Constructs targeting BTNL3 / 8 for payload delivery to gastrointestinal system

Abstract

To provide protein constructs for topical drug delivery.SOLUTION: Provided herein is a protein construct comprising a BTNL3 / 8 targeting moiety, a payload and a linker linking the targeting component to the payload.SELECTED DRAWING: None

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 62 / 680,932, filed June 5, 2018. No. 6,399,413, which is incorporated herein by reference in its entirety.

[0002] (2. Sequence Listing) This application contains a Sequence Listing submitted via EFS-Web, the entirety of which is incorporated herein by reference. The ASCII copy was created on June 4, 2019 and bears the name "GDT-P2577PCT-sequence ce listing.txt" and is 17,284 bytes in size. [Background technology]

[0003] (3.Background) Most drugs rely on systemic exposure to achieve sufficient concentrations at the site of disease. However, exposure of non-target tissues to drugs often results in significant toxicity. Therefore, there is an urgent need to develop methods for delivering pharmaceutical agents to specific tissues.

[0004] Tissue-selective homing of T cells is a key factor during the integration of normal immune responses. Such “tissue-homing” or “tissue-resident” T cells are considered to be the basis of One very unique class is the γδ T cell. γδ T cells are highly compartmentalized T cells. For example, murine Vγ5 T cells are found only in the epidermis, whereas murine Vγ 7T cells show a subset localization to specific tissues, such as the intestine. The murine and human IEL compartments express butyrophilin (BTN) and brunhoflin (BBN) through mechanisms that remain unclear. Proteins expressed by epithelial cells at steady state, such as the thyrofilin-like (Btnl / BTNL) gene They depend on proteins for their development and survival. (Di Marco Barros et al., Cell. 2016; 167(1) : 203-218; Kabelitz et al., F1000 Faculty Rev: 782; June 5, 2017).

[0005] Butyrophilin and butyrophilin-like proteins (BTN / BTNL) play important roles in immune regulation, including T cell selection, and immunoglobulin superfamily members that influence developmental processes such as differentiation and cell fate determination. -family of family members (Arnett and Viney, Nature Reviews, 20 14(14)pp. 559-569). BTNL proteins (specifically, BTNL3 and BTNL8) are expressed in human intestinal enterocytes. Disproportionately highly expressed, BTNL3 / 8 can specifically regulate human Vγ4+γδ T cells (Di Marco Barros et al., Cell. 2016; 167(1): 203-218). BTN and BTN L proteins have been reported to regulate multiple T cell responses in a negative or positive manner. How such signals are transmitted to T cells is unclear (Kabelitz et al., 2003). F1000 Faculty Rev: 782; June 5, 2017).

[0006] There is a need for localized delivery of drugs to the gastrointestinal system for the treatment of diseases such as inflammatory bowel disease. Therefore, it is important to target therapeutic payloads to BTNL3 / 8 expressing cells, particularly those in the intestinal epithelium. There is a need for compounds that can target BTNL3 / 8 expressing cells, including compounds that can target BTNL3 / 8 expressing cells. Summary of the Invention

[0007] (4. Overview) Further investigation of these interesting (albeit poorly characterized) tissue / immune compartments is needed. To further explore the “shaping” and “homing” of γδ T cells, We attempted to explore the mechanism by which tissue targeting and specificity is achieved. A panel of telodimeric and homodimeric γδ T cell receptor proteins (TCRs) was developed using human γ chain and We attempted to genetically engineer the δ chain sequence starting from the beginning of the δ chain sequence. Various fusion partners were explored, and the resulting recombinant heterodimerized and homodimerized The γδ chains are expressed (e.g., in HEK293 cells), kept intact, and purified (size It was confirmed that almost no aggregation occurred (measured by exclusion chromatography). These experiments demonstrated that (i) the use of antibody Fc fusion domain partners and (ii) engineered leucine zippers and Some more preferred fusions include the use of combined TCR α-β chain constant domain partners. (Xu et al., PNAS, 2011, Vol. 108; pp. 2414-2419). If these successful experiments were not exhaustive, the art would now be able to identify the homodimers of recombinant γδ. Identify alternative approaches to generate dimers, heterodimers, and monomeric subunits. They can recognize that there will be incentives to do so.

[0008] Create a panel or library of T cell receptor recombinant proteins derived from this human γδ sequence This can then be used to determine which (if any) recombinant γδ pairs are either tissue-specific or Therefore, we investigated whether these proteins could be selectively expressed in cells overexpressing BTNL3 / 8. Unprecedentedly, in a controlled study, the BTNL3 / 8 protein was Certain recombinant T cell receptors that specifically recognize the protein (e.g., recombinant γ4δ1 TCR and recombinant While identifying recombinant γ4δ2 TCRs, it also identified other receptors that it did not recognize (e.g., recombinant γ2δ1 TCRs and This is the first time that γ4 containing γδ TCRs are expressed on the cell surface. These results indicate that it directly interacts with expressed BTNL3 / 8.

[0009] Furthermore, TCR deep sequencing revealed that within the fraction that was BTNL3 / 8 responsive, γ4 TCR expression The results showed that there was selective enrichment of γδ T cells that expressed the α-terminal β-blocking factor 1 (β-blocking factor 1) and γδ T cells that expressed the β-blocking factor 1 (β-blocking factor 1) in the thymic thymic . Recombinant TCR experiments demonstrated that TCR pairings containing the γ4 subunit selectively bind to BTNL3 / 8. It was shown that.

[0010] Thus, in a first aspect, there is provided a protein construct comprising: A BTNL3 / 8 targeting moiety, a payload, and a method for attaching the targeting moiety to the payload. The linker may be any linker.

[0011] In one embodiment, the BTNL3 / 8 targeting moiety comprises a Vγ domain, and the sequence of the Vγ domain The amino acid at position 87 is aspartic acid or histidine, and is the Vγ domain position The amino acid at position 90 is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are At each position, a residue is independently selected from the corresponding residue in the human or murine Vγ domain. do.

[0012] In one embodiment, the remaining residues of the Vγ domain CDR4 are, at each residue position, a human Vγ In one embodiment, the corresponding residues in Vγ4, human Vγ2, or mouse Vγ7 are independently selected. The amino acid sequence of positions 87 to 90 of the γ domain is SEQ ID NO: 1. The amino acid sequence of positions 87 to 90 of the Vγ domain is SEQ ID NO:2. The remaining residues in the Vγ domain CDR4 all correspond to the corresponding residues in human Vγ4, human Vγ2, or mouse Vγ7. In one embodiment, the remaining residues of the Vγ CDR4 are selected from the corresponding residues in human Vγ4. In one embodiment, the remaining residues of Vγ CDR4 are selected from the corresponding residues in human Vγ2. In one embodiment, the remaining residues of Vγ CDR4 are selected from the corresponding residues of mouse Vγ7. In one embodiment, the Vγ domain is a human Vγ2 domain, in which CDR4 is The amino acid is substituted with aspartic acid or histidine at amino acid position 87, In one embodiment, the amino acid at position 90 of the amino acid sequence is substituted with glycine or glutamic acid. The Vγ domain is a human Vγ4 domain.

[0013] In one embodiment, the Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence. In an embodiment, the Vγ domain CDR3 comprises a human Vγ4 CDR3 sequence. In one embodiment, the Vγ domain CDR3 comprises a human Vγ2 CDR3 sequence. In one embodiment, the J region comprises a mouse Vγ7 CDR3 sequence. In one embodiment, the J region is a Vγ J region. In one embodiment, the J region is a mouse Vγ J region. In one embodiment, the J region is selected from the group consisting of SEQ ID NOs: 15-18. The sequence includes a sequence selected from the group consisting of:

[0014] In one embodiment, the BTNL3 / 8 targeting component of the protein construct further comprises a pair of In one embodiment, the Vγ domain and the Vδ domain comprise at least one In one embodiment, the Vγ domain and the V The δ domains are paired through specific heterodimeric interactions. In one embodiment, the heterodimeric interaction is leucine zipper complementarity. In one embodiment, the targeting moiety comprises SEQ ID NO:9. In one embodiment, the targeting moiety comprises SEQ ID NO:1. 0. In one embodiment, the targeting moiety is SEQ ID NO: 11. In one embodiment, The targeting moiety comprises a single chain in-frame fusion of a Vγ domain and a Vδ domain. In an embodiment, the Vγ domain is N-terminal to the Vδ domain. In one embodiment, the Vγ domain and the Vδ domain are C-terminal. The single chain in-frame fusion comprises an internal linker sequence. In one embodiment, the Vδ domain In one embodiment, the human V5 domain is V51, V52 or V55. In one embodiment, the human V5 domain is V51.

[0015] In one embodiment, the protein construct further comprises a first T cell receptor constant region, The first T cell receptor constant region is fused in-frame to the C-terminus of the Vγ domain. In one embodiment, the first T cell receptor constant region is a human T cell receptor constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor beta constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor alpha constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor gamma constant region. The targeting component further comprises a second T cell receptor constant region, In one embodiment, the Vδ domain is fused in-frame to the C-terminus of the paired Vδ domain. In one embodiment, the second T cell receptor constant region is a human T cell receptor alpha constant region. The second T cell receptor constant region is a human T cell receptor β constant region. The two T cell receptor constant regions are human T cell receptor δ constant regions. In one embodiment, the Vδ In-frame fusion of the Vδ domain with a second T cell receptor constant region results in a Vδ domain and a second T cell receptor constant region. It contains an internal linker sequence between the receptor constant region.

[0016] In one embodiment, the payload is a protein fused in-frame to the targeting moiety. In one embodiment, the payload is a polypeptide. In one embodiment, the payload is a peptide. In one embodiment, the payload is a cytokine. In one embodiment, the payload is an antibody. In one embodiment, the payload is a In one embodiment, the antibody is a single chain variable fragment (scFv) specific for a cytokine antigen. In one embodiment, the antibody is specific for the CD3 antigen. In one embodiment, the antibody comprises at least one antigen binding site (ABS) that is specific for tumor necrosis factor. In one embodiment, the antibody comprises at least one ABS specific for tumor necrosis factor alpha (TNFα) antigen. In one embodiment, the antibody comprises an Fc domain capable of interacting with an Fc receptor. In one embodiment, the payload is a small molecule. In one embodiment, the payload is a hormone. In one embodiment, the payload is a nucleic acid. In one embodiment, the payload is an inhibitory RNA (RNAi).

[0017] In one embodiment, the optional linker comprises a peptide fused in-frame to the targeting moiety. In one embodiment, the optional linker is in-frame to the C-terminus of the targeting moiety. In one embodiment, the optional linker is fused to the N-terminus of the targeting moiety. In one embodiment, the optional linker is a conjugate that is fused in frame to the targeting moiety. It is a jugate molecule.

[0018] In one aspect, described herein is any of the above protein constructs and In one embodiment, the pharmaceutical composition comprises a pharma- ceutical carrier. The composition is suitable for parenteral administration. In one embodiment, the administration is intravenous. In one embodiment, the administration is intramuscular. In one embodiment, the administration is subcutaneous.

[0019] In one aspect, described herein is a digestive tract assay in which the digestive tract tissue expresses BTNL3 / 8. 67. A method for treating a vascular condition comprising administering to a patient a therapeutically effective amount of the compound according to any one of claims 62 to 66. administering the pharmaceutical composition to a patient with a condition in which gastrointestinal tissue expresses BTNL3 / 8. In one embodiment of this method, the payload of the protein construct is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an anti-inflammatory agent. The anti-inflammatory agent is a steroid. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is prednisone. The glucocorticoid is hydrocortisone. In one embodiment, the payload is an immunomodulatory It is a money saver.

[0020] In one aspect, described herein is a method for treating a chronic inflammatory bowel disease, comprising administering to a patient a therapeutically effective amount of any of the above-described A method for treating inflammatory bowel disease comprising administering the pharmaceutical composition to a patient suffering from inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis. The disease is Crohn's disease. In one embodiment, the payload of the protein construct is an anti-inflammatory In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. tokine, optionally interleukin 10 (IL-10), interleukin 22 (IL-22) or trans In one embodiment, the anti-inflammatory payload is anti-transforming growth factor beta (TGFβ). In one embodiment, the anti-inflammatory payload is a steroid. In an embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is hydrocortisone. In one embodiment, the payload of the protein construct is an antibiotic. In an embodiment, the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, In one embodiment, the protein is dazole, moxifloxacin or amoxicillin. The payload of the polypeptide construct is a calcineurin inhibitor. The phosphorylation inhibitor is cyclosporine A or tacrolimus. The payload of the polypeptide construct is an immunomodulatory agent. In one embodiment, the immunomodulatory agent is an immunosuppressant. In one embodiment, the immunosuppressant is azathioprine, 6-mercaptopurine, methadone, In one embodiment, the payload of the protein construct is In one embodiment, the protein payload is an antibody. , an antibody fragment or a single chain variable fragment. In one embodiment, the protein payload is TN The antibody is specific for the Fα antigen and comprises at least one ABS. In embodiments, the protein payload is adalimumab, infliximab, or certolizumab. In one embodiment, the protein payload comprises the complementarity determining regions (CDRs) of the interleukin. In one embodiment, the antibody comprises at least one ABS specific for an interleukin antigen. The inflammatory cytokine is IL-12, IL-23, or a combination thereof. The polypeptide payload comprises the CDRs of ustekinumab or brikinumab. In an embodiment, the biologic payload comprises at least one antibody specific for an integrin antigen. In one embodiment, the integrin is an α4 integrin. In embodiments, the protein payload is infliximab, natalizumab or vedolizumab. In one embodiment, the protein construct comprises an analgesic payload. In one embodiment, the protein construct comprises a nutraceutical payload.

[0021] In certain aspects, described herein is a therapeutically effective amount of any of the pharmaceutical agents described above. A method for treating irritable bowel syndrome comprising administering the composition to a patient suffering from irritable bowel syndrome. In some embodiments, described herein is a therapeutically effective amount of any of the above-mentioned pharmaceutical compositions. A method for treating diverticulitis comprising administering the pharmaceutical composition to a patient suffering from diverticulitis. In one embodiment, the payload is an antibiotic. Ciprofloxacin, metronidazole, moxifloxacin or amoxicillin It's Rin.

[0022] In certain aspects, described herein is a therapeutically effective amount of any of the pharmaceutical agents described above. A method for treating celiac disease comprising administering the composition to a patient suffering from celiac disease. In certain embodiments, the payload is an immunosuppressant. The thiopurine is zathioprine, 6-mercaptopurine, methotrexate or thiopurine.

[0023] In certain aspects, described herein is a therapeutically effective amount of any of the pharmaceutical agents described above. A method of treating a microbial infection comprising administering the composition to a patient suffering from a microbial infection. In some embodiments, the payload is an antibacterial agent. In some embodiments, the antibacterial agent is an antiparasitic agent. The agent is an antimicrobial, an antibiotic, an antifungal agent or an antiviral agent.

[0024] In certain aspects, described herein is a therapeutically effective amount of any of the pharmaceutical agents described above. A method for treating a metabolic disorder or deficiency comprising administering the composition to a patient suffering from a metabolic disorder or deficiency. In one embodiment, the payload is a dietary supplement. In an embodiment, the dietary supplement is an enzyme or a vitamin.

[0025] In certain aspects, described herein is a therapeutically effective amount of any of the pharmaceutical agents described above. A method of regulating the immune system comprising administering the composition to a patient suffering from an immune-related condition. In an embodiment, the payload is an immunosuppressant. In one embodiment, the immunosuppressant is an azathioprine. In one embodiment, the thiopurine is 6-mercaptopurine, methotrexate or thiopurine. In one embodiment, the payload is an immunostimulant. In one embodiment, the immunostimulant is a cytokine. It is. [Brief description of the drawings]

[0026] 5. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows an amino acid sequence alignment of human Vγ4 (hVγ4), human Vγ2 (hVγ2) and mouse Vγ7 (mVγ7) variable (V) domains. The numbering of amino acid positions shown herein is based on the complete T cell receptor (TCR) protein sequence, including the 18 amino acid leader sequence (not shown). The dashes at positions 46 and 117 in the alignment indicate gaps in the mVγ7 sequence that were introduced to optimize the alignment. The CDR regions are shown in bold font. CDR1 is located at amino acid positions 45-50 in the alignment. CDR2 is located at amino acid positions 68-75 in the alignment. The first five amino acids of CDR3 are located at amino acid positions 114-118 in the alignment. CDR4 is located at amino acid positions 85-100 in the alignment. The underlined amino acids in human Vγ4-CDR4 at alignment positions 87 and 90, when replaced with the corresponding amino acids in hVγ2, abolish function (see FIG. 7).

[0027] [Diagram 2]We present the experimental approach used to identify, clone and test γδ T cell receptors isolated from responding intraepithelial lymphocytes (IELs).

[0028] [Diagram 3] FIG. 3A is a diagram of the lentiviral vector backbone used to express isolated IEL-derived γδ TCR variable domains in a TCR construct in TCR-deficient Jurkat cells.

[0029] FIG. 3B shows the cloned C γδ TCR variable domain in TCR-deficient Jurkat (J76 cells). Expression 72 hours after transduction of TCR constructs with DR3 pairs is shown.

[0030] [Figure 4] Figure 4A shows an example representation of BTNL3 / 8-induced responses of Vy4V51-transduced J76 cells. A positive control with anti-CD3 stimulation is also shown (vs. isotype control).

[0031] Figure 4B shows that three independent Vγ4Vδ1-transduced J76 lines differ from Vγ9Vδ2 lines in that The results show that B3, C11, and H7 responded to BTNL3 / 8 expressing cells. Each of the CDR3 pairs represents a different CDR3 pair.

[0032] [Diagram 5]Fold change (FC) in % CD69 expression in transduced cells (+ve cells) normalized to cells expressing empty vector (EV) and percent TCR downregulation in J76 cells expressing Vγ4 or Vγ2 TCR are shown. TCR activation by BTNL3 / 8 expressing cells was lost when the entire V domain of the responding Vγ4 H7 TCR was replaced with Vγ2 coding sequence (Vγ2H7) (but not CDR3γ and the entire delta chain). However, TCR activation by BTNL3 / 8 expressing cells was maintained when CDR1 (H7 CDR1Vγ2) and / or CDR2 (H7 CDR2Vγ2) of the responding Vγ4 H7 TCR were replaced with Vγ2 coding sequence.

[0033] [Figure 6] Figure 6A shows a partial V domain sequence alignment of human Vγ2 and human Vγ4. CDR1, CDR2 and CDR3 are shown in shaded boxes. A total of nine (9) amino acids differ. Four of the differing amino acids are located within framework region 3 between CDR2 and CDR4, defined herein as "CDR4."

[0034] FIG. 6B shows the Vγ4 / Vδ1 paired variable domains and Vγ5 / Vδ1 aligned using Cn3D. The previously published structures of paired variable domains are shown. The CDR4 region is distinct from the conventional CDRs. Of particular note is the CDR4 loop of Vγ4 and the CDR4 loop of Vγ5. The difference between the two groups is that they show significant differences in three-dimensional structure.

[0035] [Figure 7]Fold change (FC) in % CD69 expression in transduced cells (+ve cells) normalized to cells expressing empty vector (EV) and percent TCR downregulation in J76 cells expressing Vγ4TCR (H7 WT), Vγ2TCR with H7 CDR3 (Vγ2 H7), and Vγ4TCR with amino acid substitutions in CDR4 are shown. YA substitutions at amino acid positions 87 and 90 abolished TCR activation by BTNL3 / 8 expressing cells, whereas NL substitutions at amino acid positions 94 and 98 did not abolish TCR activation by BTNL3 / 8 expressing cells.

[0036] [Figure 8] Figure 8 is a schematic diagram of the design of the polypeptide chains of a soluble heterodimeric γδ TCR in an embodiment of the invention, where the two chains heterodimerize via leucine zipper complementation. The Vγ or Vδ domains are fused in frame to TCR α or TCR β constant regions, respectively, lacking the transmembrane domain, followed by a leucine zipper sequence and a histidine tag / linker. The Vγ and Vδ containing polypeptides were expressed and post-translationally dimerized.

[0037] [Figure 9] FIG. 9A shows flow cytometry results of HEK293T cells transduced with BTNL3 and BTNL8 constructs or empty vector after staining with soluble His-tagged Vγ4δ2 TCR and APC anti-His tag antibody.

[0038] FIG. 9B shows the BTNL3 and BTNL8 constructs or the empty vector after parallel staining with anti-FLAG and anti-HA antibodies. Flow cytometry results of HEK293T cells transduced with - are shown.

[0039] [Figure 10]Figure 10 shows staining of soluble TCRs constructed as described in Figure 8. Vy4V51 and Vy4V52 soluble TCRs show strong binding to cell lines expressing BTNL3+BTNL8 but not the empty vector (EV) control cell line.

[0040] [Figure 11] FIG. 11A is a schematic diagram showing BTNL3+BTNL8 expressing cells incubated with soluble His-tagged TCR and anti-His tag antibody at 4° C.

[0041] FIG. 11B shows BT cells incubated with soluble His-tagged TCR and anti-His-tag antibody at 37° C. FIG. 1 is a schematic diagram showing NL3+BTNL8 expressing cells.

[0042] [Figure 12] Time course of internalization of soluble TCR by BTNL3+BTNL8 expressing cells at 37° C.

[0043] [Figure 13] FIG. 13A shows a comparison of anti-BTNL3 antibody and soluble TCR on staining cells expressing BTNL3 and BTNL8 constructs.

[0044] FIG. 13B shows the fluorescence of cells incubated with anti-BTNL3 antibody compared to soluble TCR. Indicates a decrease.

[0045] [Figure 14] FIG. 1 is a schematic showing a method for assessing payload delivery by soluble TCRs.

[0046] [Figure 15]Figure 15A shows the results of an experiment assessing the internalization of soluble TCR+α-His antibody complexes in cells expressing BTNL3 and BTNL8 constructs, and Figure 15B is a graph showing the remaining fluorescent signal after trypsin treatment in cells incubated with soluble TCR+α-His antibody complexes at 4° C. and 37° C.

[0047] [Figure 16] FIG. 16A shows imaging cytometry results of DMEM-treated 293T.L3RIL8 cells incubated with soluble TCR+α-His antibody complexes at 4° C. and 37° C.

[0048] FIG. 16B shows the results of incubation with soluble TCR+α-His antibody complexes at 4° C. and then in DMEM or Tris 4 shows the results of imaging cytometry of psin-treated 293T.L3L8 cells.

[0049] FIG. 16C shows the results of incubation with soluble TCR+α-His antibody complexes at 37° C. and then in DMEM or TMEM. 4 shows the results of imaging cytometry of liposin-treated 293T.L3L8 cells.

[0050] The above drawings depict various embodiments of the present invention for illustrative purposes only. From the discussion, it is understood that the structures shown herein may be modified without departing from the principles of the invention described herein. It will be readily appreciated that alternative embodiments of the structures and methods may be employed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] 6. Detailed Description (6.1.Definition) Unless otherwise specified, all technical and scientific terms used herein are understood to be within the meaning of the present invention. The following terms have the meanings commonly understood by those of ordinary skill in the art: Words have the meanings set forth below.

[0052] As used herein, a "protein construct" refers to a protein comprising at least two functional elements, B Shown is one or more polypeptide chains comprising a TNL3 / 8 targeting moiety and a payload. The payload may or may not be a protein payload.

[0053] As used herein, "BTNL3 / 8" refers to butyrophilin protein 3 (BTNL3) and butyrophilin protein 8 (BTNL9). "BTNL3 / 8" refers to BTNL3 without BTNL8, BTN It may also refer to BTNL8 without L3, or a heterodimer of BTNL3 and BTNL8.

[0054] As used herein, a "BTNL3 / 8 targeting moiety" refers to a moiety that specifically binds to BTNL3 / 8. In one embodiment, the BTNL3 / 8 targeting moiety is an antigen binding protein. In one embodiment, the BTNL3 / 8 targeting moiety comprises at least one Vγ domain polypeptide. is also a part of it.

[0055] As used herein, "Vγ domain" refers to the variable domain of the T cell receptor (TCR) γ chain. The Vγ domain contains the J region and the complementarity determining regions (CDRs), CDR1, CDR2, CDR3, and CDR4. The numbering of Vγ domain residues is shown in FIG. 1, where residue 19 is the residue that is generated after signal sequence cleavage. For Vγ domains not shown in FIG. The numbering of the Vγ domains is assigned after best alignment to the sequences in FIG. A "corresponding residue" is an amino acid at the same numbered position as the residue it is referred to as corresponding to. .

[0056] As used herein, a "Vγ domain CDR4" refers to a Vγ domain located between the CDR2 and CDR3 regions. This refers to a continuous 16 amino acid portion of the γ domain, which corresponds to amino acid positions 85 to 100 in Figure 1. The amino acid sequence of CDR4 of the human Vγ4 domain has the sequence of SEQ ID NO:3. The amino acid sequence of the main CDR4 is SEQ ID NO: 5. The acid sequence is SEQ ID NO:4.

[0001] As used herein, a "payload" refers to a compound that is capable of delivering a payload to a target cell of interest (e.g., a compound that contains BTNL3 / 8). The payload refers to any molecule that is delivered to a target cell (cells expressing the target cell and / or intestinal epithelial cells). , nucleotides, nucleotides (e.g., containing detectable moieties or toxins or capable of inhibiting transcription) Inhibitory nucleotides), nucleic acids such as DNA and RNA (e.g., mRNA, RNAi, miRNA, siRNA, snRN A, snoRNA, piRNA, exRNA, scaRNA, and lncRNA), amino acids (e.g., detectable moieties or contains toxins or inhibits translation), polypeptides (e.g., enzymes, biologics) , lipids, carbohydrates, small molecules (e.g., small molecule drugs and small molecule toxins), and combinations thereof. In some embodiments, the payload is a therapeutic agent. The therapeutic agent may include: Non-limiting examples include chemotherapeutic agents, imaging agents (e.g., radioisotopes), immunomodulatory agents, (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., , cytotoxic agents). In some embodiments, the payload is an antibody.

[0057] As used herein, a "linker" refers to a functional element of a protein construct, e.g. , targeting moiety and payload). In some embodiments, the linker is a functional element of the protein construct (e.g., a targeting To allow site-specific conjugation of molecules to the In some embodiments, the linker can be used to synthesize the protein construct in vitro or in vivo. It can be used to identify or detect

[0058] As used herein, a "peptide linker" refers to a linker that is a polypeptide. In some embodiments, the peptide linker is a peptide linker that is linked to a protein construct (e.g., a targeting moiety and In one embodiment, the payload is fused in-frame to a functional element of the payload. The peptide linker allows for site-specific conjugation of molecules to elements of a protein construct. The peptide linker allows the functional element to which it is fused. Any length and variety of amino acid sequences may be used, provided that they allow the desired conformation of the stomach.

[0059] As used herein, an "internal linker" refers to a linker that is at least one amino acid at both its N-terminus and C-terminus. 1 shows a polypeptide sequence covalently linked to two additional polypeptides. The internal linker is an internal linker between a targeting moiety (e.g., an internal linker between a Vγ domain and a Vδ domain). Internal linkers are polypeptide chains within a polypeptide chain. Internal linkers can be of any length and type of amino acid sequence. Alternatively, the internal linker may be fused to an additional polypeptide, which may be fused to the additional polypeptide in the desired conformation. It is possible to maintain the structure.

[0060] As used herein, an "antibody" refers to at least one antibody that contains at least one antigen-binding site (ABS). The antibody may comprise any antibody protein construct comprising at least one antibody variable domain. These include, but are not limited to, variable domain only molecules, single chain variable fragments (scFv), single chain Fab fragments (scFab), bispecific antibodies, hybrid IgG, Fab fusion proteins, Fc-modified IgG, Adjunct IgG, diabodies, single chain diabodies, DART, tandem diabodies (tandAb) and Includes mini body.

[0061] As used herein, "single-chain in-frame fusion" refers to a single-chain in-frame fusion T cell Receptor variable domain (scTv) is a polypeptide variable domain of at least two polypeptides of a T cell receptor. At least a portion of the polypeptide is produced as a single fusion polypeptide chain, the variable domains of which are The sequences are fused in frame. scTv) can contain more than one variable domain and / or constant region and can be derived from T cells of any origin. Thus, scTvs contain two or more in-frame variable domains that may pair with a cell receptor. It contains tandem scTvs that are fused together.

[0062] As used herein, an "antigen-binding site" (ABS) is a site that specifically binds a given antigen or epitope. ABS refers to the region of an antibody molecule that recognizes and binds to a specific antigen or epitope. The term "affinity" as used herein refers to the ability of a molecule to bind with a specific affinity. It indicates the strength of the non-covalent intermolecular forces between the molecules of a molecule. The dissociation equilibrium constant (K D ) and low K D The value indicates that the interaction between molecules is strong. The K of the antibody construct is shown. DValues ​​may be measured by methods well known in the art and are limited to the following: Although not a standard technique, biolayer interferometry (e.g. Octet / FORTEBIO®), surface plasma These include spectral resonance (SPR) techniques (e.g., Biacore®), and cell binding assays. The affinity between the cognate antigen or epitope is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M , or 10 -10 K less than M D It has a value.

[0063] As used herein, "small molecule" refers to a molecule having a low molecular weight of less than 900 Daltons. Small molecules have the potential to modulate biological processes and are The organic molecule may have the size of a .alpha.-tocopherol.

[0064] As used herein, an "anti-inflammatory cytokine" refers to any cytokine that has anti-inflammatory activity. Anti-inflammatory cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-2 (IL-2), and inflammatory cytokines. Ikin (IL) IL-1ra, IL-4, IL-6, IL-10, IL-11, IL-13, and transforming proliferation Contains transforming growth factor beta (TGFβ).

[0065] As used herein, an "anti-inflammatory agent" refers to an agent that inhibits the activity or expression of proinflammatory cytokines. Anti-inflammatory agents refer to any molecule or agent that inhibits the production of proinflammatory cytokines. These include, but are not limited to, interleukin-1 (IL-1), IL-12, I L-18, tumor necrosis factor alpha (TNFα), interferon gamma (INF-γ) and granulocyte-macrophage Colony stimulating factors. Anti-inflammatory agents include soluble tumor necrosis factor receptor p55, soluble tumor necrosis factor receptor p55, Death factor, p75, soluble IL-1 receptor type II, IL-18 binding protein, etc., which may have anti-inflammatory activity. Anti-inflammatory agents also include soluble cytokine receptors, such as membrane-bound IL-1 receptor type II, which are involved in inflammation-inducing cytokine responses. Also included are cytokine receptors that lack intracellular signaling that competes with the sex cytokine receptor.

[0066] As used herein, an "immunomodulatory agent" refers to an agent that modifies the immune response and / or activity of cells of the immune system. In one embodiment, the immunomodulatory agent refers to any molecule that inhibits or inhibits the function of the immune system. An "immunosuppressant" is an agent that reduces the immune response and / or activity of cells of the immune system. "Immunosuppressant" refers to any molecule or agent that inhibits the immune response and / or the cells of the immune system. or any molecule or agent that increases activity.

[0067] As used herein, an "immune-related condition" refers to any condition associated with altered activity of the immune system. Immune-related conditions also refer to conditions or disorders associated with an increased or decreased immune response. Immune-related conditions include, but are not limited to, autoimmune diseases, inflammatory conditions, allergies, and These include immune reactions, immunodeficiencies, hematopoietic cancers and other hematopoietic disorders.

[0068] As used herein, an "inflammatory condition" refers to any condition associated with increased or present inflammation in inflamed tissue. Inflammatory conditions include, but are not limited to, asthma, atherosclerosis, and other conditions or disorders. Atherosclerosis, autoimmune diseases, autoinflammatory diseases, cancer, celiac disease, chronic prostatitis, Colitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, flatulence Lichen planus, mast cell activation syndrome, mastocytosis, otitis, pelvic inflammatory disease, reperfusion injury, Includes rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, transplant rejection and vasculitis.

[0069] As used herein, a "gastrointestinal condition" refers to any condition associated with any tissue of the gastrointestinal system. Gastrointestinal symptoms include, but are not limited to, the following: immune-related symptoms, inflammatory symptoms of the gastrointestinal system, microbial infection of the gastrointestinal tissue, and feeding disorders, metabolic Gastrointestinal symptoms include symptoms caused by sexual disorders or metabolic deficiencies. Although not commonly used, it is used in inflammatory bowel disease, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, and other conditions. and cancer (e.g., colon cancer, rectal cancer, gastric cancer).

[0070] As used herein, the term "treatment" or "therapy" refers to both therapeutic treatment and prophylactic or preventative treatment. The purpose of the present invention is to prevent and treat undesirable diseases such as multiple sclerosis, arthritis, or the progression of cancer. The objective of the study is to prevent or slow down (alleviate) unwanted physiological changes or disorders. Clinical outcomes may include, but are not limited to, detectable or undetectable: Alleviation of symptoms, reduction in extent of disease, stabilized (i.e., not worsening) disease state, disease progression "Treatment" includes slowing or slowing the progression of a disease, amelioration or remission of a disease, and relief (local or global). It also has the potential to prolong survival compared to expected survival without treatment. A person who needs treatment should be given the same treatment for that condition or disorder as a person who already has that condition or disorder. It also includes those who are susceptible to harm or those whose symptoms or disorders must be prevented.

[0071] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject. "Mammalian Subject" means a mammalian subject, particularly for whom diagnosis, prognosis, or therapy is sought. is for humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc. Other animals, including domestic animals, livestock, zoo animals, sports animals, or pets. nothing.

[0072] The term "sufficient amount" refers to an amount sufficient to produce a desired effect, e.g., an intracellular protein. By "adjusted dosage" is meant an amount sufficient to control lipid aggregation.

[0073] The term "therapeutically effective amount" refers to an amount effective to ameliorate symptoms of a disease. Prevention is synonymous with therapy. Thus, a therapeutically effective amount can also be a "prophylactically effective amount."

[0074] The term "recombinant human γδ TCR protein" or any derivative of this term is referred to throughout this specification. The expression can be achieved by standard genetic engineering of the human γδ TCR sequence or a functional derivative or homologue thereof. It refers to any recombinant protein produced by molecular engineering methodology. The protein can also include additional fusion elements, such as a dimerization domain. Non-limiting examples include fusion, transmembrane domains to support correct folding of the TCR. fusion to act as an interface (e.g., to support accurate presentation of the TCR at the surface of the cell membrane) to increase half-life or size (e.g., by fusion with human blood). albumin fusion domain) or a payload fusion as otherwise described herein. These additional fusion elements include sequences derived from γδ TCR sequences or their derivatives and homologs. The fusion sequence may be generated in tandem or alternatively may be non-TCR in origin.

[0075] The terms "recombinant γδ TCR sequence" or "recombinant human TCR live" referred to throughout this specification "recombinant human TCR panel" or "recombinant human TCR panel" or any derivative thereof means more than one A collection of recombinant human γδ TCR proteins, or 2, or 3, or 4, or A collection of 5 or more than 10 recombinant human γδ TCR proteins is presented. A collection may also include a collection of sequences that differ in at least one amino acid. This collection of recombinant TCRs can also be presented in a soluble form. For display purposes, the collection also includes inorganic or organic materials (non-limiting examples include Examples include "beads" or "plates" or "columns" or "phages." Alternatively, such collections may also be combined, fused, or joined together. on or in a membrane, such as that found in wounded or living cells, or of non-living membranes such as micelles The proteins may also be presented or displayed on a collection of membranes. When an expression vector is expressed and displayed on one or more living cells, it is usually A collection of homologous expression vectors is also generated. This collection of homologous expression vectors is used to or a plurality of cells are engineered to express the recombinant human γδ TCR protein or a label of the protein. The TCR protein is displayed in a library, panel, or collection. A library of expressing cells can be generated.

[0076] The terms "cognate binding partner" or "cognate binding partner candidate" or equivalents are used throughout this specification. Derived terms derived from these include those that bind to recombinant human γδ TCR protein in a sequence-specific manner. The discovery of such a cognate binding partner or a derivative thereof is thus indicated. For screening or validation, they may be grown in their natural environment (e.g., one or more cells). They can also be presented or displayed on the surface of such cells. as extracts or secretions from the cells, purified derivatives thereof, or in recombinant form; It can be presented or displayed.

[0077] (6.2. Other Interpretation Rules) Unless otherwise specified, all references to sequences herein refer to amino acid sequences.

[0078] Throughout this disclosure, the terms "comprises," "including," "equipped with," "containing", "having", "includes", "containing "Including," and linguistic variations thereof, have the meaning set forth in the U.S. Patent Act, including, but not limited to, the following: It permits the presence of additional elements beyond those explicitly recited.

[0079] Ranges provided herein are abbreviations for all values ​​within the range, inclusive of the recited endpoints. For example, the range 1 to 50 is understood to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. It is understood that the term includes any number, combination of numbers, or subrange from the group listed.

[0080] Unless otherwise stated or apparent from the context, the term "or" is used herein to refer to an inclusive or. Unless otherwise specified or clear from the context, as used herein, The terms "a," "an," and "the" are understood to be singular or plural. will be done.

[0081] Unless otherwise specified or clear from the context, the term "about" as used herein means any substance or mixture that is within the scope of the invention. This is understood to be within the normal tolerances in the field, for example, within two standard deviations of the mean. "Approximately" means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or In the present invention, the range of 0.1%, 0.05%, or 0.01% is understood to be within the range of 0.1%, 0.05%, or 0.01%. All numerical values ​​provided herein are modified by the term "about."

[0082] 6.3. Protein Constructs Containing BTNL3 / 8 Targeting Moieties In a first aspect, a protein construct is provided. The protein construct comprises BTNL3 / 8 A targeting moiety, a payload, and an optional linker that connects the targeting moiety to the payload. Including anchors.

[0083] 6.3.1.1 BTNL3 / 8-targeting components The BTNL3 / 8 targeting component specifically targets human BTNL3, human BTNL8 and / or human BTNL3 / 8. In one embodiment, the BTNL3 / 8 targeting component further comprises: At least a portion of a T cell receptor (TCR) Vγ domain polypeptide, as described in detail in In another embodiment, the BTNL3 / 8 targeting moiety is an antigen-binding site of an antibody. In one embodiment, the BTNL3 / 8 targeting moiety is a BTNL3 / 8 targeting moiety. When bound to BTNL3 / 8, it inhibits or partially inhibits the function of BTNL3 / 8. When a component that targets BTNL3 / 8 binds to BTNL3 / 8, it inhibits the function of BTNL3 / 8. In one embodiment, the BTNL3 / 8 targeting component stimulates or activates the function of the BTNL3 Binding of a component that targets / 8 to the BTNL3 / 8 heterodimer inhibits the BTNL3 / 8 heterodimer function. In one embodiment, the component that targets BTNL3 / 8 inhibits or partially inhibits the BTN Binding of L3 / 8-targeting components to the BTNL3 / 8 heterodimer inhibits the BTNL3 / 8 heterodimer mechanism. Stimulate or activate the ability.

[0084] 6.3.1. TCR gamma variable domains In one embodiment, the BTNL3 / 8 targeting moiety is a T cell receptor (TCR) Vγ domain polypeptide. In a typical embodiment, the BTNL3 / 8 targeting moiety comprises at least a portion of a Vγ peptide. Contains the main polypeptide.

[0085] In one embodiment, the BTNL3 / 8 targeting moiety comprises a CDR4 region from Vγ4. In an embodiment, the BTNL3 / 8 targeting component comprises a Vγ domain and a sequence position of the Vγ domain The amino acid at position 87 is aspartic acid or histidine, and the amino acid at position 87 is The amino acid at number 90 is glycine or glutamic acid, and the remaining residues in Vγ CDR4 are are independently selected from the corresponding residues in the human or murine Vγ domains.

[0086] In one embodiment, the remaining residues of the Vγ domain CDR4 are, at each position, human Vγ4, In some embodiments, the corresponding residues are selected from human Vγ2 or mouse Vγ7. The remaining residues in the Vγ domain CDR4 all correspond to the corresponding residues in human Vγ4, human Vγ2, or mouse Vγ7. In one embodiment, all remaining residues of the Vγ CDR4 are selected from the corresponding residues of human Vγ4. In one embodiment, all remaining residues of Vγ CDR4 are selected from the corresponding residues of human Vγ2. In one embodiment, all remaining residues of the Vγ CDR4 are selected from the corresponding Vγ7 residues. In one embodiment, the amino acid sequence of positions 87 to 90 of the Vγ domain is selected from In one embodiment, the amino acid sequence of positions 87 to 90 of the Vγ domain is SEQ ID NO: 1. The column is SEQ ID NO:2.

[0087] In one embodiment, the Vγ domain is a Vγ domain sequence shown in Figure 1. In one embodiment, the Vγ domain is a human Vγ domain. In one embodiment, the Vγ domain is a human Vγ2 domain, In the CDR4, the amino acid at position 87 of the amino acid sequence is substituted with aspartic acid or histidine. and is substituted at amino acid position 90 with glycine or glutamic acid.

[0088] In certain embodiments, the Vγ domain is human Vγ3 or human Vγ5. the Vγ domain is human Vγ3 or human Vγ5, in which the amino acid of CDR4 is At amino acid position 87, a substitution with aspartic acid or histidine is made. At amino acid position 90, a substitution with In one embodiment, the Vγ domain is a human Vγ 4 is a human Vγ domain that shares at least 70% sequence identity with Vγ domain 1 of

[0089] In one embodiment, the Vγ domain is a Vγ domain that is a non-human mammalian Vγ domain. In one embodiment, the Vγ domain is a non-human Vγ4 domain having at least 70% identity to human Vγ4. Human mammalian Vγ domain sequences.

[0090] In some embodiments, the Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence. In certain embodiments, the Vγ domain CDR3 comprises a human CDR3 sequence. The gamma domain CDR3 comprises a human Vγ4 CDR3 sequence. In a particular embodiment, the Vγ domain CDR3 comprises , human Vγ2 CDR3 sequence. In one embodiment, the Vγ domain CDR3 comprises a non-human mammalian CDR3. In one embodiment, the Vγ domain CDR3 comprises a mouse Vγ7 CDR3 sequence.

[0091] In some embodiments, the J region is a Vγ J region. In certain embodiments, the J region is a human V In one embodiment, the J region is a mouse Vγ J region. The J region has a polypeptide sequence selected from the group consisting of SEQ ID NOs: 15-18.

[0092] (6.3.1.1.1 Pair with Vδ) In one embodiment, the BTNL3 / 8 targeting component of the protein construct further comprises a pair of In one embodiment, the protein construct does not include a Vδ domain. The component comprises a Vδ domain paired with at least one additional Vγ domain. In an embodiment, the BTNL3 / 8 targeting moiety is a Vγ4 homodimer. The BTNL3 / 8 targeting component of the protein construct further comprises paired Vδ domains. In one embodiment, the V5 domain is a human V5 domain. The domain is V51, V52, V53, V55, or V58. In one embodiment, the human V5 domain is The main Vδ domain is Vδ1. In one embodiment, the Vδ domain is a non-human mammalian Vδ domain. do.

[0093] ((a) Heterodimer format) In some embodiments, the Vδ domain is a heterodimer of the first and second polypeptide. One of the polypeptides contains a Vγ domain, and One of the polypeptides comprises a Vδ domain.

[0094] Heterodimeric interactions involve multiple polypeptides containing Vγ and Vδ domains. In a typical embodiment, the interactions may include covalent and / or non-covalent interactions between the In fact, Vγ and Vδ domains form homodimers rather than heterodimers. They are paired by orthogonal features that are difficult to distinguish.

[0095] In some embodiments, a polypeptide comprising a Vγ domain and a Vδ domain, , covalently linked by at least one engineered disulfide bridge. The disulfide bridges formed by the synthesis of the non-native disulfides are known to occur when two or more domains associate. providing a non-endogenous cysteine ​​amino acid in two or more domains so that cysteine ​​bonds can be formed; In some of these embodiments, the amino acid sequence is a sequence of at least one disulfide. The cross-links are designed into the Vγ and Vδ domains. Another disulfide bridge can be located in a variable region, such as in a constant region fused in frame with a variable region. It is designed within the domain outside the variable region.

[0096] In some embodiments, the heterodimeric interaction is leucine zipper complementarity. .

[0097] In one embodiment, one or more polypeptides of a paired Vγ / Vδ heterodimer further comprises In one embodiment, the first T cell receptor constant region comprises a pair In one embodiment, the first T cell is fused in-frame to the C-terminus of the Vγ domain. In one embodiment, the first T cell receptor constant region is a human TCR constant region. In one embodiment, the first T cell receptor constant region is a human TCR β constant region. In one embodiment, the first T cell receptor constant region is a human TCR gamma constant region. In one embodiment, the paired Vγ / Vδ heterodimer polypeptide further comprises: a second T cell receptor constant region that is associated with a paired Vδ domain; In one embodiment, the second T cell receptor constant region is fused in-frame to the C-terminus of the second T cell receptor constant region. is a human TCR alpha constant region. In one embodiment, the second T cell receptor constant region is a human TCR beta constant region. In one embodiment, the second T cell receptor constant region is a human TCR delta constant region. do.

[0098] In some embodiments, the in-frame fusion of the Vγ domain with the first constant region is In some embodiments, the internal linker sequence between the domain and the first TCR constant region. In-frame fusion of the Vδ domain with a second TCR constant region results in the Vδ domain and a second T cell receptor constant region. The antibody contains an internal linker sequence between the receptor constant region and the ribozyme constant region.

[0099] In one embodiment, the BTNL3 / 8 targeting moiety comprises SEQ ID NO:9. In one embodiment, the BTNL3 / 8 targeting moiety comprises SEQ ID NO: 10. The component comprises SEQ ID NO:11.

[0100] In one embodiment, the BTNL3 / 8 targeting moiety comprises more than one Vγ domain and / or Vδ domain. In one embodiment, the one or more Vγ domains and / or Vδ domains comprise In one embodiment, more than one Vγ domain and / or Vδ domain is multimerized. All or part of the T cell receptor constant region is fused in-frame. , wherein more than one multimerized Vγ domain and / or Vδ domain comprises one or more internal linkers .

[0101] In a further aspect there is provided a recombinant γδ TCR construct. , a recombinant γδ TCR protein comprising SEQ ID NO:9 (optionally without the C-terminal His tag) is provided. In another aspect, there is provided a recombinant γδ TCR protein comprising SEQ ID NO: 10. is provided a recombinant γδ TCR protein comprising SEQ ID NO: 11 (optionally without the C-terminal His tag). In another aspect, there is provided a recombinant γδ TCR protein comprising SEQ ID NO: 12. There is provided a recombinant γδ TCR protein comprising SEQ ID NO:13.

[0102] (b) Single-stranded in-frame fusion In one embodiment, the BTNL3 / 8 targeting moiety comprises a Vγ domain and a paired Vδ domain. In some embodiments, the Vγ domain comprises a Vδ domain. In some embodiments, the Vγ domain is N-terminal to the Vδ domain. In various embodiments, the Vγ domain and the Vδ domain are each independently C-terminal to each other. The genome fusion includes an internal linker sequence.

[0103] In some embodiments, the V5 domain is a human V5 domain. The human V5 domain is V51, V52 or V55. In one embodiment, the human V5 domain is In one embodiment, the single chain in-frame fusion further comprises at least one T cell receptor agonist, In one embodiment, the single chain in-frame fusion further comprises a first T cell receptor constant region. The first T cell receptor constant region is inflated to the C-terminus of the Vγ domain. In one embodiment, the first T cell receptor constant region is fused to a human T cell receptor constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor β constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor alpha constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor gamma constant region. In one embodiment, the single chain in-frame fusion further comprises a second T cell receptor constant region. The second T cell receptor constant region is inflated C-terminally to the paired Vδ domains. In one embodiment, the second T cell receptor constant region is fused to the human T cell receptor α In one embodiment, the second T cell receptor constant region is a human T cell receptor β constant region. In one embodiment, the second T cell receptor constant region is a human T cell receptor δ constant region. In one embodiment, the Vδ domain is an inflated region of the second T cell receptor constant region. The fusion comprises an internal linker sequence between the Vδ domain and the second T cell receptor constant region.

[0104] In one embodiment, the single chain in-frame fusion comprises more than one Vγ domain and / or Vδ domain. In one embodiment, the one or more Vγ domains and / or Vδ domains comprise In one embodiment, more than one multimerized Vγ domain and / or Vδ domain is In one embodiment, the domain comprises one or more internal linkers. and / or all or a portion of the Vδ domain is inflated into at least one T cell receptor constant region. It's a fusion of the two.

[0105] 6.3.2. Antibody-Based Targeting Moieties In one embodiment, the BTNL3 / 8 targeting moiety specifically targets human BTNL3, human BTNL8, and and / or human BTNL3 / 8 heterodimer. In one embodiment, the antibody is , full length antibody fragments or antibody formats including but not limited to Fab fragments. Single, Fv, scFv, tandem scFv, diabody, sc diabody, DART, tandem diabody tandAbs, minibodies, camelid VHHs, and other antibody fragments or phosphatase known to those skilled in the art. Exemplary antibody and antibody fragment formats include those described in Brinkmann et al., MABS, 2017, Vol. 9, No. 2, pp. 182-212, the entire teachings of which are incorporated herein by reference. will be incorporated into the subsection.

[0106] In one embodiment, the antibody comprises an Fc domain capable of interacting with an Fc receptor. In one embodiment, the antibody comprises an Fc domain that is incapable of interacting with an Fc receptor. has one or more genetically engineered mutations in the amino acid sequence of an antibody domain that is naturally The effector function associated with antibody binding to the antibody is reduced. Functions include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC, antibody-dependent cellular mediator). (also called cytotoxicity), complement fixation (e.g., C1q binding), antibody-dependent cell-mediated Cellular mechanisms resulting from Fc receptors binding to the Fc portion of antibodies, such as phagocytosis (ADCP) and opsonization. The genetically engineered mutations that reduce effector function are described in U.S. Patent Application Publication No. 2017 / 0 137530, Armour et al. (Eur. J. Immunol. 29(8)(1999)2613-2624), Shields et al. (J. Biol. Chem. 276(9)(2001)6591-6604), and the paper by Oganesyan et al. (Acta Crystallograp hica D64(2008)700-704), which are hereby incorporated by reference in their entireties. In a particular embodiment, the antibody binds the Fc portion of the ROR-binding molecule via the FcR receptor. It has one or more genetically engineered mutations in the amino acid sequence of the antibody domain that reduce binding. In some embodiments, the FcR receptor is an FcRγ receptor. The effector receptors are FcγRIIa and / or FcγRIIIA receptors. One or more of the engineered mutations that reduce function are mutations in the CH2 domain of the antibody.

[0107] (6.3.2.1 Payload) The protein construct comprises a payload.

[0108] In various embodiments, the payload comprises a nucleotide and further comprises a detectable moiety or a toxin. nucleotides that contain or inhibit transcription, DNA, mRNA that codes for polypeptides such as enzymes molecules, other RNA molecules (e.g., RNAi, miRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA A and lncRNA), nucleic acids, amino acids (e.g., containing detectable moieties or toxins, or translated (amino acids that inhibit The therapeutic agent can include small molecule drugs and small molecule toxins, as well as combinations thereof.

[0109] In some embodiments, the payload is a therapeutic agent, including but not limited to: However, chemotherapy drugs, immunomodulatory agents (e.g., cytokines, chemokines, or chemoreceptors) These include receptor inhibitors, hormones and toxins (e.g., cytotoxic agents).

[0110] In one embodiment, the payload is an antibody. In one embodiment, the antibody is directed against the CD3 antigen. In one embodiment, the antibody is a tumor-specific antibody. In one embodiment, the antibody comprises at least one ABS specific for the tumor necrosis factor alpha (TNFα) antigen. In one embodiment, the antibody comprises an Fc domain capable of interacting with an Fc receptor. It contains an Fc domain that is unable to interact with

[0111] In some embodiments, the payload is a hormone. In some embodiments, the payload is an antibacterial agent.

[0112] In some embodiments, the protein construct carries multiple payloads, which may be the same or different. include.

[0113] In particular embodiments, the payload is attached to the C-terminus of the BTNL3 / 8 targeting moiety. In another embodiment, the payload is attached to the N-terminus of the BTNL3 / 8 targeting moiety.

[0114] 6.3.1. Polypeptides In various embodiments, the payload is a polypeptide. A polypeptide is an in-frame fusion to a BTNL3 / 8 targeting moiety. In an embodiment, the payload is fused in-frame to the C-terminus of the BTNL3 / 8 targeting moiety. In particular embodiments, the payload is fused in-frame to the N-terminus of the BTNL3 / 8 targeting moiety. It fits together.

[0115] In some embodiments, the polypeptide payload is a cytokine. The payload may be interleukin 10 (IL-10), interleukin 22 (IL-22) or trans and anti-inflammatory cytokines such as transforming growth factor beta (TGFβ).

[0116] In some embodiments, the payload is an anti-inflammatory polypeptide. Anti-inflammatory polypeptides are inhibitors of one or more pro-inflammatory cytokines. In an embodiment, the anti-inflammatory polypeptide is one or more of interleukin-1 (IL-1), IL-6, I L-12, IL-18, tumor necrosis factor alpha (TNFα), interferon gamma (INF-γ) or granulocyte-macrophage In one embodiment, the anti-inflammatory polypeptide is an inhibitor of phage colony stimulating factor. The receptors are soluble tumor necrosis factor receptor p55, soluble tumor necrosis factor receptor p75, and soluble IL-1 receptor type II. and soluble cytokine receptors with anti-inflammatory activity, such as IL-18 binding protein. In one embodiment, the anti-inflammatory polypeptide specifically binds to a proinflammatory cytokine. The antibody comprises an antigen-binding site.

[0117] In some embodiments, the payload is a peptide. The peptide is fused in-frame to a BTNL3 / 8 targeting moiety.

[0118] (6.3.2.1.1 Antibody antigen binding site) In one embodiment, the payload is at least one antibody antigen-binding site (ABS). In one embodiment, the antibody antigen-binding portion is a Fab fragment, an Fv, an scFv, a tandem scFv, a diabody, Di, sc diabody, DART, tandem diabody (tandAb), minibody, camelid VH H, nanobodies, or other antibody fragments or formats known to those of skill in the art. Exemplary antibody and antibody fragment formats are described in Brinkmann et al., MABS, 2017, Vol. 9, No. 2, pp. 182-212, the entire teachings of which are incorporated herein by reference. can be.

[0119] In various embodiments, at least one antibody antigen-binding site is specific for a cytokine. In some embodiments, the antigen-binding site is specific for a proinflammatory cytokine. In a particular embodiment, at least one antigen-binding site binds to interleukin-1 (IL-1). 1), IL-6, IL-12, IL-18, tumor necrosis factor α (TNFα), interferon γ (INF-γ) or condylar It is specific for granulocyte-macrophage colony-stimulating factor.

[0120] In one embodiment, the antibody is selected from the group consisting of interleukin 10 (IL-10), interleukin 22 (IL-22), or anti-inflammatory cytokines such as transforming growth factor β (TGFβ), In one embodiment, the antibody is specific for an anti-inflammatory agent. The antibody comprises at least one antigen-binding site (ABS) that is specific for the antibody.

[0121] In some embodiments, the antibody comprises at least one antibody specific for a cytokine antigen. In one embodiment, the antibody is specific for the tumor necrosis factor alpha (TNFα) antigen. In principle, the present invention is intended to include at least one ABS.

[0122] 6.3.2. Small Molecule Payloads In some embodiments, the payload is a small molecule. In some embodiments, the small molecule therapeutic is an immunomodulatory agent. In certain embodiments, the small molecule binds to a cellular protein (e.g., a receptor, other signaling molecule, an enzyme, In some embodiments, the small molecule therapeutic is a toxin. It is.

[0123] In some embodiments, the payload comprises a chemical compound that targets BTNL3 / 8. It is a drug that binds by inductive binding.

[0124]

[0102] The protein constructs disclosed herein can be used to conjugate drugs. Methods for preparing antibody drug conjugates (ADCs) that can be used are described, for example, in U.S. Pat. No. 8,624,003 ( Pot method), U.S. Patent No. 8,163,888 (single stage method), U.S. Patent No. 5,208,020 (two stage method) , U.S. Patent No. 8,337,856, U.S. Patent No. 5,773,001, U.S. Patent No. 7,829,531, U.S. Patent No. 5,208,020, U.S. Pat. No. 7,745,394, International Publication WO2017 / 136623, International Publication WO2017 / 0155 02, International Publication WO2017 / 015496, International Publication WO2017 / 015495, International Publication WO2004 / 010957, International Publication Publication WO2005 / 077090, International Publication WO2005 / 082023, International Publication WO2006 / 065533, International Publication WO2007 / 03 0642, International Publication WO2007 / 103288, International Publication WO2013 / 173337, International Publication WO2015 / 057699, International Publication WO2015 / 095755, International Publication WO2015 / 123679, International Publication WO2015 / 157286, International Publication WO2017 / 165851, International Publication WO2009 / 073445, International Publication WO2010 / 068759, International Publication WO2010 / 138719, International Publication WO2012 / 171020, International Publication WO2014 / 008375, International Publication WO2014 / 093394, International Publication WO201 4 / 093640, International Publication WO2014 / 160360, International Publication WO2015 / 054659, International Publication WO2015 / 195925, International Publication WO2017 / 160754, Storz's literature (MAbs. 2015 Nov-Dec; 7(6): 989-1009), Lambert (AdvTher, 2017 34: 1015), and Diamantis et al. (British Journal of Cancer, 2 016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007. 3: 321-2), We et al. (P roc Natl Acad Sci USA, 2009. 106: 3000-5), and Rabuka et al. (Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7:1052-67), and Agarwal et al. (Proc Natl Acad Sci USA., 20 13, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfiel et al. (Drug Dev. and D., 2014, 14:34-41), Drake et al. (Bioconjugate Chem., 2 014, 25:1331-41), Liang et al. (J Am Chem Soc., 2014, 136:10850-3), Drake et al. (Curr Opin Chem Biol., 2015, 28:174-80) and York et al. (BMC Biotechnology, 2 016, 16(1):23, the entire teachings of each of which are incorporated herein by reference. Be absorbed.

[0125] 6.3.3. Nucleic Acid Payloads In one embodiment, the payload is a nucleic acid. The nucleic acid may be, but is not limited to, However, dsDNA, mRNA, miRNA, lncRNA and siRNA, piRNA, snoRNA, snRNA, exRNA and scaR It may be DNA or RNA, such as NA.

[0126] (6.3.3.1 Optional Linker)

[0104] The protein constructs described herein can optionally be used to deliver a targeting component to a payload. The linker includes a linker that bonds to the

[0127] In some embodiments, the optional linker is in-frame fused to the targeting moiety. In one embodiment, the optional linker is a peptide linked to the C-terminus of the targeting moiety. In one embodiment, the optional linker is fused in frame to the N It is fused in frame to the terminus.

[0128] In some embodiments, the optional linker is conjugated to a targeting moiety. In various embodiments, the protein construct comprises additional functional elements. downstream processing, such as conjugating the target molecule (e.g., payload and BTNL3 / 8 targeting moiety) and downstream purification processes. In certain embodiments, the linker is a cleavable molecule (e.g., a site-specific protease or a ligase). The anchor may be cleaved into two or more fragments by another molecule (eg, a peptide that can be cleaved by another molecule). In one embodiment, the modification includes, but is not limited to, reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemistry" groups (e.g., reactive alkyne groups), and formyl groups. In one embodiment, the modified aldehyde-containing chemically reactive group is glycine (FGly). The mutations include, but are not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GS, In some embodiments, the functional group or The chemically reactive group comprises a cleavable peptide sequence. The peptides can be cleaved by, but are not limited to, photocleavage, chemical cleavage, protease cleavage, reduction, etc. In particular embodiments, the cleavage is performed by means of a protease, which may include, for example, the enzyme, the cleavage site ... Cleavage is carried out by an intracellular protease. In a particular embodiment, protease cleavage AD is mediated by extracellular or membrane-bound proteases. The C therapy is described in detail in Choi et al. (Theranostics, 2012; 2(2): 156-178.) , the entire teachings of which are incorporated herein by reference.

[0129] In one embodiment, in addition to attaching the targeting moiety to the payload, the linker comprises: Molecules into functional elements (e.g., targeting moieties and payloads) of protein constructs In one embodiment, the nucleotide sequence can be used to enable site-specific conjugation. The linker is used to identify or detect the protein construct in vitro or in vivo. In one embodiment, the protein construct may be used in addition to any one of Including cars.

[0130] 6.4. Other Target Components In one aspect, described herein are recombinant homodimers and heterodimers. A panel or library of human γδ TCRs, and the panel or library is used to detect γδ T Identifying specific cognate human binding domains or partners that determine or promote tissue-specific distribution of cells. This binding partner is often identified as a natural human “self-antigen.” Further determination or characterization is highly anticipated until the discovery described herein has been made. In one embodiment of the present invention, the present invention provides a method for detecting the cellular components of a cellular protein that is expressed in its natural cellular environment. Recombinant human γδ TCR proteins that exhibit similar tissue specificity or binding properties are described. In an embodiment of the invention, the method comprises: (i) generating at least one recombinant γδ TCR; (ii) generating one or more of said recombinant γδ TCRs; A potential one or more recombinant proteins, preferably expressed on the cell surface. (iii) displaying or presenting or mixing with a cognate binding partner; and In this invention, a method is described for identifying or validating a specific γδ TCR / binding partner interaction. In one embodiment, the resulting identified γδ TCR or a sequence derived therefrom is used as a targeting component. and employing them to deliver a therapeutic payload to a target tissue or cell expressing its cognate binding partner. In a further embodiment, the present invention provides a method for the identification of a compound or ... Cells expressing the cognate binding partner can then be targeted with another targeting moiety, such as an antibody or its derivative. It will be turned into.

[0131] (6.5. How to Create) The protein constructs described herein are useful in the production of T cell receptors or antibodies. Standard cell-free translation, transient transfection, and stable It can be easily produced by protein expression using transfection approaches.

[0132] (6.6. Purification method)

[0110] Any suitable purification method known to those skilled in the art can be used to purify the protein construct. The bound protein is attached to an affinity resin (e.g., an affinity tag on the protein construct). (combined with 100% glycerol) to easily separate unwanted proteins and protein complexes Further purification can be achieved by ion exchange chromatography, as is routinely used in the art. This can be done using roughy.

[0133] Methods for assessing the effectiveness and efficiency of a purification step are well known to those of skill in the art and include but are not limited to the following: Although not widely used, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography, etc. Purity can also be assessed by a variety of criteria. Examples of criteria include, but are not limited to: 1) a fully assembled protein structure; 2) evaluation of the percentage of total protein in the eluate obtained from the construct; The enrichment fold or percentage increase of the method for producing the protein is evaluated, e.g., the number of fully assembled proteins in the eluate is evaluated. 3) comparing the total protein obtained from the protein construct with that of the starting sample; The percentage of the product or the percentage of undesired products, e.g., the incomplete complexes, reduced. Assessment of the presence or absence of certain undesired products (e.g., unassociated single-chain polypeptides, Any combination of polypeptide chain dimers or any combination of polypeptides and determining the percent or percent reduction in tide chain trimers.

[0134] 6.7. Pharmaceutical Compositions In another aspect, the BTNL3 / 8 targeting moiety and payload described herein are and a pharma- ceutical product comprising a protein construct comprising the peptide and a pharma- ceutical acceptable carrier or diluent. Pharmaceutical compositions are provided. In typical embodiments, the pharmaceutical compositions are sterile. In some aspects, Described herein is any of the above protein constructs and pharma- ceutically acceptable carriers. The pharmaceutical composition comprises a carrier capable of administering the compound of the present invention.

[0135] In one embodiment, the pharmaceutical composition is suitable for parenteral administration. The administration is intravenous. In one embodiment, the administration is intramuscular. In one embodiment, In this case, administration is subcutaneous.

[0136] In various embodiments, the pharmaceutical composition comprises a protein construct at a concentration of 0.1 mg / ml to 100 mg / ml. In particular embodiments, the pharmaceutical composition contains the protein construct at a concentration of 0.5 mg / ml, 1 mg / ml, ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml, or 10 mg / ml concentrations. In some embodiments, the pharmaceutical composition comprises the protein construct at a concentration of greater than 10 mg / ml. In certain embodiments, the protein construct is present at 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml , 40 mg / ml, 45 mg / ml, or even 50 mg / ml or higher. In an embodiment, the protein construct is present at a concentration of greater than 50 mg / ml.

[0137] In various embodiments, the pharmaceutical compositions are as described in U.S. Pat. No. 8,961,964, U.S. Pat. No. 8,945,865, U.S. Patent No. 8,420,081, U.S. Patent No. 6,685,940, U.S. Patent No. 6,171,586, U.S. Patent No. 8,821,865, U.S. Patent No. 9,216,219, U.S. Patent Patent application Ser. No. 10 / 813,483, International Publication No. WO2014 / 066468, International Publication No. WO2011 / 104381, and International Publication No. No. WO 2016 / 180941, the entire teachings of which are incorporated herein by reference. To be incorporated.

[0138] 6.8. Compositions for Use

[0116] In one aspect, compositions for use are also provided. A protein construct comprising a BTNL3 / 8 targeting moiety and a payload as described herein. The compositions are provided for use in therapy, for example, in treating inflammatory conditions, Inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, metabolic disorders, cancer, immune-related disorders, Treatment of autoimmunity, transplant rejection, post-traumatic immune responses, graft-versus-host disease, ischemia, stroke, and infection can be used for

[0139]

[0117] In one aspect, the use of the composition for the manufacture of a medicament is also provided. In an embodiment, a polypeptide comprising a BTNL3 / 8 targeting moiety and a payload as described herein is provided. The present invention provides a use of a composition containing a protein construct for the manufacture of a medicament, the medicament comprising: For example, inflammatory conditions, inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, metabolic disorders, Cancer, immune-related disorders, autoimmunity, transplant rejection, post-traumatic immune response, graft-versus-host disease, ischemia, stroke , and for the treatment of infections.

[0140] (6.9. Treatment) In one aspect, a therapeutic method is provided that targets BTNL3 / 8 as described herein. A protein construct comprising the moiety and the payload is administered to the patient in an amount effective to treat the patient. The protein constructs of the present disclosure may be used by themselves or as a therapeutic agent. In the form of pharmaceutical compositions, for example, inflammatory conditions, inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, diseases, metabolic disorders, cancer, immune-related disorders, autoimmunity, transplant rejection, post-traumatic immune response, graft-versus-graft It may also be administered to a subject for the treatment of host disease, ischemia, stroke, and infection.

[0141] (6.9.1.1 Gastrointestinal symptoms)

[0119] In certain aspects, described herein are methods for treating gastrointestinal conditions. Gastrointestinal symptoms include, but are not limited to, immune-related symptoms of the gastrointestinal system, gastrointestinal Inflammatory conditions, microbial infection of the gastrointestinal tissues, and feeding disorders, metabolic disorders or deficiencies can cause Gastrointestinal symptoms include, but are not limited to, inflammatory Bowel disorders, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, and cancer (e.g., colon cancer) In some embodiments, the present disclosure provides a method for treating gastrointestinal cancer, including the treatment of ... A method for treating a gastrointestinal condition expressing TNL3 / 8, comprising administering a therapeutically effective amount of any one of claims 62 to 66. The pharmaceutical composition according to any one of claims 1 to 6 is administered to a patient under conditions in which gastrointestinal tissue expresses BTNL3 / 8. In one embodiment of the method, the method comprises administering to the patient a payload of the protein construct. The lorde is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. The anti-inflammatory agent is optionally interleukin 10 (IL-10), interleukin 22 (IL-22) or trans Transforming growth factor β (TGFβ) is an anti-inflammatory cytokine. In one embodiment, the anti-inflammatory agent is an anti-pro-inflammatory agent. In one embodiment, the anti-inflammatory agent is a steroid. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is hydrocorticoid. Lutisone. In one embodiment, the payload is an immunomodulatory agent.

[0142] (6.9.2.1 Inflammatory bowel disease) In one aspect, described herein is a therapeutically effective amount of any of the above. A method for treating inflammatory bowel disease, comprising administering the pharmaceutical composition described above to a patient suffering from inflammatory bowel disease. In one embodiment, the inflammatory bowel disease is ulcerative colitis. In one embodiment, the infectious bowel disease is Crohn's disease. In one embodiment, the payload of the protein construct is In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug. a cytokine, optionally interleukin 10 (IL-10), interleukin 22 (IL-22) or Transforming growth factor β (TGFβ). In one embodiment, the anti-inflammatory payload is an anti-inflammatory agent. In one embodiment, the anti-inflammatory payload is a steroid. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is hydrocorticoid. In one embodiment, the payload of the protein construct is an antibiotic. In one embodiment, the antibiotic payload is rifaximin, ciprofloxacin, meviral, or cyclosporine. In one embodiment, the agonist is tronidazole, moxifloxacin, or amoxicillin. The payload of the protein construct is a calcineurin inhibitor. The sinurin inhibitor is cyclosporine A or tacrolimus. The payload of the protein construct is an immunomodulatory agent. In one embodiment, the immunomodulatory agent is an immunomodulatory agent. In one embodiment, the immunosuppressant is azathioprine, 6-mercaptopurine, In one embodiment, the peptide of the protein construct is The erode is a protein payload. In one embodiment, the protein payload is , an antibody, an antibody fragment or a single chain variable fragment. In one embodiment, the protein payload The antibody comprises at least one ABS that is specific for the TNFα antigen. In one embodiment, the protein payload is adalimumab, infliximab or certoxin. In one embodiment, the protein payload comprises the complementarity determining regions (CDRs) of limumab. In one embodiment, the antibody comprises at least one ABS specific for an interleukin antigen. The teleukin is IL-12, IL-23, or a combination thereof. The protein payload contains the CDRs of ustekinumab or brikinumab. In one embodiment, the biologic payload comprises at least one integrin specific for the integrin antigen. In one embodiment, the integrin is an α4 integrin. In one embodiment, the protein payload is infliximab, natalizumab or vedorin. In one embodiment, the protein construct comprises the CDRs of tafamidis. In one embodiment, the protein construct comprises a nutraceutical payload.

[0143] (6.9.3.1 infection) In one aspect, described herein is a therapeutically effective amount of any of the above. A method for treating a microbial infection comprising administering to a patient suffering from a microbial infection a pharmaceutical composition comprising the steps of: In some embodiments, the payload is an antimicrobial agent. It is an antiparasitic, an antibiotic, an antifungal or an antiviral agent.

[0144] 6.9.4.1 Metabolic Disorder or Deficiency In one aspect, described herein is a therapeutically effective amount of any of the above. A method for treating a metabolic disorder or deficiency comprising administering a pharmaceutical composition comprising the compound of claim 1 to a patient suffering from a metabolic disorder or deficiency. In one embodiment, the payload is a nutritional supplement. In some embodiments, the dietary supplement is an enzyme or a vitamin.

[0145] 6.9.5.1 Regulation of the immune system In one aspect, described herein is a therapeutically effective amount of any of the above. A method for regulating the immune system, comprising administering a pharmaceutical composition of the formula (I) to a patient suffering from an immune-related condition. In some embodiments, the payload is an immunosuppressant. The agent is azathioprine, 6-mercaptopurine, methotrexate or thiopurine. In some embodiments, the payload is an immunostimulant. is a cytokine. EXAMPLES

[0146] (6.10. Example)

[0124] The following examples are not intended to limit the invention and are provided for illustration only.

[0147] (6.10.1.1 method) Design and analysis of protein constructs containing BTNL3 / 8 targeting components containing TCR Vγ domains Non-limiting exemplary methods for isolating primary lymphocytes, BTNL3 / 8 expression The current co-culture with HEK293 cells and deep sequencing methods are also described by Di Marco Barros et al. This is described in the literature, Cell. 2016,(167), pp. 203-218.

[0148] (Human samples and primary lymphocyte isolation)

[0126] Endoscopic biopsies were taken from the ascending colon of adult donors undergoing routine colonoscopy. Primary intestinal lymphocytes were obtained from the culture of Clark et al., 2006, J. Invest. Dermatol.(126), pp. 10 The biopsy was obtained by applying the method described in 59-1070. The biopsy was then washed with 5 mL of washing medium (RPMI 1640 10% FCS, β-methyl Aminopropyl ether, penicillin [500U / ml], streptomycin [500 mg / ml], metronidazole [100 mg / ml], Dazol [5 mg / ml, Guy's Hospital, Medicine Department], gentamicin [100 mg / ml, Sigma-Aldrich The cells were washed with 100 mM NaCl, 100 mM NaCl, and 12.5 mg / ml amphotericin [Thermo Fisher Scientific] for 20 minutes. An endoscopic biopsy was placed onto each matrix, which was then inverted and pressure was applied to insert the biopsy into the matrix. The matrix was placed in a 24-well plate (one per well) and 2 mL RPMI 1640 (10% FCS, β-mercaptoethanol, penicillin [100 U / ml], streptomycin 100 mg / ml, metronidazole, 1 mg / ml, gentamicin, 20 mg / ml, amphotericin IL-15 (10 ng / mL, Novartis Pharmaceutical, UK) and IL-2 (100 U / mL, Novartis Pharmaceutical, UK). The plates were covered with 1 ml of medium supplemented with 2x concentrated 100% glycerol (Biolegend). The medium was replaced with complete medium containing apoptotic cytokines. Cells were harvested and the remaining biopsies and empty wells were The cell suspension was passed through a 70 mm nylon cell strainer and centrifuged at 400 g. The cells were centrifuged at 4 °C for 5 min and resuspended in complete medium without additional cytokines and immediately Lymphocytes were used after 5–7 days in culture. PBMCs were obtained from the blood donation service. The cells were isolated from blood by Ficoll gradient. (HEK293T co-culture assay) 5 × 10 transduced with either empty vector (EV), BTNL3, BTNL8 or BTNL3+8 5 HEK293T Cells and freshly harvested 2 × 10 5 Primary human lymphocytes were cultured in 96-well plates and supplemented with The cells were co-cultured in complete medium without cytokines and incubated at 37°C, 5% CO2 for 16 hours.

[0149] (Deep Sequencing)

[0127] Mouse TRDV gene, TCRδ CDR3 from RNA purified from sorted Vγ7+ IELs Amplification and sequencing were performed using the Amp2Seq Platform (iRepertoire). Amplification and sequencing of the human TCRγ CDR3 gene was performed using the immunoSEQ Platform. The experiment was carried out using Adaptive Biotechnologies.

[0150] (Design of soluble γδ TCR heterodimer)

[0128] The soluble T cell receptor α and T cell receptor β constant regions used in the following examples The design of the γδTCR heterodimer was based on the literature review by Xu et al., PNAS, 2011 Vol. 108; pp. 2414-241. 9 was followed.

[0151] (6.10.2.1 Example 1: γδ TCR variable regions isolated from intraepithelial lymphocytes from human intestinal tissue , which induced TCR activity in BTNL3 / 8)

[0129] Cloning of TCR variable regions of human intestinal-derived and BTNL3 / 8-responsive intraepithelial lymphocytes (IELs) The IELs were isolated from human intestinal tissue and transfected with HEK293T cells. Next, TCR activation (high expression of CD25 and dapagliflozin) was observed in the 14- and 15-cell cultured mice. Responsive IELs that showed downregulation of γ and δ chains were single-cell sorted. The variable region was amplified and cloned into a lentiviral expression vector (Figure 3A). T cells (J76 cell line) were transduced and co-cultured with HEK293T cells expressing BTNL3 / 8 (Figure 3B). Next, J76 cells were sorted for TCR activation (CD69 expression and TCR downregulation) ( Anti-CD3 antibody was used as a positive control for TCR activation. When cultured, J76 cells expressing a transduced TCR (H7 TCR) with Vγ4 and Vδ1 domains They showed increased CD69 expression and downregulation of the γδ TCR. Three independent J76 lines, B3, C11, and H7, were identified as the three distinct CD40 clones obtained using the method shown in Figure 2. representative of the R3 pair, but unlike the Vγ9Vδ2 lineage (Vγ9Vδ2), responded to BTNL3 / 8-expressing cells (Fig. 4B). These results suggest that the Vγ4Vδ1 domain of human IELs is essential for conferring TCR responsiveness to BTNL3 / 8. Show that it is sufficient.

[0152] 6.10.3.1 Example 2: CDR4 of Vγ4 is required for TCR responsiveness to BTNL3 / 8 To identify the Vγ4 region important for responsiveness to BTNL3 / 8, all Vγ4 The γ4 domain was replaced with the Vγ2 region (Figure 5). When co-cultured with HEK293T expressing BTNL3 / 8, Percentage of CD69 expression in transduced J76 cells expressing Vγ4 TCR (H7 WT) or Vγ2 substituted TCR The fold change (FC) and percent TCR downregulation of responding Vγ4 H7 T cells were determined. The entire V region of CR is replaced with the Vγ2 coding sequence (Vγ2 H7) (except for CDR3γ and the entire δ chain). In contrast, CD4+ / -expressing TCRs were not activated by BTNL3 / 8-expressing cells, and CD4+ / -expressing TCRs were not activated by BTNL3 / 8-expressing cells. R1(H7 CDR1 Vγ2 ) and / or CDR2 (H7 CDR2 Vγ2 ) with the Vγ2 coding sequence, These results suggest that TCR activation by BTNL3 / 8 cells is dependent on CDR4. Indicates that it is necessary.

[0153] To further elucidate the region within Vγ4 that is essential for response to BTNL3 / 8, we cloned the nucleotide sequence located in CDR4. The two pairs of amino acids corresponding to Vγ2 were replaced with Vγ2 sequences (Figures 6 and 7). The fold change (FC) in CD69 expression in transduced cells was determined when the cells were cultured with Vγ4 TCR. expressing a Vγ2 TCR with the CDR3 of H7 (H7 WT), and a Vγ2 TCR with an amino acid substitution in CDR4 (Vγ2H7). The percent TCR downregulation was determined in J76 cells expressing Vγ4 TCR ( Figure 7). YA substitutions at amino acid positions 87 and 90 abolished TCR activation by BTNL3 / 8-expressing cells. On the other hand, NL substitutions at amino acid positions 94 and 98 did not abolish TCR activation by BTNL3 / 8 expressing cells. These results suggest that amino acids 87 and 90 in the CDR4 region of Vγ4TCR mediate TCR-mediated TC to BTNL3 / 8. We have established that it is essential for R responsiveness.

[0154] 5.10.4.1 Example 3: Soluble TCR Vγ4 / Vδ heterodimers bind to BTNL3 / 8 expressing cells Soluble Vγ / Vδ TCR heterodimers are expressed by leucine zipper complementation The Vγ or Vδ domains were stabilized by TCRα or TCRβ constant domains lacking the transmembrane domain (Figure 8). The region is fused in-frame to the leucine zipper sequence and then to a histidine tag / phosphoryl The Vγ4 / Vδ1 heterodimer corresponds to SEQ ID NOs: 10 and 9. The Vγ4 / Vδ2 heterodimer corresponds to SEQ ID NOs: 10 and 9. The Vγ2 / Vδ1 heterodimer corresponds to SEQ ID NO: 10 and 11. The Vγ2 / Vδ1 heterodimer corresponds to SEQ ID NO: 1 2 and 9. The Vy8 / V51 heterodimer corresponds to SEQ ID NOs: 13 and 9.

[0155]

[0133] HEK2 transduced with Flag-BTNL3+HA-BTNL8 or empty vector using soluble TCR 93T cells were stained (Figure 10). Vγ4 / Vδ1 soluble TCR and Vγ4 / Vδ2 soluble TCR were stained for BTNL3+BTNL8 The results show strong binding to cell lines expressing IgG but not to the empty vector (EV) control cell line. Soluble TCRs expressing the Vγ4 / Vδ1 domain or the Vγ4 / Vδ2 domain were expressed in BTNL3 / 8-expressing cells. We show that the BTNL3 / 8-deficient cells bind to the BTNL3 / 8-deficient cells but not to the BTNL3 / 8-deficient cells. showed that Vγ4 CDR4 is essential for BTNL3 / 8-induced TCR responses and These results also suggest that it interacts with 8.

[0156] Soluble Vγ4 in cells expressing BTNL3+8 + To further evaluate the binding of TCR constructs HEK293T cells were cultured with either the indicated BTNL3 and BTNL8 constructs or an empty vector (EV) encoding The cells were then transduced with soluble His-tagged Vγ4δ2 TCR for 45 min at 4 °C. Stained, washed twice, stained with APC anti-His tag antibody (α-His) for 45 minutes at 4°C, washed twice again, The cell populations shown in Figure 9B were then analyzed by flow cytometry (Figure 9A). Parallel staining with antibodies demonstrated that the lack of soluble TCR binding was due to failure of expression of the BTNL3+8 construct. This result confirmed that soluble TCR binding to cells expressing BTNL3+BTNL8 is not a The construct is capable of expressing Vγ4 + Inability to induce a T cell response and IgV domain variants previously described in the literature (e.g., L3 GQFSS , L3 RI , L3 YQKAI ) See Melandri et al., Nat. Immunol. 2018, which It is incorporated herein by reference in its entirety.

[0157] 6.10.5.1 Example 4: Soluble TCRs that bind to BTNL3 / 8 expressing cells are internalized

[0135] We investigated whether soluble TCR bound to the surface of cells expressing BTNL3+BTNL8 is internalized. To determine whether BTNL3 and BTNL8 could be expressed in HEK293T cells, we transduced them with wild-type BTNL3 and BTNL8 (293T.L3L8). were expressed and stained with soluble His-tagged Vγ4Vδ2 TCR for up to 120 min at 37°C (Figure 11B) 293T.L3L8 cells were stained with soluble His-tagged Vγ4Vδ2 TCR for 120 min at 4°C and then incubated at low temperature. The cells were then incubated with an APC α-His tag antibody (α-Hi The cells were stained with 0.05% ethanol at 4°C for 45 minutes. The results showed that the decrease in APC signal was consistent with the cells incubated at 37°C. This occurs over time within the cell population, indicating that cells rapidly internalize the soluble TCR construct ( Figure 12).

[0158]

[0136] Is internalization of soluble TCR specific or is it due to rapid cycling of cell surface BTNL molecules? To determine whether this was the result of cycling, the experiment was repeated and soluble TCR was incubated with anti-BT Compared with NL3 antibody (rabbit polyclonal, Aviva Biosystems) (references included). HEK29 3T cells were transduced with an empty vector (293T.EV) as a negative control for staining with α-BTNL3. HEK293T cells were transfected with the BTNL3 construct L3. RI L8(293T.L3 RI L8) and transduced with soluble TCR The results showed that α-BTNL3 staining was identical at 4°C and 37°C. The results showed that α-BTNL3 specifically binds to cells expressing BTNL3 / 8, whereas the antibody does not specifically bind to cells expressing BTNL3 / 8. It was shown to persist on the surface of the cells (Figure 13A). Quantification of the results is shown in Figure 13B.

[0159] 6.10.6.1 Example 5: Soluble TCRs deliver payload to BTNL3 / 8 expressing cells Payloads can be delivered intracellularly via binding of soluble TCR to BTNL3 / 8 expressing cells. To determine whether 293T.L3L8 or 293T.L3 RI L8 cells were incubated with APCα-His tag antibody The soluble TCR construct was incubated at 4°C for 1 h with soluble TCR pre-labeled on the carboxy terminus of the soluble TCR construct. The complexes were incubated at either 3 μg / mL or 10 μg / mL for 1 h at either 4°C or 37°C. The cells were then washed and incubated for 15 min with trypsin or DMEM (control), as shown in FIG. The results showed that the fraction of soluble TCR+α-His signal (APC fluorescence) was was incubated with the complex at 37°C to protect it from trypsin by internalization of the complex. The results show that trypsinization is more effective than incubation at 4 °C. Thus, the results suggest that internalization of soluble TCRs results in the intracellular expression of BTNL3 / 8 payloads. The results are shown in Figure 15B. .

[0160] Imaging cytometry was used to determine the binding of the APCα-His antibody payload to soluble T Visualization of intracellular delivery via CR. 293T.L3 RIL8 or 293T.L3L8 cells were treated with soluble TCR+α-His anti- The cells were then incubated with the antibody complex for 1 hour at 4°C or 37°C. The cells were either trypsinized or fixed and permeabilized (Figure 16A) or fixed and permeabilized and then incubated at 37°C for 24 h. The cells were stained with the endosomal marker CD107a (Figures 16B and 16C). By immunofluorescence spectrometry, it was possible to assess the background APC signal (Figure 16A). Imaging cytometry results of cells incubated with is-antibody complexes showed that the complexes When incubated with cells at 4°C, complexes were visualized around the cells after DMEM treatment. The majority of the complex was shown to be bound to the cell surface, whereas after trypsinization, The signal was completely lost (Fig. 16B). However, the complexes incubated with cells at 37°C , CD107a even after trypsinization + It is detected in the intracellular region proximal to the compartment (Figure 16C).

[0161] (6.11. Arrays) > Human Vγ4 amino acids 87-90 [SEQ ID NO:1] [ka] >Mouse Vγ7 amino acids 87-90 [SEQ ID NO:2] [ka] > Human Vγ4 domain CDR4 amino acids 85-100 [SEQ ID NO:3] [ka] > Mouse Vγ7 domain CDR4 amino acids 85-100 [SEQ ID NO:4] [ka] > Human Vγ2 domain CDR4 amino acids 85-100 [SEQ ID NO:5] [ka] >Human Vγ4 amino acids 19-118 [SEQ ID NO:6] [ka] Human Vγ2 amino acids 19-118 [SEQ ID NO:7] [ka] >Mouse Vγ7 amino acids 19-118 [SEQ ID NO:8] [ka] > Human Vδ1, CDR3 from crystal structure 30MZ, and TCRα constant with in-frame fusion of CDR3 region, leucine zipper and C-terminal His tag [SEQ ID NO: 9] [ka] > Human Vγ4, CDR3 from crystal structure 4MNH, TCRβ constant region to which CDR3 is fused, leucine Zipper [SEQ ID NO: 10] [ka] > Human Vδ2 and CDR3 from crystal structure 30MZ, TCRα constant with in-frame fusion of CDR3 region, leucine zipper and C-terminal His tag [SEQ ID NO: 11] [ka] > Human Vγ2, CDR3 from crystal structure 4MNH, TCRβ constant region to which CDR3 is fused, leucine Zipper [SEQ ID NO: 12] [ka] > Human Vγ8, CDR3 from crystal structure 4MNH, TCRβ constant region to which CDR3 is fused, leucine Zipper [SEQ ID NO: 13] [ka] >Leader sequence of human Vγ4 [SEQ ID NO:14] [ka] >Human Vγ J region, TRFJP [SEQ ID NO: 15] [ka] >Human Vγ J region, TRFJP1 [SEQ ID NO: 16] [ka] >Human Vγ J region, TRFJP2 [SEQ ID NO: 17] [ka] >Human Vγ J region, TRFJP1 / 2 [SEQ ID NO: 18] [ka]

[0162] (7. Incorporation by Citation) All publications, patents, patent applications, and other documents cited in this application are hereby expressly incorporated by reference in their entirety. , patent application or other document is separately indicated as being incorporated by reference for all purposes. No. 6,313,635, filed on Oct. 13, 2003, and is hereby incorporated by reference in its entirety for all purposes to the same extent as if fully set forth herein. Be absorbed.

[0163] (8. Equivalents) Although various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. Various modifications may be made without departing from the spirit and scope of the invention. It will be understood that there are many variations of the present invention. The answer is clear.

Claims

1. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating a gastrointestinal condition in a patient having a condition in which gastrointestinal tissue expresses BTNL3 / 8, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

2. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating inflammatory bowel disease in a patient with inflammatory bowel disease, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

3. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating irritable bowel syndrome in a patient with irritable bowel syndrome, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

4. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating diverticulitis in a patient with diverticulitis, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

5. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating celiac disease in a patient with celiac disease, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

6. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating a microbial infection in a patient suffering from a microbial infection, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

7. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating a metabolic disorder or metabolic deficiency in a patient with a metabolic disorder or deficiency, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

8. 1. Use of a pharmaceutical composition for the manufacture of a medicament for regulating the immune system in a patient with an immune-related condition, the pharmaceutical composition comprising: (i) components targeting BTNL3 / 8; payload; and an optional linker that connects the BTNL3 / 8 targeting moiety to the payload; A protein construct comprising: the BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG; and The protein construct, wherein the payload is a protein payload or a small molecule; and (ii) a pharmaceutical carrier; The use.

9. The use of any one of claims 1 to 8, wherein the CDR3 of the TCR Vγ domain of the BTNL3 / 8 targeting component comprises a human Vγ CDR3.

10. 9. The use of any one of claims 1 to 8, wherein the J region of the TCR Vγ domain comprises a human Vγ J region, a mouse Vγ J region, or an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 15 to 18.

11. The use according to any one of claims 1 to 8, wherein the BTNL3 / 8 targeting component of the protein construct further comprises a paired Vδ domain.

12. The use of claim 11, wherein the Vγ domain and Vδ domain of the BTNL3 / 8 targeting component are covalently linked by at least one disulfide bond.

13. The use of claim 11, wherein the Vγ and Vδ domains of the BTNL3 / 8 targeting component are paired by a specific heterodimeric interaction.

14. 14. The use of claim 13, wherein the heterodimeric interaction is leucine zipper complementarity.

15. The use according to any one of claims 1 to 8, wherein the BTNL3 / 8 targeting component of the protein construct comprises the amino acid sequence set forth in SEQ ID NO:

10.

16. The use of claim 11, wherein the BTNL3 / 8 targeting component of the protein construct comprises a single-chain in-frame fusion of the Vγ and Vδ domains.

17. The use of claim 16, wherein the Vγ domain is N-terminal to the Vδ domain.

18. The use of claim 16, wherein the Vγ domain is C-terminal to the Vδ domain.

19. The use of claim 16, wherein the single-chain in-frame fusion of the Vγ and Vδ domains comprises an internal linker sequence.

20. The use of claim 11, wherein the Vδ domain comprises a human Vδ domain selected from Vδ1, Vδ2 or Vδ5.

21. The protein construct further comprises a first T cell receptor constant region; The use of claim 11, wherein the first T cell receptor constant region is fused in-frame to the C-terminus of the Vγ domain.

22. The use according to claim 21 , wherein the first T cell receptor constant region is a human T cell receptor beta constant region, a human T cell receptor alpha constant region, or a human T cell receptor gamma constant region.

23. the BTNL3 / 8 targeting component of the protein construct further comprises a second T cell receptor constant region; 22. The use of claim 21, wherein the second T cell receptor constant region is fused in-frame to the C-terminus of the paired Vδ domain.

24. The use of claim 23, wherein the second T cell receptor constant region is a human T cell receptor alpha constant region, a human T cell receptor beta constant region, or a human T cell receptor delta constant region.

25. The use of claim 23, wherein the in-frame fusion of the Vδ domain and the second T cell receptor constant region comprises an internal linker sequence between the Vδ domain and the second T cell receptor constant region.

26. The use according to any one of claims 1 to 8, wherein the payload of the protein construct is fused in-frame to the BTNL3 / 8 targeting moiety.

27. The use according to any one of claims 1 to 8, wherein the payload of the protein construct comprises a polypeptide, a peptide, a cytokine, or an antibody.

28. The use according to any one of claims 1 to 8, wherein the small molecule payload of the protein construct comprises a hormone, a nucleic acid, or an inhibitory RNA (RNAi).

29. The use of any one of claims 1 to 8, wherein the optional linker of the protein construct is a peptide fused in-frame to the BTNL3 / 8 targeting moiety or comprises a molecule conjugated to the BTNL3 / 8 targeting moiety.

30. The use according to any one of claims 1 to 8, wherein at least a portion of the BTNL3 / 8 targeting component and / or the payload is internalized into cells.

31. The use according to any one of claims 1 to 8, wherein the BTNL3 / 8 targeting component and / or the payload are not internalized into cells.

32. The use according to claim 1 or 2, wherein the payload of the protein construct comprises an anti-inflammatory agent.

33. 33. The use of claim 32, wherein the anti-inflammatory agent is an aminosalicylate, a nonsteroidal anti-inflammatory agent, an anti-inflammatory cytokine, an anti-inflammatory agent, or a steroid.

34. 34. The use of claim 33, wherein the steroid is a glucocorticoid.

35. 35. The use of claim 34, wherein the glucocorticoid is prednisone or hydrocortisone.

36. The use according to any one of claims 2 to 4, wherein the payload of the protein construct comprises an antibiotic.

37. 37. The use of claim 36, wherein the antibiotic is rifaximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin.

38. The use of claim 2, wherein the payload of the protein construct comprises a calcineurin inhibitor.

39. 39. The use of claim 38, wherein the calcineurin inhibitor is cyclosporin A or tacrolimus.

40. The use according to claim 2, wherein the protein payload of the protein construct comprises an antibody, an antibody fragment or a single-chain variable fragment.

41. The use of claim 2, wherein the protein payload of the protein construct comprises an antigen binding site (ABS) specific for a tumor necrosis factor alpha (TNFα) antigen, an interleukin antigen, or an integrin antigen.

42. 42. The use of claim 41, wherein the protein payload comprises the CDRs of adalimumab, infliximab, or certolizumab.

43. 42. The use of claim 41, wherein the interleukin is IL-12, IL-23, or a combination thereof.

44. 44. The use of claim 43, wherein the protein payload comprises the CDRs of ustekinumab or brikinumab.

45. 42. The use of claim 41, wherein the integrin is alpha4 integrin.

46. 46. ​​The use of claim 45, wherein the protein payload comprises the CDRs of infliximab, natalizumab, or vedolizumab.

47. The use according to claim 1 or 2, wherein the payload of the protein construct comprises an immunomodulatory agent.

48. 9. The use of any one of claims 2, 5 and 8, wherein the payload of the protein construct comprises an immunosuppressant.

49. 49. The use of claim 48, wherein the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine.

50. The use of claim 6, wherein the payload of the protein construct comprises an antibacterial agent.

51. 51. The use of claim 50, wherein the antimicrobial agent is an antiparasitic, antibiotic, antifungal, or antiviral agent.

52. 10. The use of claim 2 or 7, wherein the payload of the protein construct comprises a nutritional supplement.

53. 53. The use of claim 52, wherein the dietary supplement is an enzyme or a vitamin.

54. The use of claim 8, wherein the payload of the protein construct comprises an immunostimulant.

55. 55. The use of claim 54, wherein the immunostimulant is a cytokine.

56. The use according to any one of claims 1 to 8, wherein the pharmaceutical composition is suitable for parenteral administration.

57. 57. The use of claim 56, wherein the parenteral administration comprises intravenous administration, intramuscular administration, or subcutaneous administration.

58. 3. The use according to claim 2, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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