A construct targeting BTNL3 / 8 for payload delivery to the gastrointestinal tract.

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-referencing of related applications) This application primarily takes priority from U.S. Provisional Patent Application No. 62 / 680,932, filed on June 5, 2018. The entire text is incorporated herein by reference.

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

[0003] (3.Background) Most drugs rely on systemic exposure to achieve sufficient concentrations at the diseased site. However, exposure of tissues other than the target tissue to the drug often causes serious toxicity. Therefore, there is an urgent need to develop methods for delivering pharmaceuticals to specific organizations.

[0004] Tissue-selective homing of T cells is a crucial element in the integration of a normal immune response. This is considered to be the basis. Such "tissue-homing" or "tissue-resident" T cells are interesting. One of the most unique classes is the γδT cell. γδT cells are highly segmented T cells. For example, mouse (murine)Vγ5T cells are found only in the epidermis, but mouse (murine)Vγ 7T cells exhibit localization of subsets to specific tissues, such as being unique to the gut. The IEL compartments of the murine and humans are linked to butyrophylline (BTN) and br Proteins expressed by stationary epithelial cells, such as tyrophyllin-like (Btnl / BTNL) genes. Its development and survival depend on the quality of the material. (Di Marco Barros et al., Cell. 2016; 167(1)) : 203-218; Kabelitz et al., F1000 Faculty Rev: 782; June 5, 2017).

[0005] Butyrophyllin and butyrophyllin-like proteins (BTN / BTNL) are involved in immunology, such as T cell selection. Furthermore, immunoglobulin superintendents that influence developmental processes such as differentiation and cell fate determination. - Family member of family member (Arnett and Viney's work, Nature Reviews, 20 14(14) pp. 559-569). BTNL proteins (specifically BTNL3 and BTNL8) are found in human intestinal cells. Disproportionately high expression, BTNL3 / 8 can specifically regulate human Vγ4+γδT cells. This has been shown (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, It is not clear how such signals are transmitted to T cells (Kabelitz et al., F1000 Faculty Rev: 782; June 5, 2017).

[0006] Local delivery of drugs to the gastrointestinal tract is required for the treatment of diseases such as inflammatory bowel disease. Therefore, targeting therapeutic agent payloads to BTNL3 / 8-expressing cells, particularly BTNL3 / 8-expressing cells in the intestinal epidermis. There is a need for compounds that can target BTNL3 / 8-expressing cells, including compounds that can be targeted. [Overview of the project]

[0007] (4. Overview) Despite such insufficient characterization of the tissues / immune compartments attracting attention, in order to further explore the "shaping" and "homing" thereof, and thereby the mechanism by which targeting and specification of γδ T cells to tissues are achieved, attempts were made to explore the recombinant he A panel of recombinant heterodimer and homodimer γδ T cell receptor proteins (TCRs) was first attempted to be engineered from the starting points of the human γ and δ chain sequences. To achieve this, various fusion partners were explored, and the resulting recombinant heterodimerized and homodimerized γδ chains were expressed (e.g., in HEK293 cells), maintained intact, and confirmed to show little aggregation (measured by size exclusion chromatography) upon purification. From these experiments, several more preferred fusion partners were identified, including (i) the use of antibody Fc fusion domain partners, and (ii) the use of engineered leucine zipper combined with TCRα-β chain constant domain partners (reference of Xu et al., PNAS, 2011 Vol. 108; pp. 2414-2419). However, even if these successful experiments were not comprehensive, one of ordinary skill in the art can now recognize that there will be a motivation to identify another approach to create recombinant γδ homodimers, heterodimers, and monomer subunits. A panel or library of T cell receptor recombinant proteins derived from these human γδ sequences was created, and then used to explore whether (if any) which recombinant γδ pairs show any tissue specificity or selectivity. Therefore, these proteins were used in cells overexpressing BTNL3 / 8 or BTNL8 or BTNL3 / 8 and BTNL8 (references of Xu et al., PNAS, 2011 Vol. 108; pp. 2414-2419). However, if these successful experiments were not comprehensive, one of ordinary skill in the art can now recognize that there will be a motivation to identify another approach to create recombinant γδ homodimers, heterodimers, and monomer subunits. If these successful experiments were not comprehensive, one of ordinary skill in the art can now recognize that there will be a motivation to identify another approach to create recombinant γδ homodimers, heterodimers, and monomer subunits. If these successful experiments were not comprehensive, one of ordinary skill in the art can now recognize that there will be a motivation to identify another approach to create recombinant γδ homodimers, heterodimers, and monomer subunits. If these successful experiments were not comprehensive, one of ordinary skill in the art can now recognize that there will be a motivation to identify another approach to create recombinant γδ homodimers, heterodimers, and monomer subunits.

[0008] A panel or library of T cell receptor recombinant proteins derived from these human γδ sequences was created, and then used to explore whether (if any) which recombinant γδ pairs show any tissue specificity or selectivity. Therefore, these proteins were used in cells overexpressing BTNL3 / 8 or BTNL8 It was incubated with the cell line. Without precedent, through a control comparison test, a certain recombinant T cell receptor that specifically recognizes the BTNL3 / 8 protein (for example, recombinant γ4δ1 TCR and recombinant γ4δ2 TCR) was identified, while other receptors that did not recognize it (for example, recombinant γ2δ1 TCR and recombinant γ8δ1 TCR) were also identified. This shows for the first time that γ4 containing γδ TCR interacts directly with BTNL3 / 8 newly discovered on the cell surface. While identifying certain recombinant T cell receptors that specifically recognize the BTNL3 / 8 protein (for example, recombinant γ4δ1 TCR and recombinant γ4δ2 TCR), other receptors that did not recognize it (for example, recombinant γ2δ1 TCR and recombinant γ8δ1 TCR) were also identified. While identifying certain recombinant T cell receptors that specifically recognize the BTNL3 / 8 protein (for example, recombinant γ4δ1 TCR and recombinant γ4δ2 TCR), other receptors that did not recognize it (for example, recombinant γ2δ1 TCR and recombinant γ8δ1 TCR) were also identified. This shows for the first time that γ4 containing γδ TCR interacts directly with BTNL3 / 8 newly discovered on the cell surface. This shows for the first time that γ4 containing γδ TCR interacts directly with BTNL3 / 8 newly discovered on the cell surface.

[0009] Furthermore, TCR deep sequencing showed that there was selective enrichment of γδ T cells expressing γ4 TCR in the fraction responsive to BTNL3 / 8. That is, the sequencing data and the recombinant TCR experiments showed that TCR pairing containing the γ4 subunit selectively binds to BTNL3 / 8. Furthermore, TCR deep sequencing showed that there was selective enrichment of γδ T cells expressing γ4 TCR in the fraction responsive to BTNL3 / 8. That is, the sequencing data and the recombinant TCR experiments showed that TCR pairing containing the γ4 subunit selectively binds to BTNL3 / 8. Furthermore, TCR deep sequencing showed that there was selective enrichment of γδ T cells expressing γ4 TCR in the fraction responsive to BTNL3 / 8. That is, the sequencing data and the recombinant TCR experiments showed that TCR pairing containing the γ4 subunit selectively binds to BTNL3 / 8. Furthermore, TCR deep sequencing showed that there was selective enrichment of γδ T cells expressing γ4 TCR in the fraction responsive to BTNL3 / 8. That is, the sequencing data and the recombinant TCR experiments showed that TCR pairing containing the γ4 subunit selectively binds to BTNL3 / 8.

[0010] Therefore, in a first aspect, a protein construct is provided. This protein construct includes a component targeting BTNL3 / 8, a payload, and any linker that binds the targeting component to the payload. Therefore, in a first aspect, a protein construct is provided. This protein construct includes a component targeting BTNL3 / 8, a payload, and any linker that binds the targeting component to the payload. Therefore, in a first aspect, a protein construct is provided. This protein construct includes a component targeting BTNL3 / 8, a payload, and any linker that binds the targeting component to the payload.

[0011] In certain embodiments, the component targeting BTNL3 / 8 includes a Vγ domain, the amino acid at sequence position number 87 of the Vγ domain is aspartic acid or histidine, the amino acid at sequence position number 90 of the Vγ domain is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are selected independently from the corresponding residues of the human or murine Vγ domain at their respective positions. In certain embodiments, the component targeting BTNL3 / 8 includes a Vγ domain, the amino acid at sequence position number 87 of the Vγ domain is aspartic acid or histidine, the amino acid at sequence position number 90 of the Vγ domain is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are selected independently from the corresponding residues of the human or murine Vγ domain at their respective positions. In certain embodiments, the component targeting BTNL3 / 8 includes a Vγ domain, the amino acid at sequence position number 87 of the Vγ domain is aspartic acid or histidine, the amino acid at sequence position number 90 of the Vγ domain is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are selected independently from the corresponding residues of the human or murine Vγ domain at their respective positions. In certain embodiments, the component targeting BTNL3 / 8 includes a Vγ domain, the amino acid at sequence position number of the Vγ domain is aspartic acid or histidine, the amino acid at sequence position number 90 of the Vγ domain is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are selected independently from the corresponding residues of the human or murine Vγ domain at their respective positions. In certain embodiments, the component targeting BTNL3 / 8 includes a Vγ domain, the amino acid at sequence position number 87 of the Vγ domain is aspartic acid or histidine, the amino acid at sequence position number 90 of the Vγ domain is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are selected independently from the corresponding residues of the human or murine Vγ domain at their respective positions.

[0012] In certain embodiments, the remaining residues of Vγ CDR4 are, at their respective residue positions, human Vγ 4. Selected independently from the corresponding residues of human Vγ2 or mouse Vγ7. In one embodiment, V The amino acid sequence at positions 87-90 of the γ domain is sequence number 1. In one embodiment, The amino acid sequence at positions 87-90 of the Vγ domain is sequence number 2. In one embodiment, All remaining residues in the Vγ domain CDR4 correspond to human Vγ4, human Vγ2, or mouse Vγ7. Selected from the base. In one embodiment, the remaining residues of Vγ CDR4 are the corresponding residues of human Vγ4. Selected from the corresponding residues of human Vγ2. In one embodiment, the remaining residues of Vγ CDR4 are selected from the corresponding residues of human Vγ2. Selected. 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 the human Vγ2 domain, and within it CDR4 The amino acid is substituted with aspartic acid or histidine at amino acid sequence position 87, The no-acid sequence position number 90 is substituted with glycine or glutamic acid. In one embodiment, The Vγ domain is the human Vγ4 domain.

[0013] In one embodiment, the Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence. In one embodiment, the Vγ domain CDR3 contains a human Vγ4 CDR3 sequence. The CDR3 domain contains a human Vγ2 CDR3 sequence. In one embodiment, the Vγ domain CDR3 is Contains the Vγ7 CDR3 sequence. In one embodiment, the J region is the Vγ J region. One embodiment Then the J region is the mouse Vγ J region. In one embodiment, the J region is from sequence numbers 15-18. Includes an array sequence selected from the group.

[0014] In one embodiment, the BTNL3 / 8 targeting component of the protein construct is further paired It includes a Vδ domain. In one embodiment, the Vγ domain and the Vδ domain are at least 1 They are covalently bonded by two disulfide bonds. In one embodiment, the Vγ domain and V The δ domains are paired by specific heterodimeric interactions. In one embodiment, the heterodimeric interaction is leucine zipper complementarity. The target component includes SEQ ID NO: 9. In one embodiment, the target component is SEQ ID NO: 1 It is 0. In one embodiment, the target component is SEQ ID NO: 11. In one embodiment, The target component includes single-strand in-frame fusions of the Vγ and Vδ domains. In one embodiment, the Vγ domain is the N-terminus to the Vδ domain. The in is the C-terminus to the Vδ domain. In one embodiment, the Vγ domain and the Vδ domain A single-stranded in-frame fusion includes an internal linker sequence. In one embodiment, the Vδ domain This is a human Vδ domain. In one embodiment, the human Vδ domain is Vδ1, Vδ2, or Vδ5. Yes, in one embodiment, the human Vδ domain is Vδ1.

[0015] In one embodiment, the protein construct further comprises the constant region of the first T cell receptor, The constant region of the first T cell receptor is in-frame fused to the C-terminus of the Vγ domain. In this implementation, the constant region of the first T cell receptor is the constant region of the human T cell receptor. In this embodiment, the first T cell receptor constant region is the human T cell receptor β constant region. In one embodiment, the first T cell receptor constant region is the human T cell receptor α constant region. In one embodiment, the first T cell receptor constant region is the human T cell receptor γ constant region. The target component further includes the constant region of the second T cell receptor, and that constant region of the second T cell receptor The region is in-frame fused to the C-terminus of the paired Vδ domain. In one embodiment, The second T cell receptor constant region is the human T cell receptor α constant region. In one embodiment, The second T cell receptor constant region is the human T cell receptor β constant region. In one embodiment, The constant region of the T cell receptor is the constant region of the human T cell receptor δ. In one embodiment, Vδ In-frame fusion of the domain and the constant region of the second T cell receptor is performed by the Vδ domain and the second T cell It includes an internal linker sequence between the receptor constant region and the receptor.

[0016] In one embodiment, the payload is a protein in frame fused to the target component. It is a qualitative payload. In one embodiment, the payload is a polypeptide. In one embodiment, the payload is a peptide. In one embodiment, the payload is a cytokine. Yes. In one embodiment, the payload is an antibody. In one embodiment, the payload is This is a variable fragment (scFv). In one embodiment, the antibody is specific to the cytokine antigen. It includes at least one antigen-binding site (ABS). In one embodiment, the antibody is specific to the CD3 antigen. It contains at least one antigen-binding site (ABS). In one embodiment, the antibody is used to destroy tumor necrosis It comprises at least one ABS specific to factor α (TNFα) antigen. In one embodiment, the antibody is F It contains an Fc domain capable of interacting with the c receptor. In one embodiment, the antibody interacts with the Fc receptor. It contains non-interacting Fc domains. In one embodiment, the payload is a small molecule. In one embodiment, the payload is a hormone. In another embodiment, the payload is a nucleic acid. In one embodiment, the payload is inhibitory RNA (RNAi).

[0017] In one embodiment, an arbitrary linker is in-frame fused to a peptide of the target component. In one embodiment, any linker is in-framed to the C-terminus of the target component. They are fused. In one embodiment, any linker enters the N-terminus of the target component. The frame is fused. In one embodiment, any linker converts to the target component. It is a jugated molecule.

[0018] In some embodiments, this specification describes any of the above protein constructs and This is a pharmaceutical composition containing a carrier that is acceptable as a pharmaceutical. In one embodiment, the pharmaceutical composition The substance is suitable for parenteral administration. In one embodiment, administration is intravenous. In one embodiment, administration is intramuscular. In another embodiment, administration is subcutaneous.

[0019] In one embodiment, the gastrointestinal tissue expressing BTNL3 / 8 is described herein. A method for treating the condition of a tubular system, comprising a therapeutically effective amount, according to any one of claims 62 to 66. A method comprising administering a pharmaceutical composition to a patient in a condition in which the gastrointestinal tissue expresses BTNL3 / 8. Yes. 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. The anti-inflammatory agent is a nonsteroidal anti-inflammatory drug. In one embodiment, the anti-inflammatory agent is an anti-inflammatory drug. It is a tokine. 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 prednisone. Glucocorticoids are hydrocortisone. In one embodiment, the payload is immunosuppressive It is a stimulant.

[0020] In some embodiments, what is described herein is a therapeutically effective amount of any of the above-described amounts. A treatment for inflammatory bowel disease, comprising administering a pharmaceutical composition to a patient with inflammatory bowel disease. In one embodiment, inflammatory bowel disease is ulcerative colitis. In one embodiment, inflammatory bowel The disease is Crohn's disease. In one embodiment, the payload of the protein construct is anti-inflammatory. It is an agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, The anti-inflammatory agent is nonsteroidal anti-inflammatory. In one embodiment, the anti-inflammatory agent is an anti-inflammatory steroid. Tokine, optionally interleukin-10 (IL-10), interleukin-22 (IL-22), or trans It is forming growth factor β (TGFβ). In one embodiment, the anti-inflammatory drug payload is anti It is a pro-inflammatory agent. In one embodiment, the anti-inflammatory agent payload is a steroid. In the application, the steroid is a glucocorticoid. In one embodiment, glucocorticoid The glucocorticoid is prednisone. In one embodiment, the glucocorticoid is hydrocortisone. In one embodiment, the payload of the protein construct is an antibiotic. In this embodiment, the antibiotic payload is rifaximin, ciprofloxacin, and metronidazole. The substance is dazole, moxifloxacin, or amoxicillin. In one embodiment, the protein The payload of the quality construct is a calcinurin inhibitor. In one embodiment, calcinurin The phosphorus inhibitor is cyclosporine A or tacrolimus. In one embodiment, the protein The payload of the construct is an immunomodulator. In one embodiment, the immunomodulator is immunosuppressive. It is a drug. In one embodiment, the immunosuppressant is azathioprine, 6-mercaptopurine, mercaptopurine, It is totrexate or thiopurine. In one embodiment, the protein construct is paid The do is a protein payload. In one embodiment, the protein payload is an antibody. , an antibody fragment or a single-stranded variable fragment. In one embodiment, the protein payload is TN ABS that is specific to the Fα antigen and includes at least one ABS. In this embodiment, the protein payload is adalimumab, infliximab, or certolizum It includes the complementarity-determining region (CDR). In one embodiment, the protein payload is an interface - Contains at least one ABS specific to the leukin antigen. In one embodiment, interlo The enzyme is IL-12, IL-23, or a combination thereof. In one embodiment, the protein The quality payload contains a CDR of ustekinumab or brikinumab. In one embodiment, the biologic payload is specific to the integrin antigen, and at least Contains one ABS. In one embodiment, the integrin is α4 integrin. In the administration method, the protein payload is infliximab, natalizumab, or vedolizumab. Includes a CDR of B. In one embodiment, the protein construct includes an analgesic payload. In this embodiment, the protein construct includes a nutritional supplement payload.

[0021] In some embodiments, what is described herein is a therapeutically effective amount of any of the above-mentioned pharmaceuticals. A treatment for irritable bowel syndrome, comprising administering a composition to patients with irritable bowel syndrome. In some embodiments, what is described herein is a therapeutically effective amount of any of the above medical A treatment for diverticulitis comprising administering a drug composition to a patient with diverticulitis. One embodiment Then, the payload is an antibiotic. In one embodiment, the antibiotic payload is a referral Ximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin It's phosphorus.

[0022] In some embodiments, what is described herein is a therapeutically effective amount of any of the above-mentioned pharmaceuticals. A treatment for celiac disease, comprising administering a composition to a patient with celiac disease. In one embodiment, the payload is an immunosuppressant. In another embodiment, the immunosuppressant is A These are zathioprine, 6-mercaptopurine, methotrexate, or thioprine.

[0023] In some embodiments, what is described herein is a therapeutically effective amount of any of the above-mentioned pharmaceuticals. A treatment for microbial infection, comprising administering a composition to a patient with a microbial infection. In one embodiment, the payload is an antimicrobial agent. In another embodiment, the antimicrobial agent is an antiparasitic agent. These are agents, antibiotics, antifungal agents, or antiviral agents.

[0024] In some embodiments, what is described herein is a therapeutically effective amount of any of the above-mentioned pharmaceuticals. The composition is administered to patients with metabolic disorders or metabolic deficiencies. It is a treatment for sexual deficiency. In one embodiment, the payload is a nutritional supplement. In one embodiment In this embodiment, the nutritional supplement is an enzyme or a vitamin.

[0025] In some embodiments, what is described herein is a therapeutically effective amount of any of the above-mentioned pharmaceuticals. A method for controlling the immune system, comprising administering a composition to a patient with immune-related symptoms. In one embodiment, the payload is an immunosuppressant. In one embodiment, the immunosuppressant is a bruise Thioprine, 6-mercaptopurine, methotrexate, or thiopurine. In one embodiment, the payload is an immunostimulant. It is. [Brief explanation of the drawing]

[0026] (5. Brief explanation of the drawing) [Figure 1] The amino acid sequence alignments of the variable (V) domains of human Vγ4 (hVγ4), human Vγ2 (hVγ2), and mouse Vγ7 (mVγ7) are shown. The amino acid position numbering shown herein is based on the complete T cell receptor (TCR) protein sequence, including an 18-amino acid leader sequence (not shown). Dashes at positions 46 and 117 in the alignment indicate gaps in the mVγ7 sequence introduced to optimize the alignment. CDR regions are shown in bold font. CDR1 is located at amino acid positions 45–50 of the alignment. CDR2 is located at amino acid positions 68–75 of the alignment. The first 5 amino acids of CDR3 are located at amino acid positions 114–118 of the alignment. CDR4 is located at amino acid positions 85–100 of the alignment. When the underlined amino acids in human Vγ4-CDR4 located at alignment positions 87 and 90 are replaced with the corresponding amino acids in hVγ2, the function is disabled (see Figure 7).

[0027] [Figure 2]This paper describes the experimental approach used to identify, clone, and test γδ T cell receptors isolated from responsive interepithelial lymphocytes (IELs).

[0028] [Figure 3] Figure 3A is a diagram of the lentiviral vector backbone used to express the isolated IEL-derived γδTCR variable domain in the TCR construct within TCR-deficient Jurkat cells.

[0029] Figure 3B shows the cloned C from the γδTCR variable domain in TCR-deficient Jurkat (J76 cells). This shows the expression of a TCR construct containing a DR3 pair 72 hours after transduction.

[0030] [Figure 4] Figure 4A shows an example of the BTNL3 / 8-induced response in J76 cells transduced with Vγ4Vδ1. Positive controls to anti-CD3 stimulation are also shown (against isotype controls).

[0031] Figure 4B shows that the three independent Vγ4Vδ1-transformed J76 lines differ from the Vγ9Vδ2 line. This indicates a response to BTNL3 / 8-expressing cells. B3, C11, and H7 were obtained using the method shown in Figure 2. This represents different CDR3 pairs.

[0032] [Figure 5]This shows the multiplicative change (FC) of CD69 expression percentage in transdextrinsed cells (+ve cells) normalized to cells expressing an empty vector (EV), and the percentage of TCR downregulation in J76 cells expressing Vγ4TCR or Vγ2TCR. When the entire V domain of the responsive Vγ4 H7 TCR was replaced with the Vγ2 coding sequence (Vγ2H7) (without substituting CDR3γ and the entire delta chain), TCR activation by BTNL3 / 8-expressing cells was lost. However, when CDR1 (H7 CDR1Vγ2) and / or CDR2 (H7 CDR2Vγ2) of the responsive Vγ4 H7 TCR were replaced with the Vγ2 coding sequence, TCR activation by BTNL3 / 8-expressing cells was maintained.

[0033] [Figure 6] Figure 6A shows the V-domain sequence alignment of portions of human Vγ2 and human Vγ4. CDR1, CDR2, and CDR3 are indicated within shaded squares. A total of nine (9) amino acids are different. Four different amino acids are located within framework region 3 between CDR2 and CDR4, and are defined herein as "CDR4".

[0034] Figure 6B shows variable domains and Vγ5 / Vδ1 pairs aligned using Cn3D. The already published structure of the paired variable domains is shown. The CDR4 region is separated from the conventional CDR. They form appropriate loops that cannot be separated. Of particular note are the Vγ4 CDR4 loop and the Vγ5 CDR4 loop. The key difference between the two is the significant difference in three-dimensional structure.

[0035] [Figure 7]This report shows the multiplicative change (FC) of CD69 expression percentage in transdextrinsed cells (+ve cells) normalized to cells expressing an empty vector (EV), as well as the percentage of TCR downregulation in J76 cells expressing Vγ4TCR (H7 WT), Vγ2TCR with H7 CDR3 (Vγ2 H7), and Vγ4TCR with amino acid substitutions in CDR4. YA substitutions at amino acid positions 87 and 90 resulted in a loss of TCR activation by BTNL3 / 8-expressing cells, while NL substitutions at amino acid positions 94 and 98 did not result in a loss of TCR activation by BTNL3 / 8-expressing cells.

[0036] [Figure 8] Figure 8 is a schematic diagram of the design of a polypeptide chain of a soluble heterodimeric γδTCR in an embodiment of the present invention, in which two chains heterodimerize by leucine zipper complementation. The Vγ domain or the Vδ domain is in-frame fused to a TCRα or TCRβ constant region lacking a transmembrane domain, followed by a leucine zipper sequence and a histidine tag / linker. The Vγ-containing polypeptide and the Vδ-containing polypeptide were expressed and dimerized post-translation.

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

[0038] Figure 9B shows the BTNL3 and BTNL8 constructs or blank vectors after parallel staining with anti-FLAG and anti-HA antibodies. The flow cytometry results of HEK293T cells transduced using the following method are shown.

[0039] [Figure 10]Figure 10 shows the staining of the soluble TCRs constructed as described in Figure 8. The Vγ4Vδ1 and Vγ4Vδ2 soluble TCRs show strong binding to cell lines expressing BTNL3+BTNL8, but not to empty vector (EV) control cell lines.

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

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

[0042] [Figure 12] This shows the time course of internal translocation of soluble TCRs at 37°C in BTNL3+BTNL8 expressing cells.

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

[0044] Figure 13B shows the fluorescence of cells incubated with anti-BTNL3 antibody compared to soluble TCRs. It shows a decrease.

[0045] [Figure 14] This is a schematic diagram illustrating a method for evaluating payload delivery by soluble TCRs.

[0046] [Figure 15]Figure 15A shows experimental results evaluating the internal translocation of soluble TCR+α-His antibody complexes within cells expressing BTNL3 and BTNL8 constructs. Figure 15B is a graph showing the residual fluorescence signal after trypsin treatment in cells incubated with the soluble TCR+α-His antibody complexes at 4°C and 37°C.

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

[0048] Figure 16B shows the soluble TCR+α-His antibody complex incubated at 4°C in DMEM or TRY The imaging cytometry results of psin-treated 293T.L3L8 cells are shown.

[0049] Figure 16C shows the soluble TCR+α-His antibody complex incubated at 37°C with DMEM or t The imaging cytometry results of liposin-treated 293T.L3L8 cells are shown.

[0050] The above drawings illustrate various embodiments of the present invention for illustrative purposes only. Those skilled in the art will see below. From the considerations, the present invention described herein can be constructed without departing from the principles of the present invention as described herein. It will be readily apparent that alternative embodiments of the construction and method can be adopted. [Modes for carrying out the invention]

[0051] (6. Detailed description) (6.1.Definition) Unless otherwise specified, all technical and scientific terms used herein are in accordance with the present invention. It has the meaning that is commonly understood by those skilled in the art to which it belongs. The word has meanings based on the following:

[0052] As used herein, "protein construct" refers to a protein construct comprising at least two functional elements, B This shows one or more polypeptide chains containing components and payloads that target TNL3 / 8. The payload may or may not be a protein payload.

[0053] As used herein, "BTNL3 / 8" refers to butyrophyllin protein 3 (BTNL3) and butyrophyllin This indicates the filin protein 8 (BTNL8) protein. "BTNL3 / 8" refers to BTNL3 without BTNL8, and BTN. It may also exhibit BTNL8 without L3, or a heterodimer of BTNL3 and BTNL8.

[0054] As used herein, "components targeting BTNL3 / 8" refers to components that specifically bind to BTNL3 / 8. This shows a child. In one embodiment, the component that targets BTNL3 / 8 is an antigen-binding protein. In one embodiment, the component targeting BTNL3 / 8 is at least one of the Vγ domain polypeptides. This is only a part of it.

[0055] As used herein, the "Vγ domain" refers to the variable domain of the T cell receptor (TCR) γ chain. This Vγ domain comprises the J region and the complementarity-determining regions (CDRs), CDR1, CDR2, CDR3, and CDR4. Includes. The numbering of Vγ domain residues is shown in Figure 1, but in the figure, residue 19 is after signal sequence cleavage. This is the N-terminal amino acid of the mature Vγ domain. Residues for the Vγ domain not shown in Figure 1. The numbering is assigned after the best alignment to the sequence in Figure 1. A "corresponding residue" is an amino acid located at the same numbered position as the residue that was identified as the corresponding residue. .

[0056] As used herein, "Vγ domain CDR4" refers to the Vγ domain located between the CDR2 and CDR3 regions. This refers to the 16-amino acid sequence within the γ domain, corresponding to amino acid sequence positions 85-100 in Figure 1. The amino acid sequence of CDR4 in the human Vγ4 domain has the sequence of SEQ ID NO: 3. The amino acid sequence of the main CDR4 is sequence number 5. The amino acid sequence of CDR4 in the mouse Vγ7 domain. The acid sequence is sequence number 4.

[0001] As used herein, “payload” refers to the target cells of interest (e.g., BTNL3 / 8) The payload is one of the molecules delivered to the expressing cells and / or intestinal epithelial cells. nucleotides, nucleotides (for example, containing detectable components or toxins, or transcriptions) Nucleic acids such as the nucleotides that inhibit DNA and RNA (e.g., mRNA, RNAi, miRNA, siRNA, snRN) A, snoRNA, piRNA, exRNA, scaRNA and lncRNA), amino acids (e.g., detectable components or (These include toxins or amino acids that inhibit translation), polypeptides (e.g., enzymes, biologics) Lipids, carbohydrates, small molecules (e.g., small molecule drugs and small molecule toxins), and combinations thereof. It may include: In one embodiment, the payload is a therapeutic agent. The therapeutic agent may include: While not limited to these, chemotherapy drugs, contrast agents (e.g., radioisotopes), and immunomodulators are also included. (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., This includes cytotoxic agents. In one embodiment, the payload is an antibody.

[0057] As used herein, "linker" refers to a functional element of a protein construct (e.g., This indicates any molecule that can be used to bind the target component and payload. In one embodiment, the linker is a functional element of the protein construct (e.g., target and To enable site-specific conjugation of molecules to the components and payload. It can be used. In one embodiment, the linker can link the protein construct in vitro or in vitro. It can be used for identification or detection.

[0058] As used herein, "peptide linker" refers to a linker that is a polypeptide. In one embodiment, the peptide linker is a protein construct (e.g., a target component and It is integrated in-frame into the functional elements of the payload. In one embodiment, Petit Drinker is a site-specific conjugate of molecules to elements of protein constructs. It enables the peptide linker to fuse with functional elements. If it enables the required three-dimensional structure, any length and type of amino acid sequence is acceptable. stomach.

[0059] As used herein, the “internal linker” has at least one at both its N-terminus and C-terminus. The polypeptide sequence is shown to covalently bond with two additional polypeptides. In one embodiment, The internal linker targets the component (for example, the internal linker between the Vγ domain and the Vδ domain). The internal linker is a polypeptide chain within a linker. The internal linker is an amino acid sequence of any length and type. However, that is fine, but the additional polypeptides that the internal linkers fuse with form the required three-dimensional structure. It is something that can maintain the structure.

[0060] As used herein, “antibody” means having at least one antigen-binding site (ABS) It also contains one antibody protein construct containing an antibody variable domain. The antibody is as follows: While not limited to these, molecules consisting only of variable domains, single-stranded variable fragments (scFv), single-stranded Fab fragments (scFab), bispecific antibodies, hybrid IgG, Fab fusion proteins, Fc-modified IgG, Additive IgG, diabodies, single-stranded diabodies, DART, tandem diabodies (tandAb), and Includes mini body.

[0061] As used herein, "single-strand in-frame fusion" refers to single-strand in-frame fused T cells. It exhibits a receptor variable domain (scTv) and at least two polypeptide variable domains of the T cell receptor. At least a portion of the in is generated as a single-stranded fusion polypeptide chain, and its variable domain The sequences are in-frame fused. Single-strand in-frame fused T cell receptor variable domain ( scTv) may contain more than one variable domain and / or constant region, and T-fine of any origin. It may also be paired with a vesicular receptor. Therefore, scTv has two or more variable domains in frame Includes fused tandem scTv.

[0062] As used herein, "antigen-binding sites" (ABS) specifically target a given antigen or epitope. This indicates the region of the antibody molecule that recognizes and binds to a specific antigen or epitope. It is said to bind with a special affinity. The term "affinity" as used herein refers to a special affinity between a molecule and another molecule. This indicates the strength of non-covalent intermolecular force interactions between molecules. Affinity, i.e., the strength of the interaction. The dissociation equilibrium constant (K D It can be expressed as, low K D The value indicates strong intermolecular interactions. This shows the K of the antibody construct. DThe value is measured by methods well-known in the art and is not limited to, but includes, biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays. The affinity between the ABS and its cognate antigen or epitope is 10 M, 10 M, 10 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or less than 10 -10 M of K D value.

[0063] As used herein, "small molecule" refers to a molecule having a low molecular weight of less than 900 daltons and does not include peptides. Small molecules can potentially regulate biological processes and include organic molecules having a size on the order of 1 nm.

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

[0065] As used herein, "anti-inflammatory agent" refers to any molecule or agent that inhibits the activity or expression of pro-inflammatory cytokines. Anti-inflammatory agents include inhibitors of pro-inflammatory cytokines, which include, but are not limited to, interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor α (TNFα), interferon γ (INF-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Contains colony-stimulating factors. Anti-inflammatory agents include soluble tumor necrosis factor receptor p55, soluble tumor necrosis Antiinflammatory agents such as death factor, p75, soluble IL-1 receptor type II, and IL-18 binding protein It contains soluble cytokine receptors. Anti-inflammatory agents also induce inflammation such as membrane-bound IL-1 receptor type II. This also includes cytokine receptors that lack intracellular signaling pathways that compete with sex cytokine receptors.

[0066] As used herein, “immunomodulators” alter the immune response and / or activity of cells in the immune system. This indicates any of the molecules that cause the reaction. In one embodiment, the immunomodulator is an immunosuppressant or an immunomodulator. It is an activator. An "immunosuppressant" reduces the immune response and / or activity of cells in the immune system. This refers to any molecule or drug, while "immunosuppressant" refers to the immune response of cells in the immune system and / Alternatively, it indicates any molecule or drug that increases activity.

[0067] As used herein, "immune-related symptoms" refers to any symptoms associated with changes in the activity of the immune system. It may also present with symptoms or disorders. Immune-related symptoms include symptoms associated with an increase or decrease in the immune response. Immune-related symptoms include, but are not limited to, autoimmune diseases, inflammatory symptoms, and allergies. This includes dysphagia, immunodeficiency, hematopoietic carcinoma, and other hematopoietic disorders.

[0068] As used herein, “inflammatory symptoms” refers to symptoms associated with increased or present inflammation of inflammatory tissue. These symptoms or disorders may also be present. Inflammatory symptoms include, but are not limited to, asthma, atelolysis. Arteriosclerosis, 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, mast cell disease, otitis, pelvic inflammatory disease, reperfusion injury, This includes rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, transplant rejection, and vasculitis.

[0069] As used herein, “gastrointestinal symptoms” refers to symptoms related to any tissue of the gastrointestinal system. These symptoms or disorders may also be present. Gastrointestinal symptoms are not limited to the following, but include gastrointestinal symptoms. Immune-related symptoms, gastrointestinal inflammation, microbial infections of gastrointestinal tissues, and feeding disorders, metabolic This includes symptoms caused by sexual disorders or metabolic deficiencies. Gastrointestinal symptoms are not limited to the following: It is not something that can be treated, but it can be used for inflammatory bowel disease, celiac disease, irritable bowel syndrome, diverticulitis, and Crohn's disease. Diseases, including cancers (e.g., colon cancer, rectal cancer, stomach cancer).

[0070] As used herein, the terms “treatment” or “therapy” refer to therapeutic treatments and preventive or protective treatments. The measures described are intended to prevent undesirable conditions such as the progression of multiple sclerosis, arthritis, or cancer. It is to protect against or slow down (reduce) physiological changes or disorders. The results may be either detectable or undetectable, but are not limited to the following: Relief of symptoms, reduction in disease extent, stable (i.e., non-worsening) disease state, disease progression This includes delay or blunting of symptoms, improvement or remission of symptoms, and (local or global) remission. "Treatment" It also has the effect of extending survival compared to the survival predicted if no treatment is received. Those who need treatment will be treated in the same way as those who already have the symptoms or disability. This also includes people who are prone to causing harm, or those who should take precautions against their symptoms or disorders.

[0071] "Subject," "individual," "animal," "patient," or "mammal" refers to all subjects. In particular, it refers to mammalian subjects for which diagnosis, prognosis, or therapy is required. This includes humans, or dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows. This includes other domestic animals, livestock, zoo animals, exercise animals, or pets. nothing.

[0072] The term "sufficient amount" refers to an amount sufficient to produce the desired effect, for example, intracellular protein This means a sufficient amount to regulate cohesiveness.

[0073] The term "therapeutic effective dose" refers to the amount that is effective in improving the symptoms of a disease. Prevention is therapy and Because it can be considered as such, the therapeutically effective dose may also be the "prophylactically effective dose."

[0074] The term “recombinant human γδTCR protein” or any derivative thereof as used throughout this specification The expression is derived from a standard genetic model derived from a human γδTCR sequence or its functional derivative or homolog. This shows any recombinant protein produced by such engineering methodologies. Proteins can also contain additional fusion elements such as dimerization domains. These non-limiting examples include fusion and transmembrane bonding to support the correct folding of TCRs. Fusion to act as an inductor (for example, supporting the precise presentation of TCRs on the surface of the cell membrane). For this purpose, or to extend the half-life or increase the size (e.g., human blood This includes a clear albumin fusion domain or a payload fusion as otherwise described herein. These additional fusion elements are derived from the γδTCR sequence or its derivatives and homologs. It can be generated in a column, and separately, this fusion sequence may have a non-TCR origin.

[0075] The terms “recombinant γδTCR sequence” or “recombinant human TCR live” are used throughout this specification. The terms "Lari" or "recombinant human TCR panel" or derived terms from them may not be more than one. A collection of recombinant human γδTCR proteins, or 2, 3, or 4, or This shows a collection of 5 or more recombinant human γδTCR proteins. The molecule may also contain a collection of different sequences in at least one amino acid. This collection of recombinant TCRs can also be presented in soluble form. Alternatively, For display purposes, this collection also includes inorganic or organic materials (not limited to the following examples). This includes "beads," "plates," "columns," or "phages." They can also be combined, fused, or bound together. Alternatively, such a collection is also without On one membrane, such as those found in wounded or living cells, or on a non-living membrane such as a micelle or This can also be presented or displayed on multiple membrane collections. When expressing and presenting a reaction on one or more living cells, typically an allogeneic expression vector is used. It also generates a collection. Using this collection of homogeneous expression vectors, first 1 Alternatively, multiple cells may be manipulated to obtain this recombinant human γδTCR protein, or the rhynchoplasmic regeneration of this protein. Display an ibrary, panel, or collection and this TCR protein It is possible to create a library of cells that express the drug.

[0076] The terms “like-type bonding partner” or “like-type bonding partner candidate” as used throughout this specification, or Derived terms derived from these refer to the binding of recombinant human γδTCR protein in a sequence-specific manner. This then indicates the identified protein or its derivative. Discovery of such a homogeneous binding partner or For screening or validation, they are used in their natural environment (e.g., one or more small details). They can be presented or displayed on the surface of cells. As extracts or secretions from cells, as purified derivatives thereof, or in recombinant form, It may be presented or displayed.

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

[0078] In this disclosure, "comprises", "comprising", "equipped with" containing), having, includes, contains "including)" and its linguistic variations have the meanings set forth in U.S. Patent Law, and that This allows for the existence of additional elements beyond those explicitly listed.

[0079] The ranges provided herein are abbreviations of all values ​​within the range, including the enumerated endpoints. It is understood that the range from 1 to 50 is 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 It consists of 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. It is understood to include any number, combination of numbers, or subrange from the group being defined.

[0080] Unless otherwise specified or evident from the context, the term "or" as used herein is inclusive. It is understood to be the case. Unless otherwise specified or evident from the context, the term used herein is used in this specification. The terms "a," "an," and "the" are understood to be singular or plural. It will be done.

[0081] Unless otherwise specified or evident from the context, the term “about” as used herein refers to the technology It is understood to be within the normal tolerance range for the field, for example, within two standard deviations of the mean. "Approximately" refers to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0.5% of the stated value. It can be understood as being within the range of 0.1%, 0.05%, or 0.01%. Unless otherwise clear from the context, the original meaning is... All figures provided in the details are subject to modification by the term "approximately".

[0082] (6.3. Protein constructs containing components that target BTNL3 / 8) In the first embodiment, a protein construct is provided. This protein construct is BTNL3 / 8 A component that targets the payload, and any component that binds the target component to the payload. Includes "nker".

[0083] (6.3.1.1 Components that target BTNL3 / 8) Components that target BTNL3 / 8 specifically target human BTNL3, human BTNL8 and / or human BTNL3 / 8. It binds to the heterodimer. In one embodiment, the component targeting BTNL3 / 8 is further described below. As described in detail, at least a portion of the T cell receptor (TCR) Vγ domain polypeptide In another embodiment, the component targeting BTNL3 / 8 is the antigen-binding site of the antibody. In this embodiment, the component that targets BTNL3 / 8 is the component that targets BTNL3 / 8 When coupled to BTNL3 / 8, it inhibits or partially inhibits the function of BTNL3 / 8. In one embodiment, BTNL3 / 8 When a component that targets BTNL3 / 8 binds to BTNL3 / 8, the BTNL3 / 8 To stimulate or activate the function. In one embodiment, a component that targets BTNL3 / 8 is used to target its BTNL3 When a component targeting / 8 binds to the BTNL3 / 8 heterodimer, the BTNL3 / 8 heterodimer function It inhibits or partially inhibits the BTN. In one embodiment, a component that targets BTNL3 / 8 targets its BTN When a component targeting L3 / 8 binds to the BTNL3 / 8 heterodimer, the BTNL3 / 8 heterodimer mechanism... To stimulate or activate the brain.

[0084] (6.3.1. TCRγ variable domain) In one embodiment, the component targeting BTNL3 / 8 is a T cell receptor (TCR) Vγ domain polyp It contains at least a portion of the ptide. In typical embodiments, the component that targets BTNL3 / 8 is Vγd Includes the main polypeptide.

[0085] In one embodiment, the component targeting BTNL3 / 8 includes a CDR4 region derived from Vγ4. In the application method, the component targeting BTNL3 / 8 contains a Vγ domain, and the sequence position of that Vγ domain The amino acid at position number 87 is aspartic acid or histidine, and the sequence position number of the Vγ domain The amino acid of no. 90 is glycine or glutamic acid, and the remaining residues of Vγ CDR4 are, respectively At this position, it is independently selected from the corresponding residue in the human or mouse (murine) Vγ domain.

[0086] In one embodiment, the remaining residues of the Vγ domain CDR4 are human Vγ4 at their respective positions. The residue is independently selected from the corresponding residues of human Vγ2 or mouse Vγ7. In some embodiments, All remaining residues in the Vγ domain CDR4 correspond to human Vγ4, human Vγ2, or mouse Vγ7. Selected from the base. In one embodiment, all remaining residues of Vγ CDR4 are the corresponding residues of human Vγ4. Selected from the base. In one embodiment, all remaining residues of Vγ CDR4 are the corresponding residues of human Vγ2. Selected from the base. In one embodiment, all remaining residues of Vγ CDR4 correspond to mouse Vγ7. Selected from residues. In one embodiment, the amino acid sequence of positions 87-90 in the Vγ domain. This is sequence number 1. In one embodiment, amino acids at positions 87-90 of the Vγ domain The column is sequence number 2.

[0087] In one embodiment, the Vγ domain is the Vγ domain sequence shown in Figure 1. In one embodiment, the Vγ domain is a human Vγ domain. It is the γ4 domain. In one embodiment, the Vγ domain is the human Vγ2 domain, and within it So, the amino acid in CDR4 is substituted with aspartic acid or histidine at amino acid sequence position 87. Furthermore, it is substituted with glycine or glutamic acid at amino acid sequence position 90.

[0088] In one embodiment, the Vγ domain is human Vγ3 or human Vγ5. In a special embodiment, The Vγ domain is human Vγ3 or human Vγ5, and within that, the amino acid of CDR4 is amino acid Substituted with aspartic acid or histidine at sequence position 87, and at amino acid sequence position 90 It is substituted with glycine or glutamic acid. In one embodiment, the Vγ domain is human Vγ This is a human Vγ domain with at least 70% sequence identity to 4.

[0089] In one embodiment, the Vγ domain is a non-human mammalian Vγ domain. Yes. In one embodiment, the Vγ domain has at least 70% identity with human Vγ4. This is a human mammalian Vγ domain sequence.

[0090] In some embodiments, the Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence. In one embodiment, the Vγ domain CDR3 contains a human CDR3 sequence. In a special embodiment, V The γ domain CDR3 contains the human Vγ4 CDR3 sequence. In a specific embodiment, the Vγ domain CDR3 is , containing a human Vγ2 CDR3 sequence. In one embodiment, the Vγ domain CDR3 is a non-human mammalian CD It includes the R3 sequence. In one embodiment, the Vγ domain CDR3 includes the mouse Vγ7 CDR3 sequence.

[0091] In some embodiments, the J region is the Vγ J region. In some embodiments, the J region is the human V This is the γ J region. In one embodiment, the J region is the mouse Vγ J region. The J region contains a polypeptide sequence selected from the group consisting of sequence numbers 15 to 18.

[0092] (6.3.1.1.1 Pair with Vδ) In one embodiment, the BTNL3 / 8 targeting component of the protein construct is further paired It does not contain the Vδ domain. In one embodiment, the BTNL3 / 8 of the protein construct is targeted. The component contains a Vδ domain paired with at least one additional Vγ domain. In the application method, the component targeting BTNL3 / 8 is the Vγ4 homodimer. In one embodiment, The component of the protein construct that targets BTNL3 / 8 further contains paired Vδ domains. In one embodiment, the Vδ domain is a human Vδ domain. The domains are Vδ1, Vδ2, Vδ3, Vδ5, or Vδ8. In one embodiment, human Vδ The main domain is Vδ1. In one embodiment, the Vδ domain is a non-human mammalian Vδ domain. ru.

[0093] ((a) Heterodimer format) In some embodiments, the Vδ domain is a heterodimer of the first and second polypeptides. They are paired by -ation, and one of the polypeptides contains a Vγ domain, One of the polypeptides contains a Vδ domain.

[0094] Heterodimeric interactions involve multiple polypeptides, including Vγ and Vδ domains. This may include shared and / or non-shared interactions between them. In a typical embodiment, The Vγ and Vδ domains form more homodimers than heterodimers. They are paired by orthogonal features that are difficult to distort.

[0095] In some embodiments, polypeptides comprising a Vγ domain and a Vδ domain are They are covalently bonded by at least one genetically modified disulfide bridge. The disulfide bridge formed when two or more domains associate is a non-native disulfide bridge. Provide non-endogenous cysteine ​​amino acids to two or more domains so that a bond is formed. This is an amino acid sequence. In some of these embodiments, at least one disulfide The crosslinks are designed within the Vγ and Vδ domains. In some embodiments, at least Another disulfide bridge is a variable region and a steady region fused in frame, It is designed within a domain outside the variable region.

[0096] In some embodiments, heterodimeric interactions are leucine zipper complementar. .

[0097] In one embodiment, one or more polypeptides of the paired Vγ / Vδ heterodimer are further It includes a T cell receptor constant region. In one embodiment, the first T cell receptor constant region is paired It is in-frame fused to the C-terminus of the Vγ domain. In one embodiment, the first T-cell The constant region of the cell receptor is the constant region of the human TCR. In one embodiment, the constant region of the first T cell receptor is The region is the human TCRβ constant region. In one embodiment, the first T cell receptor constant region is the human TCR This is the α constant region. In one embodiment, the first T cell receptor constant region is the human TCRγ constant region. Yes. In one embodiment, the paired Vγ / Vδ heterodimer polypeptide is further, It contains the constant region of the second T cell receptor, and this constant region of the second T cell receptor is paired with a Vδ domain It is in-frame fused to the C-terminus of the molecule. In one embodiment, the constant region of the second T cell receptor. This is the human TCRα constant region. In one embodiment, the second T cell receptor constant region is human TCRβ This is the constant region. In one embodiment, the second T cell receptor constant region is the human TCRδ constant region. ru.

[0098] In some embodiments, in-frame fusion of the Vγ domain with the first constant region occurs. It includes an internal linker sequence between the domain and the first TCR constant region. In some embodiments, In-frame fusion of the Vδ domain with the constant region of the second TCR is performed by the Vδ domain and the second T cell receptor. It includes an internal linker sequence between the constant-state region and the state.

[0099] In one embodiment, the component targeting BTNL3 / 8 includes SEQ ID NO: 9. The component targeting BTNL3 / 8 includes SEQ ID NO: 10. In one embodiment, BTNL3 / 8 is targeted and The component includes SEQ ID NO: 11.

[0100] In one embodiment, the component targeting BTNL3 / 8 has more than one Vγ domain and / or Vδ Includes domains. In one embodiment, one or more Vγ domains and / or Vδ domains are It is polymerized. In one embodiment, more than one Vγ domain and / or Vδ domain In all or part of it, it is in-frame fused to the constant region of the T cell receptor. In one embodiment, , more than one multimerized Vγ domain and / or Vδ domain contains one or more internal linkers .

[0101] In a further embodiment, recombinant γδTCR constructs are provided. Therefore, in a further embodiment, Recombinant γδTCR proteins including sequence number 9 (optional, without C-terminal His tag) are provided. In another embodiment, a recombinant γδTCR protein containing SEQ ID NO: 10 is provided. This provides recombinant γδTCR protein including SEQ ID NO: 11 (optional, without C-terminal His tag). In another embodiment, a recombinant γδTCR protein containing SEQ ID NO: 12 is provided. The recombinant γδTCR protein containing sequence number 13 is provided.

[0102] ((b) Single-strand in-frame fusion) In one embodiment, the component targeting BTNL3 / 8 has a Vγ domain and a paired Vδ domain. Includes a main single-stranded in-frame fusion. In some embodiments, the Vγ domain is Vδ domain It is N-terminal relative to the main domain. In some embodiments, the Vγ domain is relative to the Vδ domain. And it is at the C-terminus. In various embodiments, the Vγ domain and Vδ domain are single-strand inflation The fusion includes an internal linker sequence.

[0103] In some embodiments, the Vδ domain is a human Vδ domain. In one embodiment, The human Vδ domain is Vδ1, Vδ2, or Vδ5. In one embodiment, the human Vδ domain is This is Vδ1. In one embodiment, the single-strand in-frame fusion is further connected to at least one T cell receptor. It includes a constant state region. In one embodiment, single-strand in-frame fusion is further administered to the first T cell receptor. It includes a constant state region, and its first T cell receptor constant region is inflated to the C-terminus of the Vγ domain. It is fused. In one embodiment, the first T cell receptor constant region is the human T cell receptor constant region. This is the constant region. In one embodiment, the first T cell receptor constant region is the human T cell receptor β constant region. This is a region. In one embodiment, the first T cell receptor constant region is the human T cell receptor α constant region. This is the region. In one embodiment, the first T cell receptor constant region is the human T cell receptor γ constant region. In one embodiment, single-strand in-frame fusion further extends to the constant region of the second T cell receptor. It includes, and its second T cell receptor constant region is inflated to the C-terminus of the paired Vδ domain. It is fused. In one embodiment, the constant region of the second T cell receptor is human T cell receptor α This is the constant region. In one embodiment, the second T cell receptor constant region is the human T cell receptor β constant region. This is the constant region. In one embodiment, the second T cell receptor constant region is the human T cell receptor δ constant region. This is the region. In one embodiment, inflation between the Vδ domain and the constant region of the second T cell receptor The fusion includes an internal linker sequence between the Vδ domain and the constant region of the second T cell receptor.

[0104] In one embodiment, the single-stranded in-frame fusion comprises more than one Vγ domain and / or Vδ domain. Includes domains. In one embodiment, one or more Vγ domains and / or Vδ domains are It is polymerized. In one embodiment, there is one or more polymerized Vγ domains and / or Vδ domains. The main includes one or more internal linkers. In one embodiment, there are more than one Vγ domain and / or all or part of the Vδ domain inflates into at least one T cell receptor constant region They are fused together.

[0105] (6.3.2. Antibody-based target components) In one embodiment, the component targeting BTNL3 / 8 specifically targets human BTNL3, human BTNL8 and The antibody comprises an antibody that binds to a human BTNL3 / 8 heterodimer. In one embodiment, the antibody is , full-length antibody fragments or antibody formats, and not limited to the following, Fab fragments One-sided, Fv, scFv, tandem scFv, diamond body, sc diamond body, DART, tandem diamond Di(tandAb), Minibody, Camel VHH, and other antibody fragments or fossils known to those skilled in the art. -Includes mat. Exemplary antibody and antibody fragment formats are from the literature of Brinkmann et al., MABS, This is described in detail in 2017, Vol. 9, No. 2, pp. 182-212, and the entire instruction is revealed by citation. It will be included in the detailed specifications.

[0106] In one embodiment, the antibody includes an Fc domain capable of interacting with an Fc receptor. Therefore, the antibody contains an Fc domain that is incapable of interacting with the Fc receptor. In one embodiment, the antibody It has one or more genetically modified mutations in the amino acid sequence of the antibody domain, which is intrinsically This reduces the effector function related to antibody binding. The functions are, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC). Also known as intercellular cytotoxicity, complement fixation (e.g., C1q binding), antibody-dependent cells Cellular mechanisms arising from Fc receptors that bind to the Fc portion of antibodies, such as phagocytosis (ADCP) and opsonization. Includes the ability. Genetic mutations that reduce effector function are included in U.S. Patent Publication No. 2017 / 0 References: Issue 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 literature by Oganesyan et al. (Acta Cristallograp) These are described in detail in hica D64 (2008) 700-704), and are fully incorporated herein by reference. The body is incorporated. In a special embodiment, the antibody is incorporated into the Fc portion of the ROR-binding molecule by the FcR receptor. The antibody domain, which reduces binding, contains one or more genetically modified mutations in its amino acid sequence. In some embodiments, the FcR receptor is the FcRγ receptor. In a particular embodiment, the FcR receptor The receptor is FcγRIIa and / or FcγRIIIA receptor. In a special embodiment, the effector One or more genetically modified mutations that reduce function are mutations within the CH2 domain of the antibody.

[0107] (6.3.2.1 Payload) This protein construct includes a payload.

[0108] In various embodiments, the payload may contain nucleotides, and further detectable components or toxins. mRNA encoding polypeptides such as nucleotides, DNA, and enzymes that contain or inhibit transcription molecules, other RNA molecules (e.g., RNAi, miRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRN) Nucleic acids such as A and lncRNA, amino acids (for example, containing detectable components or toxins, or translation amino acids that inhibit [something], polypeptides (e.g., enzymes, biologics), lipids, carbohydrates, oligosaccharides This may include offspring (e.g., small molecule drugs and small molecule toxins) and combinations thereof.

[0109] In one embodiment, the payload is a therapeutic agent. The therapeutic agent is not limited to the following: However, chemotherapy drugs, immunomodulators (e.g., cytokines, chemokines, or checkpoints) are not used. This includes inhibitors, hormones, and toxins (e.g., cytotoxic agents).

[0110] In one embodiment, the payload is an antibody. In another embodiment, the antibody is a CD3 antigen. It includes a specific antigen-binding site (ABS) of at least one. In one embodiment, the antibody is a tumor It comprises at least one ABS that is specific to necrosis factor α (TNFα) antigen. In one embodiment, anti The body contains an Fc domain capable of interacting with the Fc receptor. In one embodiment, the antibody interacts with the Fc receptor. It contains an Fc domain that cannot interact with it.

[0111] In one embodiment, the payload is a hormone. In another embodiment, the payload is nutrition It is a nutritional supplement food. In certain embodiments, the payload is an antibacterial agent.

[0112] In certain embodiments, the protein construct contains a plurality of payloads that may be the same or different. including.

[0113] In a particular embodiment, the payload binds to the C-terminus of a component that targets BTNL3 / 8. In another embodiment, the payload binds to the N-terminus of a component that targets BTNL3 / 8.

[0114] (6.3.1. Polypeptide) In various embodiments, the payload is a polypeptide. In certain embodiments, the payload is a polypeptide in-frame fused to a component that targets BTNL3 / 8. In a particular embodiment, the payload is in-frame fused to the C-terminus of a component that targets BTNL3 / 8. In a particular embodiment, the payload is in-frame fused to the N-terminus of a component that targets BTNL3 / 8. combined.

[0115] In certain embodiments, the polypeptide payload is a cytokine. In certain embodiments , the payload is an anti-inflammatory cytokine such as interleukin 10 (IL-IO), interleukin 22 (IL-22) or transforming growth factor β (TGFβ).

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

[0117] In some embodiments, the payload is a peptide. In some embodiments, the payload is a peptide. D is a peptide that is in-frame fused to a component that targets BTNL3 / 8.

[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 site is a Fab fragment, Fv, scFv, tandem scFv, diabole Diabody, sc diabody, DART, tandem diabody (tandAb), minibody, camel type VH H, nanobody, or other antibody fragments or formats known to those skilled in the art are configured. Exemplary antibody and antibody fragment formats are described in Brinkmann et al., MABS, 2017, Vol. 9. This is described in detail in No. 2, 182-212, and the entire teaching is incorporated herein by reference. It can be done.

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

[0120] In one embodiment, the antibody comprises at least one antigen-binding site (ABS) specific for an anti-inflammatory cytokine such as interleukin 10 (IL-10), interleukin 22 (IL-22) or transforming growth factor β (TGFβ). In one embodiment, the antibody comprises at least one antigen-binding site (ABS) specific for an anti-proinflammatory agent.

[0121] In some embodiments, the antibody comprises at least one antigen-binding site (ABS) specific for a cytokine antigen. In one embodiment, the antibody is at least one ABS specific for a tumor necrosis factor α (TNFα) antigen and comprises (is comprises).

[0122] (6.3.2. Small molecule payload)

[0100] In certain embodiments, the payload is a small molecule. In certain embodiments, the small molecule is a therapeutic agent (i.e., a small molecule drug). In certain embodiments, the small molecule therapeutic agent is an immunomodulatory agent. In certain embodiments, the small molecule is an inhibitor or activator of a cellular protein (e.g., a receptor, other signaling molecule, enzyme or transcription factor). In certain embodiments, the small molecule therapeutic agent is a toxin.

[0123]

[0101] In some embodiments, the payload is a drug that binds by chemical conjugation to a component that targets BTNL3 / 8.

[0124] ​​​​​​​​​

[0102] To use the protein constructs disclosed herein to conjugate drugs A method for preparing antibody-drug conjugates (ADCs) that can be used is, for example, U.S. Patent 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 Patent No. 5,208,020, U.S. Patent 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 Open WO2005 / 077090, International release WO2005 / 082023, International release WO2006 / 065533, International release WO2007 / 03 0642, International Publication WO2007 / 103288, International Publication WO2013 / 173337, International Publication WO2015 / 057699, International Public release WO2015 / 095755, International release WO2015 / 123679, International release WO2015 / 157286, International release WO2017 / 165851, International publication WO2009 / 073445, International publication WO2010 / 068759, International publication WO2010 / 138719, Country International publication WO2012 / 171020, International publication WO2014 / 008375, International publication WO2014 / 093394, International publication WO201 4 / 093640, International release WO2014 / 160360, International release WO2015 / 054659, International release WO2015 / 195925, International publication WO2017 / 160754, Storz's literature (MAbs. 2015 Nov-Dec; 7(6): 989-1009), Lambert (AdvTher, 2017 34: 1015), Diamantis et al. (British Journal of Cancer, 2 016, 114, 362-367), Carrico et al.'s paper (Nat Chem Biol, 2007. 3: 321-2), We et al.'s paper (P roc Natl Acad Sci USA, 2009. 106: 3000-5), Rabuka et al.'s paper (Curr Opin Chem Biol., (2011 14: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (References: Nat Protoc., 2012 7:1052-67, Agarwal et al. (Proc Natl Acad Sci USA., 20 13, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfiel See the literature by d et al. (Drug Dev. and D., 2014, 14:34-41) and the literature by Drake et al. (Bioconjugate Chem., 2 014, 25:1331-41), Liang et al.'s paper (J Am Chem Soc., 2014, 136:10850-3), Drake et al.'s paper References (Curr Opin Chem Biol., 2015, 28:174-80), and the literature by York et al. (BMC Biotechnology, 2 As described in 016, 16(1):23), each of the above teachings is incorporated herein by reference in its entirety. It gets included.

[0125] (6.3.3. Nucleic Acid Payload)

[0103] In one embodiment, the payload is nucleic acid. The nucleic acid is not limited to the following: However, dsDNA, mRNA, miRNA, lncRNA and siRNA, piRNA, snoRNA, snRNA, exRNA and scaR DNA or RNA, such as NA, is also acceptable.

[0126] (6.3.3.1 Any linker)

[0104] The protein constructs described herein may optionally target the component Peyrot Includes a linker that connects to the 'd'.

[0127]

[0105] In some embodiments, any linker fuses in frame to the target component. It is a combined peptide. In one embodiment, an optional linker is the C-terminus of the target component. The linker is fused with the N of the target component. In one embodiment, any linker is N of the target component. It is fused into the frame at the end.

[0128]

[0106] In some embodiments, any linker conjugates the target component It is a molecule that has been modified. In various embodiments, the protein construct is an additional functional element. Downstream processes such as coupling (for example, the payload and components targeting BTNL3 / 8) It has been modified to include a functional group or a chemically reactive group that can be used in downstream purification processes. In this embodiment, the linker is a cleavable molecule (e.g., site-specific protease or ligature). It contains a peptide that can be cleaved by other molecules that allow the linker to be cleaved into two or more fragments. In one embodiment, the modification is, but is not limited to, a reactive thiol (e.g., Maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based Reactive groups (e.g., reactive alkyne groups), and holmi It is a chemically reactive group containing an aldehyde having glycine (FGly). In one embodiment, modified The mutation is not limited to the following, but is a result of affinity peptide sequences (e.g., HA, HIS, FLAG, GS). It is a functional group containing T, MBP, and Strep systems, etc. In one embodiment, the functional group or The chemically reactive group has a cleavable peptide sequence. In a special embodiment, the cleavable peptide Butids are cleaved by, but are not limited to, photocleavage, chemical cleavage, protease cleavage, and It is cleaved by means including the original conditions and pH conditions. In a special embodiment, a protease The cleavage is carried out by intracellular proteases. In a specific embodiment, protease cleavage occurs. This is carried out by extracellular or membrane-bound proteases. AD employs protease cleavage. C therapy is described in detail in the literature by Choi et al. (Theranostics, 2012; 2(2): 156-178.) The entire instruction is incorporated herein by reference.

[0129]

[0107] In one embodiment, in addition to binding the target component to the payload, the linker, The molecule is incorporated into the functional elements of the protein construct (e.g., the target component and payload). It can be used to enable site-specific conjugation. In one embodiment, Linkers are used to identify or detect protein constructs in vitro or in vivo. It can be used in addition to the following. In one embodiment, the protein construct is any more than one phosphorus Includes car.

[0130] (6.4. Other Target Components)

[0108] In some embodiments, recombinant homodimers and heterodimers are described herein. A panel or library of Mahitt γδTCR, and this panel or library, γδT A specific allohuman binding domain or partner that determines or promotes the tissue-specific distribution of cells. This is a method used to determine the binding partner. The binding partner is often a human-derived "autoantigen". Until the findings described herein are made, further determination or characterization is extremely difficult. It was difficult / impossible. In one aspect of the present invention, the one shown in its natural cellular environment is different from the one shown in its natural cellular environment. This document describes recombinant human γδTCR proteins exhibiting similar tissue specificity or binding affinity. In aspects of the present invention, (i) to generate at least one recombinant γδTCR, and (ii) to generate one or more recombinant γδTCRs. A number of recombinant proteins, preferably expressed on the cell surface, potentially one or more (iii) displaying, presenting, or mixing with a similar bonding partner, and (iii) next This paper describes methods for identifying or verifying specific γδTCR / binding partner interactions. In a clear embodiment, the consequently identified γδTCR or its derived sequence is used as the target component. The drug payload is then deployed to target tissues or cells expressing the allogeneic binding partner. In further embodiments, the methods described herein are used to identify the specified Cells expressing the same-type binding partner are then targeted by another target component, such as an antibody or its derivative. It will be transformed.

[0131] (6.5. How to Create)

[0109] The protein constructs described herein are currently used in the production of T cell receptors or antibodies. Standard cell-free translation, transient transfection, and stable transfection methods are used. It can be easily produced by protein expression using a transfection approach.

[0132] (6.6. Purification method)

[0110] Suitable purification methods known to those skilled in the art can be used to purify the protein construct. The resulting protein is bound to affinity resin (for example, affinity tags on protein constructs). Using (a suitable material), unwanted proteins and protein complexes can be easily separated. Yes, it is possible. Further purification can be done using ion exchange chromatography, as is commonly used in this field. This can be done using Laffey.

[0133]

[0111] Methods for evaluating the effectiveness and efficiency of the purification step are well known to those skilled in the art and are not limited to the following: Although it is not possible to do so, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography This includes tography and mass spectrometry. Purity can also be evaluated according to various criteria. Yes, it is possible. Examples of criteria include, but are not limited to: 1) a fully assembled protein structure 1) Evaluation of the percentage of total protein in the eluate obtained from the structure, 2) Precision of the desired product The enrichment factor or increase rate of the manufacturing method is evaluated, for example, the fully assembled eluate Comparison of total protein obtained from protein constructs with that of the starting sample, 3) Total protein Percentage of the substance or undesirable products, for example, a decrease in the percentage of the incomplete complex. Evaluation of association, for example, specific undesirable products (e.g., non-associative single-chain polypeptides, Polypeptide chain dimers of any combination, or polyp This includes determining the percentage or reduction rate of the cytoplasmic chain trimmer.

[0134] (6.7. Pharmaceutical Compositions)

[0112] In another embodiment, the components that target BTNL3 / 8 and paylow as described herein A protein construct containing a drug, and a pharmaceutically acceptable carrier or diluent, A pharmaceutical composition is provided. In a typical embodiment, the pharmaceutical composition is sterile. In a certain embodiment, This specification describes any of the above protein constructs and pharmaceutically acceptable substances. It is a pharmaceutical composition containing a carrier.

[0135]

[0113] In one embodiment, the pharmaceutical composition is suitable for parenteral administration. In one embodiment, Administration is intravenous. In one embodiment, administration is intramuscular. In one embodiment The drug is administered subcutaneously.

[0136]

[0114] In various embodiments, the pharmaceutical composition contains a protein construct in a concentration of 0.1 mg / ml to 100 mg / ml Contains in concentrations. In special embodiments, the pharmaceutical composition contains the protein construct at 0.5 mg / ml, 1 mg / It is contained in concentrations of ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml, or 10 mg / ml. In some embodiments, the pharmaceutical composition contains a protein construct at a concentration greater than 10 mg / ml. In one embodiment, the protein constructs are 20 mg / ml, 25 mg / ml, 30 mg / ml, and 35 mg / ml. It is present in concentrations of 40 mg / ml, 45 mg / ml, or even 50 mg / ml or higher. In this application method, the protein construct is present at a concentration exceeding 50 mg / ml.

[0137]

[0115] In various embodiments, the pharmaceutical composition is U.S. Patent No. 8,961,964, U.S. Patent No. U.S. Patent No. 8,945,865, U.S. Patent No. 8,420,081, U.S. Patent No. 6,685,940, U.S. Patent Patent No. 6,171,586, U.S. Patent No. 8,821,865, U.S. Patent No. 9,216,219, U.S. Patent Application No. 10 / 813,483, International Publication WO2014 / 066468, International Publication WO2011 / 104381, and International Publication Details are provided in WO2016 / 180941, and each instruction in its entirety is included herein by reference. It will be incorporated.

[0138] (6.8. Compositions for use)

[0116] In one embodiment, a composition for use is also provided. Protein constructs containing components and payloads that target BTNL3 / 8 as described in the specification. A composition for therapeutic use containing is provided. The composition is, for example, for inflammatory symptoms. Inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, metabolic disorders, cancer, immune-related disorders, Treatment of autoimmunity, transplant rejection, post-traumatic immune response, graft-versus-host disease, ischemia, stroke, and infections. It can be used for this purpose.

[0139]

[0117] In one embodiment, the use of the composition for the manufacture of pharmaceuticals is also provided. In one embodiment, a tank containing a component and payload targeting BTNL3 / 8 as described herein. The use of a composition containing a protein construct for the manufacture of a pharmaceutical is provided, and the pharmaceutical is For example, inflammatory symptoms, 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 It is used for treating infections.

[0140] (6.9. Treatment)

[0118] In one embodiment, a treatment method which targets BTNL3 / 8 as described herein The protein construct, including its components and payload, is administered to the patient in an effective dose for treatment. A method is provided which includes giving. The protein constructs of this disclosure can be used by themselves or as a medical aid. In the form of a drug composition, for example, for inflammatory symptoms, inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease Diseases, metabolic disorders, cancer, immune-related disorders, autoimmunity, transplant rejection, post-traumatic immune response, graft pairs It may be administered to the subject for the treatment of host disease, ischemia, stroke, and infection.

[0141] (6.9.1.1 Gastrointestinal symptoms)

[0119] In one embodiment, the foregoing describes a treatment for gastrointestinal symptoms. Gastrointestinal symptoms are not limited to the following, but include immune-related symptoms of the gastrointestinal system, and gastrointestinal symptoms. Inflammatory symptoms, microbial infections of gastrointestinal tissues, and feeding disorders, metabolic disorders, or metabolic deficiencies. Symptoms include those caused by inflammatory gastrointestinal symptoms, but are not limited to those caused by inflammatory gastrointestinal symptoms. Intestinal diseases, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, and cancer (e.g., colon cancer) This includes rectal cancer, gastric cancer, etc. In some embodiments, what is described herein is gastrointestinal tissue B A method for treating a gastrointestinal condition expressing TNL3 / 8, wherein a therapeutically effective amount is used, as described in claims 62-66. The pharmaceutical composition described in any one of the above items is administered to a patient under conditions in which the gastrointestinal tissue expresses BTNL3 / 8. A method that includes administering. In one embodiment of this method, the payment of a protein construct The drug 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 drug. The anti-inflammatory agent may optionally be interleukin-10 (IL-10), interleukin-22 (IL-22), or tran. Forming growth factor β (TGFβ) is an anti-inflammatory cytokine. One embodiment Therefore, anti-inflammatory agents are anti-pro-inflammatory agents. In one embodiment, the anti-inflammatory agent is a steroid. In one embodiment, the steroid is a glucocorticoid. The corticoid is prednisone. In one embodiment, the glucocorticoid is hydrocorticoid. It is a lutizone. In one embodiment, the payload is an immunomodulator.

[0142] (6.9.2.1 Inflammatory bowel disease)

[0120] In one embodiment, the foregoing describes a therapeutically effective amount of any of the above. A treatment for inflammatory bowel disease comprising administering the described pharmaceutical composition to a patient with inflammatory bowel disease. In one embodiment, inflammatory bowel disease is ulcerative colitis. In one embodiment, inflammation The symptomatic enteric disease is Crohn's disease. In one embodiment, the payload of the protein construct is It 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 drug. Sexual cytokines, optionally interleukin-10 (IL-10), interleukin-22 (IL-22), or This is growth-forming growth factor β (TGFβ). In one embodiment, the anti-inflammatory payload is It is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent payload is a steroid. In one embodiment, the steroid is a glucocorticoid. The corticoid is prednisone. In one embodiment, the glucocorticoid is hydrocorticoid. It is a lutisone. In one embodiment, the payload of the protein construct is an antibiotic. In one embodiment, the antibiotic payload is rifaximin, ciprofloxacin, and The active ingredients are tronidazole, moxifloxacin, or amoxicillin. In one embodiment, The payload of the protein construct is a calcinurin inhibitor. In one embodiment, The synulin inhibitor is cyclosporine A or tacrolimus. In one embodiment, The payload of the protein construct is an immunomodulator. In one embodiment, the immunomodulator is immunomodulatory. It is an epidemic suppressant. In one embodiment, the immunosuppressant is azathioprine, 6-mercaptopurine. It is methotrexate or thiopurine. In one embodiment, the protein construct is The iload is a protein payload. In one embodiment, the protein payload is The protein payload is an antibody, an antibody fragment, or a single-stranded variable fragment. In one embodiment, the protein payload is an antibody. It includes an ABS that is specific to the TNFα antigen and (comprises) at least one. In this embodiment, the protein payload is adalimumab, infliximab, or certo It contains the complementarity-determining region (CDR) of lizumab. In one embodiment, the protein payload is It comprises at least one ABS that is specific to the interleukin antigen. In one embodiment, Tahloikin is IL-12, IL-23, or a combination thereof. In one embodiment, The protein payload contains a CDR of ustekinumab or brikinumab. In this embodiment, the biologic payload is specific to the integrin antigen, with a small amount of It contains at least one ABS. In one embodiment, the integrin is an α4 integrin. In one embodiment, the protein payload is infliximab, natalizumab, or vedo Contains lizumab CDR. In one embodiment, the protein construct contains an analgesic payload. In one embodiment, the protein construct includes a nutritional supplement payload.

[0143] (6.9.3.1 infection)

[0121] In one embodiment, the foregoing describes a therapeutically effective amount of any of the above. A treatment for microbial infection, comprising administering the pharmaceutical composition to a patient with a microbial infection. Yes. In one embodiment, the payload is an antimicrobial agent. In one embodiment, the antimicrobial agent is an antimicrobial agent. These are parasitic agents, antibiotics, antifungal agents, or antiviral agents.

[0144] (6.9.4.1 Metabolic disorders or metabolic deficiencies)

[0122] In one embodiment, what is described herein is any of the above-mentioned therapeutically effective amounts This includes administering the pharmaceutical composition to patients with metabolic disorders or metabolic deficiencies. It is a treatment for metabolic deficiencies. In one embodiment, the payload is a nutritional supplement. In this embodiment, the nutritional supplement is an enzyme or a vitamin.

[0145] (6.9.5.1 Regulation of the immune system)

[0123] In one embodiment, what is described herein is any of the above-mentioned therapeutically effective amounts A method for controlling the immune system, comprising administering the pharmaceutical composition to a patient having immune-related symptoms. Yes. In one embodiment, the payload is an immunosuppressant. The antidepressants are azathioprine, 6-mercaptopurine, methotrexate, or thioprine. In one embodiment, the payload is an immunostimulant. These are cytokines. [Examples]

[0146] (6.10. Examples)

[0124] The following embodiments are provided for illustrative purposes only and are not intended to limit the present invention.

[0147] (6.10.1.1 method)

[0125] Design and analysis of protein constructs containing BTNL3 / 8 target components including the TCR Vγ domain. The following are non-restrictive and illustrative methods for this purpose: Isolation of primary lymphocytes, BTNL3 / 8 cells. The co-culture and deep sequencing methods with current HEK293 cells were also described by Di Marco Barros et al. This is described in the literature, Cell. 2016, (167), pp. 203-218.

[0148] (Isolation of human samples and primary lymphocytes)

[0126] Endoscopic biopsy of the ascending colon of an adult donor undergoing a colonoscopy as part of routine examinations. We obtained primary intestinal lymphocytes from Clark et al.'s paper, 2006, J. Invest. Dermatol. (126), pp. 10. The biopsy sample was obtained by applying the method described in 59-1070. The biopsy sample was placed in 5 mL of washing medium (RPMI 1640 10% FCS, β-methylcellulose). Lucaptoethanol, penicillin [500 U / ml], streptomycin [500 mg / ml], metronidazole Dazole [5 mg / ml, Guy's Hospital, Pharmacy Department], Gentamicin [100 mg / ml, Sigma-Aldri] Washed for 20 minutes with [ch] and amphotericin 12.5 mg / ml [Thermo Fisher Scientific]. 1 The endoscopic biopsy samples are placed on each matrix, inverted, and pressure is applied to transfer the biopsy samples to the matrix. It was pushed inside. The matrix was placed in a 24-well plate (one per well) and 2 mL was used. RPMI 1640 (10% FCS, β-mercaptoethanol, penicillin [100U / ml], streptomycin) Syn [100 mg / ml], metronidazole [1 mg / ml], gentamicin [20 mg / ml], amphoteric acid Syn (2.5 mg / ml), IL-2 (100 U / mL, Novartis Pharmaceutical, UK), and IL-15 (10 ng / mL) The culture medium was covered with a solution supplemented with Biolegend. 1 ml of culture medium was aspirated every other day (on the second day each day) and concentrated to 2x concentration. The culture medium was replaced with complete medium containing condensation cytokines. The cells were harvested, and the remaining biopsy samples and empty wells were removed. Washed with PBS 0.02 mM HEPES. Passed the cell suspension through a 70 mm nylon cell strainer and filled with 400 g. Centrifuge for 5 minutes, resuspend in complete medium without additional cytokines, and immediately They were placed in a co-culture. Lymphocytes were used after 5-7 days of culture. PBMCs were obtained from a blood donation service. It was isolated from the blood using a Ficoll gradient. (HEK293T co-culture assay) 5 × 10⁻¹⁰ cells transduced using either an empty vector (EV), BTNL3, BTNL8, or BTNL3+8. 5 HEK293T Cells, and newly collected 2 × 10 5 The first generation human lymphocytes were supplemented in a 96-well plate. The cells were co-cultured in a complete medium without cytokines and incubated at 37°C and 5% CO2 for 16 hours.

[0149] (Deep sequencing)

[0127] TCRδ CDR3 derived from RNA purified from sorted Vγ7+IEL of the mouse TRDV gene. Amplification and sequencing were performed using the Amp2Seq Platform (iRepertoire). The amplification and sequencing of the human TCRGVγ gene:TCRγ CDR3 was performed using the immunoSEQ Platform. The procedure was performed using m (Adaptive Biotechnologies).

[0150] (Design of soluble γδTCR heterodimers)

[0128] Soluble containing the constant regions of T cell receptor α and T cell receptor β used in the following examples The design of the γδTCR heterodimer is described in Xu et al., PNAS, 2011 Vol. 108; pp. 2414-241. I followed the instructions in 9.

[0151] (6.10.2.1 Example 1: The γδTCR variable region isolated from interepithelial lymphocytes derived from human intestinal tissue is (This induced TCR activity in BTNL3 / 8.)

[0129] The TCR variable region of human intestinal interepithelial lymphocytes (IELs) that are BTNL3 / 8 responsive was cross-sectionalized. The cells were modified and expressed in TCR-deficient cells (Figure 2). IEL was isolated from human intestinal tissue and expressed in HEK293T cells. Co-culture was performed in cells, and BTNL3 / 8 was simultaneously overexpressed. Next, TCR activation (high expression of CD25 and da Responsive IELs exhibiting unregulation (Vγ) were sorted in single cells. The mutation region was amplified and cloned into a lentiviral expression vector (Figure 3A). TCR-deficient Jurkha T cells (J76 cell line) were transduced and co-cultured with HEK293 T cells expressing BTNL3 / 8 (Figure 3B). J76 cells were sorted for TCR activation (CD69 expression and TCR downregulation). Figure 4A). Anti-CD3 antibody was used as a positive control for TCR activation. Co-cultured with BTNL3 / 8-expressing cells. When cultured, J76 cells express a transduced TCR (H7 TCR) having Vγ4 and Vδ1 domains. This showed increased CD69 expression and downregulation of γδTCR. 3 transduced with Vγ4Vδ1 B3, C11, and H7, which are two independent J76 lines, are three different CDs obtained in the manner shown in Figure 2. Although representative of the R3 pair, unlike the Vγ9Vδ2 lineage (Vγ9Vδ2), it responded to BTNL3 / 8 expressing cells (Figure). 4B). These results indicate that the Vγ4Vδ1 domain of human IEL contributes to TCR responsiveness to BTNL3 / 8. To indicate that it is sufficient.

[0152] (6.10.3.1 Example 2: CDR4 of Vγ4 is required for TCR responsiveness to BTNL3 / 8)

[0130] In order to identify the Vγ4 region which is important for responsiveness to BTNL3 / 8, all Vγ4 regions of the H7-responsive TCR system The γ4 domain was replaced with the Vγ2 region (Figure 5). When co-cultured with HEK293T expressing BTNL3 / 8, CD69 expression % in transduced J76 cells expressing Vγ4 TCR (H7 WT) or Vγ2-substituted TCR The multiple change (FC) and TCR downregulation percentage were determined. Responsive Vγ4 H7 T If the entire V region of CR is replaced with the Vγ2 coding sequence (Vγ2 H7), then CDR3γ and the entire δ chain are replaced. (Not done), TCR activation by BTNL3 / 8-expressing cells was lost. However, responsive Vγ4 H7 TCR CD R1 (H7 CDR1) Vγ2 ) and / or CDR2 (H7 CDR2) Vγ2 ) replaces with the Vγ2 code sequence, resulting in BTNL3 / 8 TCR activation by the present cells was maintained. These results suggest that CDR4 is involved in the TCR response to BTNL3 / 8. To indicate that it is necessary.

[0153]

[0131] In order to further elucidate the region within Vγ4 that is essential for the response to BTNL3 / 8, located in CDR4 Two pairs of amino acids were substituted with the Vγ2 sequence (Figures 6 and 7). Co-cultured with HEK293T expressing BTNL3 / 8. The multiplicative change (FC) of CD69 expression percentage in transduced cells during nourishment was determined, and the Vγ4 TCR was also determined. Expression (H7 WT), expression of Vγ2 TCR with H7 CDR3 (Vγ2H7), and with amino acid substitutions within CDR4 We determined the percentage of TCR downregulation in J76 cells expressing Vγ4 TCR. Figure 7). YA substitutions at amino acid positions 87 and 90 resulted in the loss of TCR activation by BTNL3 / 8-expressing cells. On the other hand, NL substitutions at amino acid positions 94 and 98 do not cause TCR activation by BTNL3 / 8-expressing cells. These results indicate that amino acids at positions 87 and 90 of the CDR4 region of Vγ4TCR are TC to BTNL3 / 8. We have confirmed that it is essential for R responsiveness.

[0154] (5.10.4.1 Example 3: Soluble TCR Vγ4 / Vδ heterodimer binds to BTNL3 / 8 expressing cells)

[0132] Soluble Vγ / Vδ TCR heterodimers are expressed via leucine zipper complementation. Stabilized (Figure 8). The Vγ or Vδ domain is stable in TCRα or TCRβ lacking a transmembrane domain. It is in-frame fused into the region, followed by a leucine zipper sequence and a histidine tag / phosphorus sequence. The car continues. The Vγ4 / Vδ1 heterodimer corresponds to sequence numbers 10 and 9. The Vγ4 / Vδ2 heterodimer The heterodimer corresponds to SEQ ID NOs: 10 and 11. The Vγ2 / Vδ1 heterodimer corresponds to SEQ ID NO: 1 Corresponds to numbers 2 and 9. The Vγ8 / Vδ1 heterodimer corresponds to sequence numbers 13 and 9.

[0155]

[0133] HEK2 transduced using a soluble TCR with Flag-BTNL3+HA-BTNL8 or an empty vector. 93T cells were stained (Figure 10). Vγ4 / Vδ1 soluble TCRs and Vγ4 / Vδ2 soluble TCRs were identified as BTNL3+BTNL8 It shows strong binding to cell lines expressing [the specified gene], but does not bind to empty vector (EV) control cell lines. Result Soluble TCRs expressing the Vγ4 / Vδ1 domain or the Vγ4 / Vδ2 domain are found in BTNL3 / 8 expressing cells. This indicates that it binds to cells but not to cells lacking BTNL3 / 8. In summary, this result This indicates that Vγ4 CDR4 is essential for the BTNL3 / 8-led TCR response, and that Vγ4 CDR4 is essential for the BTNL3 / This also suggests interaction with 8.

[0156]

[0134] Soluble Vγ4 to cells expressing BTNL3+8 + To further evaluate the binding of TCR constructs HEK293T cells are encoded with the presented BTNL3 and BTNL8 constructs or empty vectors (EVs). The cells were transduced using pCSIGPW. Next, the cells were incubated with soluble His-tagged Vγ4δ2 TCR at 4°C for 45 minutes. Stain, wash twice, stain with APC anti-His tag antibody (α-His) at 4°C for 45 minutes, wash twice again, The cells were then analyzed by flow cytometry (Figure 9A). The cell populations shown in Figure 9B were identified as anti-FLAG and anti-HA. Parallel staining with antibodies revealed a lack of soluble TCR binding, which is attributed to the failure of BTNL3+8 construct expression. This was not confirmed. This result indicates that soluble TCRs bind to cells expressing BTNL3+BTNL8. The construct demonstrates its capabilities, and this soluble TCR construct is Vγ4 + Unable to induce a response by T cells As previously described in literature, IgV domain variants (e.g., L3 GQFSS , L3 RI , L3 YQKAI ) It did not bind to any of the following. Referring to Melandri et al., Nat. Immunol. 2018, this indicates that... The entire text is incorporated herein by reference.

[0157] (6.10.5.1 Example 4: Soluble TCRs that bind to BTNL3 / 8-expressing cells undergo internal migration.)

[0135] Is the soluble TCR bound to the surface of cells expressing BTNL3+BTNL8 moving internally? To determine the difference, HEK293T cells were transduced to produce wild-type BTNL3 and BTNL8 (293T.L3L8). The cells were expressed and stained with soluble His-tagged Vγ4Vδ2 TCR at 37°C for up to 120 minutes (Figure 11B). 293T.L3L8 cells were stained with soluble His-tagged Vγ4Vδ2TCR at 4°C for 120 minutes and then incubated at low temperature. The cells were used as a control in which translocation was inhibited (Figure 11A). Next, the cells were subjected to APCα-His tag antibody (α-Hi Staining was performed at 4°C for 45 minutes. As a result, the decrease in the APC signal was observed when the sample was incubated at 37°C. This indicates that it occurs over time within a cell population and that cells rapidly transport soluble TCR constructs internally. Figure 12).

[0158]

[0136] The internal migration of soluble TCRs is specific, or the rapid cycling of cell surface BTNL molecules To determine if it was a result of cycling, the experiment was repeated to test for soluble TCR against BT Compared with NL3 antibody (rabbit polyclonal, Aviva Biosystems) (including references). HEK29 3T cells were transduced using an empty vector (293T.EV) and used as a negative control in staining with α-BTNL3. HEK293T cells were used in the BTNL3 construct L3. RI L8 (293T.L3 RI Transduction was performed at L8, and in soluble TCR It was used as a negative control in staining. The results showed that α-BTNL3 staining was identical at 4°C and 37°C. This shows that α-BTNL3 specifically binds to cells expressing BTNL3 / 8, while the antibody does not bind to cells expressing BTNL3 / 8. It was shown that the cells remained on the surface (Figure 13A). The quantification of the results is shown in Figure 13B.

[0159] (6.10.6.1 Example 5: Soluble TCR delivers payload to BTNL3 / 8 expressing cells)

[0137] The payload may be delivered into cells via binding of soluble TCRs to BTNL3 / 8 expressing cells. To determine whether to do so, use 293T.L3L8 or 293T.L3 RI L8 cells, APCα-His tagged antibody Then, the soluble TCR construct is incubated with a pre-labeled soluble TCR on the carboxyl terminus. The complex was administered at a concentration of either 3 μg / mL or 10 μg / mL at either 4°C or 37°C for 1 hour. The cells were incubated. Next, the cells were washed and treated with trypsin or DMEM (control) for 15 minutes as shown in Figure 14. The cells were processed in this manner. The result was that the fraction of soluble TCR + α-His signaling (APC fluorescence) was found in the cells. The complex was incubated with the sample at 37°C to protect it from trypsin by internal migration of the complex. In this case, the results were larger after trypsin treatment compared to incubation at 4°C. (Figure 15A). Therefore, the result is that the internal translocation of soluble TCRs allows the payload to be ejected from BTNL3 / 8 cells within the cell. This demonstrates that it can be used as a means of delivery to living cells. The quantification of the results is shown in Figure 15B. .

[0160]

[0138] Using imaging cytometry, the soluble T of the APCα-His antibody payload was determined. Intracellular delivery via CR was visualized. 293T.L3 RIL8 or 293T.L3L8 cells were treated with soluble TCR+α-His anti- The body complex was incubated with the DMEM for 1 hour at 4°C or 37°C. Next, (as described above) DMEM or Alternatively, the cells are treated with trypsin, fixed and permeabilized (Figure 16A), or fixed and permeabilized, and then... Stained with endosomal marker CD107a (Figures 16B and 16C). Negative control imaging site. Metrics allowed for evaluation of the background APC signal (Figure 16A). TCR+α-H The imaging cytometry results of cells incubated with the antibody complex showed that the complex When incubated with cells at 4°C, after DMEM treatment, the complex was visualized around the cells. While it was shown that the majority of the complex is bound to the cell surface, after trypsin treatment... Gunar was completely lost (Figure 16B). However, the complex incubated with cells at 37°C CD107a + It is detected in the intracellular region near 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, and CDR3 derived from a 30MZ crystal structure, and TCRα stationary with in-frame fusion of CDR3. Region, leucine zipper, and C-terminal His tag [SEQ ID NO: 9] [ka] >Human Vγ4, and CDR3 derived from crystal structure 4MNH, the TCRβ constant region fused with CDR3, leucine Zipper [Sequence ID: 10] [ka] Human Vδ2, and CDR3 derived from a 30MZ crystal structure, and TCRα stationary with in-frame fusion of CDR3. Region, leucine zipper, and C-terminal His tag [SEQ ID NO: 11] [ka] >Human Vγ2, and CDR3 derived from crystal structure 4MNH, the TCRβ constant region fused with CDR3, leucine Zipper [Sequence ID: 12] [ka] >Human Vγ8, and CDR3 derived from crystal structure 4MNH, the TCRβ constant region fused with CDR3, leucine Zipper [Sequence ID: 13] [ka] >Human Vγ4 leader sequence [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 citing literature) All publications, patents, patent applications and other documents referenced in this application are subject to the individual publications, patents, patent applications and other documents. , when a patent application or other document is incorporated by reference for all its purposes, it is indicated separately. To the same extent as it is incorporated herein by reference in its entirety for all purposes, To be absorbed.

[0163] (8. Equals) While various specific embodiments are illustrated and described, the description in the above specification is limited. No. Various modifications can be made without departing from the spirit and scope of the present invention. It will be understood that there are many variations of the present invention. It should be clear. This application provides the invention in the following embodiments. (Aspect 1) Components that target BTNL3 / 8; Payload; and Any linker that binds the target component to the payload A protein construct that includes [this component]. (Aspect 2) The component targeting BTNL3 / 8 includes a Vγ domain. The amino acid at sequence position number 87 of the Vγ domain is aspartic acid or histidine, V The amino acid at sequence position number 90 of the γ domain is glycine or glutamic acid, and The remaining residues of the Vγ CDR4 are, at their respective positions, human or mouse (murine) Vγ domains. A protein construct according to embodiment 1, independently selected from the corresponding residues of n. (Aspect 3) The remaining residues of the Vγ domain CDR4 are human Vγ4, human Vγ2, at their respective residue positions. Alternatively, a protein construct according to embodiment 2, independently selected from the corresponding residues of mouse Vγ7. (Aspect 4) The amino acid sequence at positions 87-90 of the Vγ domain is sequence number 1, as described in embodiment 2. Protein constructs. (Aspect 5) The amino acid sequence at positions 87-90 of the Vγ domain is sequence number 2, as described in embodiment 2. Protein constructs. (Aspect 6) All remaining residues of the Vγ domain CDR4 are paired with human Vγ4, human Vγ2, or mouse Vγ7. A protein construct according to embodiment 3, selected from the corresponding residues. (Aspect 7) The remaining residues of Vγ CDR4 are selected from the corresponding residues of human Vγ4, as described in embodiment 6. Plain structure. (Pattern 8) The remaining residues of Vγ CDR4 are selected from the corresponding residues of human Vγ2, according to embodiment 6. Plain structure. (Aspect 9) The remaining residues of Vγ CDR4 are selected from the corresponding residues of mouse Vγ7, as described in embodiment 6. A structure made of protein. (Aspect 10) The aforementioned Vγ domain is the human Vγ2 domain, and within it, the amino acid of CDR4 is an amino acid Substituted with aspartic acid or histidine at sequence position 87, and at amino acid sequence position 90 The protein construct according to embodiment 2, which is substituted with glycine or glutamic acid. (Aspect 11) The protein construct according to embodiment 2, wherein the Vγ domain is a human Vγ4 domain. (Aspect 12) The Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence, as in any of embodiments 1 to 11. Protein construct as described in item 1. (Aspect 13) The protein construct according to embodiment 12, wherein the Vγ domain CDR3 comprises a human Vγ4 CDR3 sequence. (Aspect 14) The protein construct according to embodiment 12, wherein the Vγ domain CDR3 comprises a human Vγ2 CDR3 sequence. (Aspect 15) The Vγ domain CDR3 comprises a mouse Vγ7 CDR3 sequence in the protein construct according to embodiment 12. 。 (Aspect 16) The protein construct according to any one of embodiments 2 to 15, wherein the J region is a Vγ J region. (Aspect 17) The protein construct according to embodiment 16, wherein the J region is a human Vγ J region. (Aspect 18) The protein construct according to embodiment 16, wherein the J region is the mouse Vγ J region. (Aspect 19) The J region includes a sequence number selected from the group consisting of sequence numbers 15 to 18, as described in embodiment 16. Protein constructs. (Aspect 20) The component targeting BTNL3 / 8 further includes a paired Vδ domain, as described in embodiments 1 to 19. A protein construct as described in any one of the items. (Aspect 21) The Vγ domain and Vδ domain are covalently connected by at least one disulfide bond. A protein construct according to embodiment 20, which is bound to [the specified protein]. (Aspect 22) The Vγ domain and Vδ domain are paired by specific heterodimeric interactions. The protein construct according to embodiment 21. (Aspect 23) The aforementioned heterodimeric interaction is leucine zipper complementation, according to embodiment 22. A concrete structure. (Aspect 24) The protein construct according to embodiment 23, wherein the target component includes Sequence ID No. 9. (Aspect 25) The protein construct according to embodiment 23, wherein the target component is Sequence ID No. 10. (Aspect 26) The protein construct according to embodiment 23, wherein the target component is Sequence ID No. 11. (Aspect 27) The target component includes a single-strand in-frame fusion of the Vγ domain and the Vδ domain. , the protein construct according to embodiment 20. (Aspect 28) The protein construct according to embodiment 27, wherein the Vγ domain is the N-terminus of the Vδ domain. (Aspect 29) The protein construct according to embodiment 27, wherein the Vγ domain is the C-terminus of the Vδ domain. (Aspect 30) The single-stranded in-frame fusion of the Vγ and Vδ domains includes an internal linker sequence. The protein construct according to any one of the embodiments 27 to 29. (Aspect 31) The Vδ domain is a human Vδ domain, as described in any one of embodiments 20-24 or 27-30. Protein constructs. (Aspect 32) The protein construct according to embodiment 31, wherein the human Vδ domain is Vδ1, Vδ2, or Vδ5. (Aspect 33) The protein construct according to embodiment 32, wherein the human Vδ domain is Vδ1. (Aspect 34) Furthermore, the protein construction according to any one of embodiments 2 to 33, which also includes the first T cell receptor constant region. The substance is such that the constant region of the first T cell receptor is in-frame fused to the C-terminus of the Vγ domain. The aforementioned protein construct. (Aspect 35) The first T cell receptor constant region is the human T cell receptor constant region, as described in embodiment 34. Plain structure. (Aspect 36) The first T cell receptor constant region is the human T cell receptor β constant region, as described in embodiment 35. A structure made of protein. (Aspect 37) The first T cell receptor constant region is the human T cell receptor α constant region, as described in embodiment 35. A structure made of protein. (Aspect 38) The first T cell receptor constant region is the human T cell receptor γ constant region, as described in embodiment 35. A structure made of protein. (Aspect 39) The target component further includes the constant region of the second T cell receptor, as in any of embodiments 20 to 38. The protein construct according to item 1, wherein the constant region of the second T cell receptor is a paired Vδ The protein construct is in-frame fused to the C-terminus of the domain. (Approach 40) The second T cell receptor constant region is the human T cell receptor α constant region, as described in embodiment 39. A structure made of protein. (Aspect 41) The second T cell receptor constant region is the human T cell receptor β constant region, as described in embodiment 39. A structure made of protein. (Aspect 42) The second T cell receptor constant region is the human T cell receptor δ constant region, as described in embodiment 39. A structure made of protein. (Aspect 43) The in-frame fusion of the Vδ domain and the constant region of the second T cell receptor is performed by the Vδ domain and Any one of embodiments 39 to 42, including an internal linker sequence between the constant region of the second T cell receptor and the second T cell receptor. The protein construct described. (Aspect 44) The payload is a protein payload in-frame fused to the target component. A protein construct according to any one of embodiments 1 to 43. (Aspect 45) The protein construct according to embodiment 44, wherein the payload is a polypeptide. (Aspect 46) The protein construct according to embodiment 45, wherein the payload is a peptide. (Aspect 47) The protein construct according to embodiment 45, wherein the payload is a cytokine. (Aspect 48) The protein construct according to embodiment 45, wherein the payload is an antibody. (Aspect 49) The protein construct according to embodiment 48, wherein the payload is a single-stranded variable fragment (scFv). (Appearance 50) The antibody comprises at least one antigen-binding site (ABS) that is specific to the cytokine antigen. , a protein construct according to any one of embodiments 47 to 49. (Aspect 51) The aforementioned antibody is at least one ABS specific to tumor necrosis factor α (TNFα) antigen, A protein construct according to any one of embodiments 47 to 49. (Appearance 52) The antibody comprises an Fc domain capable of interacting with the Fc receptor, according to any of embodiments 47- or 49-51. A protein construct as described in any one of the items. (Aspect 53) The antibody comprises an Fc domain that is incapable of interacting with the Fc receptor, according to any of embodiments 47 or 48-51. A protein construct as described in any one of the items. (Aspect 54) The payload is a small molecule, as described in any one of embodiments 1 to 42. (Aspect 55) The protein construct according to embodiment 54, wherein the payload is a hormone. (Aspect 56) The protein construct according to embodiment 54, wherein the payload is nucleic acid. (Aspect 57) The protein construct according to embodiment 56, wherein the payload is inhibitory RNA (RNAi). (Pattern 58) The aforementioned linker is a peptide in-frame fused to the target component, Embodiment 1 Protein constructs as described in any one of items ~57. (Aspect 59) The aforementioned linker is in-frame fused to the C-terminus of the target component, Embodiment 5 Protein constructs as described in 8. (Appendix 60) The aforementioned arbitrary linker is in-frame fused to the N-terminus of the target component, Embodiment 5 Protein constructs as described in 8. (Aspect 61) The aforementioned linker is a molecule conjugated to the target component, as in embodiments 1-5. A protein construct as described in any one of item 7. (Aspect 62) Protein constructs and pharmaceutically acceptable carriers according to any one of embodiments 1 to 61 A pharmaceutical composition containing the following: (Aspect 63) The pharmaceutical composition according to embodiment 62, wherein the pharmaceutical composition is suitable for parenteral administration. (Personal aspect 64) The pharmaceutical composition according to embodiment 63, wherein the administration is intravenous. (Patent 65) The pharmaceutical composition according to embodiment 63, wherein the administration is intramuscular. (Aspect 66) The pharmaceutical composition according to embodiment 63, wherein the administration is by subcutaneous injection. (Personal aspect 67) A method for treating a gastrointestinal condition in which gastrointestinal tissue expresses BTNL3 / 8, and the therapeutically effective dose. The pharmaceutical composition according to any one of embodiments 62 to 66 is used in a state in which gastrointestinal tissue expresses BTNL3 / 8. The method, which includes administering the medication to a patient in a certain condition. (Pattern 68) The method according to embodiment 67, wherein the payload of the protein construct is an anti-inflammatory agent. (Patent 69) The method according to embodiment 68, wherein the anti-inflammatory agent is an aminosalicylate. (Aspect 70) The method according to embodiment 68, wherein the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. (Aspect 71) The aforementioned anti-inflammatory agent is an anti-inflammatory cytokine, optionally interleukin-10 (IL-10), inter - Leukin-22 (IL-22) or transforming growth factor β (TGFβ) as described in Embodiment 68 method. (Aspect 72) The method according to embodiment 68, wherein the anti-inflammatory agent is an anti-inflammatory agent. (Aspect 73) The method according to embodiment 68, wherein the anti-inflammatory agent is a steroid. (Aspect 74) The method according to embodiment 73, wherein the steroid is a glucocorticoid. (Aspect 75) The method according to embodiment 74, wherein the glucocorticoid is prednisone. (Aspect 76) The method according to embodiment 74, wherein the glucocorticoid is hydrocortisone. (Aspect 77) The method according to embodiment 67, wherein the payload is an immunomodulator. (Pattern 78) A therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 62 to 66 is administered to a patient with inflammatory bowel disease. Treatment for inflammatory bowel disease, including administration to the patient. (Aspect 79) The method according to embodiment 78, wherein the inflammatory bowel disease is ulcerative colitis. (Aspect 80) The method according to embodiment 78, wherein the inflammatory bowel disease is Crohn's disease. (Aspect 81) The payload of the protein construct is an anti-inflammatory agent, as described in any one of embodiments 78 to 80. Method of loading. (Aspect 82) The method according to embodiment 81, wherein the anti-inflammatory agent is an aminosalicylate. (Aspect 83) The method according to embodiment 81, wherein the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug. (Pattern 84) The method according to embodiment 81, wherein the anti-inflammatory agent is an anti-inflammatory cytokine. (Pattern 85) The method according to embodiment 81, wherein the anti-inflammatory agent payload is an anti-inflammatory agent. (Pattern 86) The method according to embodiment 81, wherein the anti-inflammatory agent payload is a steroid. (Pattern 87) The method according to embodiment 86, wherein the steroid is a glucocorticoid. (Pattern 88) The method according to embodiment 87, wherein the glucocorticoid is prednisone. (Pattern 89) The method according to embodiment 87, wherein the glucocorticoid is hydrocortisone. (Aspect 90) The payload of the protein construct is an antibiotic, as described in any one of embodiments 78 to 80. Method of loading. (Aspect 91) The aforementioned antibiotic payload consists of rifaximin, ciprofloxacin, and metronidazole. The method according to embodiment 90, wherein the solution is moxifloxacin or amoxicillin. (Patent 92) The payload of the protein construct is a calcinurin inhibitor, according to embodiments 78-80. Either one of the methods described in item 1. (Aspect 93) The calcinurin inhibitor is cyclosporine A or tacrolimus, as described in Embodiment 92. The method. (Aspect 94) The payload of the protein construct is an immunomodulator, according to any one of embodiments 78 to 80. Method of description. (Aspect 95) The method according to embodiment 94, wherein the immunomodulator is an immunosuppressant. (Personal aspect 96) The immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or thio The method described in embodiment 95, which produces pudding. (Patent 97) The payload of the protein construct is a protein payload, as in embodiments 78-80. Either one of the methods described in item 1. (Pattern 98) The protein payload is an antibody, an antibody fragment, or a single-stranded variable fragment, as described in Embodiment 97. The method. (Pattern 99) The protein payload is specific to the TNFα antigen and (comprises and) is small The method according to embodiment 97 or 98, comprising ABS which is one. (Aspect 100) The protein payload is adalimumab, infliximab, or certolizumab. The method according to embodiment 99, including a complementarity determination region (CDR). (Aspect 101) The protein payload contains at least one ABS specific to interleukin antigens. The method according to embodiment 97 or 98, including the method described in embodiment 97 or 98. (Aspect 102) The interleukin is IL-12, IL-23, or a combination thereof, as described in Embodiment 101. Method of loading. (Aspect 103) The protein payload is a CDR of ustekinumab or brikinumab. The method described in embodiment 102. (Aspect 104) The biologic payload comprises at least one ABS specific to the integrin antigen. , the method described in aspect 97 or 98. (Aspect 105) The method according to embodiment 104, wherein the integrin is α4 integrin. (Aspect 106) The protein payload is a CD of infliximab, natalizumab, or vedolizumab. The method according to embodiment 105, including R. (Aspect 107) The protein construct includes an analgesic payload, as described in any one of embodiments 78 to 80. method. (Aspect 108) The protein construct includes a nutritional supplement payload, any one of embodiments 78 to 80. Method of description. (Aspect 109) A therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 62 to 66 is administered to a patient with irritable bowel syndrome. Treatment for irritable bowel syndrome, including administration to the patient. (Aspect 110) Administer a therapeutically effective amount of the pharmaceutical composition described in any one of embodiments 1 to 66 to a patient with diverticulitis. Treatment for diverticulitis, including [specific treatment]. (Aspect 111) The method according to embodiment 109 or 110, wherein the payload is an antibiotic. (Aspect 112) The aforementioned antibiotic payload consists of rifaximin, ciprofloxacin, and metronidazole. The method according to embodiment 111, wherein the solution is moxifloxacin or amoxicillin. (Aspect 113) A therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 62 to 66 is administered to a patient with celiac disease. Treatment for celiac disease, including administration to the patient. (Aspect 114) The method according to embodiment 113, wherein the payload is an immunosuppressant. (Aspect 115) The immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or thio The method described in embodiment 114, which produces pudding. (Aspect 116) A therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 62 to 66 is used to treat a microbial infection. Treatment methods for microbial infections, including administration to patients. (Aspect 117) The method according to embodiment 116, wherein the payload is an antimicrobial agent. (Aspect 118) The antibacterial agent is an antiparasitic agent, an antibiotic, an antifungal agent, or an antiviral agent, as described in Embodiment 117. Method of loading. (Aspect 119) A therapeutically effective amount of the pharmaceutical composition described in any one of embodiments 62 to 66 is used to treat metabolic disorders or metabolic disorders. Treatment for metabolic disorders or metabolic deficiencies, including administration to patients with sexual deficiency. (Aspect 120) The method according to embodiment 119, wherein the payload is a nutritional supplement. (Aspect 121) The method according to embodiment 120, wherein the nutritional supplement is an enzyme or a vitamin. (Aspect 122) A therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 62 to 66 is administered to a patient with immune-related symptoms. A method for controlling the immune system, including administration to [a specific body part]. (Aspect 123) The method according to embodiment 122, wherein the payload is an immunosuppressant. (Aspect 124) The immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or thio The method described in embodiment 123, which produces pudding. (Aspect 125) The method according to embodiment 122, wherein the payload is an immunostimulant. (Aspect 126) The method according to embodiment 125, wherein the immunostimulator is a cytokine. (Aspect 127) At least a portion of the component and / or payload that targets BTNL3 / 8 is transferred into the cell. The method described in any one of the embodiments 67 to 126, which is carried out. (Aspect 128) The component and / or payload that targets BTNL3 / 8 is not transferred into the cell, embodiment 6 The method described in any one of items 7 to 126. (Aspect 129) It consists of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. A set of sequences including at least one sequence that has at least 90% sequence identity with a sequence from the group Replacement γδTCR protein. (Aspect 130) The aforementioned protein is sequence number 9, sequence number 10, sequence number 11, sequence number 12, and sequence number Two or more sequences that have at least 90% sequence identity with a sequence from the group consisting of number 13. Recombinant γδTCR protein according to embodiment 129, comprising: (Aspect 131) The protein contains a sequence having at least 90% sequence identity with sequence number 10. Recombinant γδTCR protein according to any one of embodiments 129 to 130. (Aspect 132) The aforementioned protein has at least 90% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 11. A recombinant γδTCR protein according to any one of embodiments 129 to 131, comprising the sequence. (Aspect 133) The aforementioned protein has at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 10. A recombinant γδTCR protein according to any one of embodiments 129 to 131, comprising the sequence. (Aspect 134) The aforementioned protein is either the His-tagged SEQ ID NO: 9 or the His-tagged SEQ ID NO: 11 Either or any one of the embodiments 129 to 133, which includes a sequence having at least 90% sequence identity. The recombinant γδTCR protein described.

Claims

1. Use of a pharmaceutical composition for the manufacture of a drug for treating a gastrointestinal condition in a patient whose gastrointestinal tissue is in a state of expressing BTNL3 / 8, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

2. Use of a pharmaceutical composition for the manufacture of a drug for treating inflammatory bowel disease in patients with inflammatory bowel disease, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

3. The use of a pharmaceutical composition for the manufacture of a drug for treating irritable bowel syndrome in patients with irritable bowel syndrome, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

4. Use of a pharmaceutical composition for the manufacture of a drug for treating diverticulitis in patients with diverticulitis, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

5. Use of a pharmaceutical composition for the manufacture of a drug for treating celiac disease in patients with celiac disease, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

6. Use of a pharmaceutical composition for the manufacture of a drug for treating a microbial infection in a patient with a microbial infection, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

7. The use of a pharmaceutical composition for the manufacture of a drug for treating a metabolic disorder or metabolic deficiency in a patient with a metabolic disorder or metabolic deficiency, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

8. Use of a pharmaceutical composition for the manufacture of a drug for controlling the immune system in patients with immune-related symptoms, wherein the pharmaceutical composition is: (i) Components that target BTNL3 / 8; Payload; and Any linker that binds the BTNL3 / 8 target component to the payload A protein construct that includes, The component targeting BTNL3 / 8 includes a T cell receptor (TCR) Vγ domain, the TCR Vγ domain includes a J region and complementarity-determining regions CDR1, CDR2, CDR3, and CDR4. The CDR4 of the TCR Vγ domain contains the amino acid sequence described in Sequence ID No.

3. The CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain. The CDR1 of the TCR Vγ domain contains an amino acid sequence selected from EGSTGY or EGSNGY, the CDR2 of the TCR Vγ domain contains an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and the CDR3 of the TCR Vγ domain contains an amino acid sequence selected from ATWDE or ATWDG, and The payload is a protein payload or a small molecule, the protein construct; and (ii) Medical career, The aforementioned use, including.

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

10. The use according to any one of claims 1 to 8, wherein the J region of the TCR Vγ domain comprises the human Vγ J region, the mouse Vγ J region, or the amino acid sequence described in sequence number 15 to 18.

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

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

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

14. The use according to claim 13, wherein the heterodimeric interaction is leucine zipper complementary.

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

10.

16. The use according to claim 11, wherein the component of the protein construct that targets BTNL3 / 8 includes a single-strand in-frame fusion of the Vγ domain and the Vδ domain.

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

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

19. The use according to claim 16, wherein the single-stranded in-frame fusion of the Vγ domain and the Vδ domain includes an internal linker sequence.

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

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

22. The use according to claim 21, wherein the first T cell receptor constant region is the human T cell receptor β constant region, the human T cell receptor α constant region, or the human T cell receptor γ constant region.

23. The component of the protein construct that targets BTNL3 / 8 further comprises the constant region of the second T cell receptor, The use according to claim 21, wherein the constant region of the second T cell receptor is in-frame fused to the C-terminus of the paired Vδ domains.

24. The use according to claim 23, wherein the second T cell receptor constant region is the human T cell receptor α constant region, the human T cell receptor β constant region, or the human T cell receptor δ constant region.

25. The use according to claim 23, wherein the in-frame fusion of the Vδ domain and the second T cell receptor constant region includes 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 in-frame fused to the component that targets BTNL3 / 8.

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, nucleic acid, or inhibitory RNA (RNAi).

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

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

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

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

33. The use according to 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. The use according to claim 33, wherein the steroid is a glucocorticoid.

35. The use according to 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. The use according to claim 36, wherein the antibiotic is rifaximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin.

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

39. The use according to claim 38, wherein the calcinurin inhibitor is cyclosporine 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-stranded variable fragment.

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

42. The use according to claim 41, wherein the protein payload comprises a CDR of adalimumab, infliximab, or certolizumab.

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

44. The use according to claim 43, wherein the protein payload comprises a CDR of ustekinumab or brikinumab.

45. The use according to claim 41, wherein the integrin is α4 integrin.

46. The use according to claim 45, wherein the protein payload comprises a CDR of infliximab, natalizumab, or vedolizumab.

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

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

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

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

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

52. The use according to claim 2 or 7, wherein the payload of the protein construct includes a nutritional supplement.

53. The use according to claim 52, wherein the nutritional supplement is an enzyme or a vitamin.

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

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

56. The pharmaceutical composition is suitable for parenteral administration, as described in any one of claims 1 to 8.

57. The use according to claim 56, wherein the parenteral administration includes intravenous, intramuscular, or subcutaneous administration.

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

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