encompasses variations of ±10%, ±5%, or ±1%.

[0032] The present disclosure includes all amino acid sequences described herein and all nucleotide sequences encoding said amino acid sequences. All antibody sequences and antigen-binding fragments thereof are included. Polynucleotide and amino acid sequences with 80-99% similarity (inclusive, including all numbers and ranges therebetween) to the sequences provided herein are encompassed by the present invention. All amino acid sequences described herein may contain amino acid substitutions (e.g., conservative substitutions) that do not adversely affect the function of the protein containing the amino acid sequence.

[0033] The present disclosure reveals, inter alia, a previously unknown feature of LAG-3's effect on T cells when it is located proximal to a component of the TCR or when it is located proximal to one or more proteins normally located on the T cell surface near the TCR. The present disclosure demonstrates the effectiveness of a bispecific binding partner that specifically binds to LAG-3 and the TCR β chain to attenuate TCR involvement in harmful immune responses. Based on this demonstration, binding partners that bind to other components of the TCR complex or other proteins near the TCR are expected to have similar effects on T cells. Accordingly, the present disclosure includes the use of binding partners that include a first binding moiety that specifically binds to LAG-3 and a second binding moiety that binds to a component of the TCR complex (such as the TCR β chain, the TCR γ chain, or the δ chain) or to a protein normally located near the TCR complex (including, but not limited to, CD3δ, CD3γ, CD3ε and CD3ζ, CD4, CD5, CD6, CD7, and CD8).

[0034] Non-limiting embodiments of the present disclosure are demonstrated using the H57 antibody as a component of the described binding partner. As used herein, the terms "H57" and "H57-597" refer to the same antibody construct that specifically binds to the mouse TCR β chain. The H57 antibody is known in the art and is commercially available (e.g., BioXCell catalog #BE0102). Additional TCR-binding antibodies are known in the art, including: BMA-031 heavy chain sequence: EVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSA (SEQ ID NO: 3), and BMA-031 light chain (κ) sequence: QIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 4) and a BMA-031 antibody having the formula: JOVI-1 heavy chain sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGQGTMVTVSS (SEQ ID NO: 5), and JOVI-1 light chain (κ) sequence: DIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIK (SEQ ID NO: 6) and a JOVI-1 antibody having the formula:

[0035] In embodiments, the component of the described construct that specifically binds to LAG-3 comprises a LAG-3-specific antibody, examples of which are known in the art and include, but are not necessarily limited to, the following antibodies: IMP761, MGD013, BI754111, XmAb 22841, Sym022, MK-4280, TSR-033, REGN3767, GSK2831781, LBL-007, LAG525, INCAGN02385, and Relatlimab (sold under the trade name OPDUALAG). In several embodiments, the use of the bispecific constructs described herein exhibits improved immunosuppressive effects compared to constructs that bind only to LAG-3 (e.g., as described in Angin et al. J Immunol (2020) 204 (4): 810-81) or LAG-3 binding agents that do not bring LAG-3 into proximity with the TCR chain. In non-limiting embodiments, the use of the described bispecific constructs induces greater inhibition of T cell activation than constructs that bind only to LAG-3 or LAG-3 binding agents that do not bring LAG-3 into proximity with the TCR chain.

[0036] In addition to known antibodies, the present disclosure includes the following antibody heavy and light chain sequences for use as anti-human LAG-3 components for use in the prevention and / or treatment of autoimmune diseases: TIFF2026508061000001.tif216169

[0037] In several embodiments, the binding partners described are bispecific antibodies, although other forms (such as trispecific antibodies) are also within the scope of this disclosure. The term "antibody" includes all forms of binding partners that specifically bind to their cognate antigen (including, but not limited to, segments of antibodies that specifically bind to their cognate antigen). Bispecific antibodies include all forms of bispecific antibodies, including, but not necessarily limited to, those described in http: / / doi.org / 10.3389 / fimmu.2021.626616, the disclosure of which is incorporated herein by reference.

[0038] With respect to bispecific antibody components that bind to the TCR α or β chain, typically the components bind to the constant region of one of the chains, however, customized bispecific antibodies that bind to the variable region (including but not necessarily limited to the complementarity determining regions (CDRs) of the TCR α or β chain) are also within the scope of this disclosure (e.g., for use in personalized medicine approaches).

[0039] LAG-3 is a T cell checkpoint receptor found primarily on activated T cells and is known to be induced by cytokines such as interleukin (IL)-2, IL-7, and IL-12. ... + and CD4 + It negatively regulates both T cell proliferation, activation, effector function, and homeostasis. The immunosuppressive activity of LAG-3 is mediated through its intracellular signaling domain. The secreted protein fibrinogen-like protein 1 (FGL1) is an MHC-II-independent, high-affinity LAG-3 ligand. FGL1 is a hepatocyte-secreted protein belonging to the fibrinogen family that has no clear association with fibrin clot formation, but is active in hepatocyte proliferation and liver metabolic function.

[0040] Despite over 30 years of research, the precise mechanism of T cell inhibition by LAG-3 remains unclear. To address this knowledge gap, the present disclosure provides a series of in vitro T cell function systems designed to specifically define the role of MHC-II / LAG-3 interactions without TCR involvement (Figures 2-4). The present disclosure reveals that proximity of LAG-3 to the TCR-CD3 complex, normally enforced by MHC-II, is essential for LAG-3 function (Figure 5). Based in part on this determination, the present disclosure provides representative, non-limiting, bispecific antibodies that position LAG-3 in closer proximity to the TCR complex. This antibody inhibits CD4 + and CD8 +The antibodies exhibited potent LAG-3-dependent in vitro activity against both T cells (Figures 10-13, 19-22). Due to the potent immunosuppressive activity of this antibody approach, the antibodies of the present disclosure are referred to herein as bispecific T cell silencers (BiTS).

[0041] In non-limiting examples, the present disclosure demonstrates that the described BiTS significantly improves disease symptoms in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) ( FIG. 13 ), protects against the development of diabetes ( FIG. 19 ), and protects against hepatitis in an anti-41BB autoimmune hepatitis model ( FIG. 20 ). Other beneficial effects in terms of mitigating autoimmune effects are also shown in the figures. Thus, the presently provided approach demonstrates the use of BiTS as a potent T cell checkpoint agonist to treat MS and diabetes and is expected to be extendable to other autoimmune diseases, as further described below.

[0042] It will be appreciated from the specification and drawings that the present disclosure provides a unique in vitro T cell function assay system that has been used to determine the specific role of MHC-II / LAG-3 interactions without concomitant TCR triggering. The importance of LAG-3 proximity to the TCR and the discovery of small molecule-driven proximity to re-enhance LAG-3 function are believed to be novel. In this regard, recent publications suggest that LAG-3 mediates T cell suppression by tonic signaling, preventing Lck from interacting with co-receptors CD8 and CD4 at the immune synapse (PMID: 35437325). FGL1 may play a role in this system, likely in an MHC-II-independent manner. In contrast, the data currently available indicate that TCR proximity, rather than coreceptor engagement, is the key factor for LAG-3-mediated immunosuppression. This mechanism can be used to design LAG-3-based checkpoint agonists, representative examples of which are described and used in the figures and data of this disclosure. While not wishing to be bound by any particular theory, it is also believed that ligand binding by MHC-II is required for the ability of MHC-II to simultaneously bind both the TCR and LAG-3. Thus, the present disclosure supports the interpretation that MHC-II, and potentially oligomeric forms of FGL1, enhance the proximity of the TCR-CD3 complex to LAG-3, allowing LAG-3 to act on this complex to carry out its inhibitory function. Thus, this disclosure challenges the traditional dogma in the co-signaling receptor field that immune receptor signals are triggered solely by trans-interaction with membrane ligands. Instead, this disclosure reveals that receptor signals require another level of TCR proximity regulation in cis. Previously available autoimmune disease treatments have focused primarily on cytokines or B cell modulators. Therefore, this disclosure provides an alternative to these approaches by demonstrating T cell-based immunotherapy by targeting activated T cells while sparing naive T cells. The described BITS therefore inhibits CD4 + and CD8 +Given its selective yet potent activity in suppressing both LAG-3-positive activated T cells (but not LAG-3-negative T cells) and the close association of LAG-3 with several autoimmune diseases, this is believed to be a unique checkpoint agonist for the treatment of autoimmune diseases. The described approach differs from the use of the T cell modulator CTLA-4-Ig abatacept, which does not activate immune checkpoint pathways but rather inhibits costimulatory pathways. Thus, the present disclosure provides data supporting the conversion of LAG-3 antibodies, which are normally antagonists, into agonists that trigger LAG-3 inhibitory function by the described BiTS. The described BiTS approach can be combined with other autoimmune therapies, such as those targeting B cells or cytokines (in the form of multispecific antibody therapy or combination therapy).

[0043] In one aspect, the present disclosure provides a novel functional system comprising artificial antigen-presenting cells (aAPCs) and mouse T cell hybridomas to distinguish the contribution of MHC-II to TCR and LAG-3. In this system, we express a membrane-tethered anti-mouse CD3ε single-chain variable fragment (scFv) that functions as a TCR-CD3 activation signal, and use MHC-II covalently linked to a non-cognate peptide that does not trigger the TCR as a LAG-3 ligand (Figure 2B). However, in this assay, we did not observe LAG-3-mediated T cell inhibition by LAG-3 antibody blockade (Figure 2B). Therefore, we also generated aAPC clones with low, intermediate, and high levels of membrane-tethered anti-mouse CD3ε expression (these clones express NFAT. The LAG-3 / noncognate peptide stimulated mouse T cell activation with different potencies, as indicated by the GFP reporter signal (Figure 3A-B). Using this system, we found that overexpression of MHC-II fused to a noncognate peptide did not trigger LAG-3-mediated immunosuppression of LAG-3-positive T cells under various intensities of TCR stimulation (Figure 4A). Furthermore, anti-LAG-3 antibodies were ineffective in either the absence or presence of MHC-II / noncognate peptide (Figure 4B). This unexpected result led us to determine that LAG-3 must be located close to the TCR-CD3 complex for its inhibitory function.

[0044] In known systems, MHC-II simultaneously binds to both the TCR-CD3 complex and LAG-3, maintaining close contact between LAG-3 and the TCR-CD3 complex. To examine the importance of TCR-CD3 proximity for LAG-3 function, we created an in vitro APC / T cell coculture system that either enforced or did not enforce proximity between the TCR-CD3 complex and LAG-3. We used a rapalog-induced heterodimer system to bridge the TCR-CD3 complex and LAG-3 by attaching the FRB domain of mTOR to MHC II (covalently linked to a noncognate peptide) and FKBP12 to membrane-tethered anti-CD3ε (Figure 5A). Incubation with a small, non-immunosuppressive rapalog forms tight interactions with both FRB and FKBP12, hypothetically enforcing proximity between MHC-II and membrane-tethered anti-CD3ε, as well as between LAG-3 and the TCR-CD3 complex. Experiments revealed that such heterodimers could trigger LAG-3-mediated inhibition (Fig. 5B), suggesting that LAG-3 indeed acts in close proximity to the TCR.

[0045] The present disclosure includes a unique LAG-3 antibody screening system, a mouse LAG-3 chimeric antigen receptor-like (CAR) NFκB-GFP reporter cell assay, to validate functional LAG-3 ligands and screen for LAG-3 antibodies with or without distinct ligand-blocking capabilities (Figure 6A). This system can be used, for example, to identify other bispecific binding partners that can be used in the described methods. To develop the screening system, the intracellular domain of CD28 / 41BB / CD3ζ was linked to the extracellular (ECD) and transmembrane (TM) domains of LAG-3, and the Jurkat We overexpressed the chimera on NFκB-GFP reporter cells and found that among all known LAG-3 ligands, only MHC-II and FGL1 (membrane-tethered or oligomeric) were able to trigger LAG-3-CAR activation (Figure 6B-C). Using the CAR reporter system, we identified several antibodies capable of functioning as the described bispecific antibodies. As a non-limiting example, data presented in this disclosure demonstrate that a hybridoma-generated anti-mouse LAG-3 antibody (M8) can block both MHC-II and FGL1-mediated signaling (Figures 7A-B). This domain 1 antibody binds to an epitope outside the typical loop region (aa 68-91) of LAG-3 and exhibited potent functionality in reversing LAG-3-mediated immunosuppression, superior to the C9B7W anti-mouse LAG-3 domain 2 antibody (Figures 7-8). Therefore, this disclosure also encompasses the design and use of other BiTS that tether the LAG-3 receptor to the TCR complex on the same T cell. This facilitates the access of LAG-3 to the TCR (in cis) upon antigen-induced TCR activation, priming the TCR complex for inhibition and enhancing LAG-3 access to the TCR. + Based on this M8 anti-mouse LAG-3 antibody clone (Figure 9), we developed an anti-mouse TCRβ (H57-597 clone, commercially available) / M8 bispecific single-chain fragment variable (ScFv) antibody (H57xM8) that induces the proximity of LAG-3 to the TCR-CD3 complex. We developed a novel fusion protein (BiTS) (Figure 10). BiTS inhibits CD4 and CD8 expression in a LAG-3-dependent manner We found that the activation of both T cells and T cells could be suppressed by the anti-mouse TCRβ (H57-597 clone) / anti-human LAG-3 (BMS relatolimab) BiTS (H57xRE BiTS) also express human LAG-3-positive CD4 or CD8 We also found that H57xM8 can potently suppress T cells (Figure 12). The results described support our approach of using anti-LAG-3 antagonists to enhance T cell activation by converting them into LAG-3 agonists in a BiTS format via fusion with an anti-TCR antibody (Figures 10-12). Furthermore, we used H57xM8 We demonstrate that BiTS inhibit the TCR-CD3 complex without affecting coreceptor signaling (Figure 14). When anti-human CD3 antibodies are used in the BiTS format, these BiTS activate rather than suppress T cell activity, suggesting the importance of the H57-597 epitope (Figures 15-16). Figure 15 uses a binding partner (not the LAG-3 binding partner) that binds to an epitope within the human CD3 complex. This binding partner is the OKT3 monoclonal antibody, which is known in the art and commercially available (e.g., BioXCell catalog #BE0001-2). Similarly, Figure 16 uses a different anti-CD3 antibody that binds to a different epitope than OKT3. This binding partner is the Ono Pharmaceutical Co., Ltd. anti-CD3 antibody described in US2022 / 0281977A1, the description of which is incorporated herein by reference.

[0046] The H57-597 antibody targets the FG loop within the TCRβ constant chain (Figure 17). Furthermore, anti-mouse TCRβ (H57-597 clone) / anti-human PD-1 (nivolumab) was found to be unable to suppress T cells, suggesting the importance of LAG-3-mediated proximity induction (Figure 18). The present disclosure also includes, for example, other available TCR-CD3 Anti-LAG-3 fused to ScFv antibodies (e.g., CD3ε and CD6, which have been suggested to be closely associated with the TCR-CD3 complex) BiTS, including ScFvs, are included. Generally, the present disclosure includes the use of anti-CD3ε antibodies that activate T cells weakly or do not activate T cells.

[0047] CD4 and CD8 in a LAG-3-dependent manner Anti-LAG-3 / TCR inhibits activation of both T cells Given the powerful functionality of BiTS, the present disclosure includes the use of the described bispecific binding partners to treat a wide range of autoimmune diseases, as further described below. As a non-limiting demonstration, in a mouse model of multiple sclerosis, MOG peptide-induced, CD4-driven experimental autoimmune encephalitis, H57xM8 BiTS was tested. The described BiTS significantly improved EAE disease symptoms even when administered at a low dose (1 mg / kg) five times daily (Figure 13). H57xM8 When BiTS were tested in an autoimmune diabetes model, the described BiTS was found to prevent the onset of diabetes (Figure 19). In an autoimmune hepatitis model induced by anti-41BB, H57xM8 BiTS was found to prevent the development of hepatitis (Figure 20). Furthermore, in this model, the described BiTS inhibited LAG-3 + T cells were not depleted (Figure 21). Compared with the BiTS mutant, H57xM8 BiTS more potently suppressed inflammatory cytokine production in the liver (Figure 22). These data demonstrate the feasibility of using the BiTS approach in T cell-mediated autoimmune diseases. LAG-3 is closely related to the pathogenesis of EAE, diabetes, and arthritis, and the soluble LAG-3 ligand FGL1 showed therapeutic effects on arthritis in mice. The described BiTS is also expected to have synergistic effects with existing therapeutic options, such as anti-BAFF blockers (for lupus) and CTLA-4-Ig (for arthritis).

[0048] While the bispecific binding partners of the present disclosure are described as such, the binding partners may be further modified, for example, to be trispecific and thus specifically bind to another target in addition to the TCR and LAG-3 components. Thus, bispecific binding partners include binding partners that bind to at least two different described targets. Thus, the components of the bispecific binding partners of the present disclosure may be provided as intact immunoglobulins or as immunoglobulin fragments, 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 (Db), 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 comprising heavy and light chain variable regions. As known in the art for previously described CARs, the scFv exists in a contiguous polypeptide that further comprises a CD3 zeta chain and a costimulatory domain. In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain or a CD28 costimulatory domain. The CAR may also comprise a co-receptor hinge sequence, such as a CD8a co-receptor hinge sequence.

[0049] In some embodiments, the binding partner of the present disclosure can include a constant region, e.g., an Fc region. Constant regions of any isotype can be included. However, the present disclosure includes the proviso that the approaches described herein do not require Fc / Fcr interactions.

[0050] In particular examples, the described bispecific binding partners can include a linking amino acid that connects a first binding moiety that specifically binds to a TCR component or other described protein in proximity to the TCR, and a second binding moiety that specifically binds to LAG-3. Suitable amino acid linkers can be composed primarily of relatively small, neutral amino acids (such as glycine, serine, and alanine) and can include multiple copies of glycine- and serine-rich sequences. In specific, non-limiting embodiments, the linker includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0051] Binding partners and pharmaceutical compositions comprising binding partners can be administered to individuals in need thereof using any suitable route, including, for example, intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, oral, topical, or inhalation, depending on the particular condition being treated. Compositions can be administered parenterally or enterally. Compositions can be administered as a single dose or multiple doses, or continuously over a period of time. For example, administration can be at a pre-specified number of doses, or daily, weekly, or monthly, and can be continuous or intermittent as therapeutically indicated.

[0052] The present disclosure includes binding partners for use in diagnostic and prophylactic approaches. For therapeutic approaches, in certain embodiments, the binding partner may be delivered as an mRNA or DNA polynucleotide encoding the binding partner. Administering DNA or RNA encoding any of the binding partners described herein is also considered a method of delivering such a binding partner to an individual or one or more cells. Methods for delivering DNA and RNA encoding proteins are known in the art and can be adapted to deliver binding partners with the benefit of the present disclosure. In several embodiments, one or more expression vectors are used, including viral vectors. Thus, in several embodiments, viral expression vectors are used. Viral expression vectors can be used as naked polynucleotides or can comprise any viral particle (including, but not limited to, defective interfering particles or other replication-defective viral constructs, and virus-like particles). In several embodiments, the expression vector comprises a modified viral polynucleotide derived from an adenovirus, herpesvirus, retrovirus, or the like.

[0053] In embodiments, the disclosure includes modified cells that have been modified to express the described binding partners. In embodiments, the modified cells are modified lymphocytes. In embodiments, the modified cells are T cells, natural killer cells, or macrophages. In embodiments, the modified cells are modified stem cells. In embodiments, the modified cells are totipotent stem cells, pluripotent stem cells, or multipotent stem cells. In embodiments, the described cells are used as therapeutic agents.

[0054] In embodiments, the individual in need of a composition of the present disclosure has been diagnosed with or is suspected of having an autoimmune disease. In 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, low-grade cancer, or inflammatory bowel disease. 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).

[0055] 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 is an amount that alleviates one or more signs or symptoms of the disease and / or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of the disease or the recurrence of the disease. The exact dosage can be selected by the individual physician in consideration of the patient being treated. Dosage and administration can be adjusted to provide a sufficient level of binding partner to maintain the desired effect. Additional factors that may be considered include the severity and type of disease state, the patient's age, weight, and sex, the desired duration of treatment, the method of administration, the time and frequency of administration, drug combinations, reaction sensitivities, and / or tolerance / response to treatment.

[0056] In some embodiments, the binding partner is produced by a host cell through a recombinant expression vector and cell culture. In some embodiments, the cell culture comprises prokaryotic 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.

[0057] Kits containing binding partners and / or cell cultures expressing the binding partners are provided by the present disclosure. Generally, the kits include one or more sealed containers containing the binding partners or cells expressing them. Instructions for using the binding partners for therapeutic and / or prophylactic purposes may be included in the kit.

[0058] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. It is intended that the specification and examples be considered as exemplary only.

Claims

1. a binding partner, a first binding moiety that specifically binds to a T cell receptor (TCR) component or to another protein in proximity to a TCR chain (where the TCR component is optionally a TCR β chain); and A second binding component that specifically binds to LAG-3 A binding partner having

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

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

4. A binding partner according to any one of claims 1 to 3, wherein the TCR component is a TCR β chain.

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

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

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

8. The method of claim 7, wherein the TCR chain is a TCR β chain.

9. The method of any one of claims 5 to 7, wherein 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, or eosinophilic steroids. Iglobulinemia, idiopathic inflammatory demyelinating diseases, thrombocytopenic purpura, myasthenia gravis, pernicious anemia, psoriasis, Sjögren's syndrome, ulcerative colitis, graft-versus-host disease (GVHD), or autoimmune diseases characterized by type II, III, or IV hypersensitivity reactions, polymyalgia rheumatica, Addison's disease, Behçet's disease, scleroderma (systemic sclerosis), autoimmune pancreatitis, autoimmune hemolytic anemia, hypoparathyroidism, Guillain-Barré syndrome, reactive arthritis, and sarcoidosis.

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

11. 10. The method of claim 9, wherein the severity of the autoimmune disease or 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 to 3.

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

14. The pharmaceutical composition of claim 13, wherein the TCR component is a TCR beta chain or a TCR-CD3 complex.

15. A modified cell modified to express the binding partner of any one of claims 1 to 3.

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

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

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

19. A polynucleotide encoding the binding partner of any one of claims 1 to 3.

20. 20. The polynucleotide of 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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